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Low response rates and immune-related adverse events limit the impact of cancer immunotherapy. To improve clinical outcomes, preclinical studies have shown that combining immunotherapies with N-terminal Hsp90 inhibitors resulted in improved efficacy, even though induction of an extensive heat shock response (HSR) with these inhibitors limited their clinical efficacy as monotherapies. We discovered that Enniatin A (EnnA) binds to the interface between the middle domains of the Hsp90 dimer and destabilizes Hsp90 client oncoproteins without inducing an HSR. EnnA induces cancer cell immunogenic cell death in aggressive breast cancer models and exhibits superior anti-tumor activity compared to Hsp90 N-terminal inhibitors. EnnA reprograms the tumor microenvironment in syngeneic mouse models to promote CD8+ T cell-dependent anti-tumor activity mediated through a reduced level of PD-L1 and activation of CX3CR1 pathway. We propose that EnnA is a promising anti-tumor agent with a mechanism of action involving immunogenic cancer cell toxicity and mobilization of CD8+ T cells into the tumor site. Citation Format: Nada Eisa, Vincent M. Crowley, Asif Elahi, Vamsi K. Kommalapati, Hasan Korkaya, Abdessamad Debbab, Brian Blagg, Ahmed Chadli. Targeting the chaperone Hsp90 to activate the immune system and eradicate the triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2272.

Metastatic potential in basal-like breast cancers typically correspond with increased enrichment of EpCAM-/CD49f- cancer stem cells (CSC). With this premise in mind, it is important to better understand the mechanistic driver of these cell populations and their distinctive potential to interact with the tumor microenvironment (TME) for cancer promotion. Previous work from our lab has compared the 450K DNA methylation profile of EpCAM-/CD49f- poor breast cancer cell lines to that of EpCAM-/CD49f- enriched breast cancer cell lines and found the IL32 promoter to be hypomethylated in EpCAM-/CD49f- enriched cell lines, a result which corresponded basal-like patient samples in TCGA. By identifying IL32 being differentially regulated in CSC-enriched cell lines, we further sought to characterize IL32’s role in breast cancer aggressiveness. We first were able to identify several overarching mechanisms altered in siIL32 treated SUM15PT cells by RNAseq differential expression analysis (FDR p-value Citation Format: Megan A. Wilson, Elayne M. Benson, Emma Gray, Paige Cairns, Maria Ouzounova, Hasan Korkaya, Austin Y. Shull. Epigenetic driven IL32 expression contributes to a JNK related cell stress response in breast cancer stem cells to promote cellular invasion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3595.

Despite the advances in early diagnostics and therapeutics, women with metastatic breast cancer have limited treatment options. Women with TNBC, who constitute 15-20% of breast cancer patients, are often diagnosed with aggressive/metastatic disease. Advanced studies implicated immunosuppressive tumor microenvironment (TME) in aggressive/metastatic properties of TNBC subtype. Alternatively activated immature myeloid cells including tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), tumor-associated dendritic cells (TADC) and myeloid derived suppressor cells (MDSC) constitute a major component of TME. However, anti-tumorigenic microenvironment is also reported and that may have clinical relevance in early TNBC patients. Therefore, our hypothesis is that myeloid cells polarize to become immunosuppressive and infiltrate tumors and pre-metastatic niches in patients with advanced disease, while patients with early TNBCs may elicit anti-tumor immune response eliminating disseminated tumor cells (DTC). The utilization of syngeneic immunocompetent mouse models has contributed to our current understanding of immunosuppressive or immunomodulatory TME. Using these models, we have demonstrated that tumor dissemination and growth at metastatic sites is facilitated by MDSC’s. Emerging technologies; single cell RNA sequencing (scRNA-Seq), mass cytometry (CyTOF) or cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) has been powerful platforms for detailed characterization of tumors and TME compartments. We performed scRNA-Seq and CyTOF analyses of the myeloid cell populations of tumors and spleens from metastatic 4T1 and non-invasive EMT6 tumor-bearing mice. Tumors and spleens from 4T1 tumor-bearing mice exhibited a marked expansion of myeloid cell subsets that are characterized by the expression of immunosuppressive as well as progenitor markers. On the contrary, indicated tissues from EMT6 mice were enriched in NK cells, T and B lymphocytes and they were lacking immunosuppressive myeloid cell subsets. Furthermore, we identified a distinct differentiation pattern of immature myeloid cell subsets from neutrophil progenitors (NP) in 4T1 tumor-bearing mice. Using the murine TNBC models in syngeneic mice, we provide evidence that early TNBC tumors may elicit anti-tumor immune responses and thus the survival outcome in those patients is substantially increased after complete surgical resection of the primary tumors. Whereas immunosuppressive tumor microenvironment contributes to the poor overall survival in patients with advanced TNBCs. Therefore, identifying an anti-tumor immune signature in early TNBC patients may be utilized as a clinical biomarker before surgical intervention as well as improve the survival outcome. Citation Format: Hasan Korkaya, Elayne Benson, Fulya Koksalar Alkan, Justin Wilson, Tulshi Patel, Hilmi K. Alkan, Virginia McEvoy, Nika Shekastehband, Nate Francois, Huidong Shi, Catherine C. Hedrick. Phenotypic & functional diversity of tumor associated neutrophils in murine breast tumor models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4600.

We previously demonstrated that upregulation of A20 in TNBC subtype in response to TNFα protects these cells from cytotoxic cell death by upregulating HSP70 protein and maintaining EMT/CSC phenotype. In contrast, luminal MCF7 or ZR75-1 cells display approximately 70% apoptosis when treated with TNFα. Overexpression of A20 in luminal cells not only protected them from TNFα-induced cytotoxicity by upregulating HSP70 and EMT/CSC phenotype, but also exhibited aggressive metastatic properties in mouse xenograft models. We determined that TNFα-induced HSP70 upregulation in TNBC cell lines was dependent on A20 de-ubiquitinase activity that protected its degradation. Interestingly, our preliminary findings also suggested that A20 protein upregulation may be dependent on HSP70 chaperone activity. We show significant overexpression of HSP70 and A20 proteins in 4T1 cell line when treated with TNFα or chemotherapeutic agents. However, A20 expression is significantly reduced when we block HSP70 activity in cells treated with TNFα or chemotherapeutic agents (Docetaxel-DTX or Doxorubicin-DOX). We proposed that A20 transcriptional upregulation upon TNFα stimulation leads to suppression of E3-ligase and accumulation of HSP70 which then stabilizes A20 with chaperone activity. Based on our reasoning, we performed the LIMBO chaperone binding assay which predicted A20 being the potential HSP70 client protein. Furthermore, we show that A20/HSP70 pathway attracts tumor-infiltrating lymphocytes (TILs) while inducing the accumulation of immunosuppressive MDSCs in syngeneic mouse models. Interestingly, pulmonary DTCs as well as the immune infiltrates from 4T1 tumor-bearing mice exhibited significantly higher HSP70 expression. Therefore, targeting HSP70 will have a dual activity on tumors and MDSCs and thus it may potentiate the efficacy of immunotherapy in preclinical models of breast cancer. As previously reported, murine 4T1 tumors fail to respond to check point inhibitors. We reasoned that this may be an appropriate model to test the efficacy of HSP70 inhibitor, JG-231. Expectedly, there was no difference in tumor growth and metastasis between control and anti-PDL1 treated animals, however, combination of anti-PDL1 antibody with JG-231 and chemotherapy (cyclophosphamide-CTX) significantly reduced primary tumor growth (>10 fold) and eliminated metastasis. Collectively, our pilot experiments provide a strong rationale for testing our hypothesis and may lead to a rapid translation into the clinical utility. Citation Format: Fulya Koksalar Alkan, Justin Wilson, Elayne Benson, Tulshi Patel, Virginia McEvoy, Nate Francois, Emma Nguyen, Hilmi Kaan Alkan, Ahmed Chadli, Jason Gestwicki, Hasan Korkaya. Heat shock proteins in immunosuppressive tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4606.

Despite the advances in early diagnostics and therapeutics, women with metastatic breast cancer have limited treatment options. Women with TNBC, who constitute 15-20% of breast cancer patients, are often diagnosed with aggressive/metastatic disease. Advanced studies implicated immunosuppressive tumor microenvironment (TME) in aggressive/metastatic properties of TNBC subtype. Alternatively activated immature myeloid cells including tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), tumor-associated dendritic cells (TADC) and myeloid derived suppressor cells (MDSC) constitute a major component of TME. However, anti-tumorigenic microenvironment is also reported and that may have clinical relevance in early TNBC patients. Therefore, our hypothesis is that myeloid cells polarize to become immunosuppressive and infiltrate tumors and pre-metastatic niches in patients with advanced disease, while patients with early TNBCs may elicit anti-tumor immune response eliminating disseminated tumor cells (DTC). The utilization of syngeneic immunocompetent mouse models has contributed to our current understanding of immunosuppressive or immunomodulatory TME. Using these models, we have demonstrated that tumor dissemination and growth at metastatic sites is facilitated by MDSC’s. Emerging technologies; single cell RNA sequencing (scRNAseq), mass cytometry (CyTOF) or cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) has been powerful platforms for detailed characterization of tumors and TME compartments. Our bulk gene expression data of the myeloid cell populations of tumor microenvironment, lung, spleen and BM from 4T1 tumor-bearing mice showed distinct MDSC gene signatures. When applied to publicly available scRNAseq data, lung gMDSCs from 4T1 metastatic tumor model appeared to show different trajectory of polarization than the tumor gMDSCs. Consistent with previous findings by Hedrick Lab, lung gMDSCs from 4T1 mice also express higher levels of NeP markers compared to BM and tumor gMDSCs as well as lung gMDSCs from EMT6 mice. However, analyses of immune cells from EMT6 tumor bearing mice exhibited an anti-tumor immune signature which is consistent with the clearance of the DTCs following complete resection of the primary tumors. Using the murine TNBC models in syngeneic mice, we provide evidence that early TNBC tumors may elicit anti-tumor immune responses and thus the survival outcome in those patients is substantially increased after complete surgical resection of the primary tumors. Whereas immunosuppressive tumor microenvironment contributes to the poor overall survival in patients with advanced TNBCs. Therefore, identifying an anti-tumor immune signature in early TNBC patients may be utilized as a clinical biomarker before surgical intervention as well as improve the survival outcome. Citation Format: Fulya Alkan, Raziye Piranlioglu, Eunmi Lee, Maria Ouzounova, Catherine C Hedrick, Huidong Shi, Hasan Korkaya. Understanding the pro- and anti-tumorigenic microenvironments in syngeneic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2223.

Molecular chaperones are guardians of the proteome and of cellular homeostasis. Dysregulation in the function of the Hsp90 chaperoning machine in particular has been implicated in metabolic, oncological, neurodegenerative, and cardiovascular diseases. Targeting Hsp90 has been shown to have a combinatorial impact on dysfunctional circuitries that underlie human cancers. Thus, several inhibitors targeting the N-terminal ATP-binding pocket of Hsp90 are undergoing clinical trials as anticancer agents. They inactivate the ATPase activity of the chaperone, causing proteasomal degradation of its “client” oncogenic proteins. Although these inhibitors provided a proof-of-principal that Hsp90 is a valuable therapeutic target, they exhibit modest activity in the clinic, in part because they concomitantly induce a heat shock response (HSR), which turns on pro-survival pathways. Consequently, new mechanisms to inhibit the Hsp90 chaperone machine that do not induce the heat shock response are needed. To address this, we have developed a unique high throughput screen (HTS) based on the progesterone receptor (PR) and the five purified chaperones required for folding steroid receptors, i.e., Hsp90, Hsp70, Hsp40, Hsp70/Hsp90 organizing protein (Hop), and p23. During our preliminary screening of natural products, we discovered that the compound AD07 kills cancer cells through inhibition of the Hsp90 machine without a significant induction of HSR. In addition, AD07 induces a powerful T cell-mediated immune response, resulting in highly efficient tumor killing in a syngeneic mouse model and long-term memory against the primary tumor. At the molecular level, AD07 reduces the protein levels of two key mediators of tumor-induced immune tolerance: programmed cell death ligand-1 (PDL-1) and indolamine 2,3 dioxygenase (IDO). We therefore propose that AD07 is a promising anti-tumor agent targeting the Hsp90 machine through a novel mechanism of action involving cancer cell toxicity, which increases its immunogenicity, and modulation of the tumor microenvironment to reduce immunotolerance. Citation Format: Ahmed Chadli, Nada Eisa, Vincent Crowley, Sumin Lu, Yasmeen Jilani, Hasan Korkaya, Brian Blagg. A novel Hsp90 machine inhibitor combining chemotherapy and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 12.

Although clinically apparent metastasis is associated with late stages of cancer development, micro-metastatic dissemination may be an early event. However, the fate of these early disseminated tumor cells (DTC) remains elusive. Using the syngeneic mouse models, we demonstrated that although both orthotopically-implanted murine 4T1 and EMT6 tumors are capable of disseminating into secondary organs, only 4T1 tumors develop overt metastasis. However, EMT6 tumors induce an anti-tumor immunity in syngeneic mice and that eradicates disseminated tumor cells (DTC) in distant organs. Following the complete removal of primary EMT6 tumors, mice do not develop detectable metastasis and generate an immunological memory that leads to complete elimination of repeatedly injected tumor cells via tail vein. Conversely, these cells readily grow and metastasize in immuno-deficient athymic or Rag2- mice, and when g-MDSCs from 4T1 tumor-bearing mice were co-injected into immunocompetent EMT6 primed mice. In contrast to complete resection, mice with residual tumors following surgery exhibited an enhanced growth of local and concomitant growth of DTCs at metastatic site with increased g-MDSCs accumulation in lung and spleen. Together, our results suggest that some tumors are capable of inducing anti-tumor immunity against the DTCs when complete resection of primary tumor cures animals. However, in the presence of residual tumors, inflammation induced by surgical procedure promote the growth of DTCs. Citation Format: Raziye Piranlioglu, Eunmi Lee, Maria Ouzuonova, Riley Rodier, Adam Greer, Feyzanur Bayraktar, Omer Can Durmus, Ali S. Arbab, Muthushamy Thangaraju, Max S. Wicha, Esteban Celis, Hasan Korkaya. Primary tumor-induced immunity eradicates disseminated tumor cells in syngeneic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4580.

Large cohorts of recent clinical studies have firmly established that increased levels of tumor-infiltrating lymphocytes (TILs) in TNBC and HER2+ subtypes predicted better clinical outcome compared to the luminal subtype. These observations led to the hypothesis that women with TNBC or HER2+ subtypes may respond to a checkpoint blockade. However, early results from these trials using check point inhibitors alone or in combination with chemotherapy have shown very little promise in breast cancer patients, despite the remarkable long-lasting responses in other hard to treat malignancies such as non-small cell lung and melanoma. Although the outcome falls short of the expectation, it has suggested that the combinations of check point blockade with therapeutics that target immunosuppression may potentiate its efficacy. TNFα exhibits paradoxical roles; it may fuel tumor cell growth, invasion and metastasis in some tumor types, while in others it induces cytotoxic cell death. We recently demonstrated that TNFα distinctly induces A20 in TNBC subtype and protects these cells from TNFα-induced cytotoxic cell death by upregulating HSP70 protein and maintaining EMT/CSC phenotype. In contrast, luminal MCF7 or ZR75-1 cells display approximately 70% apoptosis when treated with TNFα. Overexpression of A20 in luminal cells not only protected them from TNFα-induced cytotoxicity by upregulating HSP70 and EMT/CSC phenotype, but also exhibited aggressive metastatic properties in mouse xenograft models. Furthermore, we show that A20/HSP70 pathway attracts tumor-infiltrating lymphocytes (TILs) while inducing the accumulation of immunosuppressive MDSCs in syngeneic mouse models. Interestingly, pulmonary DTCs as well as the immune infiltrates from 4T1 tumor-bearing mice exhibited significantly higher HSP70 expression. Therefore, we proposed that targeting HSP70 may potentiate the efficacy of immunotherapy in preclinical models of breast cancer. As previously reported, murine 4T1 tumors do respond to check point inhibitors. We reasoned that this may be an appropriate model to test the efficacy of HSP70 inhibitor, JG-231. Expectedly, there was no difference in tumor growth and metastasis between control and anti-PDL1 treated animals, however, combination of anti-PDL1 antibody ed with JG-231 and chemotherapy (cyclophosphamide-CTX) significantly reduced primary tumor growth (>10 fold) and eliminated metastasis. Collectively, our pilot experiments provide a strong rationale for testing our hypothesis and may lead to a rapid translation into the clinical utility. Citation Format: Hasan Korkaya, Eunmi Lee, Raziye Piranioglu, Maria Ouzounova, Ahmet Korkaya, Jason Gestwicki, Max S. Wicha, Esteban Celis. Improving the effectiveness of immunotherapy in breast cancer by targeting the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2245.

Metastatic basal-like breast cancers are believed to correspond with EpCAM-/Cd49f- cancer stem cell (CSC) enrichment. As well, basal-like breast cancers typically correspond with tumor inflammation and immunoediting phenotypes. However, the exact interplay between CSCs and the inflammatory signature of basal-like breast cancers is not well understood. To provide insight regarding the clinical overlap between breast cancer stem cells and tumor inflammation, we compared the 450K DNA methylation profile of EpCAM-/CD49f- CSCs from the isogenic MCF10A p53-/PTEN- breast cell line against the corresponding EpCAM+/CD49f+ and EpCAM-/CD49f+ subpopulations to determine whether differential DNA methylation occurred within the promoters of immune-related genes in CSCs. In addition, we also overlapped the 450K DNA methylation profile from 16 established breast cancer cell lines of varying EpCAM-/CD49f- concentrations to compare against the isolated CSCs. Based on our results, we identified 1432 differentially methylated promoter regions overall (ANOVA FDR p-value Citation Format: Emma V. Gray, Caroline E. Dyar, Maria Ouzounova, Max S. Wicha, Hasan Korkaya, Austin Y. Shull. IL32 expression is epigenetically regulated in EpCAM-/Cd49f- basal-like breast cancers and can be suppressed by the bromodomain inhibitor JQ1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3683.

Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 10-50% of lung adenocarcinomas. EGFR tyrosine kinase inhibitors (TKIs) are the mainstay of treatment for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, acquired EGFR mutations are the main mechanism of on-target resistance to TKIs. T790M mutation that occurs after first generation TKI treatment, is a cytosine to thymine (C>T) single nucleotide transition leading to a threonine to methionine amino acid change at position 790 (i.e. T790M). Our data suggest that resistant mutations are acquired events secondary to cytosine deamination through Activation Induced Cytosine Deamination enzyme (AICDA). Interestingly, treatment with Osimertinib, that overcomes the T790M mutation, leads to other acquired resistant mutations (C797S, G796S/R and L792F/H) that are mostly cytosine based. Results: Sub clones of the lung adenocarcinoma cell line PC9 with no evidence of T790M mutation by digital droplet PCR (ddPCR) at baseline, were treated with EGFR TKI. After serially increasing the treatment dose, T790M mutation was detected by ddPCR associated with a significant increase in AICDA expression. Similarly, when the resistant T790M PC9 clones were treated with Osimertinib, the expression of AICDA was also induced. Knocking down AICDA by shRNA or CRISPR-Cas9, decreases the development of T790M in PC9 cell lines after TKI exposure. Using mass spectrometry, we established that cytosine at codon 790 is methylated; thus, deamination of 5-methylcytosine leads to thymine directly, explaining the T790M C>T mutation. In addition, using ChIP assay and pharmacological inhibition we confirm that upon TKI exposure, NFκB binds AICDA promoter and induces its expression. In a mouse xenograft model, the induction of NFκB and AICDA after EGFR TKI exposure is abrogated by concurrent use of an NFκB inhibitor. Finally, patients treated with EFR TKI had an increased expression of AICDA upon progression. Conclusion: In EGFR driven lung adenocarcinoma, NFκB pathway is activated upon exposure to EGFR TKIs which induces AICDA expression. AICDA deaminates cytosine into other nucleotides leading to treatment resistance. Citation Format: Najwa El Kadi, Luo Wang, April Davis, Alexander Cooke, Varun Vadnala, Hasan Korkaya, Gregory P. Kalemkerian, Khaled A. Hassan. The T790M mutation is acquired through 5-methylcytosine deamination after EGFR TKI treatment in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 931.

The development of HER2 targeting agents has dramatically altered the natural history of HER2-positive breast cancer and is often cited as a prime example of the effectiveness of molecularly targeted therapy. Emerging data suggest that the remarkable clinical efficacy of these agents may be related to their ability to target the breast cancer stem cell (CSC) population. A new study suggests that the regulation of BCSCs by HER2 may extend to breast cancers that do not display HER2 gene amplification. In these tumors, HER2 is selectively expressed in the CSC population, and this expression is regulated by the tumor microenvironment. In mouse models, trastuzumab blocked growth of these HER2-negative tumors when administered in the adjuvant setting but had no effect on established tumors. These studies provide a potential biologic explanation for retrospective analysis of clinical trials, which surprisingly suggest that the clinical benefits of adjuvant trastuzumab may extend to women currently classified as HER2-negative. In addition to having significant implications for breast cancer therapy, these studies suggest the need to reevaluate the role of HER2 in regulating CSCs in other tumor types. Furthermore, these studies suggest that effective adjuvant therapies may need to target the CSC population. Cancer Res; 73(12); 3489–93. ©2013 AACR.

Although current breast cancer treatment guidelines limit the use of HER2-blocking agents to tumors with HER2 gene amplification, recent retrospective analyses suggest that a wider group of patients may benefit from this therapy. Using breast cancer cell lines, mouse xenograft models and matched human primary and metastatic tissues, we show that HER2 is selectively expressed in and regulates self-renewal of the cancer stem cell (CSC) population in estrogen receptor-positive (ER+), HER2− luminal breast cancers. Although trastuzumab had no effects on the growth of established luminal breast cancer mouse xenografts, administration after tumor inoculation blocked subsequent tumor growth. HER2 expression is increased in luminal tumors grown in mouse bone xenografts, as well as in bone metastases from patients with breast cancer as compared with matched primary tumors. Furthermore, this increase in HER2 protein expression was not due to gene amplification but rather was mediated by receptor activator of NF-κB (RANK)-ligand in the bone microenvironment. These studies suggest that the clinical efficacy of adjuvant trastuzumab may relate to the ability of this agent to target the CSC population in a process that does not require HER2 gene amplification. Furthermore, these studies support a CSC model in which maximal clinical benefit is achieved when CSC targeting agents are administered in the adjuvant setting. Cancer Res; 73(5); 1635–46. ©2012 AACR.

We recently demonstrated that infiltration of granulocytic MDSCs in distant organs is a critical step in metastatic process. The mouse transcriptome analysis of in vitro co-cultures and samples from syngeneic mouse model revealed that granulocytic subset of myeloid-derived suppressor cells (gMDSCs) from metastatic 4T1 tumor bearing mice are regulated a distinct set of genes. Two genes, S100A8 and S100A9 were highly upregulated in gMDSCs isolated from mice with metastatic 4T1 tumors compared to mice with non-invasive tumors. Using the recombinant single S100A8, S100A9 proteins or the S100A8/S100A9 heterotetramer (calprotectin), we demonstrated that calprotectin is a critical player in gMDSC induction and infiltration in distant organs particularly in lungs. Furthermore, injection of recombinant calprotectin but not single S100A8 or S100A9 proteins via tail vein enhanced the metastatic ability of nan-invasive EMT6 tumors in mice. We therefore performed computational screen of NCI compounds against the crystal structure of calprotectin and identified over 100 lead compounds. In vitro gMDSC differentiation assay identified 3 compounds with strong gMDSC inhibitory activity. We therefore designed a pilot experiment to test these compounds in preclinical mouse models. We also compared the activity of these compounds against tasquinimod, a small molecule targeting S100A9 only. However, tasquinimod treatment of 4T1 tumor-bearing mice had a moderate anti-tumor activity which may be due to a limited activity on granulocytic MDSC accumulation. In addition, we determined that there was a significant upregulation of S100A8 in MDSCs from tasquinimod treated mice. This data suggested that inhibiting only S100A9 leads to activation of S100A8 and thus may be ineffective targeting of MDSCs. In line with these data, one of the compounds (#10) showed a potent activity against gMDSCs in 4T1 tumor-bearing mice and significantly inhibited pulmonary metastasis. Moreover, this compound eliminated the cytotoxicity caused by cyclophosphamide and animals were free of cancer. In summary, we have identified a molecular target which regulates gMDSC induction and play critical role in metastatic process and our findings from in vitro screens and preclinical testing provide a strong rationale for targeting MDSCs. Citation Format: Hasan Korkaya, Eunmi Lee, Raziye Piranioglu, Thomas Albers, Maria Ouzounova, Charlie Weeks, Riley Rodier, Ahmet K. Korkaya, Khaled A. Hassan. Screening the chemical library against granulocytic MDSCs: Critical player in breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1100.

The metastatic potential of breast cancer cells is believed to correspond with the preferential enrichment of a tumor population known as cancer stem cells (CSCs). Thus, evaluating the unique molecular characteristics of breast CSCs is of great interest from a prognostic and therapeutic standpoint. In order to provide a more detailed profile of breast cancer stem cells, we compared the 450K DNA methylation landscape of the EpCAM-/CD49f- cancer stem cell subpopulation from the isogenic MCF10A p53-/PTEN- breast cell line against the corresponding EpCAM+/CD49f+ and EpCAM-/CD49f+ subpopulations to determine how DNA methylation varies within the different genomic regions of CSCs. In addition, we also overlapped the 450K DNA methylation profiles from 16 established breast cancer cell lines of varying subtypes and aggressiveness to determine how these cell lines relate epigenetically with the isolated CSCs. Based on unsupervised PCA and matrix dissimilarity clustering, we were able to identify 3 distinct groups that appeared to independently cluster based on EpCAM-/CD49f- enrichment status. It is also interesting to note that the aggressive cell lines SUM149 and MDA-MB-231 would cluster away from the EpCAM-/CD49f- subset when utilizing strictly promoter probes for clustering, whereas SUM149 and MDA-MB-231 would cluster more closely with the EpCAM-/CD49f- subset when utilizing gene body probes, potentially indicating a more sensitive correlation between gene body methylation and CSC-associated aggressiveness. To further investigate the differing promoter and gene body DNA methylation patterns in CSCs, we performed differential methylation analysis between the 3 previously defined groups. Based on our results, we discovered 1432 differentially methylated promoter probes and 7243 differentially methylated gene body probes (ANOVA FDR p-value Citation Format: Austin Y. Shull, Caroline E. Dyar, Maria Ouzounova, Nicole L. Hudson, Max S. Wicha, Hasan Korkaya. Deciphering the DNA methylation signature of EpCAM-/CD49f- breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5321.

Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 10-50% of lung adenocarcinomas. This leads to constitutive activation of EGFR, which triggers multiple downstream survival and proliferation pathways. EGFR tyrosine kinase inhibitors (TKIs) are the mainstay of treatment for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. Acquired EGFR mutations are the main mechanism of on-target resistance to TKIs. T790M mutation that occurs after first line TKI treatment, is a cytosine to thymine (C>T) single nucleotide transition leading to a threonine to methionine amino acid change at position 790 (i.e. T790M). Interestingly, treatment with Osimertinib, that overcomes the T790M mutation, leads to other acquired resistant mutations, C797S, G796S/R and L792F/H. Our data suggest that resistant mutations are acquired events secondary to cytosine deamination through Activation Induced Cytosine Deamination enzyme (AICDA). Results: Sub clones of the lung adenocarcinoma cell line PC9 with no evidence of T790M mutation by droplet digital PCR (ddPCR) at baseline, were treated with EGFR TKI. After serially increasing the treatment dose, T790M mutation was detected by ddPCR associated with a significant increase in AICDA expression. Knocking down AICDA by shRNA, decreases the development of T790M in PC9 cell lines after TKI exposure. Similarly, when the resistant T790M PC9 clones were treated with Osimertinib, the expression of AICDA was also induced. Using mass spectrometry, we established that cytosine at codon 790 is methylated; thus, deamination of 5-methylcytosine leads to thymine directly, explaining the T790M C>T mutation. In addition, using ChIP assay and pharmacological inhibition we confirm that upon TKI exposure, NFĸB binds AICDA promoter and induces its expression. In a mouse xenograft model, the induction of NFĸB and AICDA after EGFR TKI exposure is abrogated by concurrent use of an NFĸB inhibitor. Finally, patients treated with EFR TKI had an increased expression of AICDA upon progression. Conclusion: In EGFR driven lung adenocarcinoma, NFĸB pathway is activated upon exposure to EGFR TKIs which induces AICDA expression. AICDA deaminates cytosine into other nucleotides leading to treatment resistance. Citation Format: Najwa El Kadi, April Davis, Alexander Cooke, Luo Wang, Hasan Korkaya, Gregory Kalemkerian, Khaled Hassan. Activation Induced Cytosine Deamination, AICDA, is induced after EGFR TKI exposure leading to secondary resistant mutations in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5836.

Metastatic disease is the end stage of extremely inefficient processes that entail overcoming multiple barriers including anti-tumor immunity. Although evidences from preclinical and clinical settings suggest that dissemination of malignant cells is an early process, majority of disseminated cells either eliminated or remain dormant in distant organs, while very few cells eventually develop successful metastasis. Therefore, it is widely accepted that dynamic and reversible tumor cell plasticity is required for metastasis, however, in vivo steps and molecular mechanisms are poorly elucidated. We provide evidence that monocytic and granulocytic subsets of myeloid derived suppressor cells (m-MDS, g-MDSC) infiltrated in primary 4T1 tumor and distant organs with different time kinetics regulate spatiotemporal tumor plasticity. Using co-culture experiments and syngeneic mouse models of murine 4T1 (metastatic) tumor, we demonstrated that tumor infiltrated m-MDSCs facilitate tumor cell dissemination from the primary site by inducing the EMT/CSC phenotype. In contrast, g-MDSCs infiltrated in the lungs support metastatic growth by reverting the EMT/CSC phenotype and thus promoting tumor cell proliferation. In contrast, less invasive EMT6 tumors fail to induce efficient pulmonary infiltration of g-MDSCs and results in clearance of disseminated tumor cells in the lungs. Gene expression analyses of tumors and MDCS subsets in primary tumor site and distant organs at different time points reveal mechanistic temporal regulation of in vivo tumor plasticity by m-MDSC and g-MDSC subsets in 4T1 tumor-bearing mice. However, the lung microenvironment of EMT6 tumor-bearing mice display a gene expression signature of anti-tumor immunity which predict better survival in breast cancer patients. In our functional studies, we demonstrate that EMT6 tumor-bearing animals efficiently eliminate disseminated tumor cells in the lungs. Furthermore, g-MDSCs isolated from 4T1-tumor bearing animals significantly enhance metastatic growth of already disseminated tumor cells. Consistent with the “seed and soil” hypothesis, our studies provide a molecular mechanism by which the immune system regulate spatiotemporal tumor plasticity and generation of permissive or anti-tumorigenic microenvironment in distant organs determining the fate of disseminated tumor cells. Citation Format: Hasan Korkaya, Eunmi Lee, Raziye Piranioglu, Maria Ouzounova, Abdeljabar El-Andaloussi, Iskander Asm, Ahmet K. Korkaya, Gang Zhou, Ali Arbab, Paulo Rodriguez. Immune regulation of disseminated tumor cell clearance versus metastatic growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3992. doi:10.1158/1538-7445.AM2017-3992

Parity and breastfeeding influence a woman’s risk of developing breast cancer. Understanding the underlying molecular changes in the breast will facilitate identification of additional factors during pregnancy and postpartum that modify breast cancer risk. Most prior studies in humans on this topic, however, have been cross-sectional studies comparing breast tissue samples from nulliparous versus post-pregnant women, and are limited in their ability to characterize longitudinal changes and within-person variations. Breast milk contains cells of the mature gland, and may have a potential as a noninvasive source for studying molecular characteristics of the pregnancy-associated changes in the breast and their impact on breast cancer risk. The objectives of this study were to (1) determine normative patterns of gene expression levels in longitudinally collected breast milk samples and (2) correlate gene expression data in relation to self-reported measures of psychosocial stress during pregnancy and postpartum period. Our preliminary results showed that the post-pregnancy gene expression levels in breast milk samples can be measured reproducibly over a short period of time. Whole-genome transcriptome analysis revealed that expression levels of the post-pregnancy genes are different in early (less than 3 month postpartum) and late lactation period (more than 6 month postpartum). Moreover cytokine expression profiling correlated expression of inflammatory cytokines with early versus late postpartum period. The unique contribution of this study is to capture longitudinal gene expression data from breast milk samples collected over the course of lactation, hence providing a valuable basis for future studies to identify differential dynamics associated with breast cancer susceptibility. Note: This abstract was not presented at the meeting. Citation Format: Maria Ouzounova, Georgina Lewis, Raziye Piranlioglu, Tiana Curry-McCoy, Wonsuk Yoo, Nita Maihle, Sangmi Kim, Hasan Korkaya. Determination of normative patterns of gene expression levels and breast cancer risk biomarkers in human breast milk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4240. doi:10.1158/1538-7445.AM2017-4240

Recent findings have shown that the Heat Shock Protein 90 (Hsp90) co-chaperone UNC45A is overexpressed in ovarian and breast cancers. Previously, we have shown that UNC45A is a centrosomal protein essential for cervical tumor cell growth through activation of the checkpoint kinase 1 (ChK1). In this report, we further examined the role of UNC45A in breast tumorigenesis using a variety of biochemical and cell biology techniques and animal models. We confirmed that UNC45A is highly overexpressed in human breast-infiltrating ductal carcinomas as compared to adjacent normal tissues. Silencing UNC45A in vitro blocked the proliferation of all breast cancer subtypes and drastically reduced tumor growth of the triple negative MDA-MB-231 cell line implanted in mammary fat pads of NOD/SCID mice. However, loss of UNC45A did not affect the proliferation of normal mammary cells. Remarkably, UNC45A becomes more nuclear in human cancer tissues and cancer cell lines as compared to normal tissues and non-transformed Hs578Bst and HME mammary cell lines, respectively. This suggests an important nuclear function for UNC45A during tumorigenesis. Microarray analysis of mRNA from Hs578T cells showed that loss of UNC45A alters the expression of 121 genes, involved in cancer and cellular development and growth networks. Relevant to cell proliferation, we found that Nek7 gene was significantly repressed upon silencing UNC45A, which was validated by RTqPCR and Western blot analyses in multiple breast cancer cell lines. Nek7 is a member of the NIMA (never in mitosis, gene A) family of serine/threonine kinases. It plays a key role in centrosomal separation during mitosis. This correlates neatly with our observation that loss of UNC45A causes a centrosomal separation defect, cell proliferation arrest and death of breast cancer cell lines. ChIP experiments showed that UNC45A binds to the promoter of the Nek7 gene, suggesting direct transcriptional regulation. Interestingly, the UNC45A sequence contains four LxxLL motifs, which are thought to be signatures for co-activator binding to nuclear receptors. Furthermore, computational analysis identified two glucocorticoid response elements (GRE) consensus sequences in the Nek7 promoter, suggesting its transcriptional regulation by the glucocorticoid receptor (GR). This hypothesis was further strengthened by a significant decrease in the mRNA and protein levels of Nek7 upon silencing GR. Thus, our data suggest that UNC45A functions as a GR co-activator to control Nek7 gene transcription. Consistent with this, immunoprecipitation experiments confirmed that UNC45A and GR form endogenous complexes, and treatment of Hs578T and MCF7 cell lines with dexamethasone upregulates Nek7 mRNA and protein levels. In conclusion our data strongly support the premise that UNC45A promotes Nek7 transcription through activation of GR, and thus controls centrosomal separation and cancer cell proliferation. Citation Format: Yasmeen Jilani, Nada H. Eisa, Kashish Kainth, Sumin Lu, Nehal M. Elsherbiny, Laila A. Eissa, Mamdouh M. Elshishtawy, Hasan Korkaya, Abdeljabar El Andaloussi, Ahmed Chadli. The co-chaperone UNC45A controls cancer cell proliferation through Nek7 and centrosomal separation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4493. doi:10.1158/1538-7445.AM2017-4493

EGFR resistant mutation, T790M, in lung adenocarcinoma is acquired through a specific cytosine deamination mechanism. Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 15% of lung adenocarcinomas. This leads to constitutive activation of EGFR, which triggers multiple downstream survival and proliferation pathways. Currently, EGFR tyrosine kinase inhibitors (TKIs) are first line therapy for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. Despite initial significant response to TKIs, most tumors develop resistance. The main mechanism of resistance detected in 50-60% of cases is a cytosine to thymine (C>T) single nucleotide transition mutation at position 2369. This causes a threonine to methionine amino acid change at position 790 (i.e. T790M). Our data suggests that the C>T mutation is an acquired event secondary to cytosine deamination by Activation Induced Cytosine Deamination enzyme (AICDA). Results: Single cell clones of lung adenocarcinoma cell line, PC9, were treated with EGFR TKI. At baseline, these clones have EGFR exon 19 deletion but no evidence of T790M mutation by digital droplet PCR (ddPCR). However, after treatment with a serial increasing dose of EGFR TKI, T790M mutation was detected by ddPCR. Assessing whether cytosine deamination enzymes were altered by this treatment, a significant increase in AICDA expression was seen. Furthermore, recombinant AICDA protein could deaminate cytosine at position 2369 in vitro. In addition, using mass spectrometry and methylation specific primers, we determined that cytosine at position 2369 is in fact methylated. This further supports our hypothesis since 5-methyl cytosine is deaminated into thymine directly. Since in germinal center B-lymphocytes, AICDA is activated through a non-canonical NFkB mechanism, we assessed NFkB pathway in PC9 cell line. RelB and p52 expression were significantly increased after TKI treatment. In addition direct interaction between RelB and AICDA promoter was confirmed by ChIP Assay. These findings were also seen in a mouse PC9 xenograft model. Daily oral gavage of EGFR TKI caused significant increase in the expression of RelB as well as AICDA. Adding NFkB inhibitor twice weekly inhibited the expression of RelB and AICDA. Finally, knocking down AICDA by shRNA prevented the development of T790M mutation in PC9 cell lines after TKI exposure. Conclusion: Our data suggest that the T790M mutation could be actively acquired after TKI treatment through a cytosine deamination process by AICDA. This would have significant implications for treatment with targeted therapy. In fact, Imatinib resistance in CML and GIST tumors have a similar C>T single nucleotide transition mutation. Citation Format: Khaled Hassan, Najwa El Kadi, April Davis, Gregory Kalemkerian, Luo Wang, Hasan Korkaya. The development of EGFR resistant mutation, T790M, in lung adenocarcinoma is acquired through a specific cytosine deamination mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4119. doi:10.1158/1538-7445.AM2017-4119

The classical model of metastasis suggests that the tumor cell dissemination occurs late in tumor development, however accumulating evidence coming from mouse studies and clinical data provide striking evidence that tumor cells start to disseminate during the initial steps of tumor development. However, the dissemination from the primary area does not always result in metastasis. Due to the non-permissive nature of microenvironment in distant sites, these early disseminated tumor cells might be cleared or maintained in a non-proliferative/dormant state. The mechanism by which some early disseminated tumor cells colonize and generate metastatic growth while some remain dormant is not well known. In order to understand the underlying factors that may contribute to the metastatic growth, we performed time course experiments by utilizing murine mammary tumors (4T1 as metastatic and EMT6 as less metastatic) in a syngeneic mouse model. Luciferase expressing 4T1 or EMT6 tumor cells were orthotopically implanted into the fat pads and tumor cell dissemination was analyzed over 3-week time points. We determined that both 4T1 and EMT tumors disseminated as early as one to two-week post implantation, however only 4T1 tumor develop metastasis in distant organs. Moreover, we also resected primary tumors 1, 2 and 3-week post implantation of EMT6-Luci or 4T1-Luci tumors and observed distant metastasis via optical imaging of luciferase expression in live animals. Although the majority of 4T1 tumor-bearing mice (>80%) develop pulmonary metastasis when 4T1 tumors resected 2 and 3 weeks post-implantation only 10% of mice develop metastasis when primary tumor resected one-week post implantation. In contrast, EMT6 tumors following resection only relapsed in the primary tumor site but failed to develop metastasis. Furthermore, EMT6 tumor-bearing mice efficiently cleared tail vein injected EMT6-luci cells in the lungs. We investigated the possible mechanism by which EMT6 tumor-bearing mice clears disseminated tumor cells in the lung. We provide evidence that pulmonary infiltrated mMDSCs mediate tumor cell killing via secretion of high levels of cytotoxic granules, granzyme A, granzyme B, perforin. This was confirmed by mouse transcriptome and qPCR analyses as well as biochemistry using in vivo samples and in vitro co-culture samples. Our studies provide a new paradigm in the understanding of the fate of disseminated tumor cells in secondary organs and the role of the immune system in this process. Citation Format: Raziye Piranlioglu, Eun Mi Lee, Maria Ouzunova, Ali S. Arbab, Paulo C. Rodriguez, Asm L. Iskander, Hasan Korkaya. Disseminated tumor cell clearance by the immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5807. doi:10.1158/1538-7445.AM2017-5807

We previously demonstrated that MDSC subsets accumulated in primary tumor and distant organs regulate tumor plasticity. The mouse transcriptome analysis of in vitro co-cultures and samples from syngeneic mouse model revealed that granulocytic subset of myeloid-derived suppressor cells (gMDSCs) from metastatic 4T1 tumor bearing mice regulate several hundred genes in tumor cells upon co-culture. The top genes are S100A8, S100A9, MMP8, FPR1, CCL3, and TGFβ2 which also predicted poor survival in human solid tumors including breast cancer. Therefore, we called these 6 genes as “metastatic gene signature”. It has been reported that S100A8/S100A9 heterotetramer, called calprotectin, play a key role in inflammation-associated cancer progression. To investigate the role of calprotectin (S100A8/A9) in tumor-mediated immunosuppression and metastasis, we first utilized Tasquinimod, a small molecule targeting S100A9 only. However, Tasquinimod treatment of 4T1 tumor-bearing mice had a moderate anti-tumor activity which may be due to a limited activity on granulocytic MDSC accumulation. In addition, we determined that there was a significant upregulation of S100A8 in MDSCs from Tasquinimod treated mice. This data suggested that inhibiting only S100A9 leads to activation of S100A8 and thus may be ineffective targeting of MDSCs. We therefore run a computational screen of the NCI chemical library against the crystal structure of calprotectin (S100A8/S100A9) and identified 40 lead compounds. We then performed in vitro screening assay to identify compounds that inhibit MDSC induction. We identified 3 compounds that significantly suppressed gMDSC differentiation. We are currently performing in vivo studies with these 3 candidate drugs in our murine breast cancer model and will present our findings at the AACR meeting. We believe that this study will provide a novel drug targeting S100A8/S100A9 heterotetramer, a key molecule in MDSC induction and its regulatory functions during tumor progression. Citation Format: Eunmi Lee, Maria Ouzounova, Raziye Piranlioglu, Abdeljabar El Andaloussi, Sena Arbag, Gang Zhou, Hasan Korkaya. Chemical library screen identifies compounds that target S100A8/S100A9 complex and MDSC accumulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2956. doi:10.1158/1538-7445.AM2017-2956

Despite recent advances and better diagnostics, the major challenge is that metastatic breast cancer is still incurable and remains leading cause of cancer related death. Cachexia is considered to be a chronic inflammatory syndrome which is defined by loss of skeletal muscle mass (with or without loss of adipose mass), negative energy and metabolic balance, and systemic inflammation. Cancer patients who develop cachexia are more susceptible to infections and sepsis. Clinical studies suggest that cachexia in cancer patients cannot be fully reversed by conventional nutritional supports, which distinguishes this condition from anorexia. Due to its complexity and lack of clinical biomarkers, currently there is no standard treatment for these patients. Therefore cachexia remains a largely underestimated and untreated condition. Nearly 60-80% of the advanced/ metastatic cancer patients experience cachexia, a condition that accounts for 20% of cancer-related deaths. Chronic inflammation has been recognized as a risk factor contributing to the etiology of many human malignancies. Accumulating evidence suggest that tumor infiltrating immune cells (mainly myeloid origin) differentiate into cells that promote tumor growth and metastasis via inducing a systemic inflammation. Our preliminary studies suggest that systemic induction and infiltration (tumor, bone marrow, spleen, liver, and lung) of myeloid derived suppressor cells (MDSC) generate a pro-inflammatory micro-environment and are a major source of inflammatory cytokines, many of which are implicated in cancer cachexia. Using a murine breast cancer in a syngeneic (immunocompetent) mouse model we show that metastatic (4T1) murine tumor produce significantly higher level of inflammatory cytokines and is able to induce systemic expansion and infiltration of MDSC compared to non-metastatic murine tumor (EMT6). Furthermore, injection of condition media from metastatic 4T1 tumor cells is also able to induce MDSC expansion in vivo suggesting that tumor-produced factors play role in this process. Moreover, we demonstrate the involvement of the inflammatory cytokines in muscle wasting as shown by co-culture experiments with C2C12 myoepithelial cells and analysed the expression of cachexia markers such as E3 ubiquitin ligases Trim63 and Fbxo3, Myh1(myosin heavy chain), Stat3 and NFkB pathway activation, and elevation of pro-cachexia cytokines. Our preliminary studies demonstrated that the monocytic MDSC induce EMT phenotype and contribute to the dissemination of tumor cells while the granulocytic MDSC promote the metastatic outgrowth, and present higher infiltration in the lungs. We therefore propose that tumor-induced inflammatory cytokines play role in induction of MDSC and further elevation of inflammatory markers leading to metastasis and cancer cachexia. Citation Format: Maria Ouzounova, EunMi Lee, Raziye Piranlioglu, Ena Novakovic, Mehmet Demirci, Sumeyye Korkaya, Alicia Hudson, Hasan Korkaya. Myeloid derived suppressor cells-mediated inflammation in metastasis and cancer cachexia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1554.

Metastatic breast cancer is the second leading cause of cancer-related death among women. Although the genetic and epigenetic differences between the metastatic versus non-metastatic breast tumors have been well studied, early events between tumor and immune system in metastatic process remain poorly understood. In order to determine early events, we utilized murine mammary tumors (4T1 as metastatic, EMT6 or 67NR as non-metastatic) in syngeneic mouse model. The 4T1 tumor contained a higher proportion of cancer stem cell (CSC) population compared to the non-metastatic EMT6 or 67NR clones. Although, both murine tumor cell lines (50K each) grow to same size tumors within 8 weeks, 4T1 tumors develop spontaneous metastasis in 100% of animals most of which do not survive more than 8 weeks due to extensive wide spread metastasis to lung, liver and bone. We observed immune infiltrates in the lungs of 4T1 tumor bearing mice as early as 1 week. We next assessed the cytokine profile of metastatic 4T1 tumor compared to non-metastatic counterparts (EMT6 or 67NR) secretes significantly higher levels of inflammatory cytokines, including the IL6, IL8, RANTES, GCSF, GM-CSF, IL12, CXCL16 and CXCL5. MDSCs are potent suppressor of anti-tumor immunity and a significant impediment to cancer therapy. We therefore hypothesized that the tumor secreted inflammatory cytokines promotes the systemic expansion of MDSCs that down regulate immune surveillance and anti-tumor immunity, thus facilitating tumor progression. We sought to determine whether 4T1 tumors could induce MDSCs in mice. Murine 4T1 or EMT6 tumor cells (at 50K cells each) were implanted into the fat pads of BALB/c mice, then sacrificed (4 mice from each group) at weeks 1, 2, 3 and 4 for subsequent evaluation of the MDSC expansion in bone marrow, spleen, lung and tumors. The MDSC induction and infiltration in bone marrow, spleen, lung and tumors were observed as early as one-week post-implantation of 4T1 tumor compared to the EMT6. Furthermore, the MDSCs isolated from 4T1 tumor bearing animals were more suppressive than that of the EMT6 tumor bearing mice. We determined that non-metastatic EMT6-Luciferase tumor growth and metastasis is robustly enhanced in pre-primed animals (in which metastatic 4T1 cells were pre-implanted in the fat pads and resected after 10 days when tumors were 2mm in size) or by IP injection of inflammatory cytokine rich 4T1 conditioned medium when compared to injection of EMT6-Luci cells into naïve animals. Our preliminary findings suggested that 4T1 tumors within 10 days of implantation created a systemic tumor-promoting microenvironment and thus promoted the metastatic spread of EMT6-Luci. Together these studies strongly suggest that metastatic 4T1 tumor with high CSC phenotype generate a permissive systemic microenvironment for successful metastasis via secretion of inflammatory cytokines in syngeneic BALB/c mice. Citation Format: Hasan Korkaya, Eunmi Lee, Maria Ouzounova, Raziye Piranlioglu, Abdeljabar El-Andaloussi, Ena Novakovic, Alicia Hudson, Sumeyye Korkaya, Mhmet F. Demirci, Gang Zhou. MDSC mediated spatiotemporal tumor plasticity in breast cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1555.

Metastatic disease -end stage of tumor progression- is the major cause of cancer-related death.It is widely accepted that malignant cell plasticity between epithelial-mesenchymal-transition (EMT) and mesenchymal-epithelial-transition (MET) is required for metastasis to occur. The classical model of metastasis suggests tumor cell dissemination occur late in tumor development, however emerging studies strongly indicates that dissemination is an early process and provide a striking evidence that tumor cells start to disseminate during the initial steps of tumor development. Late appearing metastases arise from these early-disseminated tumor cells. The mechanism by which some early-disseminated tumor cells colonize and generate metastatic growth while some remain dormant is not well known. In order to understand the underlying factors, we utilized murine mammary tumors (4T1 as metastatic and EMT6 as less metastatic) in a syngeneic mouse model. We performed time course experiments to determine the early factors that may contribute to the metastatic growth. 4T1 or EMT6 tumor cells were implanted orthotopically into the fat pads and tumor cell dissemination was analyzed over 3 weeks time points. We determined that both 4T1 and EMT tumor cells disseminated as early as one week post-implantation, however only 4T1 tumor cells develop metastasis in distant organs. Furthermore, we also resected primary tumors 1,2 and 3 week post implantation of EMT6-Luci or 4T1-Luci tumors and observed distant metastasis via optical imaging of luciferase expression in live animals. Although the majority of 4T1 tumor bearing mice (>80%) develop pulmonary metastasis when 4T1 tumors resected 2 and 3 weeks post-implantation, only 10% of mice develop metastasis when primary tumor resected one week post implantation. In contrast, EMT6 tumors following resection only relapsed in the primary tumor site but failed to develop metastasis. We investigated the possible mechanism of efficient pulmonary metastatic growth following the resection of tumors 2-3 week post implantation. Interestingly we found a significant infiltration of granulocytic subset of myeloid derived suppressor cells (g-MDSC) in 4T1 tumor bearing mice by week 2 and 3. Furthermore, we found that lung infiltrated g-MDSCs promote tumor cell growth via paracrine factors. In co-culture studies we found that there is a reciprocal secretion of panel of inflammatory cytokines, growth factors and matrix metalloproteases between tumor cell and g-MDSCs suggesting that these cells in the lung microenvironment support the metastatic growth. Our studies provide a new paradigm in the understanding of metastatic growth and the role of microenvironment in distant organs. Citation Format: Raziye Piranlioglu, Maria Ouzounova, Eunmi Lee, Alicia Hudson, Sumeyye Korkaya, Ali Arbab, Hasan Korkaya. Immune regulation of tumor dormancy in syngeneic mouse model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 908.

It is widely accepted that the epithelial-mesenchymal plasticity of malignant cells is required during cancer metastatic cascade. The complex phenotypic changes highly depend on the collaboration of various molecular signaling and extracellular cues originating from wide range of stromal cells in the tumor microenvironment. However, the specific mechanisms of how EMT plasticity spatiotemporally regulates metastasis are poorly defined. Myeloid-derived suppressor cells (MDSCs) have been identified in most cancer patients and animal models due to their immune suppressive functions, but recent studies implicate their direct role in promoting metastasis by activating tumor-angiogenesis. To determine the roles of MDSCs in breast cancer metastasis, we utilized murine breast cancer cells, non-metastatic EMT6 and metastatic 4T1 cells. We showed that the metastatic 4T1 murine breast tumors induced early systemic expansion and mobilization of MDSCs in distant sites as well as in the primary tumor. We investigated the direct functions of MDSCs in tumor progression by isolating monocytic and granulocytic MDSCs from primary tumor, lung and bone marrow of tumor-bearing mice and then they were co-cultured with non-metastatic EMT6 cells. We found that tumor infiltrating m-MDSCs from 4T1 tumor-bearing mice increased the expression of Vimentin, Twist1, TGF-â and IL-6 in EMT6 tumor cells. In contrast, flow cytometry sorted lung infiltrating MDSCs from 4T1 tumor-bearing mice enhanced the EpCAM expression and proliferation in EMT6 cells. Cell invasion assay showed that invasive ability of EMT6 cells were significantly increased when they were co-cultured with m-MDSCs while g-MDSCs slightly decreased the number of invaded cells, compared to control group. We utilized immunofluorescence staining and confirmed the increased expression of Vimentin, CK14 (cytokeratin 14) in EMT6 cells co-cultured with m-MDSCs. In contrast, g-MDSCs induced down-regulation of these markers while they increased cell proliferation as assessed by Ki67 staining. Furthermore, flow cytometry analysis showed the increased CD24+CD29+ population, a marker of murine cancer stem cell (CSC) phenotype, in EMT6 cells when co-cultured with m-MDSCs from 4T1 tumor-bearing mice. Tumor sphere assay confirmed that m-MDSCs enhanced sphere forming ability of tumor cells. Taken together, these data suggest that m-MDSCs derived from metastatic 4T1 tumor-bearing mice are able to confer EMT/CSC phenotype on tumor cells while g-MDSCs are more potent in inducing epithelial phenotype and proliferation in tumor cells. Citation Format: Eunmi Lee, Maria Ouzounova, Raziye Piranlioglu, Abdeljabar El Andaloussi, Mehmet Demirci, Ena Novakovic, Alicia Hudson, Sumeyye Korkaya, Gang Zhou, Hasan Korkaya. Monocytic and granulocytic MDSCs display distinct molecular properties and coordinate the dynamic switches between EMT-MET in breast cancer model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 717.

An intrinsic claudin-low subtype within the TNBC was recently identified based on low/absent expression of luminal differentiation markers, enrichment of epithelial-to-mesenchymal transition (EMT) and stem cell markers. Interleukin-6 (IL6), a potent pleiotropic cytokine, is induced in response to acute and chronic inflammation. IL6 is a key regulator of inflammatory responses and orchestrates these physiological functions by controlling the Suppressor of cytokine signaling 3 (SOCS3) mediated Stat3/NF-κB pathway. SOCS3 is a critical negative regulator of IL6-mediated pathways. We have showed that constitutive activation of inflammatory loop in transformed cells but not in their untransformed counterparts depends on proteolytic degradation of SOCS3. Several studies indicate that activation of the tryptophan-degrading enzyme indoleamine-2,3-dioxygenase (IDO) represents a key pathway suppressing anti-tumor immunity. Moreover a recent study in dendritic cells found that SOCS3 binds IDO via phosphotyrosine-containing peptides and target the IDO/SOCS3 complex for ubiquitination and subsequent proteasomal degradation. IDO is shown to be constitutively expressed by many tumors. We analyzed a panel of breast cancer cell lines and found an inverse correlation between SOCS3 and IDO1 protein levels. Interestingly IDO protein and transcript levels were significantly increased in TNBC compared to luminal cell lines, thus confirming that IL6-JAK-STAT inflammatory loop is responsible for IDO regulation. Enforced expression of stable form of SOCS3, lacking the PEST and SOCS box domain in TNBC cell lines resulted in decreased expression of IDO. Therapeutic targeting of the IDO-IL6-SOCS3 inflammatory loop would revert the immune suppression mediated by IDO in this breast cancer subtype. These studies would provide a strong rationale for development of inflammatory pathway targeted agents for the treatment of TNBC, an aggressive disease that currently lacks molecularly targeted therapeutics. Citation Format: Maria Ouzounova, EunMi Lee, Ena Novakovic, Satish Kumar Noonepalle, Raziye Piranlioglu, Huidong Shi, Max Wicha, Hasan Korkaya. SOCS3 regulates IDO proteasomal degradation and inflammatory signaling in triple negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1984. doi:10.1158/1538-7445.AM2015-1984

Metastatic breast cancer is the second leading cause of cancer-related death among women in the United States. Although the genetic and epigenetic differences between the metastatic versus non-metastatic breast tumors have been well studied, early events between tumor and immune system in metastatic process remain poorly understood. In order to determine early events, we utilized murine mammary tumors (4T1 as metastatic, EMT6 or 67NR as non-metastatic) in syngeneic mouse model. The 4T1 tumor contained a higher proportion of cancer stem cell (CSC) population compared to the non-metastatic EMT6 or 67NR clones. Although, both murine tumor cell lines (50K each) grow to same size tumors within 8 weeks, 4T1 tumors develop spontaneous metastasis in 100% of animals most of which do not survive more than 8 weeks due to extensive wide spread metastasis to lung, liver and bone. We observed immune infiltrates in the lungs of 4T1 tumor bearing mice as early as 1 week. We next assessed the cytokine profile of metastatic 4T1 tumor compared to non-metastatic counterparts (EMT6 or 67NR) secretes significantly higher levels of inflammatory cytokines, including the IL6, IL8, RANTES, GCSF, GM-CSF, IL12, CXCL16 and CXCL5. MDSCs are potent suppressor of anti-tumor immunity and a significant impediment to cancer therapy. We therefore hypothesized that the tumor secreted inflammatory cytokines promotes the systemic expansion of MDSCs that down regulate immune surveillance and anti-tumor immunity, thus facilitating tumor progression. We sought to determine whether 4T1 tumors could induce MDSCs in mice. Murine 4T1 or EMT6 tumor cells (at 50K cells each) were implanted into the fat pads of BALB/c mice, then sacrificed (4 mice from each group) at weeks 1, 2, 3 and 4 for subsequent evaluation of the MDSC expansion in bone marrow, spleen, lung and tumors. The MDSC induction and infiltration in bone marrow, spleen, lung and tumors were observed as early as one-week post-implantation of 4T1 tumor compared to the EMT6. Furthermore, the MDSCs isolated from 4T1 tumor bearing animals were more suppressive than that of the EMT6 tumor bearing mice. We determined that non-metastatic EMT6-Luciferase tumor growth and metastasis is robustly enhanced in pre-primed animals (in which metastatic 4T1 cells were pre-implanted in the fat pads and resected after 10 days when tumors were 2mm in size) or by IP injection of inflammatory cytokine rich 4T1 conditioned medium when compared to injection of EMT6-Luci cells into naïve animals. Our preliminary findings suggested that 4T1 tumors within 10 days of implantation created a systemic tumor-promoting microenvironment and thus promoted the metastatic spread of EMT6-Luci. Together these studies strongly suggest that metastatic 4T1 tumor with high CSC phenotype generate a permissive systemic microenvironment for successful metastasis via secretion of inflammatory cytokines in syngeneic BALB/c mice. Citation Format: Hasan Korkaya, Eunmi Lee, Maria Ouzounova, Abdeljabar El Andaloussi, Ena Novakovic, Raziye Piranlioglu, Mehmet F. Demirci, Shawn G. Clouthier, Max S. Wicha. Systemic early immune priming via tumor-secreted cytokines facilitates breast cancer metastasis in syngeneic mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 466. doi:10.1158/1538-7445.AM2015-466

Indoleamine 2, 3-dioxygenase 1, encoded by IDO1, is a key immunosuppressive enzyme that catabolizes essential amino acid tryptophan to kynurenine in the tumor microenvironment. Severe depletion of tryptophan by IDO1 affects proliferation of T cells and tryptophan metabolites cause T-cell anergy and induce apoptosis. In this study, we investigated the epigenetic regulation of IDO1 expression by DNA methylation in breast cancer cells. Bisulfite pyrosequencing analysis of IDO1 promoter methylation performed on a panel of 10 breast cancer cell lines revealed that triple negative breast cancer (TNBC) cell lines (i.e. MDA-MB-231, Hs578t, SUM159) are hypomethylated compared to estrogen receptor positive (ER+) cell lines (i.e. MCF7, BT474, T47D). The same analysis was extended to 30 primary breast tumor and normal control samples and the results demonstrated that TNBC tumors had lower IDO1 promoter methylation compared to ER+ tumors. RT-PCR analysis showed that IDO1 mRNA expression is higher in TNBC cell lines than ER+ cell lines. This inverse correlation between IDO1 promoter methylation and mRNA expression can also be observed in TCGA 450K methylation and RNA-seq data sets in which promoter is hypomethylated and mRNA is up-regulated in basal-like molecular subtypes as compared to other breast cancer subtypes. IDO1 promoter is relatively CpG poor with most CpG sites concentrated near interferon γ (IFNg) responsive GAS and ISRE transcription factor binding sites. To investigate the role of CpG methylation in regulating IDO1 induction by IFNg, we cloned either methylated or unmethylated IDO1 promoter DNA into a luciferase reporter plasmid. Methylated promoter reporter exhibited significantly lower luciferase activity at basal or with IFNg stimulation compared to unmethylated reporter plasmid when transfected into MCF-7 or MDA-MB-231 cell lines. Treatment with a demethylating agent, 5-aza-deoxycytidine, synergistically up-regulated IDO1 mRNA expression with IFNg in MCF7 cells which have hypermethylated IDO1 promoter further supporting the influence of CpG methylation on IDO1 expression. IFNg stimulation or co-culture with activated T-cells significantly induced IDO1 protein expression in MDA-MB-231 cells, but not in MCF7 cells. We found no difference in IDO1 mRNA stability and IFNg induced JAK/STAT signaling pathway between MDA-MB-231 and MCF7 cell lines except promoter methylation. Furthermore, RNA-seq analysis of MDA-MB-231 and MCF7 cell lines co-cultured with activated T-cells revealed an active immune signaling mediated through JAK/STAT pathway shared by both cell lines, but also differential induction of IDO1 transcription between these two cell lines. These findings indicate IDO1 promoter methylation status as an important factor that regulates anti-immune responses by tumor cells towards tumor infiltrating lymphocytes and it could be used as a useful biomarker for IDO inhibitor based immunotherapy. Citation Format: Satish Kumar Reddy Noonepalle, Eun Joon Lee, Maria Ouzounova, Jaejik Kim, Jeong-Hyeon Choi, Austin Shull, Lirong Pei, Ravindra Kolhe, Pei-Yin Hsu, Nagireddy Putluri, Tim Hui-Ming Huang, Arun Sreekumar, Hasan Korkaya, David Munn, Huidong Shi. Promoter methylation regulates interferon-γ induced indoleamine 2,3-dioxygenase expression in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4060. doi:10.1158/1538-7445.AM2015-4060

We demonstrated that knock-down of tumor suppressors p53 and PTEN in mammary epithelial MCF10A cells (called MCF10A-p53-PTEN-) results in generation of aggressive tumors. Furthermore MCF10A-p53-PTEN- cells also display an increased EMT phenotype as well as expansion of cancer stem cell (CSC) population. Utilizing Affymetrix gene expression analyses, we confirmed that malignant MCF10Ap53-PTEN- cells displayed a gene expression pattern which closely resembled that of the basal/claudin molecular subtype with the TN. This subtype is previously characterized by the expression of EMT and stem cell genes. The Inflammatory cytokines IL6 and TGF-β have been found to regulate stem cells and EMT. Consistent with these data, we found that MCF10A-p53-PTEN- cells secreted significantly higher levels of IL6 (>1000 fold) and TGF-β (>200 fold) compared to parental or single gene deleted cells. Furthermore combined deletion of p53 and PTEN compared to single gene knockdown resulted in transformation of MCF10A cells generating aggressive metastatic tumors in NOD/SCID mice. IL6 is a key regulator of inflammatory responses and orchestrates these physiological functions by controlling the SOCS3 mediated Stat3/NF-κB pathway. Consistent with these reports, we demonstrated that SOCS3 is abundantly expressed in MCF10A cells where it negatively regulates the IL6 feedback loop. Unexpectedly, we found that SOCS3 protein was undetectable in MCF10A-p53-PTEN- cells. Basal/claudin-low breast cancer cell lines that display an activated IL6 feedback loop also had low SOCS3 protein levels. Furthermore, we found a similar discordance between SOCS3 protein and mRNA levels in primary basal/claudin-low breast cancer samples as demonstrated by immunochemistry utilizing a primary breast cancer tissue array. Together these findings suggest that loss of SOCS3 protein expression allows for maintenance of the IL6 mediated inflammatory feedback loop in aggressive/metastatic TN breast cancers. We found that MCF10A-p53-PTEN- cells displayed increased proteasome activity as evidenced by SOCS3 protein accumulation upon proteasome inhibition. Proteolytic degradation of SOCS3 protein in turn is controlled by constitutive activation of the IL6 mediated Stat3/NF-κB pathway. We demonstrate that enforced expression of SOCS3 or IL6 pathway inhibition interfering with this loop through IL6R blockade repress the CSC population, reducing the tumor growth and metastasis in mouse xenografts. These studies provide a strong rationale for development of IL6 pathway-targeting agents for the treatment of TNBC, an aggressive disease that currently lacks molecularly targeted therapeutics. Citation Format: Hasan Korkaya, Maria Ouzounova, Gwangil Kim, Ali Quraishi, April Davis, Nader Tawakkol, Sumeyye Korkaya, Max S. Wicha. SOCS3-mediated regulation of inflammatory cytokines in triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3869. doi:10.1158/1538-7445.AM2014-3869

Recent studies by us and others suggested that activation of inflammatory feedback loop play critical role in metastasis and therapeutic resistance by regulating EMT/CSC phenotype. Of these cytokines, IL6 is a key regulator of inflammatory responses and orchestrates these physiological functions by controlling the Stat3/NF-kB pathway which is negatively regulated by the suppressor of cytokine signaling 3 (SOCS3). Consistent with these reports, we demonstrated in our preliminary data that SOCS3 is abundantly expressed in non-malignant MCF10A cells as well as in luminal and HER2+ tumors where it negatively regulates the IL6/Stat3/NF-kB signaling. In contrast, SOCS3 protein was undetectable in basal cluadin-low breast cancer cell lines and primary tumors which express substantially higher levels of IL6. We developed transformed MCF10A model by simultaneous knockdown of p53 and PTEN. In this model single p53 and PTEN deletions result in hyperplasia or DCIS-like lesions. Interestingly, SOCS3 protein expression was undetectable in MCF10A-p53-PTEN- cells compared to parental MCF10A, single p53 (MCF10A-p53-) or PTEN (MCF10A-PTEN-) deleted cells. We demonstrated that IL6 mediated Stat3/NF-kB signaling is negatively regulated by SOCS3 that strongly inhibited the IL6/Stat3/NF-kB pathway when overexpressed in MCF10A-p53-PTEN- cells. Moreover, enforced SOCS3 expression in these cells reduced the tumor growth and inhibited metastasis. Furthermore, loss of SOCS3 expression is strongly associated with increased risk of recurrent disease in breast cancer patients. We hypothesized that loss of SOCS3 in TNBCs results in activation of IL6 feedback loop which drives aggressive metastatic phenotype. Interestingly Stat3/NF-kB signaling drives expression of SOCS3 in response to inflammation and serves as negative feedback loop, although molecular mechanism of SOCS3 expression in cancer has not been investigated. IL6 signaling has been previously reported to be involved in epigenetic regulation. We therefore correlated the gene expression and methylation profile after enforced SOCS3 expression. Our analysis revealed that expression levels of a significant number of genes involved in transcription, migration and metabolism were reverted to those of the parental non transformed cells and their transcription was regulated by methylation. These results suggest that interplay between genetic and epigenetic changes during malignant transformation of mammary epithelial cells may activate multiple signaling cascades and reconstitution of SOCS3 may have therapeutic utility in TNBC. Citation Format: Maria Ouzounova, Gwangil Kim, April Davis, Ahmed A. Quraishi, Nader Tawakkol, Shalini Kota, Max S. Wicha, Hasan Korkaya. Inactivation of p53/PTEN confers a specific epigenetic profile regulated by IL6-SOCS3 signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3866. doi:10.1158/1538-7445.AM2014-3866

Introduction: Previous studies showed that 60-80% of Her2+ breast cancers develop Trastuzumab-resistance after one year treatment and more than 50% of Her2+ Trastuzumab-resistant breast cancers have PTEN deletion and increased populations of cancer stem cells (CSCs). The purpose of this study is to explore novel targets to regulate cancer stem cells and to test treatment option for Her2+ Trastuzumab-resistant breast cancer. Method: Trastuzumab resistance of Her2+ breast cancer cells (BT474) was induced by shRNA knockdown of PTEN and long term treatment of Trastuzumab (LTT) in comparison with Trastuzumab-sensitive BT474. The whole transcriptome RNA sequencing was performed and the differentially expressed genes were evaluated using dChip analyzer and the bioinformatics tool DAVID. Colony formation in soft agar and mammosphere formation were used to evaluate the effect of candidate genes on breast CSCs. Immunohistochemistry (IHC) was used to assess the protein expression levels in xenograft animal tissues and human breast cancer tissues. MTS assay was used to evaluate the effect of drug treatment to inhibit proliferation of BT474 and LTT. A xenograft model was used to determine whether drug efficacy to inhibit tumor formation. The secondary implantation was used to monitor cancer stem cells growth in vivo after treatment. Result: In comparison of BT474 and LTT under treatment of Sulforaphane (2-10 uM, 8-24 hr), RNA-Seq analysis revealed that 51 genes, including 23 up-regulated and 28 down-regulated by Sulforaphane treatment. DAVID functional clustering analysis revealed that 6 significantly altered genes were associated with 9 distinct functional categories. Among them, MEOX-1 was identified as top candidate to regulate Trastuzumab-resistance phenotype. siRNA knockdown of MEOX-1 in LTT cells inhibited mammosphere formation by 60.4% and colony formation by 86.7%. Further, in the IHC of 68 primary breast cancer samples, expression of MEOX-1 was positive in 34 primary breast cancer tissues (34/68, 50%), especially in triple negative breast cancer tissues (P Sulforaphane inhibited the proliferation of LTT cells by 20% to 80%, while it showed no effect on BT474 cells in vitro. Also Sulforaphane decreased MEOX-1 mRNA level by 6-fold LTT cells. Daily injection with 50 mg/kg Sulforaphane reduced the growth of mouse xenografts by >50% in SCID xenograft tumors (P Conclusion: MEOX1 is a novel target to regulate cancer stem cells in Her2+ Trastuzumab-resistant breast cancer cells. Drug treatment to down-regulate Meox1 provides treatment option for Trastuzumab-resistant breast cancer by eliminating breast cancer stem cells. Citation Format: Lichao Sun, Joseph P. Burnett, Mari Gasparyan, Hasan Korkaya, Hui Jiang, Yajing Liu, Jamie Connarn, Max Wicha, Duxin Sun. MEOX-1 as a novel cancer stem cell target for treatment of trastuzumab-resistant Her2+ breast cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 218. doi:10.1158/1538-7445.AM2014-218

Estrogen (E2) stimulation promotes proliferation in estrogen receptor-positive (ER+) breast cancer, which accounts for 60-75% of all breast cancer. E2 signals through the ER via three pathways: the classical genomic pathway involving transcriptional activation of the ER, crosstalk between ER and other transcription factors, and “non-genomic” signaling cascades, which do not involve the transcriptional activity of the ER. This “non-genomic” pathway likely involves Src family kinases (SFKs) and has been implicated in cell cycle progression in fibroblasts engineered to express the ER (Castoria 1999). While not typically amplified or mutationally activated in ER+ breast cancer, Src is hyperactive in most breast cancers. To test whether SFK activity is required for G1/S cell cycle progression in breast cancer cells, we pretreated quiescent ER+ MCF7 cells with a selective SFK inhibitor (SU11333) prior to stimulation with E2. Inhibition of SFK activity blocked progression from G1 to S phase. Previous data from our laboratory has demonstrated that G1/S progression in response to peptide growth factor stimulation of quiescent fibroblasts is dependent upon SFK-mediated stabilization of myc mRNA (Bromann 2005). We therefore tested whether SFK activity also regulates myc expression in breast cancer cells. Our data suggest that SFK activity is required for accumulation of myc mRNA in quiescent MCF7s after E2 stimulation, and that E2 stimulated stabilization of myc mRNA is dependent on SFK activity. One potential myc mRNA stabilizer is IMP1 (IGF2BP1, ZBP1, CRDBP) an RNA binding protein that is primarily expressed during development and in stem cells, and re-expressed in some cancer types, including breast cancer. IMP1 has previously been shown to stabilize myc mRNA in stem cells as well as osteosarcoma cells. While the signaling pathways that mediate this stabilization have not been well studied, previous work has identified a Src phosphorylation site on IMP1 which regulates IMP1 function (Sasaki 2010). We hypothesized that IMP1 may play a role in E2-mediated stabilization of myc mRNA. Indeed, MCF7 cells require IMP1 to accumulate myc mRNA after E2 stimulation. In summary, E2 stimulation activates SFKs, which then promote G1/S cell cycle progression in part by stabilizing myc mRNA levels, through the activity of IMP1. Bromann et al JBC, 2005 Castoria et al. EMBO J, 1999 Sasaki et al. J Neurosci, 2010 Citation Format: Christopher Abdullah, Hasan Korkaya, Sara A. Courtneidge. Estrogen stimulates cell cycle progression dependent on Src kinase activity and IMP1 stabilization of myc mRNA in estrogen receptor-positive breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3300. doi:10.1158/1538-7445.AM2014-3300

The natural product sulforaphane (SF) has demonstrated efficacy against multiple forms of cancers and cancer stem cells. However, it is unclear what are the molecular targets for sulforaphane to inhibit breast cancer stem cells and how efficacy varies among four subtypes of breast cancers. In vitro assay showed that sulforaphane preferentially inhibited triple negative (TNBC) breast cancers, but did not show any efficacy to inhibit Her2+ breast cancer cells (even at >100 uM). However, when Her2+ breast cancer cells (with PTEN deletion) developed resistance to trastuzumab, sulforaphane showed more than 20-fold higher activity. Low concentration of sulforaphane (1 uM) also preferentially eliminates breast cancer stem cells from triple negative breast cancers. Her2+ breast cancer cells (with PTEN deletion and long term treatment of trastuzumab) induced resistance to trastuzumab and increase breast cancer stem cell populations by activating a positive feedback loop of IL-6 and NF-kB. Luciferase reporter assay and immunostaining showed that sulforaphane (1 uM) disrupted NF-kB translocation from cytosol to nucleus and inhibited its activity. Sulforaphane (1 uM) also inhibited endogenous IL-6 production by 50%. Sulforaphane (50 mg/kg, adjuvant therapy) reduced tumor formation rate by 70% and inhibit tumor volume by more than 90% of trastuzumab resistant Her2+ breast cancers in mouse fat pad. These data suggest that sulforaphane inhibits trastuzumab-resistant Her2+ breast cancers and breast cancer stem cells by disrupting NF-kB and IL6 positive feedback loop. Citation Format: Joseph Burnett, Hasan Korkaya, Bryan Newman, Sarah Conley, Jamie Connarn, Lichao Sun, Hsiu-Fang Lee, Maria Ouzounova, Max Wicha, Duxin Sun. Natural product sulforaphane selectively inhibits breast cancer stem cells in basal and trastuzumab resistant Her2+ breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 913. doi:10.1158/1538-7445.AM2013-913

Backgroung: The Epithelial-Mesenchymal Transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. EMT cells possess the characteristics of cell motility, invasiveness, and have stem cells properties. EMT expression profile correlates with resistance to chemotherapy and poor outcome in multiple tumors and there is evidence suggesting an integral role of Notch pathway in mediating an EMT status. In this study, we investigated Notch activity and EMT status and the correlation with chemoressitance in lung adenocarcinoma. Results: Transduction of multiple lung adenocarcinoma cell lines, NCI-H1299, NCI-H460, NCI-H358, NCI-H441 with a Notch GFP-reporter construct identified a subset of cells with high Notch activity (GFP-positive). We compared cell migration capacity of GFP-positive and -negative cells utilizing a transwell invasion assay. Cells with high Notch activity, as evidenced by GFP expression, had a significantly higher migration capability as compared to GFP-negative cells. To evaluate resistance to chemotherapy, we treated GFP-pos and GFP-neg cells with docetaxel or cisplatin and measured apoptosis by Annexin V expression with flow cytometry. GFP-pos cells showed major resistance to both agents as compared to GFP-neg cells. When GFP-pos cells were exposed to a Notch activity blocker, gamma secretase inhibitor (GSI), these cells were rendered sensitive to cisplatin or docetaxel exposure as evident by an increase in Annexin V expression. Using RNA microarray analysis and RT-PCR assays, there was an increased expression of EMT related genes (Vimentin, N-Cadherin, Snail-1,….) in GFP-pos cells as compared to GFP-neg cells. Furthermore, NOD/SCID mice with subcutaneous primary lung tumor xenografts treated with GSI showed decreased expression of downstream effectors of Notch as as well as vimentin. Mice treated with combination of GSI and docetaxel had better tumor control as compared to those treated with GSI and docetaxel as single agents. Conclusion: These studies suggest that the Notch pathway is an important regulator of EMT status and that its inhibition induces mesenchymal to epithelial transition overcoming chemoresistnace. Notch inhibitors in combination with standard chemotherapy present a potential effective approach to achieve better disease control in lung cancer. Citation Format: Khaled Hassan, Luo Wang, Hasan Korkaya, Paige O'Dowd, Sarah Dubaisi, Rayyan Kadi, Rawad Nasr, Binghui Zhang, April Davis, Gregory Kalemkerian, Max Wicha. Blocking the Notch pathway induces mesenchymal-to-epithelial transition in lung adenocarcinoma: A novel approach to overcoming chemoresistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 960. doi:10.1158/1538-7445.AM2013-960

The development of HER2 targeting agents such as trastuzumab and lapatinib represents one of the greatest achievements in clinical oncology demonstrating the effectiveness of molecularly targeted therapeutics (2). Despite the clinical benefits of trastuzumab in improving progression free and overall survival, majority of patients demonstrate “intrinsic” resistance or develop “acquired” resistance within one-to-two years. Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab, the mechanisms mediating this resistance remain to be investigated. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrated that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of breast cancer stem cell (CSC) population. We and others previously demonstrated that cytokines including IL6 and IL8 regulates breast cancer stem cell self-renewal. Furthermore, elevated serum levels of these cytokines in breast cancer patients strongly correlated with metastatic disease and therapeutic resistance. We therefore hypothesized that activation of IL6 feedback loop may mediate trastuzumab resistance. Utilizing these models, we demonstrated that long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. Recombinant IL6 or conditioned medium from high-IL6 secreting cells resulted in trastuzumab resistance in parental PTEN wild type cells expanding CSCs with EMT phenotype characterized by the CD44+/CD24- expression and EMT gene signature. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies have important clinical implications demonstrating that trastuzumab resistance may be mediated by an IL-6 inflammatory loop and suggest that blocking this loop represents a novel strategy to overcome trastuzumab resistance. . Citation Format: April L. Davis, Hasan Korkaya, Maria Ouzounova, Fayaz Malik, Gwangil Kim, Ahmed A. Quraishi, Suling Liu, Shawn G. Clouthier, Max S. Wicha. IL6 feedback loop regulates breast cancer stem cells which mediate trastuzumab resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3736. doi:10.1158/1538-7445.AM2013-3736

Gene expression analysis predicted strong activation of HDACs, MYC and NF-kappaB with potential sensitivities to vorinostat in breast cancer stem cells. Vorinostat was subsequently confirmed to inhibit mammosphere formation of the ALDH+ subpopulation of SUM159. In the present study, we conducted microarray experiments with RNA extracts from stem cells (ALDH+) and non-stem cell (ALDH-) populations of SUM159 in the absence or presence of 1uM vorinostat for 12h and 24h. The action of vorinostat was modeled by a series of computational tools integrating chemical genomics, gene signatures, gene promoters, microRNAs, pathways and gene networks. In the stem-cell population (ALDH+ SUM159), promoter and gene signature analysis revealed that MYC and NF-kappaB were strongly down-regulated by vorinostat at 12h and more prominently at 24h. MicroRNAs predicted to be most likely down-regulated by vorinostat at 12h were known transcriptionally regulated by either MYC or NF-kappaB. Furthermore, Notch1 and its ligand JAG2 were identified as key factors in modulating MYC activities in response to vorinostat. The predicted MYC down-regulation by vorinostat was confirmed by western blot and RT-PCR and NF-kappaB down-regulation by luciferase reporter assay. Other pathways such as beta-catenin and SRC were not significantly modulated by vorinostat. Surprisingly, the RAS pathway was activated by vorinostat with up-regulation of a RAS-activation gene signature, over-expression of KRAS/MEK/ERK and potential down-regulation of let-7, a KRAS targeting microRNA. In addition, epithelial-to-mesenchymal transition (EMT) was predicted to be activated by vorinostat with up-regulation of an EMT gene signature (TGFB1, SNAIL, SLUG, TWIST, ZEB1, ZEB2, alpha-SMA, FN1, VIM, N-cadherin etc.) and potential down-regulation of miR-200, an EMT antagonizing microRNA. Activation of RAS and EMT could render drug resistance in bCSCs. In the non-stem cell population (ALDH- SUM159), vorinostat down-regulated MYC and NF-kappaB at 12h but to a lesser extent at 24h. Unlike the ALDH+ cells, the RAS pathway was down-regulated at both 12h and 24h with no significant change in EMT. In summary, vorinostat exhibited differential activities in the stem cell and non-stem cell populations of SUM159 with prominent down-regulation of MYC and NF-kappaB, and divergent activities in RAS and EMT. A gene network was constructed to model the dynamics of vorinostat action in bCSCs. Further investigations were warranted including potential drug combinations. The computational framework proved complementary to the wet-lab approaches and capable of providing guidance in therapeutic discovery in targeting breast cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-100. doi:1538-7445.AM2012-LB-100

Recent studies have suggested similarities between cancer stem cells and the epithelial mesenchymal transition (EMT) state. In contrast, other studies suggest that these “states” are mutually exclusive. Our studies suggest that these divergent views may be explained by the existence of multiple stem cell states, which are regulated by microRNAs. Utilizing primary breast tissue and established cell lines, we demonstrate that both normal and malignant breast stem cells exist in distinct, inter-convertible states. The EMT-like state is characterized by expression of vimentin and N-cadherin, slug, snail and twist transcription factors. EMT-like CSCs have a mesenchymal morphology, are largely quiescent, invasive and characterized by expression of the CSC markers CD44+CD24− and are EpCAM−CD49f+. In contrast, the MET (mesenchymal epithelial transition) state of CSCs is characterized by an epithelial morphology and expression of E-cadherin and EpCAM. MET-like CSCs undergo self-renewal and express the CSC marker Aldehyde dehydrogenase (ALDH) and are EpCAM+CD49F+. A subpopulation of cells expressing both CD44+CD24− and ALDH may represent cells in transition between these states. This transition is regulated by signals in the microenvironment which in turn modulate microRNA networks. Expression induction of mir100 in MCF10A cells and several cancer cell lines resulted in a decrease of ALDH-positive CSC population with a concomitant increase in the CD24−CD44+ population accompanied by induction of EMT. We demonstrated mir100 effects are mediated by targeting BMPR2, SMARCA5 and SMARCD1, all of which may contribute to induction of EMT. Moreover, we show that mir100 overexpression induces cellular quiescence as shown by Ki67 and Brdu staining. Induction of mir100 expression immediately upon orthotopic implantation or after the tumors are established significantly reduced the subsequent tumor growth in NOD/SCID mice. In contrast of overexpression of mir100 stimulates cell invasion in vitro matrigel assay. The existence of multiple cancer stem cell states has important implications for understanding stem cell plasticity as well as tumor growth and metastasis. In addition, the existence of these states has implications for the development of CSC-targeting therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3310. doi:1538-7445.AM2012-3310

Background: The Epithelial-Mesenchymal Transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. EMT cells have stem cells properties and possess the characteristics of cell motility, invasiveness and chemotherapy resistance. EMT expression profile correlates with poor outcome in multiple tumors and there is evidence suggesting an integral role of Notch pathway in mediating EMT. In this study, we investigated Notch activity and EMT status and the correlation with chemoressitance in non-small cell lung cancer (NSCLC). Results: Transduction of multiple NSCLC cell lines, NCI-H1299, NCI-H460, NCI-H358, NCI-H441 with a Notch GFP-reporter construct identified a subset of cells with high Notch activity (GFP-pos). We compared the tumor invasivness capacity of GFP-pos and GFP-neg cells utilizing a transwell invasion assay. Cells with high Notch activity, as evidenced by GFP expression, had multiple fold higher invasion capability compared to GFP-neg cells (p=0.0001). To evaluate whether Notch activity correlates with resistance to chemotherapy, we treated GFP-pos and GFP-neg cells with docetaxel or cisplatin and assessed Annexin V expression using flow cytometry. GFP-pos cells had five fold less apoptotic cells as compared to GFP-neg cells indicating resistance of GFP-pos cells to chemotherapy. When GFP-pos cells were exposed to a Notch signaling inhibitor inhibitor, gamma secretase inhibitor (GSI), these cells were rendered sensitive to cisplatin and docetaxel. RNA microarray analysis and RT-PCR analysis revealed an increased expression of EMT related genes (Vimentin, N-Cadherin, Snail-1,….) in GFP-pos cells as compared to GFP-neg cells. Furthermore, NOD/SCID mice with subcutaneous lung tumor xenografts treated with GSI showed decreased expression of downstream effectors of Notch as well as vimentin. This combination of GSI and docetaxel reduced tumor grwoth in mouse xenographs compared to GSI and docetaxel as single agents. Conclusion: These studies suggest that the Notch pathway is an important regulator of EMT status and that its inhibition may alter EMT chemo-resistance properties. The addition of a Notch inhibitor may enhance the efficacy of chemotherapy in the treatment of NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3309. doi:1538-7445.AM2012-3309

Breast cancer can be grouped by expression profiling into categories that differ in biological and clinical characteristics. Molecular characterization of these subtypes on the basis of stem cell markers has shown that basal and claudin-low tumor bear stem cell like characteristics with a higher population of CD44+/CD24- and CD49f+/EpCAM- cells, which also display characteristics of epithelial mesenchymal tumor transition (EMT). There are several genetic and epigenetic signaling pathways that regulate the stem cell like characteristics of these subtypes. We examined the role of microRNA221 (mir-221) in sub populations of normal breast cells isolated from reduction mammoplasties as well as in cancer cell lines from different molecular subtypes of breast cancer. We found that mir-221 drives cells towards more basal subtype and induces EMT in both normal mammary cells and breast cancer cell lines. On the basis of CD49f and ESA staining in the cells isolated from normal mammary tissue, we found high mir-221 expression in the CD49f+/EpCAM- population. Furthermore, we showed that ALDH+ cells from mammospheres expressed significantly higher mir-221 compared to ALDH- cells. In order to determine the effect of mir-221 on the stem cell population, we overexpressed mir-221 in cells from primary mammospheres and showed that overexpression of mir-221 increases the CD49f+/EpCAM- population with increased expression of mesenchymal biomarkers, an effect also seen in a non-transformed breast cell line MCF10A. Overexpression of mir-221 in luminal breast cancer cell lines MCF-7 and T47D cell lines increased the proportion of CD44+/CD24- cells. Furthermore, overexpression of mir-221 significantly stimulated the tumor growth of MCF7 xenographs in NOD/SCID mice. These studies suggest that mir-221 regulates breast CSCs by promoting EMT. This may play an important role in tumor behavior and treatment resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3327. doi:1538-7445.AM2012-3327

Although current breast treatment guidelines limit the use of HER2 blocking agents such as trastuzumab to women whose tumors display HER2 gene amplification, recent retrospective analysis by Paik et al., have suggested that a wider group of patients may benefit from this therapy. Utilizing breast cancer cell lines, mouse xenograft models and primary human tumors and metastatic tissues, we provide a potential molecular explanation for these unanticipated clinical finding by demonstrating that HER2 is selectively expressed in the cancer stem cell (CSC) population in luminal ER+ breast cancers that do not display HER2 amplification. Furthermore, we demonstrate that the effects of trastuzumab on mouse tumor xenografts are highly dependent on the timing of administration. While the effects of trastuzumab on established tumors are limited to those with HER2 amplification, when this HER2 blocker is given immediately after tumor inoculation simulating an adjuvant setting, it significantly inhibits the growth of luminal tumors in the absence of HER2 amplification. In addition, HER2 expression is increased in luminal tumors propagated in mouse tibias as compared to primary tumors grown in mammary fat pads. Utilizing co-culture experiments with human osteocytes, we provide evidence that this regulated expression of HER2 is mediated by RANK ligand present in the bone microenvironment. The clinical relevance of these studies is demonstrated in breast cancer tissues by the co-expression of HER2 and CSC marker ALDH1 in a subset of cells which are selectively found at the invasive tumor front in luminal breast cancers. Recapitulating the mouse xenograft studies, we demonstrate increased expression of HER2 in bone metastasis compared to matched primary tumors in 8 patients with luminal breast cancer. Our results have significant clinical implications since they suggest that the benefits of adjuvant HER2 targeting therapies may extend to patients with luminal breast cancers who are currently classified as HER2-negative. Furthermore, these results support the cancer stem cell hypothesis and emphasize the clinical importance of targeting cancer stem cells in the adjuvant setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3470. doi:1538-7445.AM2012-3470

Background: The cancer stem cell hypothesis postulates that tumors contain a subset of cells with stem cell properties that can initiate and recapitulate original tumor heterogeneity in serial xenotransplantation assays. A number of developmental signaling pathways, including Notch, have been implicated in regulating stem cell properties of self-renewal and differentiation. In this study, we investigated the role of Notch activity in lung cancer stem cells utilizing a Notch GFP-reporter construct and a gamma secretase inhibitor (MRK003), which inhibits the activation of Notch through preventing the cleavage of its intracellular domain (NICD). Results: Transduction of lung cancer cell line NCI-H1299 with a Notch GFP-reporter construct identified a subset of cells with high Notch activity (GFP-positive). We compared the tumor initiating capacity of GFP-positive and -negative cells and only cells with high Notch activity, as evidenced by GFP expression, were tumorigenic in serial transplantation assays in NOD/SCID mice. Furthermore, GFP-positive cells were capable of generating both GFP-positive and GFP-negative cell populations. In contrast, GFP-negative cells generated primary xenograft tumors only and failed to generate the GFP-positive population or secondary tumors in serial xenotransplantation. Furthermore, a single GFP-positive cell but not a GFP-negative cell was able to generate a xenograft in NOD/SCID mice. In vitro, the gamma secretase inhibitor MRK003 induced cell cycle inhibition and apoptosis in lung cancer cell lines NCI-H1299, NCI-H460, NCI-H358, and NCI-H82. To determine the specificity of MRK003, we introduced NICD expressing vectors that rescued the effects of MRK003, thus demonstrating a direct role in Notch inhibition. MRK003 treatment of mice with lung tumor xenografts (100mg/kg, 3 days per week) decreased the expression of downstream effectors of Notch. Furthermore, cells derived from MRK003 treated tumors failed to induce tumors upon secondary reimplantation in NOD/SCID mice, consistent with effects of this treatment on the cancer stem cell population. Utilizing multivariate analysis, we assessed the association of Notch pathway components with clinical outcome of 441 lung adenocarcinoma patients. We found a statistically significant correlation between poor overall survival and increased Notch ligands expression (p=0.042) or decreased expression of negative modulators (p=0.00016). Conclusion: These studies suggest that the Notch pathway is an important regulator of lung cancer stem cells and that its inhibition may specifically target the cancer stem cell population. The correlation between Notch pathway activation and patient survival, suggests a potential role for gamma secretase inhibitors in the treatment of lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 496. doi:10.1158/1538-7445.AM2011-496

Recent identification of breast cancer stem cells (CSCs) suggest that a subpopulation of tumor cells with a stem cell phenotype may initiate and maintain tumors. Breast CSCs are characterized by their ability to self-renew and generate tumors in serial xenotransplantation assays in mice. The Notch pathway is an evolutionally conserved signaling pathway and Notch activity has been implicated in normal and malignant mammary stem cell maintenance. Therefore, we investigated the role of Notch in breast CSCs by utilizing a number of CSC specific assays and a mouse xenograft model. Upon activation, the Notch intracellular domain (NICD) is cleaved by the gamma secretase complex and translocates to the nucleus. Subsequently, it binds to the transcriptional complex (RBP-jk), to activate target genes such as the HES and HEY family of proteins. The purpose of this study is to test the Notch activity in CSCs from breast cancer cell lines including basal, luminal, and HER2-amplified subtypes. We hypothesize that cells with higher Notch activity may be enriched for cells with stem cell properties. To study the Notch pathway in breast cancer cell lines we used a Notch reporter that contains multiple copies of the Notch response elements upstream of the mCMV promoter that turns on GFP and luciferase expression upon Notch activation. We measured Notch activity by flow cytometry of cells or by bioluminescent imaging of tumors in mice utilizing Notch responsive GFP and luciferase expression, respectively. In addition, we utilized a gamma secretase inhibitor (GSI) which inhibits the cleavage of NICD and nuclear translocation. We treated mice with GSI, docetaxel, or GSI and docetaxel in combination. We have found that basal cell lines such as Sum 159 and MDA-MB-436 have a higher expression of Notch activity compared to the ER+ luminal cell lines MCF7, T47D, and ZR-75-1. We demonstrated that Notch activated cells show a stem cell phenotype with co-localization of expression of the Notch target gene HES1 and the stem cell marker ALDH1. In addition, Notch active cells were able to generate tumors in serial xenotransplantations in mice and to generate both Notch GFP-positive and Notch-GFP negative cells in vitro and in vivo. In contrast, Notch-GFP negative cells neither gave rise to Notch-GFP positive cells nor did they generate tumors in serial xenotransplantation assays. GSI treatment of mice with breast tumors decreased the percentage of Notch-GFP positive cells in the tumor, reducing the cancer stem cell population and decreasing tumorigenesis in serial passages. Furthermore, the combination of docetaxel with GSI further reduced tumor size by targeting the bulk tumor cell populations. These studies strongly suggest that the Notch pathway plays an essential role in breast CSC maintenance and may be an attractive target for more effective treatment. The combination therapy of GSI with cytotoxic chemotherapy is currently being evaluated in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3352. doi:10.1158/1538-7445.AM2011-3352

There is increasing evidence that many human cancers, including breast cancer, are driven and maintained by a cellular subcomponent that displays stem cell properties. These “cancer stem cells” (CSCs) may also mediate tumor metastasis and contribute to treatment resistance and relapse. Our laboratory has identified cellular markers and developed in vitro and mouse models to isolate and characterize normal and malignant human mammary stem cells. We have previously demonstrated that breast cancer cell lines contain subpopulations with stem cell properties that can be isolated by virtue of their expression of Aldehyde dehydrogenase (ALDH) as assessed by the Aldefluor assay. We demonstrated, among microRNAs differently expressed in Aldefluor-positive and Aldefluor-negative populations of breast cancer cell lines we identified micrRNA100 (mir100). We found that mir100 is significantly increased in Aldefluor-negative compared to Aldefluor-positive populations. Utilizing a tetracycline inducible lentivirus driving mir100 expression, we found that induction of mir100 expression decreased the ALDH-positive population of SUM159 cells in vitro as well as in mouse xenografts where this reduction was associated with decreased tumor growth. Furthermore, induction of mir100 expression immediately upon orthotopic implantation or intracardiac injection completely blocked subsequent tumor growth and metastasis formation. These studies demonstrate that mir100 plays a functional role in the self-renewal and differentiation of breast cancer stem cells. Furthermore, the TET-inducible mir100 system allows for controlled regulation of the cancer stem cell population providing a valuable model to simulate the effects of CSC-directed therapies on breast cancer growth and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3330. doi:10.1158/1538-7445.AM2011-3330

Sulforaphane, a Dietary Component of Broccoli/Broccoli Sprouts, Inhibits Breast Cancer Stem Cells The existence of cancer stem cells (CSCs) in breast cancer has profound implications for cancer prevention. The purpose of this study was to evaluate sulforaphane, a naturally occurring dietary compound from broccoli/broccoli sprouts, for its efficacy to inhibit breast CSCs as well as to elucidate the potential mechanisms of its effect. Aldefluor and mammosphere formation assays were used to assess the effect of sulforaphane on breast CSCs in vitro. A NOD/SCID xenograft model was employed to determine the efficacy of sulforaphane in targeting breast CSCs in vivo. The potential mechanism of sulforaphane was investigated utilizing Western blotting and β-catenin reporter assays. The results showed that sulforaphane (1∼5 µM) decreased aldehyde dehydrogenase (ALDH)-positive cell population by 65%∼80% in human breast cancer cell line (P < 0.01), and reduced the size and number of primary mammospheres by 8∼125-fold and 50%∼75% (P < 0.01), respectively. Daily injection with 50 mg/kg sulforaphane for two weeks reduced ALDH-positive cells by more than 50% in tumors (P = 0.003). Furthermore, sulforaphane eliminated breast CSCs in vivo, thereby abrogating tumor re-growth in secondary mice (P < 0.01). Western blotting showed that sulforaphane down-regulated the Wnt/β-catenin self-renewal pathway. These data suggest that sulforaphane targets breast CSCs through the down-regulation of the Wnt/β-catenin pathway as a potential mechanism. These findings provide a scientific rationale for use of sulforaphane and potentially broccoli/broccoli sprout dietary interventions for breast cancer chemoprevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4271.

There is increasing evidence that the growth and metastasis of many tumors, including breast cancer, are driven by a cellular population displaying stem cell properties. Like their normal counterparts, these breast cancer stem cells may be regulated by MicroRNAs (miRNAs). We have previously demonstrated that breast cancer cell lines contain subpopulations with stem cell properties that can be isolated by virtue of their expression of Aldehyde dehydrogenase (ALDH) as assessed by the Aldefluor assay. We compared miRNA expression in Aldefluor-positive and Aldefluor-negative populations in a series of five breast cancer cell lines. We identified specific miRNA expression profiles for each population. Among the differentially expressed miRNAs was miR-93 whose expression was significantly increased in Aldefluor-negative compared to Aldefluor-positive populations. To confirm the regulation of miR-93 during cell differentiation we constructed a miR-93 sensor tagged with GFP and demonstrated that sensor-positive (miR-93-negative) cells had significantly increased tumor initiating capacity in NOD/SCID mouse xenografts compared to sensor-negative (miR-93-positive cells). Furthermore, miR-93-negative cells gave rise to tumors containing both miR-93-negative and miR-93-positive cell populations. Utilizing a tetracycline inducible lentivirus driving miR-93 expression, we found that induction of miR-93 expression decreased the ALDH-positive population in vitro as well as in mouse xenografts where this reduction was associated with decreased tumor growth. Furthermore, induction of miR-93 expression immediately upon orthotopic implantation or intracardiac injection completely blocked subsequent tumor growth and metastasis formation. These studies demonstrate that miR-93 plays a functional role in the self-renewal and differentiation of breast cancer stem cells. Furthermore, the TET-inducible miR-93 system allows for the controlled regulation of cancer stem cell function providing a valuable model to simulate the effects of CSC-directed therapies on breast cancer growth and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 13.

Background: We showed previously that tumorigenic, mammosphere-forming human breast cancer cells characterized by high CD44 and low or undetectable CD24 levels (CD44+/CD24-/low) are intrinsically resistant to conventional chemotherapy, and therefore may be responsible for cancer relapse. Our goal is identify novel drugs that selectively target these chemotherapy-resistant, tumor-initiating cells. Gene expression analysis of CD44+/CD24-/low cells vs. non-tumorigenic cells implicated the Notch, PI3K, and Hedgehog signaling pathways in regulating CD44+/CD24-/low cells. Thus, Notch, PI3K-AKT, and/or Hedgehog inhibitors may eliminate this unique subpopulation of cancer cells, either alone or in combination with chemotherapy, and could improve patient outcome. To test this hypothesis, we are carrying out a series of preclinical and clinical studies using a gamma-secretase inhibitor (GSI) to target the Notch pathway. Methods: For preclinical studies, stable xenograft lines were generated by transplantation of human tumor biopsy fragments into immunocompromised mice. Mice with tumors (n = 32, 150-300 mm3) were randomized to four treatment groups: 1) vehicle control, 2) chemotherapy: docetaxel 3) drug: GSI (MRK-003, Merck) or 4) combination: docetaxel + MRK-003. During treatment, mice were monitored for tumor volume and body weight. At the end of the treatment cycle, residual tumors were characterized by FACS for the percentage of CD44+/CD24-/low cells, as well as for mammosphere-forming efficiency (MSFE) and tumor-initiating capacity. In a complementary clinical trial, breast cancer biopsies taken before and after treatment with GSI (MK-0752, Merck) were characterized for expression of CD44, CD24, and ALDH by FACS and for MSFE. Results and Conclusions: In preclinical studies using two independent triple negative xenograft lines, Notch pathway inhibition reduced mammosphere formation but did not affect tumor volume, with no consistent change in marker expression by FACS. In patient samples, MSFE also declined after the first cycle of GSI/chemotherapy and remained low after subsequent cycles. This response corresponded with a stasis of metastatic growth during five cycles of treatment, but metastatic burden began to increase coincident with the sixth cycle of treatment. Marker analysis suggests that GSI treatment chemo-sensitizes a significant proportion of the otherwise chemo-resistant CD44+/CD24-/low cell population indicating that they are dependent on the Notch pathway for survival. The decrease of MSFE in both preclinical and clinical studies suggests that inhibition of the Notch pathway by GSI may reduce the number of tumorigenic cancer cells that would otherwise remain after chemotherapy. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 48.