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In the United States, a woman has a 12% chance of developing breast cancer, and current treatments offer little relief to patients diagnosed with metastatic disease. Tumorigenesis and successful establishment of metastases depend upon tumor cell interactions with the surrounding immune microenvironment. Elevated tumor infiltration of immunosuppressive (M2) macrophages correlates with poor prognosis of breast cancer patients. The tumor microenvironment remarkably orchestrates molecular mechanisms that program these macrophages toward the M2 phenotype. Also, metabolic programming is instrumental in orchestrating the polarization of macrophages to assume an M1 (tumor-eradicating) or an M2 (tumor-promoting) phenotype. Aberrant activation of Hedgehog (Hh) signaling in breast cancer cells enables them to survive, proliferate, and metastasize, thus making it a promising target for breast cancer treatment. Hh signaling also enables a crosstalk between breast cancer cells and cells in their milieu, thus contributing to M2 macrophage polarization. We used two immunocompetent orthotopic mouse models of mammary tumors to test the effect of inhibiting Hh signaling on tumor-associated macrophages, and discovered that treatment with the pharmacologic Hh inhibitor, Vismodegib, induced a significant shift in the profile of tumor-infiltrating macrophages. We hypothesized that Hh activity calibrates the metabolism in macrophages, leading to enhanced M2 phenotype and function within the tumor microenvironment. Using a mass spectrometry-enabled untargeted metabolomics approach, we identified that inhibiting Hh signaling reduces flux through the hexosamine biosynthetic pathway, resulting in reduced cellular O-GlcNAcylation in M2 macrophages. This impinges upon diminished STAT6 O-GlcNAcylation, which consequently decreases fatty acid oxidation and ultimately enacts a metabolic cascade including lipid utilization, cellular bioenergetics, and mitochondrial dynamics. As such, inhibiting Hh activity mitigates the metabolomic and bioenergetic underpinnings of the immunosuppressive program of M2 macrophages, resulting in macrophages that are functionally and phenotypically reminiscent of inflammatory, anti-tumor macrophages. In conclusion, we discovered a novel role for Hh signaling in promoting polarization of tumor-associated macrophages to the M2 type through recalibrating their metabolic circuitries, ultimately leading to diminished M2 phenotype and function within the tumor microenvironment. This is the first evidence highlighting the relevance of Hh signaling in controlling a complex metabolic network in immune cells. This knowledge will help us to better understand how to target and diminish the pro-tumorigenic functions of tumor-infiltrating macrophages. Citation Format: Dominique C. Hinshaw, Ann Hanna, Tshering Lama-Sherpa, Brandon Metge, Sarah C. Kammerud, Gloria A. Benavides, Atul Kumar, Heba A. Alsheikh, Mateus Mota, Dongquan Chen, Scott Ballinger, Jeffrey C. Rathmell, Selvarangan Ponnazhagan, Victor Darley-Usmar, Rajeev S. Samant, Lalita A. Shevde. Hedgehog signaling regulates metabolism and polarization of mammary tumor-associated macrophages [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 2103.

Delicate balance between cellular plasticity and differentiation is critically maintained during mammary development. Disruption of this balance leads to breast cancer initiation and metastatic progression. Recent findings from our lab have revealed that N-Myc and STAT Interactor (NMI) protein is decreased in 70% of primary patient specimens with metastatic breast cancer. Mammary specific Nmi knock out mouse model revealed that conditional Nmi loss disrupts luminal differentiation in the mammary gland affecting alveologenesis and prompted the progression of tumors with aggressive metastatic characteristics. Our studies showed that Nmi is induced at the onset of pregnancy and its expression remains throughout lactation. Furthermore, prolactin stimulation and differentiation of HC11 murine mammary epithelial cells is accompanied by up-regulation of Nmi. STAT5a is one of the downstream effector of prolactin and is essential for differentiation of secretory alveolar epithelium. However, functional relationship of NMI and STAT5a was not known. Here we present our finding that NMI and STAT5a expression has a direct relationship in normal mammary development as well as in breast primary and metastatic tumor specimens. We demonstrate that loss of Nmi in vivo caused a decrease in STAT5a activity and a subsequent transcriptomic shift in mammary epithelial and breast cancer cells. Detailed examination of STAT5a mammary specific controlled genetic program in the context of transcription profiles of NMI knockout and overexpressing cell lines as well as mammary tumors revealed ISG20, interferon stimulated exonuclease gene 20, as a unique negatively regulated transcript. Here we show that expression of ISG20 is kept in check by NMI by miR17-92 cluster and that ISG20 has a positive influence on tumor progression and metastasis. Citation Format: Rajeev Sharad Samant, Heba A Alsheikh, Brandon J Metge, Dongquan Chen, Shi Wei, Lalita A Shevde. N-Myc and STAT interactor regulates STAT5a in mammary development and metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-14.

In the tumor microenvironment, breast cancer cells participate in crosstalk with the surrounding stroma. This tumor-stromal interaction forms a balance that dictates tumor suppressing or promoting response mechanisms. Macrophages in the tumor microenvironment are plastic and can mediate several functions depending on their activation states. Tumor-associated macrophages co-exist as two major phenotypes: anti-tumorigenic and immune-eliciting classically activated M1 as well as tumor-promoting and immune-suppressive alternatively activated M2 macrophages. Alternatively activated macrophages are associated with more aggressive stages and poor clinical outcomes in breast cancer patients as they suppress the tumoricidal properties of the immune system, thus facilitating tumor cell proliferation and dissemination. Hedgehog (Hh) signaling is a critical developmental pathway; its activation modulates vital cellular processes such as cell proliferation, stem-cell differentiation, and angiogenesis. While the Hh signaling pathway is normally tightly controlled, it can often be dysregulated which fosters tumorigenesis and tumor progression. Aberrant Hh signaling activation is particularly implicated in breast cancer progression and metastasis. In this study, we investigate the role of Hh signaling in polarizing breast cancer-associated macrophages toward the detrimental M2 subtype. We report that targeting different steps of the Hh signaling cascade attenuates the cytokine profile associated with M2 macrophages. Additionally, we demonstrate that Hh signaling enables the molecular mechanism responsible for alternative macrophage polarization. We furthermore identify significant shifts in immune cell populations infiltrating the primary tumor when we administer an FDA-approved Hh inhibitor to an in vivo mammary tumor model. This altered immune profile is dynamically characterized by a reduction in immune-suppressive cells concomitant with enhanced antigen presentation properties and cytotoxic immune cell infiltration, overall culminating in reduced metastasis. Thus, we describe a novel role for Hh signaling in enabling pro-tumorigenic immunity by alternatively polarizing breast cancer-associated macrophages. This study identifies a novel strategy, which has potential clinical implications to treat breast cancer through targeting aberrant tumor cell Hh activation and eliciting a robust anti-tumorigenic immune response. Citation Format: Ann Hanna, Dongquan Chen, Darshan S. Chandrashekar, Sooryanarayana Varambally, Rajeev S. Samant, Lalita A. Shevde. A novel role for Hedgehog signaling in macrophage-mediated immune evasion [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 4043.

The process of development requires a delicate balance between plasticity and differentiation. This balance is disrupted in cancer initiation and progression, ultimately ending in metastasis and plummeting rates of patient survival. Recent findings from our lab have revealed that protein expression of evolutionarily conserved, N-Myc and STAT Interactor (NMI) is decreased in 70% of primary patient specimens with metastatic breast cancer. To study how Nmi loss facilitates metastatic behavior and gain insight into its role in normal mammary biology, we created a novel mammary specific Nmi knock out mouse model, which we then challenged with carcinogen and oncogene induced tumorigenesis. Our studies show that Nmi is induced at the onset of pregnancy and its expression remains throughout lactation. Furthermore, prolactin stimulation and differentiation of HC11 murine mammary epithelial cells is accompanied by up-regulation of Nmi. STAT5 is the downstream effector of prolactin and is essential for differentiation of secretory alveolar epithelium. NMI interacts with STAT5 and this interaction appears to be critical to normal differentiation of mammary epithelium. Indeed, loss of Nmi in vivo caused a decrease in STAT5 activity and a subsequent transcriptomic shift in mammary epithelial and breast cancer cells. Moreover, knockdown of Nmi in HC11 cells impedes the expression of beta-casein upon differentiation with prolactin. Concurrently, Nmi knock out alveoli exhibit an extensive presence of nuclear beta-catenin, a mediator of stem cell maintenance in the mammary gland. As a result, mice exhibit an increased number of alveoli and more proliferating mammary epithelial cells (MECs). The Nmi knock out pubertal ductal tree extends into the mammary fat pad at an accelerated rate and exhibits enhanced terminal end bud numbers, a phenotype that mirrors mice altered for Wnt signaling. Tumors derived from Nmi null mammary epithelium contain significantly more cells with stem and progenitor markers, indicating that these tumors are less differentiated than their littermate counterparts. In addition, Nmi null mammary tumors exhibit invasive morphology as well as enhanced distant metastasis. We observe that conditional Nmi loss disrupts differentiation in the mammary gland and promotes the progression of tumors with aggressive metastatic characteristics. Citation Format: Hawley Christine Pruitt, Brandon J. Metge, Shannon E. Weeks, Dongquan Chen, Shi Wei, Lalita A. Shevde, Rajeev S. Samant. Conditional knockout of N-Myc and STAT Interactor disrupts normal mammary development and enhances metastatic ability of mammary tumors [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 1046. doi:10.1158/1538-7445.AM2017-1046

The transcriptional regulator TRIM28 plays an important role in development, stem cells self-renewal, chromatin organization and DNA damage response. KAP1 is an essential co-repressor for KRAB zinc finger proteins (KRAB-ZNFs). Though KRAB-ZNFs represent the largest family of human transcription factors, their biological functions are largely unknown. We showed that KAP1 and certain KRAB-ZNFs were overexpressed in breast tumors and breast cancer cell lines at both mRNA and protein levels. More significantly, an active SUMOylated form of KAP1 was markedly increased in breast cancer cells. KAP1 depletion in several breast cancer cell lines slowed cell proliferation and inhibited primary tumor growth and metastasis of MDA-MB-231LN xenografts, while KAP1 overexpression conversely stimulated cell proliferation and tumor growth. KAP1 knockdown led to down-regulation of PTGS2/COX2, EREG, CD44, MMP1, MMP2 and ID transcription factors, previously implicated in cancer progression and metastasis. These results indicate that KAP1 and KRAB-ZNFs are up-regulated in breast cancer cells and contribute to increased cell proliferation, tumor growth and metastasis. Citation Format: Joseph Addison, Colton Koontz, Sarah McLaughlin, Ryan Livengood, Elena Pugacheva, Dongquan Chen, Chad Creighton, Alexey V. Ivanov. TRIM28 is overexpressed in breast cancer and regulates the expression of multiple cancer-associated genes. [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 LB-2. doi:10.1158/1538-7445.AM2014-LB-2

Objective: To determine gene expression profile of Stage II colorectal cancers (CRCs) based on disease recurrence and patient race/ethnicity. Background: About 25-35% of Stage II CRCs recur after potentially curative surgery. Furthermore, the incidence, recurrence, and mortality rates for CRC are higher in African Americans (AAs) relative to Caucasian Americans (CAs), even when they are diagnosed with early stage disease. Differences in tumor biology may contribute to this race-based disparity. Thus, identification of these ‘high-risk’ patients is an unmet medical need. Methods: Using GeneChip microarray technology, we obtained gene expression profiles for Stage II CRCs from 16 AA and 30 CA patients. Samples from recurrent disease, within 5 years post-surgery (n=5 in AA, n= 9 in CA) were compared against non-recurrent, within 10 years post-surgery, (n=11 in AA, and n=21 in CA) samples. The data were analyzed for expression differences at the gene level comparing AA with CA patients and further analyzed for functions and pathways. Results: In the samples of recurrent CRCs, a significant number of genes (n=561) were differentially expressed, with a false-discovery rate of ≤5%. Further analyses showed 253 genes differentially expressed within the AA recurrent subgroup and 317 genes in CA recurrent subgroup. In recurrent CRCs, there were 9 genes common to AA and CA patients, suggesting a common gene signature specific for Stage II disease. Canonical pathway analyses and interaction network analyses for these differentially expressed genes revealed that ERK/MAPK, protein kinase A, and CDK5 signaling were the top three affected signaling pathways in recurrent samples from AAs. In contrast, RhoA, integrin, and cell cycle control signaling were the top three affected pathways in Stage II CRCs of CAs. This suggests, for samples from CAs and AAs, a distinct race-based gene expression and downstream effects on pathways. Conclusion: A common gene expression signature, specific for stage II, together with a race-specific expression profile may have therapeutic implications for AA and CA patients. Citation Format: Trafina Jadhav, Dongquan Chen, Balananda-Dhurjati Kumar Putcha, Temesgen Samuel, James Posey, Martin Heslin, Upender Manne. Distinct gene expression profile of recurrent stage II colorectal cancers in African American and Caucasian American patients. [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 4031. doi:10.1158/1538-7445.AM2013-4031

Background and Objective: Chromatin remodeling is fundamental for normal development and is universally affected in cancer cells. The largest family of human transcription factors, KRAB-ZNF repressors, and their essential cofactor KAP1 play important roles in chromatin organization and regulate multiple cellular processes such as cell differentiation, pluripotency, apoptosis, DNA repair and imprinting. Here, we explored the role of KAP1 and KRAB-ZNFs in breast cancer. Methods: Using cancer patient samples, in vitro and animal xenograft model we analyzed KAP1 role in cancer cell proliferation and primary tumor formation. Results: Patient sample analysis showed KAP1 and KRAB-ZNFs overexpression in significant proportion of breast tumors. KAP1 depletion in a panel of breast cancer cell lines inhibited cancer cell proliferation, while KAP1 overexpression stimulated cell growth. Concordantly, KAP1 knockdown in MDA-MB-231LN human breast cancer cells inhibited primary tumor growth in orthotopic xenograft mouse model, while KAP1 overexpression had an opposite, growth stimulatory effect. KAP1 depletion resulted in decreased expression of a number of cancer-associated genes. Functional in vitro studies provided evidence that KAP1 positively regulates expression of multiple KRAB-ZNFs. Discussion and Conclusions: These results thus establish KAP1 as an oncogenic factor in breast cancer. Grant support: NIH NCRR grant P20 RR16440 and Susan G. Komen for the Cure grant KG110350. Citation Format: Joseph B. Addison, Renata Galeeva, Dongquan Chen, Elena N. Pugacheva, Alexey V. Ivanov. KAP1 promotes malignant properties of breast cancer cells and regulates the expression of multiple KRAB-ZNFs. [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 3125. doi:10.1158/1538-7445.AM2013-3125

Background and Objective: The Epithelial-to-Mesenchymal Transition (EMT) is a developmental program which is often reactivated in aggressive metastatic breast cancer. Several transcription factors (TFs), such as Snail and ZEB can act as master regulators of EMT, directly repressing the gene for E-cadherin. We sought to identify common gene expression signatures characteristic to both EMT and a reverse process, Mesenchymal-to-Epithelial Transition (MET). Methods: Using Affymetrix Exon microarrays we characterized global gene expression changes in two cellular models: normal mammary epithelial HMLE cells induced to undergo EMT by expression of Snail1, Snail2 or ZEB1; and MDA-MB-231LN breast cancer cells induced to undergo MET by depletion of ZEB1 and ZEB2. Results: We identified two large gene sets: 1) 1017 genes down-regulated during EMT and up-regulated during MET, which we designated as MET-specific; and 2) 438 genes up-regulated during EMT and down-regulated during MET, which we designated as EMT-specific. 14 TFs were found in the EMT category. Their functional validation showed that individual overexpression of several of these TFs in HMLE cells was sufficient to elicit partial EMT. Discussion and Conclusions: Our comprehensive analysis of gene expression signatures common to EMT and MET identifies the EMT transcriptional program characteristic to invasive metastatic breast cancer. Grant support: NIH NCRR grant P20 RR16440 and Susan G. Komen for the Cure grant KG110350. Citation Format: Maria Voronkova, Joseph Addison, Renata Galeeva, James Denvir, Dongquan Chen, Alexey Ivanov. Gene expression profiling during EMT and MET identifies multiple transcription factors involved in the EMT transcriptional reprogramming. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B94.

Background: Microsatellite (MS) instability and p53 mutations have been implicated in development, progression and metastasis of colorectal cancer (CRCs). Thus, to identify aggressive tumor subsets, the gene expression profiles of Stage- III MS-stable (MSS), sporadic CRCs were analyzed based on their p53 gene status. Methods: DNA samples extracted from 136 Stage-III formalin-fixed paraffin embedded CRC tissues were analyzed for MSS and for p53 mutations. Further, mRNA samples extracted from 20 (10 each of p53-mutated and wild-type) corresponding snap-frozen Stage-III-MSS-CRCs were profiled for differential gene expression. Affymetrix Human Genome U133 Plus 2.0 arrays were used for gene expression profiling; and Affymetrix Microarray Suite (MAS 5.0), gene ontology annotation, and KEGG pathways were used for data analysis. Differentially expressed (DE) genes were confirmed by measuring mRNA levels by RT-PCR and by the quantitative nuclease protection assay. Results: A higher incidence of p53 mutations was found in MSS (65 of 113, 58%) than in MSI (6 of 23, 30%) phenotypes. Kaplan-Meier analyses demonstrated that patients with MSS-CRCs exhibiting p53 mutations had poor overall survival when compared to p53-wt-phenotypes (log rank, p=0.031). Gene expression analyses identified 116 DE genes (73 down-regulated and 43 up-regulated). All of these passed the stringent background filter (intensity p-value ≤0.001 and intensity ≥3 SD above mean background) and DE ratio (χ2 p-value ≤0.01 & fold change ≥2.0). GBP1, PSMB9, TRIM29, BST2 and TFF1, that have prognostic value in CRCs, were up-regulated in p53-mutated-MSS-CRCs but down-regulated in p53-wt-MSS-CRCs. Further, genes that regulate cell-cycle arrest (CDKN1B) and tumor growth suppression (ACVR1B, SESN1, WFDC1 and IRF7) were down-regulated in p53-mutated-MSS-CRCs, but up-regulated in p53-wt-MSS-CRCs. The genes presented here are the top-ranked (top-5) genes in their respective groups. The mRNA expression levels of these DE genes in all 136 CRCs were confirmed on two different platforms. Conclusion: Our findings suggest that mutant- and wt-p53 Stage-III-MSS-CRCs have distinct gene expression profiles and appear to be useful in identifying aggressive Stage III subsets and for the assessment of clinical outcomes. These studies are supported by grants from the National Institute of Health/National Cancer Institute (U54-CA118948, RO1-CA98932, and R03CA139629). 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 4655.