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Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by lack of ER, PR, and Her2. Up to 85% of TNBCs have a p53 mutation as their oncogenic driver. TP53 is a tumor suppressor known as the “guardian of the genome” for its roles in regulating growth and death after genomic insult. Due to this high frequency of p53 mutations in TNBCs, targeting p53 mutants in a clinical setting is highly attractive. However, there are currently no FDA-approved drugs that can directly target p53 mutant TNBCs. We propose pathways exist that, when inhibited, will induce the specific death of p53-mutant breast cancer cells, but not p53-wild type breast cells. To investigate this hypothesis, we performed high-throughput drug screening to identify drugs that induce death in p53-mutant TNBCs. We then characterized the identified drugs for mechanism of death induction and in vivo drug effect. Methods: In vitro and in silico drug screens were conducted with small-molecule libraries of drugs with known protein targets, and screens were integrated and common drugs identified. P53-wild type and mutant cells were treated with identified drugs and stained with DAPI and DRAQ7 to identify growth-suppressive and death-inductive effects. One of these drugs, the GPX4 inhibitor ML-162, was selected for further study. Mechanism of death induction by ML-162 was determined with AnnexinV/PI, caspase cleavage, and ferroptosis assays. To confirm these results, GPX4 was knocked out and replicated the effect of ML-162. To determine the reliance of this phenotype on p53 mutational status, a series of inducible p53-mutant cell lines were created in ER+ and TNBC cell lines and then treated with small molecule inhibitors. ML-162 in vivo effect was demonstrated by treating p53-mutant TNBC xenografts in nude mice. Results: Integration of in vitro and in silico screens identified 6 common drugs, representing cell cycle inhibitors, cell division inhibitors, a proteasome inhibitor, and a peroxidase inhibitor. Identified drugs were demonstrated to reduce growth or induce death of p53-mutant TNBC cell lines. The GPX4 inhibitor ML-162 was further characterized, and found to induce death through ferroptosis, and not apoptosis or necroptosis. GPX4 knockout in p53-mutant TNBC cell lines induced ferroptosis and can be blocked with an anti-ferroptosis drug. Inducible p53-mutations in ER+ and TNBC cell lines were treated with ML-162. P53 mutations in TNBC, and not ER+, cell lines showed increased sensitivity to ML-162. To demonstrate in vivo effect of ML-162, TNBC cell line xenografts were grown in nude mice and treated with ML-162. This treatment significantly reduced tumor volume and also induced lipid peroxidation, a hallmark of ferroptosis. Conclusion: Our high-throughput screening demonstrated several of the identified drugs suppress growth or induce death preferentially in p53-mutant breast cancers. One of these drugs, ML-162, induces death of p53-mutant triple-negative breast cancer cells through induction of ferroptosis, both in vitro and in vivo. These studies provide the basic science foundation to further develop ferroptosis inducers for the targeted treatment of p53-mutant breast cancers. This work was funded by the John Charles Cain Endowment. Citation Format: William M Tahaney, Jing Qian, Reid Powell, Cassandra L Moyer, Yanxia Ma, Nghi Nguyen, Jamal Hill, Clifford Stephan, Abhijit Mazumdar, Peter JA Davies, Powel H Brown. Inhibition of GPX4 induces preferential death of p53-mutant triple-negative breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS1-09.

Background: Triple-negative breast cancers (TNBCs) are the most aggressive breast cancers and have a very poor prognosis. Since TNBCs lack the expression of the estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and progesterone receptor (PR), development of new TNBC treatment strategies is an urgent clinical need. Our previous studies have demonstrated that high expression and activity of SOX9 transcription factor positively related to TNBC cell growth and tumor metastasis in vivo. It may be possible to target the SOX9 transcription factor by targeting upstream SOX9-activating proteins. Hypothesis: SOX9-binding protein, S100A10, regulates SOX9 activity and control TNBC growth. Material and Methods: Immunoprecipitation (IP) in combination with mass spectrometry (IP-MS) analysis was used to identify SOX9 binding proteins, with support from MD Anderson’s proteomics core. The Mascot Score is a statistical score used as a measure of the reliability of identifying a specific protein by IP-MS. The data was summarized based on the Mascot Score. IP-Western Blotting analysis was used to confirm that S100A10 bound SOX9 in TNBC cells. We then treated TNBC cells with or without siRNAs of S100A10 to evaluate its effect on TNBC growth. Cell growth was measured using an automated cell counting assay. Protein and mRNA levels were examined by western blotting and qRT-PCR assays. SOX9 activity on regulating β-catenin pathway was measured using TOP/FOP luciferase activity analysis. Data are presented as mean values ± SD. Statistical significance was calculated using the Student’s t-test unless otherwise indicated. Results: Using immunoprecipitation combined with mass spectrometry (IP-MS), we identified multiple SOX9-binding proteins. One of the identified SOX9-binding proteins was S100A10 (S100A10). Knockdown of S100A10 decreased TNBC cell growth in vitro. Using TOP/FOP luciferase activity analysis, we demonstrated that S100A10 regulates the β-catenin pathway. Thus, S100A10 is a potential activator of SOX9 that can be targeted to inhibit SOX9 downstream signaling and TNBC growth. Conclusion: Our results demonstrate that S100A10 is a SOX9-binding protein, that regulates SOX9 downstream signaling and controls TNBC cell growth. Implications: Inhibition of S100A10 represents a novel strategy to treat TNBCs. S100A10 inhibitors should be developed as promising new drugs to treat these aggressive breast cancers. Grant Support: These studies were supported by a Breast Cancer Research Foundation (BCRF) grant (PB). The Proteomics and Metabolomics Facility was supported in part by Cancer Prevention Research Institute of Texas (CPRIT) grant number RP130397 and NIH grant numbers 1S10OD012304-01 and P30CA016672. Citation Format: Yanxia Ma, Jing Qian, Cassandra Moyer, Amanda Lanier, Jamal Hill, Darian Coleman, David H. Hawke, Abhijit Mazumdar, Powel H. Brown. Targeting S100A10, a SOX9 binding protein, for the treatment of 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 3966.

Background: Triple-negative breast cancer (TNBC) is an aggressive cancer that lacks expression of the estrogen receptor (ER), progesterone receptor, and Erb-B2 receptor tyrosine kinase 2. TNBC patients exhibit a poor prognosis, even if treated with chemotherapy. Although studies using selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs) have shown that breast cancer prevention is feasible, these drugs do not prevent ER-negative or TNBC tumors. It has been shown by our laboratory and others that retinoid X receptor (RXR)-specific ligands (rexinoids) can prevent breast cancers that are both ER-positive and ER-negative in mice. In our previous studies in MMTV-erbB2 mice, IRX-4204, a fourth generation rexinoid, prevented the development of most HER2/erbB2-positive tumors in these mice. For this study, we hypothesized that by targeting RXR pathway, we can prevent the development of triple negative, BRCA1-mutant mammary tumors in mice. To test the hypothesis, we treated MMTV-Cre/BRCA1co/co/p53+/- mice prior to their developing tumors with IRX-4204 to determine whether this RXR agonist effectively prevents triple-negative breast tumors. Methods: We tested the tumor preventative effect of IRX-4204 using the established MMTV-Cre/BRCA1co/co/p53+/- mouse model. These mice were produced by breeding MMTV-Cre/BRCA1co/co/p53+/- males and MMTV-Cre/BRCA1co/co/p53+/+ females, and female pups were PCR genotyped. All mice were separated into 4 groups: 1) sesame oil control, 2) IRX-4204 (10 mg/kg), 3) IRX-4204 (20 mg/kg) and 4) 9-cis-UAB-30 (5 mg/kg). All treatments were given by oral gavage, five days a week from 4 months of age. Mice were observed daily for tumor formation and toxicity. The percentage of tumor free mice were recorded, from which tumor incidence and time to tumor formation was visualized using Kaplan Meier curves and analyzed using the Log-rank test. Results: In MMTV-Cre/BRCA1co/co/p53+/- mice, IRX-4204 reduced tumor incidence and was associated with an increase in median tumor free survival time from 209 days to 336 days at 10 mg/kg dose (p=0.005). At the higher dose (20 mg/kg) IRX-4204 also delayed tumor formation with median tumor free survival from 209 days to 260 days (p=0.039). The rexinoid 9-cis-UAB 30 also significantly delayed tumor formation in MMTV-Cre/BRCA1co/co/p53+/- mice with median survival from 209 days to 270 days (p=0.04). Long term treatment of IRX-4204 was not associated with any toxicity. Conclusion: RXR agonist IRX-4204 delayed ER-negative mammary tumor formation in BRCA1co/co; MMTV-Cre; p53+/- mice. Based on our results, IRX-4204 is an effective cancer preventive drug without observed toxicity. Our results suggest that studies with reduced IRX-4204 dose alone or in combination with other targeted therapies such as selective estrogen receptor modulators are warranted. In the future, clinical trials of the IRX-4204 should be considered for the prevention of breast cancer in high-risk patients. (Supported by NCI-PREVENT contract to P. Brown and A. Mazumdar HHSN26100008). Citation Format: Cassandra Moyer, Jamal Hill, Darian Coleman, Shizuko Sei, Altaf Mohammed, Martin Sanders, Powel Brown, Abhijit Mazumdar. Targeting the RXR pathway for the prevention of triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P004.

Phase III cancer prevention clinical trials have shown that breast cancer prevention is feasible using anti-estrogen drugs. However, these drugs have not been widely accepted because of concerns about toxicity. In addition, anti-estrogen drugs do not prevent estrogen-negative breast cancers, including the most aggressive form of breast cancer, triple-negative breast cancer (TNBC), that does not express the estrogen receptor (ER), progesterone receptor, or the human epidermal growth factor receptor 2 (HER2). Our laboratory is focused on identifying growth-regulatory molecules that are essential for the growth of TNBCs. For this study, we identified mTOR as an essential growth-regulatory molecule that is highly expressed in TNBCs. We then investigated whether the mTOR inhibitor everolimus can prevent mammary tumors in transgenic mouse models including 4 models of TNBC and one model of ER-negative/HER2-positive breast cancer. Everolimus treatment significantly delayed mammary tumor formation but with a varying degree in all five mouse models. Everolimus treatment for up to 1 year was well tolerated with no observable toxicity. These results suggest that mTOR inhibitors may be promising drugs for the prevention of ER-negative and triple-negative breast cancers in women at risk of these aggressive breast cancers. Grant support: This research was supported by an NCI-PREVENT contract to P. Brown and A. Mazumdar (HHSN261201500018I/HHSN26100006). Citation Format: Powel H. Brown, Abhijit Mazumdar, William Tahaney, Jamal Hill, Yun Zhang, Sumankalai Ramachandran, Jitesh Kawedia, Jing Qian, Alejandra Contreras, Michelle Savage, Lana Vornik. Targeting the mTOR pathway for the prevention of triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr IA017.

Background: Triple-Negative breast cancer (TNBC) is an aggressive cancer that lacks expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. These cancers have a poor prognosis and are treated predominantly with chemotherapy. Therefore, we are working to find preventive therapies for these potentially lethal cancers. Dysregulation of PI3K-mTOR pathway is commonly associated with TNBC and other ER-negative breast cancers. We hypothesized that targeting mTOR may prevent development of ER-negative breast cancers and in this study tested whether the mTOR inhibitor, everolimus, prevents cancer in several mouse models of ER-negative breast cancer. Methods: MMTV-erbB2 mice were purchased from the Jackson Laboratory. C3(1)/SV40TAg and BRCA1co/co;MMTV-Cre;p53+/- were generated through breeding. P53-null and p53-mutant mammary gland mice were generated by transplanting p53-null or p53-mutant mammary glands into cleared fat pads of p53-wild type mice. The mice were treated with either control or everolimus (5mg/kg), administered by oral gavage daily (5X/week). All mice develop palpable mammary tumors within 8-12 months. Mice were observed daily for tumor formation and drug toxicity. The percentage of tumor free mice was recorded, and time to tumor development was visualized using Kaplan-Meier curves and analyzed using the generalized Wilcoxon test. Results: Treatment with everolimus significantly increased median survival of all mouse models. Everolimus treatment was also able to completely prevent mammary tumor formation in 27% of C3(1)/SV40Tag mice and 44% p53-mutant mammary gland mice. In p53-null mammary gland mice, 46% of control mice had developed tumors after 420 days, compared to 7% of everolimus treated mice. Long term treatment of everolimus was associated with mild toxicity that includes slight weight loss ( Conclusions: Everolimus significantly delayed mammary tumorigenesis in all five breast cancer mouse models. Our results suggest that everolimus is a promising cancer preventive drug for ER-negative tumors, and that further studies of everolimus in combination with other targeted therapies are warranted. In the future, clinical trials of the everolimus should be considered for the prevention of breast cancer in high-risk patients. Supported by NCI PREVENT Cancer Preclinical Drug Development Program (HHSN-2612015000-18I (PB). Citation Format: Abhijit Mazumdar, Jamal Hill, William Tahaney, Yanxia Ma, Alejandro Contreras, Shizuko Sei, Altaf Mohammed, Powel Brown. Targeting the mTOR pathway for the prevention of er-negative breast cancer [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 715.

Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and lacks effective targeted treatment strategies. Previously, we identified 33 transcription factors highly expressed in TNBC. Here, we focused on six sex determining region Y-related HMG-box (SOX) transcription factors (SOX4, 6, 8, 9, 10, and 11) highly expressed in TNBCs. Our siRNA screening assay demonstrated that SOX9 knockdown suppressed TNBC cell growth and invasion in vitro. Thus, we hypothesized that SOX9 is an important regulator of breast cancer survival and metastasis, and demonstrated that knockout of SOX9 reduced breast tumor growth and lung metastasis in vivo. In addition, we found that loss of SOX9 induced profound apoptosis, with only a slight impairment of G1 to S progression within the cell cycle, and that SOX9 directly regulates genes controlling apoptosis. On the basis of published CHIP-seq data, we demonstrated that SOX9 binds to the promoter of apoptosis-regulating genes (tnfrsf1b, fadd, tnfrsf10a, tnfrsf10b, and ripk1), and represses their expression. SOX9 knockdown upregulates these genes, consistent with the induction of apoptosis. Analysis of available CHIP-seq data showed that SOX9 binds to the promoters of several epithelial–mesenchymal transition (EMT)- and metastasis-regulating genes. Using CHIP assays, we demonstrated that SOX9 directly binds the promoters of genes involved in EMT (vim, cldn1, ctnnb1, and zeb1) and that SOX9 knockdown suppresses the expression of these genes. Implications: Our studies identified the SOX9 protein as a “master regulator” of breast cancer cell survival and metastasis, and provide preclinical rationale to develop SOX9 inhibitors for the treatment of women with metastatic triple-negative breast cancer.

Heightened secretion of pro-tumorigenic effector proteins is a feature of malignant cells. Yet the molecular underpinnings and therapeutic implications of this feature remain unclear. Here we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIβ (PI4KIIIβ). Molecular, biochemical, and cell-biological studies show that PI4KIIIβ-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIβ-dependent secreted factors maintain 1q-amplified cancer cell survival and influence pro-metastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIβ antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIβ-dependent secretion for cancer cell survival and tumor progression.

Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIβ (PI4KIIIβ). Molecular, biochemical, and cell biological studies show that PI4KIIIβ-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIβ-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIβ antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIβ-dependent secretion for cancer cell survival and tumor progression.

Background: Triple negative breast cancers (TNBCs) are the most aggressive ER negative breast cancers with limited therapy strategies and poor prognosis. P53 gene is frequently mutated in approximately 80% of TNBCs. To identify novel molecular targets for ER negative breast cancer, particularly the more aggressive TNBC, we conducted a human kinome screen and identified death-associated protein kinase 1 (DAPK1) as one of the kinases that are highly expressed in ER negative breast cancer. Deletion or inhibition of DAPK1 suppresses growth of p53-mutant but not p53-wildtype breast cancer cells. Here we investigate whether DAPK1 inhibition will enhance chemotherapy action against p53-mutant TNBCs. Experimental design and methods: We performed experiments to test cell growth of p53-mutant TNBCs that were treated with DAPK1 siRNA or DAPK1 inhibitors in combination with different doses of chemotherapy drugs including 5-FU (5-Fluorouracil), doxorubicin, cisplatin, PARP inhibitor (BMN673), paclitaxel, gemcitabine and vinorelbine. Results: Our results show that DAPK1 inhibitors enhance the growth inhibitory effects of cisplatin and PARP inhibitor in p53-mutant TNBCs. Furthermore, combined DAPK1 inhibition (via siRNA knockdown) with cisplatin synergistically inhibits cell growth of p53-mutant TNBCs. Conclusion: DAPK1 is a novel, promising target for the treatment of triple-negative p53-mutant breast cancer. Our studies demonstrate that DAPK1 inhibition sensitizes TNBCs to the cytotoxic effects of cisplatin or the PARP inhibitor. We are now conducting studies to determine whether DAPK1 inhibition will sensitize TNBC tumors and patient-derived TNBC xenografts to the effects of cisplatin and PARP inhibition. These studies suggest that the combination of DAPK1 inhibition with drugs that interfere with DNA repair will be useful for the treatment of the most aggressive form of breast cancer, triple-negative breast cancer. Funding: This study was funded by a Susan G. Komen Promise grant (SAB12-00006 to P.H. Brown), a MD Anderson Knowledge Gap Moonshot grant (to P.H. Brown) and a Breast Cancer Research Foundation grant (BCRF 15101807, 2015–2016 to P.H. Brown). Citation Format: Zhao D, Zhao J, Mazumadar A, Bollu L, Shepherd J, Ma Y, Zhang Y, Hill JL, Savage MI, Brown PH. Inhibition of death-associated protein kinase 1 enhances chemotherapy action against triple-negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-07.

Background: SRY (Sex Determining Region Y)-related HMG-box (SOX) genes belong to a super-family of genes, which is characterized by a homologous sequence called the HMG-box residing on the Y-chromosome. There are 20 SOX genes present in humans and mice. We performed a siRNA screen of SOX transcription factors, and found that SOX9 was essential for breast cancer cell growth. The SOX9 protein recognizes the sequence CCTTGAG along with other members of the HMG-box class DNA-binding proteins and has been shown to be required for development, differentiation and lineage commitment. Moreover, SOX9 is expressed in adenocarcinomas, and is highly expressed in the most aggressive cancers. Our previous data shows SOX9 is highly expressed in “triple negative breast cancer” (TNBC) than in non-TNBC. Thus, we hypothesized that the SOX9 transcription factor acts as an essential molecule regulating TNBC growth and invasion. To test the hypothesis, we used SOX9-overexpressed, or SOX9-knockdown/knockout breast cancer cell models to determine whether SOX9 is necessary and/or sufficient to regulate TNBC cell proliferation, migration and invasion. Methods: We measured the cell growth using an automated cell counting assay. Cell migration and invasion were detected by transwell migration & invasion assays in ER-positive (MCF7 and ZR75-1) and ER-negative (MDA231 and MDA468) breast cancer cells. DOX-inducible SOX9-knockout cell lines were established in MDA231, MDA468, and LM2 cell lines using an inducible Cas9-CRISPR system. A SOX9 expressing lentivirus was used to overexpress SOX9, and siRNAs was used to knockdown SOXs in the different breast cancer cells. Protein and mRNA levels of SOX9 in TNBC, non-TNBC, immortalized human breast epithelial cell lines were examined by western blotting and qRT-PCR assay. Results: Knockdown of SOXs by siRNA caused decreased cell proliferation of MDA231 by ≥50% and of MDA468 by 30%-50% in siSOX4, siSOX6, siSOX9, siSOX10 and siSOX11 treatment groups (but not in siSOX8 and siSOX17 treatment groups). However, in MCF7 and T47D cell lines, treatment with siRNA to these SOX factors did not cause significant cell growth reduction. We demonstrated that SOX9 is more highly expressed in TNBC cells at both the mRNA and protein levels. Knockdown of SOX9 decreased cell migration and invasion of MDA231 to 25% and 50% respectively. The same effect also was observed in MDA468 cells, with approximately a 50% decline in migration and invasion. In SOX9-knockout MDA231, MDA468, and LM2 cells, cell proliferation, migration, and invasion were significantly reduced. In contrast, overexpression of SOX9 in MCF7 and ZR75-1 cells increased cell migration and invasion. We are now conducting in vivo studies to determine the effect of SOX9 on breast cancer cell metastasis. Conclusion: SOX9 is a critical regulator of TNBC cell proliferation, migration and invasion. These studies suggest that regulating SOX9 transcription factor and its signaling pathway will be a promising therapeutic strategy to treat TNBC and prevent metastasis. This work was supported by a Susan G. Komen Scientific Advisory Board Grant, SAB1600006 (PB), and a grant from the Breast Cancer Research Foundation 2015-2016 BCRF grant(PB), and by the Charles Cain Endowment (PB). Citation Format: Ma Y, Shepherd J, Mazumdar A, Zhao D, Bollu L, Hill J, Zhang Y, Brown P. SOX9 is a critical regulator of triple-negative breast cancer cell growth and invasion [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-04.

Estrogen receptor (ER)-negative, progesterone receptor (PR)-negative and HER2-negative, or “triple negative,” breast cancer (TNBC) is a poor prognosis clinical subtype that occurs more frequently in younger women and is commonly treated with toxic chemotherapy. Effective targeted therapy for TNBC is urgently needed. Our previous studies have identified several kinases critical for TNBC growth. Since phosphatases regulate the function of kinase signaling pathways, we sought to identify critical growth-regulatory phosphatases that are expressed differentially in ER-negative, as compared to ER-positive, breast cancers. In this study, we examined the role of one of these differentially expressed phosphatases, the protein phosphatase Mg + 2/Mn + 2 dependent 1A (PPM1A) which is underexpressed in ER-negative breast cancer as compared to ER-positive breast cancers, in regulating TNBC growth. We found that PPM1A is deleted in ~40% of ER-negative breast cancers, and that induced expression of PPM1A suppresses in vitro and in vivo growth of TNBC cells. This study demonstrates that induction of PPM1A expression blocks the cell cycle and reduces CDK and Rb phosphorylation. These results suggest PPM1A is a crucial regulator of cell cycle progression in triple negative breast cancer. Our results also suggest that PPM1A loss should be explored as a predictive biomarker of CDK inhibitor sensitivity.

Background: Triple-negative breast cancers (TNBC) represent 15% of all breast cancers, and are highly aggressive cancers with poor clinical outcome. We have previously conducted a whole phosphatase RNA microarray analysis of TNBCs compared to estrogen receptor (ER)-positive breast cancers. These studies identified phosphatases that were either over-expressed or under-expressed in TNBCs as compared to ER-positive breast cancers. One of the phosphatases highly expressed in TNBCs was NUDT5, which hydrolyzes 8-oxo-dGDP and ADP-ribose (ADPR). Recent studies have shown that NUDT5 regulates cell cycle progression, and increases cellular ATP in response to hormone stimulation in cancer cells. However, the biological function of NUDT5 in TNBCs is largely unknown. Hypothesis: We hypothesized that NUDT5 is a critical phosphatase over-expressed in TNBCs that regulates the growth of these breast cancer cells. Methods: We analyzed TNBC NUDT5 mRNA expression and clinical outcome using publicly available TCGA, METABRIC, and the CCLE datasets. Overall survival was evaluated using Kaplan-Meier curves and compared using log-rank analysis (* p Results: Analysis of NUDT5 mRNA expression demonstrated that NUDT5 is overexpressed in triple-negative breast cancers, specifically in the basal-like subtype of TNBC. Analysis of publically available breast cancer datasets showed that high NUDT5 expression is associated with poor overall survival of breast cancer patients. Knockdown of NUDT5 decreased TNBC growth, but did not decrease the growth of non-TNBC cells. Conclusions: Our results demonstrate that NUDT5 is a critical regulator of TNBC growth, and raise the possibility of targeting this critical phosphatase for the treatment of TNBC tumors. Future studies will investigate the function of NUDT5 in regulating nucleotide pool metabolism in TNBCs. Understanding the role of NUDT5 in controlling TNBC growth will provide the basic science foundation for the future development of NUDT5 inhibitors for the treatment of women with these aggressive, potentially life-threatening cancers. This work was supported by Susan G Komen Promise Grant (PB), and the John Charles Cain Endowment (PB). Citation Format: Jing Qian, William Tahaney, Yanxia Ma, Lakshmi Bollu, Abhijit Mazumdar, Jamal Hill, Yun Zhang, Powel Brown. NUDT5 is a critical regulator of triple-negative breast cancer growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1439.

Background: Breast cancer is the most common cancer in women, and about 12% U.S. women will develop invasive breast cancer over the lifetime. Triple-negative breast cancers (TNBCs) which lack expression of the estrogen and progesterone receptors and the HER2 protein.have a poor prognosis and are typically treated with chemotherapy. Our previous studies have demonstrated that SOX9 is essential for survival and metastasis of TNBCs via suppressing apoptosis and promoting epidermal to mesenchymal transition (EMT). Hypothesis: SOX9-regulated proteins control TNBC growth, and represent promising targets for the treatment of these potentially lethal breast cancers. Material and Methods: Several breast cancer datasets were used to analyze the association between overall survival and SOX9 expression. A doxycycline-inducible Cas9-CRISPR knockout or siRNA knockdown system was used to inhibit SOX9 in cells. RNA-Seq analysis was used to identify SOX9 downstream targets in TNBC cells (MDA MBA-232 and MDA MB-468 with SOX9 knockdown by siRNA transfection) or non-TNBC cells (MCF-7 with SOX9 overexpression by transfection of SOX9-expression plasmid), with support from MD Anderson's genomic biostatistics core. After identification of SOX9 downstream genes, we treated TNBC cells with or without siRNAs of these identified genes to evaluate their effect on TNBC growth. Cell growth was measured using an automated cell counting assay. Protein and mRNA levels were examined by western blotting and qRT-PCR assays. Significant genes are defined by using a false discovery rate (FDR) cut-off of 0.01 and log2 fold change of 2. Significant common genes are defined by using a FDR cut-off of 0.05 and log2 fold change of 1 among the cell lines. Data are presented as mean values ± SD. Statistical significance (p-values) was calculated using the Student's t-test unless otherwise indicated. Results: TNBCs and late stage breast cancers showed higher SOX9 expression compared to non-TNBCs and early stage breast cancers. Using SOX9 inhibited cells, we demonstrated that SOX9 is critical for TNBC cell growth in vitro and in vivo. Using mRNA-seq analysis, we identified downstream genes regulated by SOX9 in breast cancer. 340 genes and 843 genes were suppressed in MDA MB-231 and MDA MB-468 respectively upon SOX9 knockdown, while 250 genes were upregulated in MCF-7 cells upon SOX9 overexpression. Between MDA MB-231 and MDA MB-468 cell line, there were 301 common genes were down-regulated upon SOX9 knockdown. 38 genes were down-regulated upon SOX9 knockdown in TNBC cell lines, and up-regulated upon SOX9 overexpression in MCF-7 cells. Among them, the top 10 genes that show largest difference between TNBCs and MCF-7 cells are MIOX, LAMB2P1, SLCO4C1, RGCC, PRTN3, LINC00173, UCN, LOC100129, VPREB3 and CTF1 genes. We then investigated the effect of these SOX9 downstream targets on cell growth in vitro. Loss of RGCC, PRTN3, and SLCO4C1 reduced cell growth in MDA MBA-231, and MDA MB-468, while having minimal effect on the growth of MCF-7 cells. Conclusion: Our results demostrate that the SOX9-regulated genes RGCC, PRTN3, and SLC04C1 are required for TNBC growth. These genes are critical regulators of TNBC growth and are potential novel targets for the treatment of these aggressive breast cancers. Grant Support: These studies were supported by a Breast Cancer Research Foundation (BCRF) grant (PB). Citation Format: Yanxia Ma, William M. Tahaney, Jing Qian, Jamal Hill, Yun Zhan, Abhijit Mazumdar, Powel H. Brown. PRTN3, RGCC and SLCO4C1 are critical SOX9-regulated genes that control TNBC growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1303.

Estrogen receptor–negative (ER-negative) breast cancers are extremely aggressive and associated with poor prognosis. In particular, effective treatment strategies are limited for patients diagnosed with triple receptor–negative breast cancer (TNBC), which also carries the worst prognosis of all forms of breast cancer; therefore, extensive studies have focused on the identification of molecularly targeted therapies for this tumor subtype. Here, we sought to identify molecular targets that are capable of suppressing tumorigenesis in TNBCs. Specifically, we found that death-associated protein kinase 1 (DAPK1) is essential for growth of p53-mutant cancers, which account for over 80% of TNBCs. Depletion or inhibition of DAPK1 suppressed growth of p53-mutant but not p53-WT breast cancer cells. Moreover, DAPK1 inhibition limited growth of other p53-mutant cancers, including pancreatic and ovarian cancers. DAPK1 mediated the disruption of the TSC1/TSC2 complex, resulting in activation of the mTOR pathway. Our studies demonstrated that high DAPK1 expression causes increased cancer cell growth and enhanced signaling through the mTOR/S6K pathway; evaluation of multiple breast cancer patient data sets revealed that high DAPK1 expression associates with worse outcomes in individuals with p53-mutant cancers. Together, our data support targeting DAPK1 as a potential therapeutic strategy for p53-mutant cancers.

Background: Women with “triple-negative breast cancer” (TNBC), are currently treated with chemotherapy, and have a very poor prognosis. TNBC tumors also frequently have p53 and BRCA1 gene mutations. Dysregulation of PI3K-mTOR pathway has been commonly associated with ER-negative breast cancers with poor prognosis. The mTOR inhibitor everolimus is used to treat ER-positive tumors that have become resistant to anti-estrogen therapy. We hypothesized that targeting mTOR may prevent development of ER-negative and BRCA1-mutant breast cancers and asked whether the mTOR inhibitor everolimus exhibited tumor preventive efficacy in several mouse models of breast cancer. Methods: p53-null mammary gland donor mice were transplanted into cleared fat pads of p53 wild-type mice. BRCA/p53-deficient mice: We produced BRCA1 mice by breeding males and females. All mice were separated into two groups 1) Control and 2) everolimus. MMTV-erbB2 and p53 null mammary gland mice were treated with everolimus (5mg/kg, by oral gavage). BRACA1 mice were given 2mg/kg and 5mg/kg 2X a week of everolimus. All of these mice spontaneously developed mammary tumors within 12 months. Mice were observed daily, toxicity and the percentage of tumor free mice were recorded. Tumor incidence and time to tumor formation was visualized using Kaplan- Meier curves and analyzed using the generalized Wilcoxon test. Results: In MMTV-erbB2 mice everolimus reduced tumor incidence and was associated with an increase in median survival time from 240 days to 410 days. At 365 days, when all mice in control group died, only half of the mice treated with everolimus had developed tumors (p=0.0001). Everolimus also reduced tumor incidence in p53 null mammary gland mice. At 420 days, 50% of the control mice developed mammary tumors compare to only 7 % of the everolimus treated mice (p=0.04). Everolimus also reduced tumor incidence in BRCA1 mice. At 262 days, when 13 (out of 18) (72%) of the control mice developed mammary tumors compare to only 7 (out of 18) (38%) in everolimus treated group developed tumors (p=0.02). Long term treatment (>150 days) of everolimus was associated with mild toxicity that includes slight weight loss ( Conclusions: The mTOR inhibitor everolimus prevented mammary tumorigenesis in all three mouse models. Our results suggest that everolimus can be an effective cancer preventive drug and that further studies with reduced everolimus dose alone or in combination with other targeted therapies are warranted. In the future, clinical trials of the everolimus should be considered for the prevention of breast cancer in high-risk patients. Supported by NCI PREVENT Cancer Preclinical Drug Development Program (HHSN-2612015000-18I (PB)). Citation Format: Abhijit Mazumdar, Jamal Hill, Yun Zhang, Lakshmi Reddy Bollu, Alejandro Contreras, Michelle Savage, Shizuko Sei, Altaf Mohammed, Powel Brown. Targeting the mTOR/TORC Pathway for the prevention of er-negative and triple-negative breast 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 5072.

Background: There are few effective targeted therapies available for triple-negative breast cancers (TNBCs), the most aggressive form of breast cancer. A better understanding of the critical molecular regulators is necessary to develop effective targeted therapies for TNBCs. Our previous study focused on SOX9 transcription factor and determined that SOX9 is highly expressed in TNBCs which is required for TNBC cell survival and metastasis. Hypothesis: SOX9 controls TNBC cell growth and metastasis via directly regulating cell apoptosis and epithelial-mesenchymal transition (EMT) genes. Material and Methods: The breast cancer datasets were used to analyze the association between overall survival and SOX9 expression. Cell growth was measured using an automated cell counting assay. Cell migration and invasion were detected by transwell migration and invasion assays. A doxycycline-inducible Cas9-CRISPR knockout or siRNA knockdown system was used to inhibit SOX9 in cells. Xenograft mice model was established to evaluate the effect of SOX9 on TNBC cell growth in vivo. Protein and mRNA levels were examined by western blotting and qRT-PCR assays. An EMT PCR array and the CHIP-seq enrichment analysis based on published SOX9-CHIP-seq datasets were performed to identify effect of SOX9 on downstream pathways. SOX9 binding peaks in gene promoter region were determined and confirmed by SOX9-CHIP-seq datasets and CHIP assay. Data are presented as mean values ± SD. Statistical significance (p-values) was calculated using the Student’s t-test unless otherwise indicated. Results: Using available gene profiling datasets, we found that high expression of SOX9 was correlated with worse overall survival in Boersma breast dataset, but not in other datasets. Late stage breast cancers showed higher SOX9 expression compared to early stage breast cancers. Using SOX9 inhibited cells, we demonstrated that SOX9 is critical for TNBC cell survival and invasion in vitro and in vivo. To identify the molecular mechanisms of these functions, we used CHIP-seq data and CHIP assays to demonstrate that SOX9 directly binds to the promoter of several extrinsic apoptosis-inducing factors (such as fadd and tnfrsf10b) and represses their expressions. Loss of SOX9 caused up-regulation of the expression of these genes, consistent with the induction of apoptosis. We then investigated the effect of SOX9 on EMT-regulating genes. Loss of SOX9 induced down-regulation of most EMT genes. Using CHIP assays, we demonstrated that SOX9 directly binds the promoters of several EMT genes (such as vim and ctnnb1) and increases the expression of these genes. Conclusion: Our results demonstrate that by directly binding and regulating cell apoptosis and EMT gene expression, SOX9 controls TNBC survival and induces EMT resulting in aggressive and potentially lethal tumors. Grant Support: These studies were supported by a Breast Cancer Research Fund (BCRF) grant (PB). Citation Format: Yanxia Ma, Lakshmi Bollu, Jamal Hill, Yun Zhang, William M. Tahaney, Abhijit Mazumdar, Powel H. Brown. SOX9 controls TNBC growth and metastasis via regulating the expression of apoptosis and EMT genes [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 2618.

Estrogen receptor (ER)-negative cancers have a poor prognosis, and few targeted therapies are available for their treatment. Our previous analyses have identified potential kinase targets critical for the growth of ER-negative, progesterone receptor (PR)-negative and HER2-negative, or “triple-negative” breast cancer (TNBC). Because phosphatases regulate the function of kinase signaling pathways, in this study, we investigated whether phosphatases are also differentially expressed in ER-negative compared to those in ER-positive breast cancers. We compared RNA expression in 98 human breast cancers (56 ER-positive and 42 ER-negative) to identify phosphatases differentially expressed in ER-negative compared to those in ER-positive breast cancers. We then examined the effects of one selected phosphatase, dual specificity phosphatase 4 (DUSP4), on proliferation, cell growth, migration and invasion, and on signaling pathways using protein microarray analyses of 172 proteins, including phosphoproteins. We identified 48 phosphatase genes are significantly differentially expressed in ER-negative compared to those in ER-positive breast tumors. We discovered that 31 phosphatases were more highly expressed, while 11 were underexpressed specifically in ER-negative breast cancers. The DUSP4 gene is underexpressed in ER-negative breast cancer and is deleted in approximately 50 % of breast cancers. Induced DUSP4 expression suppresses both in vitro and in vivo growths of breast cancer cells. Our studies show that induced DUSP4 expression blocks the cell cycle at the G1/S checkpoint; inhibits ERK1/2, p38, JNK1, RB, and NFkB p65 phosphorylation; and inhibits invasiveness of TNBC cells. These results suggest that that DUSP4 is a critical regulator of the growth and invasion of triple-negative breast cancer cells. Electronic supplementary material The online version of this article (doi:10.1007/s10549-016-3892-y) contains supplementary material, which is available to authorized users.

Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and women diagnosed with TNBC currently lack targeted treatment options. To identify novel targets for TNBC, we evaluated phosphatase expression in breast tumors and characterized their contributions to in vitro and in vivo growth of TNBC. Using Affymetrix microarray analysis of 102 breast cancers, we identified 146 phosphatases that were significantly differentially expressed in TNBC compared with estrogen receptor (ER)-positive tumors. Of these, 19 phosphatases were upregulated (0.66-fold; FDR = 0.05) in TNBC compared with ER-positive breast cancers. We knocked down 17 overexpressed phosphatases in four triple-negative and four ER-positive breast cancer lines using specific siRNAs and found that depletion of six of these phosphatases significantly reduced growth and anchorage-independent growth of TNBC cells to a greater extent than ER-positive cell lines. Further analysis of the phosphatase PTP4A3 (also known as PRL-3) demonstrated its requirement for G1–S cell-cycle progression in all breast cancer cells, but PTP4A3 regulated apoptosis selectively in TNBC cells. In addition, PTP4A3 inhibition reduced the growth of TNBC tumors in vivo. Moreover, in silico analysis revealed the PTP4A3 gene to be amplified in 29% of basal-like breast cancers, and high expression of PTP4A3 could serve as an independent prognostic indicator for worse overall survival. Collectively, these studies define the importance of phosphatase overexpression in TNBC and lay the foundation for the development of new targeted therapies directed against phosphatases or their respective signaling pathways for TNBC patients. Cancer Res; 76(7); 1942–53. ©2016 AACR.

Background: Triple-negative breast cancers (TNBCs) have the worst prognosis of all breast cancers, and have few available therapies other than non-specific and toxic chemotherapy. To identify novel targets for TNBCs, we investigated expression levels of transcription factors (TFs) in TNBCs compared to those in Non-TNBCs. Our previous analyses identified the TFs highly expressed in TNBCs. Of these, six SRY (Sex Determining Region Y)-related HMG-box (SOX) TFs were highly expressed in TNBCs as compared to non-TNBCs. SOX genes belong to a superfamily of gene. There are approximately 20 SOX genes in humans and mice. Hypothesis: SOX TFs are important regulators of TNBC cell growth and metastasis. Material and Methods: We measured breast cancer cell growth using an automated cell counting assay. Cell migration and invasion were detected by transwell assays in non-TNBC (MCF7 and ZR75-1) and TNBC (MDA231 and MDA468) cells. DOX-inducible SOX9-knockout (KO) cell lines were established in MDA231, MDA468, and LM2 cell lines using an inducible Cas9-CRISPR system. A SOX9 expressing lentivirus was used to overexpress SOX9, and siRNAs were used to knockdown (KD) SOXs in the different breast cancer cells. Cell cycle phase and apoptosis were detected using flow cytometric analysis. Protein and mRNA levels of SOX9 in cell lines were examined by western blotting and qRT-PCR assays. SOX9 RNA expression data were obtained from the Oncomine Database. The Curtis dataset was used to analyze survival according to SOX9 expression using Kaplan-Meier survival curves and the statistical significance was determined using the log-rank test. Results: We performed a screen using specific siRNA targeting the 6 SOX transcription factors (SOX4, 6, 8, 9, 10 and 11) that are high-expressed in TNBC, and measured the effect of loss on SOX expression on TNBC cell growth and invasion. SOX4, 6, 8, 9, and 10-KD using siRNA caused decreased cell proliferation (by 30% or more) of MDA231 and MDA468 TNBC cells, but not of MCF7 and T47D cell lines (non-TNBC). SOX11 or 17-KD had no effect on breast cancer growth. SOX9-KD and SOX9-KO decreased cell migration and invasion of MDA231 and MDA468 cells. Reduced expression of SOX9 also inhibited the in vivo growth and metastasis of MDA MB-231 and LM2 cells in mice. In contrast, overexpression of SOX9 in MCF7 and ZR75-1 cells increased cell migration and invasion. Our studies also demonstrated that loss of SOX9 induced a G1 to S cell cycle arrest and apoptosis. In addition, high expression of SOX9 was correlated with worse overall survival and 5-year disease-free survival in patients with TNBC breast cancer. Conclusion: Our results demonstrate that the SOX9 acts as an essential molecule regulating TNBC growth and invasion. In the future, it may be possible to target SOX9 and its downstream genes to treat TNBC and prevent its metastasis. Grant Support: These studies were supported by a Susan G. Komen for the Cure Promise Grant (PB). Citation Format: Yanxia Ma, Jonathan Shepherd, Dekuang Zhao, Lakshmi Bollu, Jamal Hill, Yun Zhang, Abhijit Mazumdar, Powel H. Brown. SOX9 is a critical regulator of triple-negative breast cancer growth and invasion [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 3347.

The chemopreventive effects of three agents, rexinoid bexarotene, tyrosine kinase inhibitor gefitinib, and celecoxib, were tested on mammary tumor development arising in p53-null mammary epithelium. The rexinoid bexarotene was the most efficacious inhibitor as it reduced mammary tumor development by 75% in virgin mice and significantly delayed mean tumor development by 98 days in hormone-stimulated mice. The tyrosine kinase inhibitor gefitinib reduced mammary tumor incidence by 50% in virgin mice but did not significantly delay mean tumor latency in hormone-stimulated mice. Celecoxib did not reduce tumor incidence or mean tumor latency in either of the two models. The high doses of the rexinoid and the tyrosine kinase inhibitor did not affect the progression of tumors arising from the premalignant mammary outgrowth line, PN8a. A comparison of these agents with tamoxifen shows the superiority of tamoxifen in preventing tumor development in p53-null mammary cells. Similarly, a comparison of the results of the p53 model with other transgenic models in their response to the chemopreventive agents showed that mammary tumors arising from different oncogenic events will respond differently to the different agents.

Genetically engineered mouse cancer models are among the most useful tools for testing the in vivo effectiveness of the various chemopreventive approaches. The p53-null mouse model of mammary carcinogenesis was previously characterized by us at the cellular, molecular, and pathologic levels. In a companion article, Medina et al. analyzed the efficacy of bexarotene, gefitinib, and celecoxib as chemopreventive agents in the same model. Here we report the global gene expression effects on mammary epithelium of such compounds, analyzing the data in light of their effectiveness as chemopreventive agents. SAGE was used to profile the transcriptome of p53-null mammary epithelium obtained from mice treated with each compound versus controls. This information was also compared with SAGE data from p53-null mouse mammary tumors. Gene expression changes induced by the chemopreventive treatments revealed a common core of 87 affected genes across treatments (P < 0.05). The effective compounds, bexarotene and gefitinib, may exert their chemopreventive activity, at least in part, by affecting a set of 34 genes related to specific cellular pathways. The gene expression signature revealed various genes previously described to be associated with breast cancer, such as the activator protein-1 complex member Fos-like antigen 2 (Fosl2), early growth response 1 (Egr1), gelsolin (Gsn), and tumor protein translationally controlled 1 (Tpt1), among others. The concerted modulation of many of these transcripts before malignant transformation seems to be conducive to predominantly decrease cell proliferation. This study has revealed candidate key pathways that can be experimentally tested in the same model system and may constitute novel targets for future translational research.

Lapatinib, a selective orally available inhibitor of epidermal growth factor receptor (EGFR) and ErbB2 receptor tyrosine kinases, is a promising agent for the treatment of breast cancer. We examined the effect of lapatinib on the development of mammary tumors in MMTV-erbB2 transgenic mice, which express wild-type ErbB2 under the control of the mouse mammary tumor virus promoter and spontaneously develop estrogen receptor (ER)–negative and ErbB2-positive mammary tumors by 14 months of age. Mice were treated from age 3 months to age 15 months with vehicle (n = 17) or lapatinib (30 or 75 mg/kg body weight; n = 16 mice per group) by oral gavage twice daily (6 d/wk). All statistical tests were two-sided. By 328 days after the start of treatment, all 17 (100%) of the vehicle-treated mice vs five (31%) of the 16 mice treated with high-dose lapatinib developed mammary tumors (P < .001). Among MMTV-erbB2 mice treated for 5 months (n = 20 mice per group), those treated with lapatinib had fewer premalignant lesions and noninvasive cancers in their mammary glands than those treated with vehicle (P = .02). Lapatinib also effectively blocked epidermal growth factor–induced signaling through the EGFR and ErbB2 receptors, suppressed cyclin D1 and epiregulin mRNA expression, and stimulated p27 mRNA expression in human mammary epithelial cells and in mammary epithelial cells from mice treated for 5 months with high-dose lapatinib. Thus, cyclin D1, epiregulin, and p27 may represent useful biomarkers of lapatinib response in patients. These data suggest that lapatinib is a promising agent for the prevention of ER-negative breast cancer.

The oncogene erbB2 is overexpressed in 20% to 30% human breast cancers and is most commonly overexpressed in estrogen receptor (ER)–negative breast cancers. Transgenic mice expressing erbB2 develop ER-negative mammary tumors, mimicking human breast carcinogenesis. Previously, we have shown that activator protein 1 (AP-1) regulates proliferation of ER-negative breast cancer cells. We hypothesized that blockade of AP-1 in mouse mammary epithelial cells will suppress ER-negative tumorigenesis induced by erbB2. Trigenic erbB2 mice were generated by crossing a bigenic pUHD-Tam67/MMTV-rtTA mouse to a MMTV-erbB2 mouse. The resulting trigenic mice develop tumors and express a doxycycline-inducible c-Jun dominant negative mutant (Tam67) in the mammary glands. In vivo AP-1 blockade by Tam67 expression started delayed mammary tumor formation in MMTV-erbB2 mice by more than 11 weeks. By 52 weeks of age, 100% (18 of 18) of the untreated animals had developed mammary tumors, whereas 56% (9 of 16) of the doxycycline-treated trigenic mice developed tumors. In addition, the tumors that arose in the AP-1–blocked erbB2 mice failed to express Tam67. Twenty-five percent of the doxycycline-treated MMTV-erbB2 mice survived more than 72 weeks of age without developing mammary tumors. Examination of normal-appearing mammary glands from these mice showed that AP-1 blockade by Tam67 also significantly prevents the development of premalignant lesions in these glands. The expression of erbB2 either in normal mammary tissue or in mammary tumors was not altered. Our results show that blocking the AP-1 signaling in mammary cells suppresses erbB2-induced transformation, and show that the AP-1 transcription factor is a critical transducer of erbB2. These results provide a scientific rationale to develop targeted drugs that inhibit AP-1 to prevent the development of ER-negative breast cancer.

Retinoids, vitamin A analogues that bind to retinoic acid receptor (RAR) or retinoid X receptor (RXR), play important roles in regulating cell proliferation, apoptosis, and differentiation. Recently, RXR-selective ligands, also referred to as rexinoids, have been investigated as potential chemopreventive agents for breast cancer. Our previous studies demonstrated that the rexinoid bexarotene significantly prevented ER-negative mammary tumourigenesis with less toxicity than naturally occurring retinoids in animal models. To determine whether bexarotene prevents cancer at the early stages during the multistage process of mammary carcinogenesis, we treated MMTV-erbB2 mice with bexarotene for 2 or 4 months. The development of preinvasive mammary lesions such as hyperplasias and carcinoma-in-situ was significantly inhibited. This inhibition was associated with reduced proliferation, but no induction of apoptosis. We also examined the regulation of a number of rexinoid-modulated genes including critical growth and cell cycle regulating genes using breast cell lines and mammary gland samples from mice treated with rexinoids. We showed that two of these genes (DHRS3 and DEC2) were modulated by bexarotene both in vitro and in vivo. Identification of these rexinoid-modulated genes will help us understand the mechanism by which rexinoid prevents cancer. Such rexinoid-regulated genes also represent potential biomarkers to assess the response of rexinoid treatment in clinical trials.

Purpose: To test whether a novel rexinoid, LG100268, prevents the development of preinvasive and invasive estrogen receptor–negative mammary tumorigenesis in MMTV-erbB2 mice. Experimental Design: For invasive breast cancer prevention, MMTV-erbB2 mice were treated with daily gastric gavage of vehicle, LG100268 (10 mg/kg), or LG100268 (100 mg/kg) for long term starting at 3 months of age. For preinvasive lesion study, mice were treated with daily gastric gavage of vehicle or LG100268 (100 mg/kg) for 4 months. Results: Long-term treatment with LG100268 significantly prevented invasive mammary tumor development. Median time (age) to tumor development was delayed from 217 days in vehicle group to 357 days in low-dose group. In high-dose group, only 2 of 20 mice developed tumors after 430 days of treatment. Short-term treatment of LG100268 significantly prevented the development of preinvasive mammary lesions including hyperplasia and ductal carcinoma in situ. The cancer prevention effect was associated with reduced expression of Ki67 and cyclin D1 in mammary glands by >80%. Conclusion: Rexinoid LG100268 is an effective chemopreventive agent in preventing the development of both malignant and premalignant mammary lesions in MMTV-erbB2 mice.

Two major unresolved issues for breast cancer prevention are the lack of effective preventive agents for estrogen receptor (ER)-negative disease and the potential toxicity of long-term pharmacologic treatment. The ability of retinoid X receptor (RXR)-selective retinoids (rexinoids) to inhibit breast cancer formation independently of ER-status was previously demonstrated in various preclinical models. The goal of our study was to identify ideal druggable targets for the synergistic amplification of the cancer preventive effect of synthetic rexinoids and test the efficacy of a low-dose combination treatment in vivo. We identified the nuclear hormone receptors essential for rexinoid-dependent growth suppression by a high throughput siRNA screen of mammary epithelial cells. Knock-down of ELP, COUP-TF II, GR, TR2, VDR and PPARgamma partially reversed the anti-proliferative effect of the rexinoid bexarotene. Conversely, expression profiling showed that the largest fraction of genes up-regulated by the synthetic rexinoid LG100268 represented regulators of lipid metabolism and putative target genes of the RXR partner receptor PPARgamma. Combination dose-response experiments showed that the PPARgamma agonist pioglitazone was synergistic with low doses (100-300 nM) of LG100268. Long-term daily treatment of MMTV-ErbB2 mice with LG100268 (25 mg/kg) or pioglitazone (20 mg/kg), or their combination resulted in a significant delay of tumor formation, while serum triglyceride levels were not significantly elevated by the combination. Suppression of cell proliferation measured by reduced Ki67 staining was enhanced by the addition of pioglitazone, in both lesions and intact mammary epithelium, while pioglitazone also increased the frequency of cells containing cleaved caspase 3. Furthermore, pioglitazone treatment was associated with a marked induction of microvesicle formation in the adipose issue. These data suggest that the combination of LG100268 and pioglitazone, while potentially targeting the same heterodimer, can invoke distinct biological processes resulting in more effective cancer prevention than achieved by either agent alone, and may be an effective strategy for the prevention of ER-negative breast cancer. Citation Format: Iván P. Uray, Jennifer M. Rodenberg, Yun Zhang, Jamal L. Hill, Powel H. Brown. The thiazolidinedione pioglitazone enhances the cancer preventive activity of the rexinoid LG100268 in Her2/neu induced 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 2804. doi:10.1158/1538-7445.AM2015-2804

Background: Estrogen receptor (ER) -negative breast cancer is a clinical subtype that is overrepresented among younger women and is associated with a poor prognosis. Current treatments for ER-negative tumors include cytotoxic chemotherapy, or for those overexpress HER2, the anti-HER2 antibody. Targeted therapy for triple-negative breast cancers is urgently needed. In this project, we investigated phosphatases that are differentially expressed in ER-negative as compared to ER-positive breast cancers. We hypothesized that: (1) specific phosphatases govern the growth of ER-negative cancers, (2) Induced expression of specific phosphatases that are under expressed in ER-negative cancers will suppress the growth of ER-negative breast cancers. Methods: Using 102 human breast tumors (57 ER-positive & 45 ER-negative) from the neo-adjuvant studies from the Baylor Breast Center tumor bank, we isolated RNA and performed Affymetrix microarray studies. Statistical analysis was done with dChip software, and phosphatases over (>1.5 fold; FDR Results: We identified 20 over-expressed and 29 under-expressed phosphatases in ER-negative breast cancers. We selected the under expressed phosphatase PPM1A, for further study. Multivariate cox regression analysis shows PPM1A is an independent predictor for breast cancer survival. Induced expression of PPM1A in ER-negative cells inhibited anchorage-dependent and independent growth but had no effect on ER-positive cell. PPM1A expression also inhibited MDA-MB 231 cells growth in vivo. Induced expression of PPM1A blocked cell cycle progression at G1 phase. PPM1A interacts with CDK6 and inhibits phosphorylation of CDK and MDM2. Conclusions: We identified a set of over- and under- expressed phosphatases in ER-negative breast cancers as compared to ER-positive cancers. Overexpression of PPM1A in ER-negative breast cancer cells inhibits growth. By identifying the molecules that regulate breast cancer cells growth we are identifying potential new targets for the treatment of these aggressive ER-negative breast cancers. Supported by Komen Promise grant KG081694 & Komen SAB grant Citation Format: Abhijit Mazumdar, Jamal Hill, Yun Zhang, Lakshmi Reddy Bollu, Anna Tsimelzon, Jenny Chang, Gordon Mills, Powel Brown. Induced expression of PPM1A in ER-negative breast cancer cells inhibits growth by suppressing CDK phosphorylation [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 5525. doi:10.1158/1538-7445.AM2017-5525

Triple-negative breast cancers (TNBC) are aggressive with no effective targeted therapies. A combined database analysis identified 32 inflammation-related genes differentially expressed in TNBCs and 10 proved critical for anchorage-independent growth. In TNBC cells, an LPA-LPAR2-EZH2 NF-κB signaling cascade was essential for expression of interleukin (IL)-6, IL-8, and CXCL1. Concurrent inhibition of IL-6 and IL-8 expression dramatically inhibited colony formation and cell survival in vitro and stanched tumor engraftment and growth in vivo. A Cox multivariable analysis of patient specimens revealed that IL-6 and IL-8 expression predicted patient survival times. Together these findings offer a rationale for dual inhibition of IL-6/IL-8 signaling as a therapeutic strategy to improve outcomes for patients with TNBCs. Cancer Res; 73(11); 3470–80. ©2013 AACR.

Background: Prevention of estrogen receptor (ER)-positive breast cancer prevention is now possible antiestrogen drugs; however, this treatment is ineffective against ER-negative breast cancers. ER-negative breast cancer occurs more often in younger women and is associated with a poor prognosis. Dysregulation of PI3K-mTOR pathway, specifically upregulation of mTOR and loss of phosphatase and tensin homolog (PTEN) has been commonly associated with ER-negative breast cancers and been shown to be associated with poor prognosis. We hypothesized that by targeting mTOR, will suppress the growth of ER-negative and triple negative breast cancers. To test the hypothesis, we used MMTV-erbB2, C3(1)/SV40T, and p53 null mammary gland mouse models to determine whether mTOR inhibitor everolimus is effective in preventing growth of ER-negative mammary tumors. Methods p53-null Balb/C donor mice were transplanted into both cleared inguinal mammary fat pads of Balb/C p53 wild-type mice. All mice were separated into 2 groups 1) sesame oil control and 2) everolimus (5 mg/kg). All treatments were given by oral gavage, five days a week from 3 months of age for MMTV-erbB2 and 2 months of age for SV40T mice, and in case of p53 mice, two months after transplantation. Mice were observed daily for tumor formation, toxicity and the percentage of tumor free mice were recorded. Tumor incidence and time to tumor formation was visualized using Kaplan Meier curves and analyzed using the generalized Wilcoxon test. Results: In MMTV-erbB2 mice the mTOR inhibitor, everolimus reduced tumor incidence and was associated with an increase in median survival time from 240 days to 410 days in. At 365 days, when all mice in control group died, only half of the mice treated with everolimus had developed mammary tumors. Long term treatment of everolimus was associated with mild toxicity that includes weight loss (180 days). At 150 days, when all mice from the control group developed tumor only 35% mice from treatment group had developed tumors. Everolimus also significantly reduced tumor incidence in p53 null mammary gland mice. At 420 days, when approximately in 50% of the control mice developed mammary tumors compare to only 7% of the everolimus treated mice developed tumors. Conclusion: The mTOR inhibitor everolimus prevented ER-negative mammary in two mouse models (MMTV-erbB2, C3 (1)/SV40, and p53 null mammary gland mice). Based on our results Everolimus is an effective cancer preventive drug. Our results suggest that, studies with reduced everolimus dose alone or in combination with other targeted therapies such as selective estrogen receptor modulators are warranted. In the future, clinical trials of the everolimus should be considered for the prevention of breast cancer in high-risk patients. Citation Format: Abhijit Mazumdar, Yun Zhang, Jamal Hill, Powel Brown. Targeting the mTOR pathway for the prevention of ER-negative breast cancer. [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 2610.

In pursuit of effective therapeutic agents for the estrogen receptor (ER)-negative breast cancer, we previously showed that bexarotene reduced mammary tumor development by 75% in ErbB2 mice. To further improve the effectiveness of breast cancer prevention, we have now investigated the effects of a combinatorial therapy consisting of two cancer preventive drugs. On the basis of the hypothesis, rexinoid LG100268 plus tamoxifen would more effectively prevent the development of both ER-positive and ER-negative breast cancer. We treated p53-null mammary gland mice with tamoxifen and LG100268, individually and in combination. By 60 weeks of age, vehicle-treated mice developed tumors in 52% of transplanted mammary glands, whereas mice treated with tamoxifen and LG100268 developed tumors in only 13% of transplanted mammary glands. To further define the mechanistic effects of this combinatorial treatment, we investigated the effects of tamoxifen and LG100268 on mammary tissue biomarkers. In mammary tissue harvested before tumor development, the proliferation markers Ki67 and cyclin D1 were significantly reduced in mice treated with the combination therapy. In addition, the rexinoid target genes ABCA1 and ABCG1 were induced in both the rexinoid and combination treatment groups, whereas expression remained constant in tamoxifen group. These results show that tamoxifen-LG100268 combinatorial treatment is more effective in preventing mammary tumors than either agent alone. In addition, these studies have identified relevant tissue biomarkers that can be used to show the effect of these agents on mammary tissue. These results support the development of clinical trials of antiestrogen and rexinoid combinatorial therapy for the prevention of patients with high-risk breast cancer. Cancer Prev Res; 5(10); 1195–202. ©2012 AACR.

Background: Anti-estrogen drugs such as selective estrogen receptor modulators and aromatase inhibitors effectively prevent ER-positive breast cancer, but do not prevent triple negative breast cancer (TNBC) which represents approximately 20% of all breast cancers. TNBCs are characterized by a high proliferation rate and a dysregulated cell cycle. Drugs that specifically activate RXR without activating RAR nuclear receptors (“rexinoids”) are being studied as potential cancer preventive drugs. One of the most promising rexinoids is the compound, LG100268. LG100268 is highly effective in preventing ER-negative/HER2-positive mammary tumors in MMTV-ErbB2 mice by inhibiting cyclin D-dependent pathways, but less preventive in models of TNBC (p53-null mice and C3(1)-SV40 Tag mice). Therefore, we hypothesized that inhibiting multiple cell cycle regulatory points by combining kinase inhibitors and LG100268 is an effective and safe strategy to prevent triple negative breast cancer. Methods: To identify kinases that inhibit cell growth we carried out a ‘whole kinome’ (636 kinases) high-throughput siRNA screen in combination with LG100268 (1µM) in immortalized, human mammary epithelial cells overexpressing telomerase (HME-tert). After 4 days of growth following LG100268 treatment and suppression of the respective kinase cells were stained with DAPI and cell number was determined by automated microscopy and image analysis. Combination “hits” were selected based on a p-value Results: The analysis of the kinome screen showed that the combination of LG100268 with each of 27 kinases had a significant growth suppressive effect compared to siRNA control and vehicle alone. Knockdown of the respective kinases was independently confirmed by qRT PCR. Growth assays of HME-tert cells after siRNA knockdown of kinases in combination with LG100268 demonstrated that in particular CDK1, 2 and 5 enhanced the chemopreventive effect of LG100268. Using a CDK inhibitor dinaciclib in combination with LG100268 showed that the combination additively suppressed mammary epithelial growth in vitro. In vivo in SV40 Tag mice, LG100268 or dinaciclib alone significantly reduced the area (mm2) of hyperplastic lesions (defined as any hyperplastic ductal foci comprised of more than three layers of epithelial cells) compared to the control group. The combination therapy did not result in a significant reduction of hyperplastic area compared to dinaciclib alone. Long-term treatment with LG100268 did not delay TNBC formation compared to vehicle (both MTTF = 144 days). However, long-term treatment with dinaciclib alone delayed mammary tumorigenesis with a median time to tumor formation (MTTF) of 167 days compared to a MTTF of 144 days of age in the control group (p < 0.001). The combination therapy did not further affect the tumor formation compared to dinaciclib alone (MTTF = 162 days). Conclusions: These studies demonstrate that dinaciclib alone or the combination therapy of LG100268 and dinaciclib causes inhibition of premalignant cell growth and delayed ER-negative mammary tumorigenesis in SV40 Tag mice. While an additive effect of dinaciclib and LG100268 was observed in vitro, the in vivo combination therapy was not significantly more effective than dinaciclib treatment alone. Future studies should investigate different combinations of dosages of dinaciclib and LG100268 in vivo. These studies were supported by an NCI/NIH R25T grant (R25CA057730, BL) and Susan G. Komen for the Cure Promise grant (KG081694) Citation Format: Beate Christiane Litzenburger, Ivan Peter Uray, Jamal Hill, Jun Zhang, Abhijit Mazumdar, Powel H. Brown. Targeting multiple cell cycle regulatory points for the prevention of triple-negative breast cancer. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr PR03.

Tamoxifen reduces the relative risk of breast cancer developing from specific premalignant lesions. Many breast cancers that arise after tamoxifen treatment are estrogen receptor-α (ER-α)–negative, although premalignant lesions such as atypical ductal hyperplasia are highly ER-α–positive. The p53 null mouse mammary epithelial transplant model is characterized by ER-α–positive premalignant lesions that give rise to both ER-α–positive and ER-α–negative tumors. Given this progression from ER-α–positive to ER-α–negative lesions, we tested the ability of tamoxifen to block or delay mammary tumorigenesis in several versions of this model. In groups 1 and 2, p53 null normal mammary epithelial transplants were maintained in virgin mice. In groups 3 to 5, the p53 null and mammary transplants were maintained in mice continuously exposed to high levels of progesterone. In groups 6 and 7, transplants of the premalignant outgrowth line PN8a were maintained in virgin mice. Tamoxifen blocked estrogen signaling in these mice as evidenced by decreases in progesterone-induced lateral branching and epithelial proliferation in the mammary epithelium. Tamoxifen did not alter the elevated levels of progesterone in the blood while significantly reducing the circulating level of prolactin. Tamoxifen reduced tumor incidence in p53 null normal mammary epithelial transplants maintained in virgin mice from 55% to 5% and in progesterone-stimulated mice from 81% to 21%. The majority of the resultant tumors were ER-α–negative. Tamoxifen also significantly delayed tumorigenesis in the ER-α–positive high premalignant line PN8a from 100% to 75%. These results show that tamoxifen delays the emergence of ER-α–negative tumors if given early in premalignant progression.

Although antiestrogens have been effective in preventing estrogen receptor (ER)-positive breast cancer, chemopreventive agents are still needed to prevent ER-negative breast cancer. Tyrosine kinase inhibitors are promising agents for the treatment and prevention of human cancers. ZD1839 (gefitinib or Iressa) is an orally active epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that blocks signal transduction pathways in epithelial cells. We examined whether ZD1839 blocks signal transduction and prevents the development of ER-negative breast cancer.The ability of ZD1839 to block signal transduction in normal, immortalized, and malignant breast cells was assessed by western blotting with specific antibodies to detect phosphorylation of cytoplasmic signaling molecules. The effect of ZD1839 on growth of these breast cells was assessed with anchorage-dependent and anchorage-independent growth assays. Its effect on ER-negative mammary tumorigenesis was assessed in MMTV-erbB2 transgenic mice. All statistical tests were two-sided.ZD1839 suppressed the phosphorylation of EGFR and mitogen-activated protein kinase in normal and malignant breast cells. ZD1839 treatment statistically significantly suppressed mammary tumorigenesis in MMTV-erbB2 transgenic mice; median time to tumor development was approximately 230 days in vehicle-treated mice and more than 310 days in mice treated with ZD1839 at 100 mg/kg (P.001). ZD1839 reduced proliferation of normal breast cells by 20.3% (95% confidence interval [CI] = -13.7% to 44.2%) and of tumor cells by 42.0% (95% CI = 20.2% to 58.2%). ZD1839 also increased expression of the cell cycle regulator p27 in normal mammary tissue by 48.7% (95% CI = 27.0% to 74.2%) and in tumor tissue by 50.3% (95% CI = 35.8% to 66.7%).This study appears to provide the preclinical rationale for the development of these EGFR tyrosine kinase inhibitors for the prevention of human breast cancer.

Background: Estrogen receptor (ER) -negative breast cancer is a clinical subtype that is overrepresented among younger women and is associated with a poor prognosis. Current treatments for ER-negative tumors include cytotoxic chemotherapy, or for those overexpress HER2, the anti-HER2 antibody. Targeted therapy for triple-negative breast cancers is urgently needed. In this project, we investigated phosphatases that are differentially expressed in ER-negative as compared to ER-positive breast cancers. We hypothesized that: (1) specific phosphatases govern the growth of ER-negative cancers, (2) Induced expression of specific phosphatases that are under expressed in ER-negative cancers will suppress the growth of ER-negative breast cancers. Methods: Using 102 human breast tumors (57 ER-positive & 45 ER-negative) from the neo-adjuvant studies from the Baylor Breast Center tumor bank, we isolated RNA and performed Affymetrix microarray studies. cDNA was synthesized and Biotin-labeled and hybridized onto an Affymetrix HGU133A GeneChipTM. Statistical analysis was done with dChip software, and phosphatases over (>1.5 fold; FDR Results: We identified 20 over-expressed, and 29 under-expressed phosphatases in ER-negative breast cancers. We selected the under expressed phosphatase PPM1A, for further study. Multivariate cox regression analysis shows PPM1A is an independent predictor for breast cancer survival. Induced expression of PPM1A in ER-negative cells inhibited anchorage-dependent and independent growth but had no effect on ER-positive cell. PPM1A expression also inhibited MDA-MB 231 cells growth in vivo. Induced expression of PPM1A blocked cell cycle progression at G1 phase. Conclusions: We identified a set of over- and under- expressed phosphatases in ER-negative breast cancers as compared to ER-positive cancers. Overexpression of PPM1A in ER-negative breast cancer cells inhibits growth. By identifying the molecules that regulate breast cancer cells growth we are identifying potential new targets for the treatment of these aggressive ER-negative breast cancers. Supported by Komen Promise grant KG081694 & SAB13-00006 Citation Format: Abhijit Mazumdar, Graham Poage, Jamal Hill, Yun Zhang, Anna Tsimelzon, Jenny Chang, Gordon Mills, Powel Brown. Induced expression of PPM1A in ER-negative breast cancer cells inhibits growth by suppressing cell cycle. [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 5279. doi:10.1158/1538-7445.AM2014-5279

Nuclear receptors are key proteins that regulate cell proliferation, death, and differentiation. In breast cells, estrogen receptors (ERs) are critical for mammary gland development, differentiation, and tumorigenesis, and selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene, and aromatase inhibitors can prevent the development of ER-positive breast cancer. Other nuclear transcription factors are critical regulators of proliferation and differentiation including retinoic acid receptors (RARs), vitamin D receptors (VDR), and peroxisome proliferator-activator receptors (PPARs). Each of these ligand activated nuclear hormone receptors bind the retinoid X receptors (RXRs) to regulate gene expression. Ligands of these nuclear hormone receptors have been shown to prevent many different types of cancer, including breast cancer. I will present results in preclinical and early clinical studies testing retinoids, rexinoids, Vitamin D analogs, and PPAR ligands for the prevention of breast cancer. Our results and the results of others have shown that ligands that bind and activate the rexinoid receptor are highly effective in preventing breast cancer in animals, particularly ER-negative breast cancer. These preclinical results supported the testing of the RXR ligand bexarotene in women at high risk of breast cancer. This early study demonstrated a biological effect of bexarotene on breast tissue in women. We and other have conducted preclinical studies testing combinations of nuclear hormone receptor ligands (RXR ligand + anti-estrogen SERMS)(RXR ligand + Vitamin D analog) and (RXR ligand + PPAR agonist). These results demonstrate that nuclear hormone receptor ligands can be used to prevent breast cancer and that rexinoids and other ligands provide an opportunity to prevent all forms of breast cancer. Supported by RO1CA078480-11 Citation Format: Abhijit Mazumdar, Ivan Uray, Beate Litzenburger, Yun Zhang, Jamal Hill, Nanjoo Suh, Powel Brown. Targeting nuclear hormone receptors for breast cancer prevention. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr ED05-01.

Background: Estrogen receptor (ER) -negative breast cancers are poor prognosis tumors that occur more commonly in young women. Current treatments for ER-negative tumors include cytotoxic chemotherapy, or for those ER-negative breast cancers that overexpress HER2, the anti-HER2 antibody herceptin. Similar therapy for ER-negative, Her2 negative tumors, particularly for (triple-negative breast cancers) is urgently needed. In this project, we investigated whether phosphatases that are differentially expressed in ER-negative as compared to ER-positive breast cancers. We hypothesized that: (1) the specific phosphatases that govern the growth of ER-negative cancers are different from those of ER-positive cancers, (2) Induced expression of some of those phosphatases that are lowly expressed in ER-negative breast cancers will suppress the growth and invasion of ER-negative breast cancers. Methods: Using 102 human breast tumors (57 ER-positive & 45 ER-negative) from the neo-adjuvant studies from the Baylor Breast Center tumor bank, we isolated RNA and performed Affymetrix microarray studies. cDNA was synthesized and Biotin-labeled and hybridized onto an Affymetrix HGU133A GeneChipTM. Statistical analysis was done with dChip software, and phosphatases more highly (>1.5 fold; FDR Results: We identified 20 highly-expressed (>1.5 fold; FDR=0.05), and 29 lowly-expressed ( Conclusions: We identified a set of highly and lowly expressed phosphatases in ER-negative breast cancers as compared to ER-positive cancers. Overexpression of DUSP4 in ER-negative breast cancer cells inhibits growth and invasion of these cells. By identifying the molecules that regulate breast cancer cells growth we are identifying potential new targets for the treatment of these aggressive ER-negative breast cancers. Supported by Komen Promise grant KG081694 (PHB). Citation Format: Abhijit Mazumdar, Petra Den Hollander, Graham Poage, Jamal Hill, Yun Zhang, Anna Tsimelzon, Susan Hilsenbeck, Jenny Chang, Gordon Mills, Powel H. Brown. High expression of DUSP4 in ER-negative breast cancer cells suppresses growth and invasion. [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 856. doi:10.1158/1538-7445.AM2013-856

Background: Breast cancers can be subgrouped into ER-positive and ER-negative breast cancers according to their estrogen receptor status. Few effective targeted treatments are available for the more aggressive ER-negative breast cancers, 80% of which carry p53 mutation. Our lab conducted a human kinome study which identified 52 kinases highly expressed in ER-negative breast tumors. One highly expressed kinase, DAPK-1, was selected for further study. Death associated protein kinase-1 (DAPK-1) transduces death signaling through P53-dependent or -independent pathways. In this study, we determined a novel role of DAPK-1 in tumorigenesis of ER-negative, p53 mutant breast cancers. Methods: To perform expression and survival analysis of DAPK-1 in breast tumors, we used data from VandeVijver, Desmedt and Ivshina datasets. Inducible DAPK-1 knockdown cell lines were constructed through pTRIPZ lentiviral system. Anchorage-independent growth was performed in soft agar and colonies were enumerated after 15-30 days. Xenograft experiments were performed in nude mice. Proteomic analysis was used to determine downstream signaling. Results: Gene expression analysis showed that high DAPK-1 expression correlates significantly with the ER-negative subgroup of breast cancer. Using an inducible knockdown system, we found that depletion of DAPK-1 strongly suppressed in vitro growth and in vivo tumorigenesis of ER-negative, P53-mutant cells. Through a siRNA knockdown study in an expanded panel of breast cancer cell lines, we established an association between p53 mutation status and sensitivity of the cells to DAPK-1 depletion. ER-negative breast cancer cell lines with P53 mutations are very sensitive to DAPK-1 knockdown, while ER-positive breast cancer cell lines with wild-type P53 are resistant to DAPK-1 knockdown. By depleting P53 in P53 wild-type cells, we then showed that P53 wild-type breast cancer cells become partially dependent on DAPK-1 upon P53 dysfunction. Through proteomic analysis, we determined that DAPK-1 regulates cell growth through phosphorylating TSC2 at Ser939 and interfering with TSC1/TSC2 complex, which is a well-known repressor of mTOR pathway. Our survival analysis confirmed that high DAPK-1 expression in breast tumors is associated with worse clinical outcome, especially in patients that carry P53 mutations. Conclusion: P53-mutant breast cancer cells are sensitive to DAPK-1 inhibition in vitro and in vivo. P53 dysfunction causes breast cancer cells to become dependent on DAPK-1 signaling for their growth. High expression of DAPK-1 in P53-mutant breast tumor activates the mTOR pathway through relieving the TSC1/TSC2-mediated repressor of mTOR signaling. Our results demonstrate a dual role of DAPK-1 in regulating breast cancer proliferation and apoptosis depending on the P53 status. This study is supported by a PROMISE grant (KG081694) from Susan G. Komen for the Cure Foundation. Citation Format: Jing Zhao, Yun Zhang, Jamal L. Hill, Zack Hartman, Abhijit Mazumdar, Gordon B. Mills, Powel H. Brown. DAPK-1, a death signaling transducer, is critical for the tumorigenicity of p53 mutant 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 2329. doi:10.1158/1538-7445.AM2013-2329

Background: ER-positive Breast cancer prevention is now possible using antiestrogen drugs; however, this treatment is ineffective against estrogen receptor (ER)-negative breast cancers. Current treatments for ER-negative tumors include chemotherapy, or for those ER-negative cancers that overexpress HER2, the anti-HER2 antibody herceptin. Targeted therapy for ER-negative tumors, particularly those that do not express HER2 (triple-negative breast cancer) is urgently needed. Epidemiological evidence from several case control and cohort studies support an inverse relationship between vitamin D intake and breast cancer incidence. Vitamin D deficiency was also linked to poor outcomes in patients with early breast cancer. Using a transgenic mouse model of ER-negative breast cancer, our laboratory previously demonstrated that rexinoid LG100268 reduced mammary tumor development in these mice. We hypothesized that the combination of a rexinoid and a vitamin D analog (BXL-0124) would more effectively prevent the development of ER-negative breast cancer. To test the hypothesis, we used MMTV-erbB2 mouse tumors that do not express ER, to determine whether LG100268 and BXL-0124 combination is more effective than either treatment alone in preventing ER-negative mammary tumors. Methods: Virgin female MMTV-erbB2 mice (Jackson Lab) were housed in the institutional animal facilities and fed a purified diet (AIN-76A; Harlan Teklad). Mice were separated into 4 treatment groups: 1) sesame oil and DMSO control 2) Rexinoid (25 mg/kg) plus DMSO 3) BXL-0124 (3ug) plus sesame oil. 4) LG100268 (25mg/kg) plus BXL-0124 (3ug). All treatments were given by oral gavage, five days a week from 3 months of age. Mice were observed daily for tumor formation, toxicity and the percentage of tumor free mice were recorded. Tumor incidence and time to tumor formation was visualized using Kaplan Meier curves and analyzed using the generalized Wilcoxon test. Results: Rexinoid LG100268 reduced tumor incidence and was associated with an increase in median survival time from 242 days to 365 days. BXL-0124 also reduced tumor incidence with an increase in median survival time from 242 days to 288 days. The combination of LG 100268 and BXL-0124 had additive effect in preventing ER-negative mammary tumor development with a median survival time increase from 242 days to 422. days. We are investigating blood and tissue biomarkers currently and that results are forthcoming. Conclusion: Rexinoid LG100268 and vitamin D analog BX-0124 reduced tumor incidence in MMTV-erbB2 mice. The combination treatment of LG100268 and BXL0124 was more effective in preventing mammary tumor development compare to either agent alone. Based on our results, more combinatorial studies of LG100268 and BXL-0124 in other ER-negative models are warranted. In future clinical trials of the combination of BXL-0124 and LG100268 should be considered for the prevention of breast cancer in high-risk patients. (Supported by NIH R01 CA078480 (PB) and MDACC CCSG P30 CA 016672) Citation Format: Abhijit Mazumdar, Yun Zhang, Jamal Hill, Nanjoo Suh, Powel Brown. Combination of BXL-0124, a vitamin D analog, and LG100268 for the prevention of ER-negative breast cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A62.

Background: Breast cancers can be subgrouped into ER-positive and ER-negative breast cancers according to their estrogen receptor status. Clinical data has shown that ER-negative breast cancers are aggressive and have a poor prognosis; however, few effective targeted treatments are available for these cancers. A human kinome study conducted in our lab has identified 52 kinases highly expressed in ER-negative breast tumors. One of these kinases, DAPK-1, was selected for further study. Death-associated protein kinase-1 (DAPK-1) mediates cell death through P53-dependent or -independent pathways. In this study, we investigated the role of DAPK-1 in regulating growth and death in ER-negative breast cancer. Methods: To perform expression and survival analysis of DAPK-1 in breast tumors, we used data from VandeVijver, Desmedt and Ivshina datasets. Inducible DAPK-1 knockdown cell lines were constructed through pTRIPZ lentiviral system. Anchorage-independent growth was performed in soft agar. Colonies were enumerated after 15-30 days. Xenograft experiments were performed in nude mice by injecting 5×106 cells per mice. Results: Our gene expression analysis found that high DAPK-1 expression correlated significantly with ER-negative breast cancers. Survival analysis showed that high DAPK-1 expression is associated with worse clinical outcome. Using an inducible DAPK-1 knockdown system, we found that depletion of DAPK-1 suppressed anchorage-dependent and -independent growth of ER-negative P53 mutant cells but not P53 wildtype cells. We next investigated whether DAPK-1 suppression inhibits tumor growth in mouse xenograft experiments. Results of in vivo experiments demonstrated that DAPK-1 suppression inhibited the growth of ER-negative breast tumors but not ER-positive breast tumors. Through a siRNA study in an expanded panel of breast cancer cell lines, we found that the sensitivity of cell lines to DAPK-1 knockdown is dependent on ER and P53 status. ER-negative breast cancer cell lines with P53 mutations are very sensitive to DAPK-1 knockdown, while ER-positive, P53 wildtype breast cancer cell lines are resistant to DAPK-1 knockdown. To determine if p53 dysfunction can sensitize breast cancer cell lines to DAPK-1 suppression, we depleted P53 expression in MCF7 and found an increased sensitivity to DAPK-1 knockdown, indicating that MCF7 growth becomes partially addicted to DAPK-1 after P53 dysfunction. Conclusion: DAPK-1 is an essential growth regulator of ER-negative breast cancer cells, but not of ER-positive, P53 wildtype breast cancer cells in vitro and in vivo. These results suggest that P53 dysfunction causes breast cancer cells to become addicted to DAPK-1 signaling pathway for their growth. Our results show that DAPK-1 has a dual role in regulating breast cancer proliferation and apoptosis depending on the status of P53. This study is supported by a PROMISE grant (KG081694) from the Susan G. Komen for the Cure Foundation. 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 82. doi:1538-7445.AM2012-82

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Breast cancer is a heterogeneous disease that can be subdivided into distinct types based upon the molecular classification of oncogenic driver mutations. While most breast cancers are driven by estrogen-mediated signaling (ER+) or HER2 expression (HER2+), the cellular signals that mediate transformation and growth of ER-HER2- (or basal-type) breast cancers are incompletely understood. To identify novel targets for the treatment of this aggressive and treatment-refractory disease, we investigated inflammatory signaling in these cancers, as inflammatory pathways have proven critical for oncogenic transformation of multiple cancers. In our primary analysis, we analyzed ER- and ER+ primary patient tumors (N=106, Baylor College of Medicine Tumor Bank) to determine inflammatory genes (in the KEGG Cytokine-Cytokine Receptor set, N=226) that are differentially expressed (FDR

Background: Breast cancer prevention is now possible using antiestrogen drugs such as tamoxifen and raloxifene; however, antiestrogens are ineffective against estrogen receptor (ER)-negative breast cancers. Since ER-negative breast cancers are highly aggressive and particularly difficult to treat, preventive agents against these life-threatening cancers are urgently needed. Metformin, a first line treatment for adult-onset diabetes, recently was shown to be associated with reduced incidence of cancer (including breast cancer) in epidemiologic studies. Metformin activates AMP-dependent kinase (AMPK) and stimulation of AMPK by metformin reduces cell proliferation in both estrogen receptor alpha (ERa)-positive and -negative human breast cancer cell lines. We hypothesized that the metformin would effectively prevent the growth and development of ER-negative breast cancer. To test this hypothesis, we tested metformin's ability to prevent mammary tumors in two mouse models: the MMTV-erbB2 transgenic mouse model that develops ER-negative mammary tumors and the p53-null mammary gland mouse model that develops both ER-positive and ER-negative mammary tumors. Methods: MCF7, BT474, MDA MB 468 and MDA MB 453 cells were treated with10mM metformin and cell proliferation was measured using an MTS assay. Virgin female MMTV-erbB2 mice (Jackson Lab) were housed in the institutional animal facilities and fed a purified diet (AIN-76A; Harlan Teklad). Mice were separated into 2 treatment groups: 1) Vehicle control (PBS via gastric gavage daily), 2) Metformin (250mg/kg via gastric gavage daily; from 3 months to 12 months of age). P53-null Balb/C mammary glands from donor mice were transplanted into both cleared inguinal mammary fat pads of Balb/C p53 wild-type mice. Mice were observed daily for tumor formation and the percentage of tumor free mice were recorded. Tumor incidence and time to tumor formation was visualized using Kaplan Meier curves and analyzed using the generalized Wilcoxon test. Results: Metformin (at 10mM) significantly reduced proliferation of breast cancer cells irrespective to their ER, Her2 and p53 status. In the MMTV-erbB2 mice, metformin treatment did not prevent or delay mammary tumor formation. However, in p53-null mammary gland mice, metformin completely prevented mammary tumor formation. Conclusions: Metformin treatment totally prevented mammary tumor development in p53-null mammary gland mice. These results suggest that metformin will be most useful in preventing breast cancers with P53 mutations such as occurs in Li-Fraumeni syndrome individuals or individuals with ERa-negative or “triple-negative” breast cancer. These results support testing metformin in clinical trials for the prevention of ER-negative and p53-mutant breast cancers. This research was supported by the NIH/NCI R01 CA78480 grant (PHB). Citation Information: Cancer Prev Res 2011;4(10 Suppl):A101.

Background: Recent research in breast biology supports the cancer stem cell hypothesis which asserts that malignancies arise in tissue stem cells through dysregulation of self-renewal. The cancer stem cell hypothesis has important implications for early detection, prevention and treatment of breast cancer. If breast cancers arise from the transformation of normal stem cells, it may be possible to target stem cells for the prevention of cancer. We have previously shown that rexinoids prevent the development of ER-negative breast cancer in transgenic mouse models. Previous studies have demonstrated a role of retinoid signaling in the regulation of normal breast stem cell self-renewal and differentiation. Bexarotene and LG100268 are rexinoids that selectively bind RXR receptors. Based on the effects of these molecularly targeted agents, we hypothesize that rexinoids prevent breast cancer development by suppressing the growth and transformation of the breast stem cell population. Methods: We performed in vivo and in vitro studies to address our hypothesis. Cell proliferation assays were used to determine whether rexinoids can inhibit the growth of normal, pre-malignant and malignant breast cell lines (HMECs, MCF10A, DCIS.COM, SUM225, HCC1937 and HCC38 cell lines). To determine whether these agents affect stem cells, we performed mammosphere assays and FACS analysis after treatment with vehicle or rexiniods. We next conducted in vivo studies using MMTV-Wnt transgenic mice treated with LG100268 (50mg/kg) or vehicle, and studied the stem cell properties of mammary epithelial cells from these mice. The stem cell population in mammary glands was quantified by staining of CD24, CD49f and using FACS analysis and mammosphere assays. Results: Our results showed that the rexionids LG100268 and bexarotene inhibit the growth of breast cell lines only at a high concentrations (the IC50 (concentration that reaches a 50% growth inhibiton) in most of the tested cell lines exceeded 10uM). Results of in vitro mammosphere assays showed that 100nM LG100268 reduced mammosphere formation efficiency by more than 40% in all breast cancer cell lines. Results from our in vivo experiments showed that LG100268 reduced the mammary epithelial stem cell population by 57% as defined by the CD24+/CD49f++ population identified by FACS and by 47% as measured by in vitro mammosphere assays. Conclusion: These results from in vitro and in vivo studies demonstrate that the rexinoid LG100268 is a potent mammary stem cell inhibitor. This study shows that rexinoids suppress stem cell expansion and suggest that mammary stem cell inhibitors will be useful breast cancer preventive agents. This work is supported by a grant from the Breast Cancer Research Foundation (to PHB) and from an NIH/NCI RO1 grant (CA078480) (to PHB). Citation Information: Cancer Prev Res 2011;4(10 Suppl):A45.

Breast cancers are now subdivided into distinct tumor types based upon molecular classification of tumor gene expression. While most breast cancers are driven by estrogen-mediated signaling (estrogen receptor-positive, (ER+) or Luminal-type breast cancers) or by addiction to the HER2 oncogene (HER2+ breast cancer), the essential cellular signals that control the growth of ER-negative and HER2-negative (or basal-type) breast cancers (approximately 15% of all breast cancers), are incompletely understood. Patients with these ER− and HER2− breast cancers have poor outcomes and few available treatment options other than non-specific chemotherapy. To identify novel targets for the treatment and prevention of basal breast cancers, we investigated the activity of inflammatory genes in these cancers, previously linked to the progression of several other types of cancer. To identify critical inflammatory genes in basal breast cancer, we performed univariate t-tests (FDR1.5 or < 0.7 in at least two of the datasets analyzed). Further analysis of inflammatory gene expression in breast cancer cell lines revealed that 24 of these genes were highly expressed in basal-like cell lines at the RNA and protein level, as compared to luminal-like ER+ breast cancer lines (showing fold-differences of >1.5 or Citation Information: Cancer Prev Res 2011;4(10 Suppl):A42.

Background: Breast cancer prevention is now possible using anti-estrogen drugs such as tamoxifen and raloxifene. However, the anti-estrogen drugs do not prevent all types of breast cancer. These drugs are useful for the prevention of estrogen receptor (ER)-positive breast cancers, but are unable to prevent the development of ER-negative breast cancer. ER-negative breast cancers are highly aggressive and are particularly difficult to treat. Thus, there is an urgent need to develop ways to prevent these life-threatening cancers. Using a transgenic mouse model of ER-negative breast cancer, our laboratory has previously demonstrated that LGD100268, a ligand to the RXR nuclear hormone receptor, prevents up to 90% of the ER-negative breast cancers in these mice. However, because this drug does not prevent all breast cancers in these mice, we have attempted to achieve more effective cancer prevention by combining two cancer preventive drugs. We hypothesized that the combination of the rexinoid LG100268 in combination with tamoxifen will more effectively prevent the development of ER-negative breast cancer. To test this hypothesis, we used the p53-null mouse model that develops both ER-positive and ER-negative mammary tumors to determine whether the combination of a rexinoid and an anti-estrogen is more effective than either treatment alone in preventing ER-positive and ERnegative mammary tumors. Methods: Using our standard transplant protocol, p53-null Balb/C donor mice was transplanted into both cleared inguinal mammary fat pads of Balb/C p53 wild-type mice. Mice were separated into 5 treatment groups (15 mice each). 1) Sham Control: 2) Vehicle Control: 3) Tamoxifen + Vehicle: 4) Sham pellet (5mg) + Rexinoid: (at 50mg/kg), 5) Tamoxifen + Rexinoid: Mice were treated for 60 days with each drug sequentially. Mice were observed daily for tumor formation and the percentage of tumor free mice were recorded. Tumor incidence and time to tumor formation was analyzed. The expression of mammary tissue biomarkers was done using immunohistochemistry and real time QRT-PCR. Results: 38% of sham and 41% vehicle control animals developed tumors. Sequential treatment with tamoxifen and LGD100268 prevented more (8% incidence) than tamoxifen (24% incidence) or LGD100268 (20% incidence) alone. Proliferation markers Ki67 and cyclin D1 were markedly reduced in the mammary tissue from the mice treated with the combination therapy. We have also observed induction of rexinoid target genes ABCA1 and ABCG1, in the retinoid group and in the combination group. Conclusions: The combination treatment of rexinoid LG100268 and tamoxifen is more effective at preventing mammary tumors than either agent alone. Based on these studies, clinical trials using the combination of tamoxifen and rexinoids should be considered for the prevention of breast cancer in high-risk patients. Supported by NIH grants P50 CA58183 (PB, DM) and R01 CA101211 (PB, DM). Citation Information: Cancer Prev Res 2010;3(12 Suppl):B56.

Background: The rexinoid bexarotene (LGD1069, Targretin) is a highly selective retinoid × receptor (RXR) agonist that inhibits the growth of pre-malignant and malignant breast cells. Bexarotene was shown to suppress the development of breast cancer in transgenic mice models without side effects. The chemopreventive effects of bexarotene are due to transcriptional modulation of cell proliferation, differentiation and apoptosis. Our goal in the present study was to obtain a profile of the genes modulated by bexarotene on mammary gland from three transgenic mouse mammary cancer models in an effort to elucidate its molecular mechanism of action and for the identification of biomarkers of effectiveness. Methods: Serial analysis of gene expression (SAGE) was employed to profile the transcriptome of p53-null, MMTV-ErbB2, and C3(1)-SV40 mammary cells obtained from mice treated with bexarotene and their corresponding controls. Results: This resulted in a dataset of approximately 360,000 transcript tags representing over 20,000 mRNAs from a total of 6 different SAGE libraries. Analysis of gene expression changes induced by bexarotene in mammary gland revealed that 89 genes were dysregulated among the three transgenic mouse mammary models. From these, 9 genes were common to the three models studied. Conclusion: Analysis of the indicated core of transcripts and protein-protein interactions of this commonly modulated genes indicate two functional modules significantly affected by rexinoid bexarotene related to protein biosynthesis and bioenergetics signatures, in addition to the targeting of cancer-causing genes related with cell proliferation, differentiation and apoptosis., Centro de Investigaciones Inmunológicas Básicas y Aplicadas