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The association of SOX transcription factors expression level and breast cancer subtypes IN Esserman dataset

Supplementary Figure Legends 1-16 and Supplementary Table Legends 1-4

H3k27AC/Me3 binding peaks in the promoter area of SOX9 targets

SOX9 regulates cell apoptosis in TNBC cells but not in non-TNBC cells

Supplementary Materials 2 from Identification of Modulated Genes by Three Classes of Chemopreventive Agents at Preneoplastic Stages in a p53-Null Mouse Mammary Tumor Model

Supplementary Table 1. Primers and probes for qRT-PCR assay. Supplementary Table 2. Primers for CHIP assay (positive binding sequence). Supplementary Table 3. Primers for CHIP assay (negative binding sequence). Supplementary Table 4. Primers for H3K27Ac/Me3 CHIP assay (binding peak and negative binding sequence).

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.

Expression level of SOXs in breast cancer cell lines

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.

Supplementary Materials 1 from Identification of Modulated Genes by Three Classes of Chemopreventive Agents at Preneoplastic Stages in a p53-Null Mouse Mammary Tumor Model

Related Article from Prevention of Tumorigenesis in p53-Null Mammary Epithelium by Rexinoid Bexarotene, Tyrosine Kinase Inhibitor Gefitinib, and Celecoxib

SOX9 controls G0/G1 to S phase cell cycle

SOX9-knockout suppresses TNBC cells xenograft growth in vivo

Expression of SOX9 in Curtis breast cancer dataset and NEVE breast cancer cell lines

The association of SOX transcription factors expression level and breast cancer subtypes

SOX9 expression level regulates cell migration and invasion in breast TNBC and Non-TNBC cells

Supplementary Materials 3 from Identification of Modulated Genes by Three Classes of Chemopreventive Agents at Preneoplastic Stages in a p53-Null Mouse Mammary Tumor Model

Related Article from Identification of Modulated Genes by Three Classes of Chemopreventive Agents at Preneoplastic Stages in a p53-Null Mouse Mammary Tumor Model

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.

Table S2 - XLSX file 77K, Verification of lentiviral shRNA knock-down in SUM159 cells by quantitative RT-PCR

Figure S5 - PDF file 1389K, Inducible inhibition of IL-6, IL-8, and CXCL1, and tandem inhibition of IL-6, IL-8, and CXCL1 in SUM159, MDA-MB-231, and MDA-MB-468 cells

Supplementary Text - PDF file 109K, Supplementary Figure Legends

Supp Fig 5 - A) PTP4A3 and Myc amplification status in Luminal A/B and Basal-like breast cancers from TCGA data set. B) PTP4A3 and Myc amplification status in ovarian cancer from TCGA data set. C) Map detailing the location of the genes analyzed for gene amplification. D) Comparative analysis of PTP4A3 gene copy number and expression levels

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.

Supp Fig 2 - Cleaved caspase 7 is selective increased in basal-like breast cancers with low PTP4A3 expression.

Supp Fig 4 - A) Curtis data set analyzed for expression levels of PTP4A3 in normal breast tissue compared to invasive ductal carcinoma (IDC). B) Curtis data set analyzed for expression levels of PTP4A3 in non-TNBCs versus TNBCs. C) Kaplan-Meier curves of overall survival of all breast cancer patients (Kao Dataset) stratified by PTP4A3 expression and ER. D) Kaplan-Meier curves of disease-specific survival of all breast cancer patients (Curtis Dataset) stratified by PTP4A3 expression.

Supp Tab 2 - Cox proportional hazard ratio analysis of the Curtis Data set.

Supplemental Figure Legend

Supp Fig 3 - A-B) Average tumor growth of MDA MB231 shPTP4A3 and control tumors treated with doxycycline or vehicle. C) mRNA expression of MDA MB231 control tumors and PTP4A3 tumors treated with doxycycline or vehicle. D) Average tumor growth of MDA MB468 control tumors and PTP4A3 tumors treated with doxycycline or vehicle. D) Tumor growth rate is not reduced in tumors from the ER-positive breast cancer cell lines.E). Tumor growth slopes of MCF-7 control tumors and PTP4A3 tumors treated with doxycycline or vehicle.

Supp Tab 1 - Tissue samples used for this study were obtained from the Lester and Sue Smith Breast Cancer Tumor bank at Baylor College of Medicine. We studied 102 breast tumors from this bank that were collected from sites in the US and Europe. Study demographics define the majority of the population to be postmenopausal women, 97% Caucasian, and the median subject age to be 53 years. In this cohort, 95% of the tumors were invasive ductal carcinomas, 47% presented no lymph node involvement, all were without metastasis, and 76% of study participants had tumors {greater than or equal to}2cm3 at the time of diagnosis. The ER and PR status of each tissue sample was determined through immunohistochemistry (IHC) analysis, and HER2 status by fluorescent in-situ hybridization (FISH). This identified 49 ER-positive tumors and 53 triple-negative tumors. Of the ER-positive breast cancers, 12 tumors were also HER2-positive (24%), and 42 PR positive (86%).

Supp Tab 3 - Excel spreadsheet containing gene list used to identify phosphatases and phosphatase-interacting proteins.

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: The most aggressive form of breast cancer is triple-negative breast cancer (TNBC) which lacks expression of the estrogen receptor (ER), progesterone receptor (PR), and does not have overexpression of the human epidermal growth factor receptor 2 (HER2). Treatment options for women with TNBC tumors are limited, unlike those with ER-positive tumors, that can be treated with hormone therapy, or those with HER2-positive tumors, that can be treated with anti-HER2 therapy. Thus, we have sought to identify novel targeted therapies for TNBC. In this study, we investigated whether a novel phosphatase, NUDT5, is a potential therapeutic target. Methods: TCGA and METABRIC (Curtis) datasets were used to investigate the mRNA expression levels of NUDT5 in breast cancers. NUDT5 ablation was achieved by targeting NUDT5 with siRNA, shRNA, and sgRNA and by inhibiting NUDT5 with the small molecule inhibitor TH5427. Xenograft animal models were used to determine the effect of NUDT5 inhibition on TNBC in vivo growth. Proliferation, death, and DNA replication assays were used to investigate the cell biologic effect of NUDT5 loss or inhibition. The accumulation of 8-oxoG and the induction of gH2AX after NUDT5 loss was determined by immunofluorescence staining. Results: In this study, we demonstrated the important role of an overexpressed phosphatase, NUDT5, in regulating oxidative DNA damage in TNBCs. We found that NUDT5 loss led to suppressed growth of TNBC in vitroand in vivo. This growth inhibition was not induced by death, but instead by suppressed proliferation. Loss or inhibition of NUDT5 induced an increase in 8-oxoG and gH2AX lesions in DNA and a stall in DNA replication, thus inhibiting proliferation. Conclusions: NUDT5 plays a critical role in preventing oxidative DNA damage in TNBC cells. Loss or inhibition of NUDT5 suppressed the growth of TNBCs. These biological and mechanistic studies provide a basic research foundation for the future development of NUDT5 inhibitors for the treatment of TNBC patients.

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 subtype of breast cancer, characterized high rates of tumor protein 53 (p53) mutation and limited targeted therapies. Despite being clinically advantageous, direct targeting of mutant p53 has been largely ineffective. Therefore, we hypothesized that there exist pathways upon which p53-mutant TNBC cells rely upon for survival. Methods In vitro and in silico drug screens were used to identify drugs that induced preferential death in p53-mutant breast cancer cells. The effects of the glutathione peroxidase 4 (GPX4) inhibitor ML-162 was deleniated using growth and death assays, both in vitro and in vivo. The mechanism of ML-162 induced death was determined using small molecule inhibition and genetic knockout. Results High-throughput drug screening demonstrated that p53-mutant TNBCs are highly sensitive to peroxidase, cell cycle, cell division, and proteasome inhibitors. We further characterized the effect of the Glutathione Peroxidase 4 (GPX4) inhibitor ML-162 and demonstrated that ML-162 induces preferential ferroptosis in p53-mutant, as compared to p53-wild type, TNBC cell lines. Treatment of p53-mutant xenografts with ML-162 suppressed tumor growth and increased lipid peroxidation in vivo. Testing multiple ferroptosis inducers demonstrated p53-missense mutant, and not p53-null or wild type cells, were more sensitive to ferroptosis, and that expression of mutant TP53 genes in p53-null cells sensitized cells to ML-162 treatment. Finally, we demonstrated that p53-mutation correlates with ALOX15 expression, which rescues ML-162 induced ferroptosis. Conclusions This study demonstrates that p53-mutant TNBC cells have critical, unique survival pathways that can be effectively targeted. Our results illustrate the intrinsic vulnerability of p53-mutant TNBCs to ferroptosis, and highlight GPX4 as a promising target for the precision treatment of p53-mutant triple-negative breast cancer.

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.