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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 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.