Background: Triple-negative breast cancer (TNBC), the most difficult subtype to treat, is defined as estrogen receptor (ER), HER2, and progesterone receptor (PR) negative and comprises roughly 15-20% of all breast cancers. Patients with a TNBC diagnosis have worse outcomes and poorer survival rates when compared to women with other breast cancers, despite adjuvant chemotherapy. One of the factors contributing to its metastatic ability is the angiogenic process it undergoes. Clinical trials with anti-angiogenic therapeutics showed to be inadequate, with moderate response rates and insignificant survival gains for patients. TRPS1, a GATA and GATA-like transcription factor, has been proven to play a role in the epithelial to mesenchymal transition (EMT) which ultimately promotes tumor growth and metastasis. It has previously been shown that TRPS1 regulates angiogenesis via the expression of VEGF proteins in breast cancer. Our purpose in this study was to further investigate the functional role of TRPS1-regulated genes involved in TNBC angiogenic pathways. Methods: Sleeping-Beauty (SB) transposons help detect cancer progression genes which participate in tumorigenesis and metastasis, not easily identified with current sequencing technology. Using the SB transposon methodology, several candidate trunk drivers were elucidated using Pten mutant mice. Validation studies further showed TRPS1 as one of the eight TNBC tumor suppressor genes discovered by this technology. Then, ChIP-seq array studies were performed which showed that TRPS1 regulates expression of genes involved in the angiogenesis pathway. To corroborate the functional role of TRPS1 in angiogenesis, tube formation and sprouting assays were performed using overexpression and inactivation of TRPS1 in MDA-MD-231 and HCC70 cells, respectively. Furthermore, we performed a ChIP qPCR human angiogenesis array to determine TRPS1-regulated angiogenic genes. A luciferase reporter assay was performed to determine whether TRPS1 is a direct transcriptional regulator of these genes. Results: Inactivation of TRPS1 expression allowed tube formation and cell branching in the sprouting assays. Tumor xenografts overexpressing TRPS1 were analyzed by immunohistochemistry, showed a significant reduction of angiogenic vasculature when stained with CD31; and a substantial increase in blood vessels when TRPS1 was shRNA inactivated. Furthermore, human ChIP qPCR angiogenesis array identified 10 top candidate genes potentially regulated by TRPS1 transcription factor. Of these candidates, JAG1 and TYMP showed to have a higher fold enrichment compared to other angiogenic related genes. Finally, we validated its direct functional binding by using luciferase reporter assay, such demonstrated that TRPS1 is a direct transcriptional regulator of JAG1 and TYMP. Discussion: It has been previously established that the NOTCH ligand, JAG1 and the thymidine phosphorylase enzyme, TYMP are powerful genes participating in angiogenesis. Furthermore, elevated expression of JAG1 mRNA and protein has been associated with poor patient outcomes in breast cancer. Similarly, TYMP activity has been shown to be higher in triple-negative breast tumors, leading to poor outcomes as well. Although several anti-angiogenic drugs have been approved, tumor-acquired resistance to therapy limit their effectiveness. An increased understanding of tumor vessel mechanisms involved in tumorigenesis and metastasis is necessary to overcome the obstacles that prevent successful control of the angiogenic response in tumors. This would require as many paths as can be unraveled regarding the angiogenic mechanisms driving TNBC. Understanding the relationship between the function of TRPS1 in regulating the expression of important genes such as JAG1 and TYMP lays a better foundation for new drugs and drug targets that control and inhibit tumor angiogenesis. Citation Format: Liliana Guzman, Camila Ayerbe, Roberto Rangel, Jenny Chang, Roberto Rosato. TRPS1 Regulates angiogenesis via JAG1 and TYMP in Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-25-01.

Background: Metaplastic breast cancer (MpBC) is a rare, lethal, and highly chemoresistant breast cancer subtype, with no FDA-approved therapeutic options. Most MpBCs are triple-negative, yet have a worse prognosis than non-metaplastic triple-negative breast cancer (non-MpTNBC). MpBC tumors are enriched for markers of epithelial-to-mesenchymal transition (EMT)/cancer stem cells (CSC), produce high nitric oxide (NO) levels, and have a hyperactive phosphoinositide 3-kinase (PI3K) signaling pathway. Increased PI3K and inducible nitric oxide synthase (iNOS) activity are poor prognostic indicators in MpBC. NO can activate multiple oncogenic pathways spatially and temporally, such as PI3K and transforming growth factor beta (TGFβ), a critical regulator of EMT. Therefore, our study evaluates whether pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA) augments the efficacy of alpha isoform-specific PI3K inhibitor alpelisib in MpBC in vitro and in vivo models. Methods: MpBC cell lines (SUM159, BT549, Hs578T, HCC1806) and Patient-Derived Xenograft (PDX) models were used in our studies. Droplet digital polymerase chain reaction (ddPCR) was conducted to evaluate the iNOS-associated mutation (RPL39 A14V) and PIK3CA hotspot mutation rates in PDX models. Cell viability (SRB/Cell Titer-Glo), combination index (CI), immunoblotting, and immunofluorescence of treated MpBC cell lines and tumor tissues were evaluated. Results: Immunostaining analysis revealed that MpBC PDX tumors had elevated co-expression of iNOS and pAkt (60% vs 23%, p=0.0495) relative to non-MpTNBC PDX tumors. MpBC PDX tumors had higher RPL39 A14V (66% vs 4.7%, p< 0.0006) and PIK3CA hotspot mutation rates (50% vs 19.1%, p=0.1247) than non-MpTNBC PDX tumors. Combining L-NMMA with alpelisib was synergistic in MpBC cell lines harboring PIK3CA/PIK3R1 mutations (CI< 1) and antagonistic in PIK3CA-wild type and PTEN-deleted models (CI>1). In vivo evaluation using MpBC PDX tumors found that L-NMMA significantly augmented the efficacy of alpelisib in reducing tumor volume in PIK3CA-mutated MpBC PDX models. Transcriptomic analysis found gene sets associated with EMT reversal, such as the formation of cornified envelope (Padj = 0.0254) and keratinization pathway (Padj = 0.048) were enriched pathways in MpBC PDX tumors that responded to combination therapy. Pharmacological and genomic inhibition of iNOS reversed EMT in MpBC cells, as shown by decreased expression of Zeb1, TGFβ, Snail, Vimentin, and increased expression of E-cadherin and ZO-1 in immunoblotting analysis. MpBC cells with NOS2 knockout acquired an epithelial-like cellular morphology and this reversal of EMT rendered MpBC cells more sensitive to alpelisib and taxane-chemotherapy. MpBC PDX tumors that responded to combination therapy also exhibited a reversal in EMT, with an associated decrease in aldehyde dehydrogenase (ALDH1), a CSC marker. L-NMMA and alpelisib therapy also resulted in the loss of tumor-initiating ability, enhanced chemosensitivity, and improved overall survival in MpBC PDX models. These studies paralleled results from a phase 1b/2 clinical trial with L-NMMA combined with taxane chemotherapy in a cohort of anthracycline-refractory MpBC patients (n=15, NCT02834403). The clinical benefit rate was 40% (6/15), the overall response rate was 20% (3/15), and one patient achieved a pathologic complete response. Relative to baseline tumors, the responder end-of-treatment tumors had undergone reversal of EMT, with enhanced expression of E-cadherin, and decreased expression of iNOS, Zeb1, and ALDH1. Conclusion: Our findings suggest that combining L-NMMA and alpelisib is a novel therapeutic strategy to treat MpBC by reversing EMT and decreasing CSCs, rendering MpBC tumors more chemosensitive. This combination therapy is being tested in a first multicenter phase 2 study targeting this orphan disease. Citation Format: Tejaswini Reddy, Akshjot Puri, Liliana Guzman, Wei Qian, Jianying Zhou, Roberto Rosato, Hong Zhao, Christoforos Thomas, Xiaoxian Li, Bijan Mahboubi, Adrian Oo, Young-Jae Cho, Baek Kim, Jose Thaiparambil, Camila Ayerbe, Noah Giese, Stacy Moulder, Helen Piwnica-Worms, Funda Meric-Bernstam, Jenny Chang. NOS inhibition reverses epithelial-to-mesenchymal transition and synergizes with alpelisib in metaplastic breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-05.

Metaplastic breast cancer (MpBC) is a rare and highly chemoresistant breast cancer subtype, with a median survival of 8 months after metastatic disease, and no standard therapeutic options. MpBCs are enriched for epithelial-to-mesenchymal transition (EMT)/cancer stem cell (CSC) markers, produce high levels of nitric oxide (NO), and have a hyperactive phosphoinositide 3-kinase (PI3K) signaling pathway. NO activates multiple oncogenic pathways, such as PI3K and transforming growth factor beta (TGFβ), a regulator of EMT. Therefore, we hypothesized that pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA) could augment the efficacy of α-specific PI3K inhibitor alpelisib in MpBC in vitro and in vivo models. Immunostaining analysis revealed that MpBC PDX tumors had elevated co-expression of iNOS and pAkt (60% vs 23%, p=0.04) relative to triple-negative breast cancer (TNBC) PDX tumors. MpBC PDX tumors had higher RPL39 A14V (66% vs 4.7%, p< 0.00) and PIK3CA hotspot mutation rates (50% vs 19.1%, p=0.31) than TNBC PDX tumors. L-NMMA was synergistic with alpelisib in MpBC cell lines with PIK3CA/PIK3R1 mutations and antagonistic in PIK3CA-wild type and PTEN-deleted models. In vivo evaluation using MpBC PDX tumors found that L-NMMA augmented the efficacy of alpelisib in reducing tumor volume in PIK3CA-mutated MpBC PDX models. Transcriptomic analysis found gene sets associated with EMT reversal, such as the formation of cornified envelope (NES = 2.04 Nom p<0.00) and keratinization pathway (NES = 2.06, Nom p<0.00), were enriched pathways in MpBC PDX tumors that responded to combination therapy. Pharmacological/genomic inhibition of iNOS reversed EMT in MpBC cells, by decreased expression of Zeb1, TGFβ, Snail, Vimentin, and increased expression of E-cadherin and ZO-1 in immunoblotting analysis. MpBC cells with NOS2 knockout acquired an epithelial-like cellular morphology, and this reversal of EMT rendered MpBC cells more sensitive to alpelisib and taxane-chemotherapy. MpBC PDX tumors that responded to combination therapy also exhibited a reversal in EMT, with a decrease in aldehyde dehydrogenase (ALDH1), a CSC marker. Combination therapy also reduced tumor-initiating ability, enhanced chemosensitivity, and improved overall survival in MpBC PDX models. These studies paralleled a phase 1b/2 clinical trial with L-NMMA+taxane chemotherapy in a cohort of anthracycline-refractory MpBC patients (NCT02834403). The clinical benefit rate was 40% (6/15), overall response rate was 20% (3/15), and one patient achieved a pathologic complete response. Relative to baseline tumors, the responder end-of-treatment tumors had undergone reversal of EMT, with decreased expression of iNOS and ALDH1. We find that combining L-NMMA and alpelisib is a novel therapeutic strategy to treat MpBC, and combination therapy is being tested in a first multicenter phase 2 study for patients with MpBC. Citation Format: Tejaswini P. Reddy, Akshjot Puri, Liliana Guzman-Rojas, Christoforos Thomas, Wei Qian, Jianying Zhou, Hong Zhao, Xiaoxian Li, Bijan Mahboubi, Adrian Oo, Cho Young-Jae, Baek Kim, Jose Thaiparambil, Maria Florencia Chervo, Roberto Rosato, Camila Ayerbe, Noah Giese, Stacy Moulder, Helen Piwnica-Worms, Funda Meric-Bernstam, Jenny C. Chang. NOS inhibition reverses epithelial-to-mesenchymal transition and synergizes with alpelisib in metaplastic 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 3447.