Loss of Neuroligin 4X Induces An Intrinsic Innate Immune Response in TNBC

Background Immune checkpoint blockade therapies, which act on T cell inhibitory receptors, including CTLA-4 and PD-1, induce durable responses across diverse cancers. However, most patients do not respond to these therapies, and initially responsive cancers may relapse. Identifying molecular mechanisms that influence therapeutic responses and resistance is critical to realize the full therapeutic potential of immune checkpoint inhibitors. The presence of immune infiltrates in the tumor microenvironment is associated with positive outcomes in breast cancer, specifically in triple-negative breast cancer (TNBC). The underlying mechanisms driving this response are unclear. We have previously identified Neuroligin 4X (NLGN4X) as a protein expressed in TNBC.Methods Bioinformatic analysis was used for pathway analysis of TCGA TNBC patient dataset. Immunohistochemistry was performed on breast cancer tissue microarray for NLGN4X protein expression. RNA-seq was performed on MDA-MB-231 breast cancer cells for differential gene expression upon gene knockdown. Cytokine array, western blot, cell adhesion array and Nanostring was performed to determine the role of NLGN4X in TNBC.Results In this study, we report that NLGN4X expression is lost in breast cancer with lymph node metastasis. Its expression negatively correlates with immune markers in vitro, The Cancer Genome Atlas (TCGA) TNBC patient dataset, and metastatic breast cancer tissues. RNA-seq analysis of the MDA-MB-231 breast cancer cell line, silenced for NLGN4X by siRNA showed more than 500 differentially regulated genes. GSEA analysis of these genes revealed upregulation of interferon signaling pathway, cytokine signaling, and downregulation of cholesterol metabolism and lipid metabolism pathways. NLGN4X knockdown induced loss of cell adhesion, epithelial to mesenchymal transition (EMT), and MAVS-IRF7 signaling in breast cancer cells. Interestingly, analysis of the TCGA dataset of 104 TNBC patients also showed interferon signaling (IFN) as one of the significant pathways downregulated in TNBC patients expressing NLGN4X.Conclusion Loss of NLGN4X leads to innate immune activation in breast cancer and coincides with an aggressive phenotype of cancer. This study identifies the role of NLGN4X in regulating interferon signaling and immune microenvironment in TNBC.

The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer

Background Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-associated mortality in women. In particular, triple-negative BC (TNBC) has the highest rate of mortality due in large part to the lack of targeted treatment options for this subtype. Thus, there is an urgent need to identify new molecular targets for TNBC treatment. RALA and RALB are small GTPases implicated in growth and metastasis of a variety of cancers, although little is known of their roles in BC. Methods The necessity of RALA and RALB for TNBC tumor growth and metastasis were evaluated in vivo using orthotopic and tail-vein models. In vitro, 2D and 3D cell culture methods were used to evaluate the contributions of RALA and RALB during TNBC cell migration, invasion, and viability. The association between TNBC patient outcome and RALA and RALB expression was examined using publicly available gene expression data and patient tissue microarrays. Finally, small molecule inhibition of RALA and RALB was evaluated as a potential treatment strategy for TNBC in cell line and patient-derived xenograft (PDX) models. Results Knockout or depletion of RALA inhibited orthotopic primary tumor growth, spontaneous metastasis, and experimental metastasis of TNBC cells in vivo. Conversely, knockout of RALB increased TNBC growth and metastasis. In vitro, RALA and RALB had antagonistic effects on TNBC migration, invasion, and viability with RALA generally supporting and RALB opposing these processes. In BC patient populations, elevated RALA but not RALB expression is significantly associated with poor outcome across all BC subtypes and specifically within TNBC patient cohorts. Immunohistochemical staining for RALA in patient cohorts confirmed the prognostic significance of RALA within the general BC population and the TNBC population specifically. BQU57, a small molecule inhibitor of RALA and RALB, decreased TNBC cell line viability, sensitized cells to paclitaxel in vitro and decreased tumor growth and metastasis in TNBC cell line and PDX models in vivo. Conclusions Together, these data demonstrate important but paradoxical roles for RALA and RALB in the pathogenesis of TNBC and advocate further investigation of RALA as a target for the precise treatment of metastatic TNBC.

Loss of Neuroligin 4X Induces an Intrinsic Innate Immune Response in TNBC

BackgroundImmune checkpoint blockade therapies, which act on T cell inhibitory receptors, including CTLA-4 and PD-1, induce durable responses across diverse cancers. However, most patients do not respond to these therapies, and initially responsive cancers may relapse. Identifying molecular mechanisms that influence therapeutic responses and resistance is critical to realize the full therapeutic potential of immune checkpoint inhibitors. The presence of immune infiltrates in the tumor microenvironment is associated with positive outcomes in breast cancer, specifically in triple-negative breast cancer (TNBC). The underlying mechanisms driving this response are unclear. We have previously identified Neuroligin 4X (NLGN4X) as a protein expressed in TNBC.MethodsBioinformatic analysis was used for pathway analysis of TCGA TNBC patient dataset. Immunohistochemistry was performed on breast cancer tissue microarray for NLGN4X protein expression. RNA-seq was performed on MDA-MB-231 breast cancer cells for differential gene expression upon gene knockdown. Cytokine array, western blot, cell adhesion array and Nanostring was performed to determine the role of NLGN4X in TNBC.ResultsIn this study, we report that NLGN4X expression is lost in breast cancer with lymph node metastasis. Its expression negatively correlates with immune markers in vitro, The Cancer Genome Atlas (TCGA) TNBC patient dataset, and metastatic breast cancer tissues. RNA-seq analysis of the MDA-MB-231 breast cancer cell line, silenced for NLGN4X by siRNA showed more than 500 differentially regulated genes. GSEA analysis of these genes revealed upregulation of interferon signaling pathway, cytokine signaling, and downregulation of cholesterol metabolism and lipid metabolism pathways. NLGN4X knockdown induced loss of cell adhesion, epithelial to mesenchymal transition (EMT), and MAVS-IRF7 signaling in breast cancer cells. Interestingly, analysis of the TCGA dataset of 104 TNBC patients also showed interferon signaling (IFN) as one of the significant pathways downregulated in TNBC patients expressing NLGN4X.ConclusionLoss of NLGN4X leads to innate immune activation in breast cancer and coincides with an aggressive phenotype of cancer. This study identifies the role of NLGN4X in regulating interferon signaling and immune microenvironment in TNBC.

Chemotherapy Shifts the Balance in Favor of CD8+ TNFR2+ TILs in Triple-Negative Breast Tumors

Triple-negative breast cancer (TNBC) is primarily treated via chemotherapy; in parallel, efforts are made to introduce immunotherapies into TNBC treatment. CD4+ TNFR2+ lymphocytes were reported as Tregs that contribute to tumor progression. However, our published study indicated that TNFR2+ tumor-infiltrating lymphocytes (TNFR2+ TILs) were associated with improved survival in TNBC patient tumors. Based on our analyses of the contents of CD4+ and CD8+ TILs in TNBC patient tumors, in the current study, we determined the impact of chemotherapy on CD4+ and CD8+ TIL subsets in TNBC mouse tumors. We found that chemotherapy led to (1) a reduction in CD4+ TNFR2+ FOXP3+ TILs, indicating that chemotherapy decreased the content of CD4+ TNFR2+ Tregs, and (2) an elevation in CD8+ TNFR2+ and CD8+ TNFR2+ PD-1+ TILs; high levels of these two subsets were significantly associated with reduced tumor growth. In spleens of tumor-bearing mice, chemotherapy down-regulated CD4+ TNFR2+ FOXP3+ cells but the subset of CD8+ TNFR2+ PD-1+ was not present prior to chemotherapy and was not increased by the treatment. Thus, our data suggest that chemotherapy promotes the proportion of protective CD8+ TNFR2+ TILs and that, unlike other cancer types, therapeutic strategies directed against TNFR2 may be detrimental in TNBC.

Functional Characterization of the Orphan Nuclear Receptor TLX in Triple Negative Breast Cancer

Despite the development of various therapeutic strategies, breast cancer persists as the second leading cause of cancer-related death among women in the United States. While endocrine modulation and monoclonal antibody therapy have proved to be indispensable modes of intervention for hormone receptor (HR)-positive and HER-2 positive patients, the triple negative breast cancer (TNBC) patient population do not respond to these therapies. As TNBC is considered one of the most challenging subtypes of breast cancer to treat, there is a significant need for the development of targeted therapeutics. Due to their well-known amenability to small-molecule modulation, we investigated whether any nuclear receptors beyond those that are traditionally studied in breast cancer (e.g. ER, PR, and AR), may represent a novel target in the TNBC patient population. Analysis of clinical data revealed that expression of the orphan nuclear receptor TLX (NR2E1) was positively correlated with relapse-free survival, distant metastasis-free survival, and overall survival in both ER-negative and basal-like breast cancer patients. Therefore, we hypothesized that TLX could influence the pathophysiology of TNBC. To interrogate this hypothesis, we established TNBC cells with stable expression of TLX in order to identify direct regulatory targets, as well as the precise physiological mechanism(s) TLX may be regulating. To date, our work has revealed that TLX inhibits proliferation, slows migration, alters chemosensitivity, and impairs cell cycle progression in TNBC cells. In agreement with these findings, our work has also revealed that TLX is capable of modulating the expression of several genes that are known to regulate the processes of growth, migration, and cell cycle. Taken together, our early work supports our hypothesis, and provides valuable insight into the potential pro-survival function of TLX in TNBC. Ongoing work will continue to probe the mechanisms by which TLX impacts breast cancer biology, and establish whether the growth-inhibitory effects translate to in vivo models. As prior work has demonstrated that TLX’s transcriptional activity can be regulated by both synthetic and natural ligands, the results of our work would provide the foundational data necessary for the development of a TLX-based therapy for a patient population with limited therapeutic options and a poor prognostic outlook.

The Small G-Protein RalA Promotes Progression and Metastasis of Triple Negative Breast Cancer

Background: Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-associated mortality in women. In particular, triple-negative BC (TNBC) has the highest rate of mortality due in large part to the lack of targeted treatment options for this subtype. Thus, there is an urgent need to identify new molecular targets for TNBC treatment. RALA and RALB are small GTPases implicated in growth and metastasis of a variety of cancers, although little is known of their roles in BC. Methods: The necessity of RALA and RALB for TNBC tumor growth and metastasis were evaluated in vivo using orthotopic and tail-vein models. In vitro, 2D and 3D cell culture methods were used to evaluate the contributions of RALA and RALB during TNBC cell migration, invasion, and viability. The association between TNBC patient outcome and RALA and RALB expression was examined using publicly available gene expression data and patient tissue microarrays. Finally, small molecule inhibition of RALA and RALB was evaluated as a potential treatment strategy for TNBC in cell line and patient-derived xenograft (PDX) models. Results: Knockout or depletion of RALA inhibited orthotopic primary tumor growth, spontaneous metastasis, and experimental metastasis of TNBC cells in vivo. Conversely, knockout of RALB increased TNBC growth and metastasis. In vitro, RALA and RALB had antagonistic effects on TNBC migration, invasion, and viability with RALA generally supporting and RALB opposing these processes. In BC patient populations, elevated RALA but not RALB expression is significantly associated with poor outcome across all BC subtypes and specifically within TNBC patient cohorts. Immunohistochemical staining for RALA in patient cohorts confirmed the prognostic significance of RALA within the general BC population and the TNBC population specifically. BQU57, a small molecule inhibitor of RALA and RALB, decreased TNBC cell line viability, sensitized cells to paclitaxel in vitro and decreased tumor growth and metastasis in TNBC cell line and PDX models in vivo. Conclusions: Together, these data demonstrate important but paradoxical roles for RALA and RALB in the pathogenesis of TNBC and advocate further investigation of RALA as a target for the precise treatment of metastatic TNBC.

Targeting Hypoxia Sensitizes TNBC to Cisplatin and Promotes Inhibition of Both Bulk and Cancer Stem Cells

Development of targeted therapies for triple-negative breast cancer (TNBC) is an unmet medical need. Cisplatin has demonstrated its promising potential for the treatment of TNBC in clinical trials; however, cisplatin treatment is associated with hypoxia that, in turn, promotes cancer stem cell (CSC) enrichment and drug resistance. Therapeutic approaches to attenuate this may lead to increased cisplatin efficacy in the clinic for the treatment of TNBC. In this report we analyzed clinical datasets of TNBC and found that TNBC patients possessed higher levels of EGFR and hypoxia gene expression. A similar expression pattern was also observed in cisplatin-resistant ovarian cancer cells. We, thus, developed a new therapeutic approach to inhibit EGFR and hypoxia by combination treatment with metformin and gefitinib that sensitized TNBC cells to cisplatin and led to the inhibition of both CD44+/CD24− and ALDH+ CSCs. We demonstrated a similar inhibition efficacy on organotypic cultures of TNBC patient samples ex vivo. Since these drugs have already been used frequently in the clinic; this study illustrates a novel, clinically translatable therapeutic approach to treat patients with TNBC.

Targeting Hypoxia Sensitizes TNBC to Cisplatin and Promotes Inhibition of Both Bulk and Cancer Stem Cells

Development of targeted therapies for triple-negative breast cancer (TNBC) is an unmet medical need. Cisplatin has demonstrated its promising potential for the treatment of TNBC in clinical trials; however, cisplatin treatment is associated with hypoxia that in turn promotes cancer stem cell (CSC) enrichment and drug resistance. Therapeutic approaches to attenuate this may lead to increased cisplatin efficacy in the clinic for the treatment of TNBC. In this report, we analyzed clinical dataset of TNBC and found that TNBC patients possessed higher levels of EGFR and hypoxia gene expression. A similar expression pattern was also observed in cisplatin-resistant ovarian cancer cells. We thus developed a new therapeutic approach to inhibit EGFR and hypoxia by combination of metformin and gefitinib that sensitized TNBC cells to cisplatin and led to the inhibition of both CD44+/CD24- and ALDH+ CSCs. We demonstrated a similar inhibition efficacy on organotypic cultures of TNBC patient samples ex vivo. Since these drugs have already been used frequently in the clinic, this study illustrates a novel, clinically translatable therapeutic approach to treat patients with TNBC.