Molecular Mechanisms Regulating Mouse Mammary Gland and Human Breast Tumor Cells

Division Of Basic Sciences - Nci

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Abstract

Inflammatory Breast Cancer (Balamurugan et al, manuscript under review). During FY2021 we have made strides in a project that started by characterizing a function for CEBPD in Inflammatory Breast Cancer and led to the discovery that The COX-2/PGE2/ GSK3beta pathway promotes tumor cell adhesion by E-cadherin and is a candidate target for inflammatory breast cancer. Inflammatory breast cancer (IBC) is an extremely aggressive subtype of breast cancer (BC; classified by NIH-GARD as Rare Disease) with pronounced racial and socioeconomic disparities. Clinical presentation includes extensive skin invasion and intralymphatic tumor cell emboli that exhibit E-cadherin (CDH1)-mediated cell-cell adhesions. The study of IBC thus offers an opportunity to understand the pathways leading to E-cadherin-associated metastasis. Through analysis of the C/EBPdelta (CEBPD) transcription factor, we found that prostaglandin-endoperoxide synthase 2 (COX-2, PTGS2) promoted E-cadherin-mediated adhesions, despite the reported role of COX-2 in epithelial-mesenchymal transition. Using an in vitro emboli formation assay, we found that COX-2, through activation of the serine-threonine kinase AKT/PKB and inhibition of GSK3beta, prevented proteasomal degradation of p120 catenin (CTNND1), which stabilizes E-cadherin cell adhesion complexes. Expression of E-cadherin and GSK3beta inhibition was confirmed in patient-derived xenografts (PDX) of metastatic breast cancers. Treatment with either prostaglandin E2 (PGE2), inhibitors of GSK3beta, or the overexpression of E-cadherin reconstituted tumor cell emboli formation and also induced clustering of non-IBC cells in suspension. Conversely, the COX-2 inhibitor celecoxib caused cell death within emboli and synergized with paclitaxel therapy in vitro and in an experimental metastasis model in vivo. These results point to PGE2 signaling and GSK3beta inhibition as a mechanism for tumor emboli formation and suggest that common oncogenic signals can converge on stabilization of E-cadherin mediated cell-cell adhesions that are relevant for breast cancer metastasis. Lastly, up-regulated expression of COX-2 and E-cadherin is common in IBC and correlates with reduced breast cancer survival, suggesting that patients with such breast cancer may specifically benefit from targeting of the PGE2 pathway. These findings describe an unexpected function of prostaglandin-E2 in promoting E-cadherin protein expression and cell-cell adhesion, suggesting that patients with COX2+/E-Cadherin+ breast cancer may specifically benefit from targeting of the PGE2 pathway. Ongoing research is testing the combination of celecoxib with standard-of0care therapeutics in vitro and in vivo including patient-derived xenograft models. Endoplasmic Reticulum Stress (Sheshadri, Poria et al, manuscript under revision) Cancer cells often experience endoplasmic reticulum (ER) stress due to activated oncogenes and microenvironmental conditions such as nutrient deprivation and hypoxia. Cells activate the unfolded protein response (UPR) mediated by ATF6, IRE1/XBP1s and PERK/ATF4 pathways to support adaption to stress, accompanied by activation of inflammatory signaling. The elevated UPR in cancer cells contributes to enhanced cell survival and therapy resistance. Therefore, it is important to understand the full complexity of cellular responses to ER stress. Using a panel of breast cancer cell lines, we found that CCAAT-enhancer binding protein delta (CEBPD) is rapidly induced in response to various chemical and physiological inducers of ER stress. Transcription factor, CEBPD is most known for its role in pro-inflammatory signaling pathways and research from our laboratory documents its dual functions in breast cancer by both attenuating or enhancing oncogenic pathways depending on context. Using pharmacological inhibitors and RNAi of specific UPR effectors, we found that PERK-mediated STAT3 activation induces CEBPD gene expression concurrent with the activation of the classical UPR effectors, XBP1s and ATF4, suggesting a potential role in stress adaptation. Examination of the global transcriptional profilee using RNAseq confirmed that CEBPD specifically supports pathways involved in ER stress adaptation, such as chaperone functions, UPR transcription factors, autophagy and cytokine signaling. While XBP1s and ATF4 are known to activate these pathways, this is the first report of a role for CEBPD in this process. Functional assays showed that knock down of CEBPD in cancer cells decreased chemokine expression, especially of CXCL8, which promotes cancer cell stemness and modulates immune cell gene expression. CEBPD is a novel proximal effector of the ER stress response in breast cancer cells that contributes to modulation of the tumor microenvironment. These data also add a novel mechanism for the tumor promoting functions of CEBPD. Myeloid lineage response to tumor development: Ongoing work is analyzing the immune response to tumor cells in mice with a focus on the myeloid lineage. We discovered that CEBPD mediates in part the massive development of myeloid-derived suppressor cells that is distinct to female mice. SARS-CoV2 Testing: We have initiated a new project to assess a novel method for detection of RNA including SARS-CoV2. A provisional patent application has been filed. We are in the process of assessing the application of the test to other pathogens.