Jin, Kideok Theses Advised
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Item Crosstalk between adipocytes and breast cancer cells with ESR mutations via adipsin enhances tumor growth.(Albany College of Pharmacy and Health Sciences Theses, 2023-08) Belyakov, Artem '23Estrogen receptor positive breast cancer is a prevalent disease traditionally treated with hormone therapy which relies on the expression of the estrogen receptor to starve the tumor of growth signaling due to estradiol. While hormone therapy generally has great outcomes, the development of endocrine resistance remains a huge hurdle, with poorer outcomes and less effective therapeutic options. Endocrine resistant tumors have been found to uniquely contain point mutations in the ligand binding domain of the Estrogen Receptor 1 (ESR1) coding region, predominantly Y537S and D538G. A growing body of literature has shown the stromal involvement in tumorigenesis, which supports resistance, survival, proliferation and eventual metastasis. Adipocytes have been found to promote breast cancer growth and invasion in coculture but without an understood mechanism. The complement system is generally associated with innate immunity but is increasingly found to promote tumor growth by dysregulation of inflammatory pathways. Since Adipsin (also known as Complement Factor D) is preferentially expressed in adipocytes, an investigation was done on the role of complement, specifically the C3a-Adipsin-C3aR axis on the impact of breast cancer cell characteristics. Using RT-qPCR and ELISA, it was found that C3a and Adipsin are both upregulated preferentially in the mutant cells as compared to wild type when cultured in adipocyte condition media. In addition, experimental inhibition of C3aR using the small molecule SB290157, had tumor attenuating effects that were limited to the mutated breast cancer cell lines. Using CyQuant for cell viability, and flow cytometry to investigate apoptosis, cell proliferation and cell cycle analysis, it was found that SB290157 had a drastic effect by decreasing cell viability and cell proliferation, increasing apoptosis, and causing cell cycle arrest at G2/M. Using knockdown of C3 and Adipsin by siRNA for the more sensitive mutant YS led to a marked decrease in cell viability as well. These findings contribute to the idea that endocrine resistant breast cancers are utilizing complement inflammatory pathways as one of their strategies to survive, proliferate and invade. As this pathway is investigated more, it may be possible to therapeutically block it for better breast cancer outcomes.Item The HOXB7 protein promotes breast cancer cell growth through activation of the CCL5/CCR5 pathway in the crosstalk of adipocytes(Albany College of Pharmacy and Health Sciences Theses, 2023-08) Akume, Ahone Gina '23Hox genes are regulatory genes that encode nuclear proteins acting as transcription factors during normal development and differentiation. One such gene, HOXB7, plays a role in various developmental processes, including hematopoietic differentiation, lymphoid development, and mammary gland development. However, the role of HOX genes in breast cancer development remains largely unexplored. Our previous studies revealed that HOXB7 expression was significantly elevated in primary cancer and distant metastasis. HOXB7 overexpression promoted cell proliferation in SKBR3 breast cancer cells, leading to robustly vascularized xenografts in immunodeficient mice. Furthermore, HOXB7 enhanced tumor growth through TGF-B signaling and recruitment of macrophages, indicating the role of HOXB7 in the crosstalk of stromal components. We found evidence that HOXB7 overexpression in ER+ breast cancer cells promote tumor cell growth through increased expression and signaling of CCL5/CCR5 in crosstalk with adipocytes. By screening secreted factors in adipocytes induced by TCM of MCF-7-HOXB7 cells, it was also found that CCL5 is highly expressed in HOXB7- overexpressing MCF-7 cells. The CCR5 inhibitor, maraviroc, sensitized MCF-7- HOXB7 cells compared to control cells. These findings suggest that blocking the interaction between CCL5 and CCR5 signaling significantly inhibits ER+ breast cancer with HOXB7 overexpression. We aim to further investigate if HOXB7 overexpression in ER+ breast cancer cells promote tumor cell growth through increased expression and signaling of CCL5/CCR5 in crosstalk with adipocytes and the role of CCL5 in breast cancer cell growth and migration.Item The role of secreted factors in crosstalk between endocrine resistant breast cancer and stroma(Albany College of Pharmacy and Health Sciences Theses, 2022-08) Galke, Daniel '22Treatments for estrogen receptor positive (ER+) breast cancer have traditionally relied on the expression of the estrogen receptor to sufficiently treat those afflicted. An issue arises within those afflicted with this type of breast cancer in that an endocrine resistant form of this type of cancer can form and lead to tumor metastasis. Within these populations, it was found that the Estrogen Receptor 1 (ESR1) coding region was mutated more frequently than in non-resistant populations, and that the predominant mutations in the region include Y537S (YS) and D538G (DG). Cadherin-13 (CDH-13) is protein that is not well understood in terms of its importance to disease markers and CXCL1 (GRO\316\261) is a chemokine found to naturally increase in abundance with age but can lead to tumorigenesis in instances of dysregulation. These two proteins were upregulated in the secretome of YS and DG mutant induced lymphatic endothelial cells (LECs) by human cytokine array analysis and were confirmed by quantification of their relative mRNA and protein levels through RT-qPCR and ELISA, in which, both were significantly upregulated. Treatment of ESR1 mutants with CXCL1 inhibitor reparixin and a CDH-13 neutralizing antibody were then used to determine effects on cellular viability and cytotoxicity through CyQUANTTM viability assay with and without the presence of ER antagonists or Cyclin-Dependent Kinases 4/6 (CDK4/6), flow cytometry and migration assay. In the ESR1 mutants, reparixin was found to decrease the viability and increase cytotoxic characteristics through apoptosis, proliferation and cell cycle assays. This study provides evidence that functional inhibition of CXCL1 signaling pathways can potentially prevent breast cancer growth and metastasis. While no small molecule inhibitor is available for CDH-13, it remains attractive to future investigations into ER+ breast cancer.Item Crosstalk between stromal components and endocrine resistant breast cancer via secreted factors enhances tumor growth and metastasis(Albany College of Pharmacy and Health Sciences Theses, 2021-07) Pandithar, Sneha Tirumani '21Breast cancer is currently targeted using various endocrine therapies that include use of Selective Estrogen Receptor modulators (SERM), Selective Estrogen Receptor Down Regulator (SERD) and Aromatase inhibitors (AI). Tamoxifen (TAM) is a primary choice in treating early-stage estrogen receptor positive breast cancer in post-menopausal women. Despite the proven therapeutic efficacy and safety profile of TAM as a SERM, resistance to the drug and reoccurrence of tumor appears to be a complication that many patients deal with. Molecular pathways underlying the development of resistance are being widely studied. The tumor microenvironment is perceived to play a vital role in multiple stages of disease progression, development of resistance and immune-escaping ability. However, the role of the tumor microenvironment in endocrine resistant breast cancer needs to be investigated. Our previous work shows that the critical molecular and cellular components secreted from a crosstalk between breast cancer cells and stromal cells can regulate tumor growth and the process of metastasis in breast cancer. We have established four different tamoxifen resistant breast cancer (TAMR) cells to simulate pre- and post-menopausal conditions. We screened the secreted factors from normal fibroblast co-cultured with TAMR cells using cytokine antibody arrays targeting 105 cytokines simultaneously and ranked the expression of each of the cytokines using real time qPCR. CXCL1 and IL-6 were our top candidates, which we further confirmed using U-Plex (MSD) assay. Our data showed increased proliferation of TAMR cells co-cultured with fibroblasts when compared to monoculture. To study the role of the cytokines in metastasis we examined cell migration of TAMR cells. TAMR cell migration, a key step in tumor metastasis, was promoted by conditioned medium (CM) from TCM-induced fibroblasts. Furthermore, inhibition of the CXCL1 and IL-6 signaling pathway by Reparixin, an inhibitor of the CXCL1 receptor CXCR1/2, and Tocilizumab, an inhibitor of the IL-6 receptor yielded diminished growth and migration of the TAMR cells.We have also been able to show that reparixin affects the phosphorylation of the ERK in fibroblasts and treatment with reparixin has been affecting the viability of TAMR cells. These findings have identified the key regulators in endocrine resistant breast cancer and support our hypothesis that CXCL1 signaling pathways support the tumor cells to bypass endocrine therapy. In this project we have been able to identify the key signaling pathways that may be activated by these cytokines and determine a therapeutic strategy that could alter such activation and induce cell death.Item Adipsin is an ERα target gene that leads to endocrine resistance in breast cancer with ESR1 mutations(Albany College of Pharmacy and Health Sciences Theses, 2020-08) Malone, Marie Kathryn '20In ER+ breast cancer, the expression of the estrogen receptor allows for the use of targeted endocrine therapies, however, some populations that undergo this treatment become endocrine resistant and can become metastatic as a result. Of this population, the ERα 1 (ESR1) gene was more frequently mutated than in non-resistant population. The most prevalent mutations, Y537S (YS) and D538G (DG), cause constitutive ER activity that is ligand independent. Adipsin is a complement system component and has been linked to increased proliferation and cancer stem cell-like properties in ER+ breast cancer when secreted by adipocytes. In this study, the cytokine adipsin was found to be upregulated in the secretome of YS and DG mutant MCF7 and T47D cells by human cytokine array analysis. Adipsin mRNA and protein levels were also found to be elevated in these cells by RT-qPCR and ELISA. The complement components C3 and C3aR had increased mRNA levels and C3a had increased protein levels in all ESR1 mutants. Treatment of ESR1 mutants with the C3aR inhibitor SB290157 on cellular viability and cytotoxicity were assessed using a CyQUANT assay and flow cytometry with annexin-V staining. Cellular viability and apoptosis were significantly decreased in all ESR1 mutants compared to WT when treated with SB290157. ESR1 mutant cells were treated with SB290157 and tamoxifen (4-OHT) in combination which revealed an increased sensitivity of these cells to tamoxifen. We have provided evidence that supports the hypothesis that functional inhibition of the Adipsin signaling pathway has the potential to circumvent breast cancer growth and metastasis.