Background: Defects in primary cilium can lead to a group of disorders termed ciliopathies, with links to both obesity and cancer. Obesity causes inflammation and downregulates anti-tumor immunity generating a protumor microenvironment and increases the risk of breast cancer (BC) development and metastasis. Also, obesity affects cilium length, which could disrupt the essential role of the cilium in detecting extracellular cues, coordinating cell signaling responses, and regulating cell fate and mitochondrial function. However, there is a gap in understanding the effects of obesity on ciliogenesis in cancer cells and in regard to the impact of cilium length and localization on mitochondrial function of tumor cells. We therefore sought to elucidate the impact of obesity on the presence, localization, and function of primary cilium, using murine models of triple-negative breast cancer (TNBC). Methods: We generated mammary tumors by orthotopic transplantation of murine metM-Wntlung or E0771 TNBC cells, and evaluated the impact of obesity on ciliary specific gene sets in tumors using GSEA analysis. We also tested the abundance of cilium in tumor sections using immunofluorescence. Additionally, we developed metM-Wntlung and E0771 expressing the ciliary transmembrane protein Smo fluorescently tagged to pHluorin. Using these cells, we performed in vitro studies to investigate the length and cellular localization of the cilium in cancer cells in 3 compartments; intracellular (in), intermediate, or extracellular (out). Results: Our GSEA analysis results indicated significant upregulation of 18/19 ciliary gene sets in metM-Wntlung mammary tumors from obese mice and 19/19 ciliary gene sets in E0771 mammary tumors from obese mice compared to the corresponding tumors developed in non-obese mice. Ongoing leading-edge analysis will determine the genes driving the enrichment of ciliary-related gene sets in tumors of obese mice. Immunofluorescence analysis showed a significant number of ciliated cells in tumor sections of obese mice and in cultured cells expressing pHluorin-Smo. Ongoing studies will reveal the specific cellular localization of the cilium on cancer cells and the effects on mitochondrial function. Our research is establishing a proof-of-concept that mammary cancer cells express cilium and will elucidate key mechanistic details related to the role of the primary cilium in the crosstalk between obesity and cancer metabolism. This research is supported by R35CA197627 (SDH), AICR Marylin Gentry Fellowship (XBM). Citation Format: Ximena Bustamante-Marin, Jenna L. Merlino, Emma J. Grindstaff, Michael F. Coleman, Erika Rezeli, Kristina K. Camp, Laura Smith, Stephen D. Hursting. Role of the primary cilium in the crosstalk between obesity and cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2388.
Background: Obesity increases the risk of triple negative breast cancer (TNBC) and its progression to metastasis, the primary cause of death in women with TNBC. In obesity, dysregulated adipose tissue secretes growth factors and hormones, promoting chronic inflammation. Adipose tissue also secretes extracellular vesicles (EVs), cell-derived nanoparticles (50 - 5000 nm diameter) containing proteins and nucleic acids that interact with and modify local and distant cellular targets. Research from our team and others demonstrated that the enzyme pyruvate carboxylase (PC) is upregulated during obesity. Moreover, PC upregulation correlates strongly with breast cancer progression to lung metastasis. We hypothesize that tumor and adipose-derived EVs from obese versus normoweight mice regulate PC activity promoting metabolic reprogramming and metastasis. Methods: Female C57BL/6 mice (n=32) were randomized to receive either control diet (CON) or a diet-indued obesity (DIO) regimen for 15 weeks and then orthotopically injected with metMWntlung cells (a murine model of metastatic TNBC). Half of the mice were sacrificed when the tumor volume reached 0.5 cm3, and the other half at 1 cm3. The EVs from mammary tumors and visceral adipose tissue of CON and DIO mice were purified by differential centrifugation in combination with a flotation Iodixanol density gradient. In parallel, serum-EVs were isolated from the same mice. After proper characterization of the purity of EV by western blot, electron microscopy, and nanotracking analysis, the EV proteomics was analyzed by global LC-MS/MS analysis and the microRNA content by small RNA sequencing. Results: Preliminary proteomic analysis of adipose-EV identified 2,844 proteins, of which 50% were associated with EV according to GOCC name. In adipose-EV, obesity but not tumor size significantly impacted protein composition. In the comparison DIO-0.5 cm3 versus CON-0.5 cm3, 613 proteins were statistically (p-value < 0.05) and biologically significant. In this comparison, 197 proteins were more abundant, and 412 were less abundant in the DIO-05 cm3 relative to CON-0.5 cm3. In addition, we detected the presence of PC in adipose and tumor-EVs. Ongoing analysis will compare the composition of EVs in more detail, determine the presence of PC regulators in EVs, and the metabolic consequences and metastatic effects of the combined action of adipose- and tumor-derived EVs. R01CA232589 to SDH and DT supports this research; an NCI diversity supplement of this grant supports XBM. Citation Format: Ximena Minerva Bustamante-Marin, Emma J. Grindstaff, Laith A. Rayyan, Dorothy Teegarden, Stephen Hursting. Proteomics and microRNA characterization of mammary tumor and adipose tissue-derived EVs and their combined impact on cancer cell metabolism [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 3054.
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