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Abstract

Despite advances in the treatment of breast cancer, it is still the second leading cause of cancer-related death in women worldwide. A large number of patients develop recurrence and die of advanced metastatic disease. More than 70% of metastatic breast cancers express androgen receptor (AR) representing a potential target for anti-hormone therapies. AR is suggested to directly interact with the lineage-specific transcription factor, SAM pointed domain-containing ETS transcription factor (SPDEF) in breast cancer however, the functional role of SPDEF and its interaction with AR remains to be elucidated. I found that AR expression highly correlates with SPDEF expression in breast cancer patients. To study its functional role in tumorigenesis and metastatic outgrowth, I utilized patient-derived xenograft (PDX) derived cell lines from liquid biopsies of metastatic breast cancer patients. Using genetically manipulated PDX cell models, I demonstrated that repression of SPDEF significantly reduced tumor growth of AR+ breast cancer cells in vivo. Downregulation of SPDEF prevented metastasis formation in the brain whereas lung metastatic lesions were not affected by SPDEF silencing. Reduced tumor growth upon downregulation of SPDEF was also observed in estrogen receptor (ER)-positive breast cancer cells. Notably, I observed enhanced tumor growth in an AR negative breast cancer model suggesting a tumor suppressive function when AR is not present. Overexpression of SPDEF in AR- breast cancer cells significantly inhibited in vivo tumor growth. To investigate the underlying mechanism on the molecular level, I established transcriptional profiles by performing tumor tissue and cell line microarray analysis in SPDEF-overexpressing and knockdown models. Mechanistically, I found that SPDEF regulates key metabolic processes: (1) Pharmacological inhibition of AR or silencing of SPDEF restricted mitochondrial respiration activity resulting in decreased energy production. AR activation by testosterone treatment enhanced basal and maximal oxygen consumption rates, as did SPDEF overexpression. However, testosterone treatment did not restore decrease in mitochondrial respiration when SPDEF was downregulated. (2) Further, I found that SPDEF regulates genes encoding enzymes involved in glucose and fatty acid metabolism in AR+ breast cancer cells. FBP1, the rate-limiting enzyme in gluconeogenesis was identified as a direct target gene of SPDEF. However, FBP1deletion did not impair in vivo tumor growth. Enzymes involved in de novo fatty acid biosynthesis were downregulated in SPDEF knockdown SPDEF-deficient cells. The fatty acid transporter CD36 was upregulated upon downregulation of SPDEF as validated by RT-qPCR and western blot analysis. Flow cytometry analysis revealed increased plasma-membrane localized CD36 expression in shSPDEF cells and vive versa, cell surface CD36 expression was decreased in SPDEF-overexpressing cells. I performed isotope tracing experiments using 13C-glucose, 13C-glutamine and 13C-acetate to functionally assess fatty acid metabolism upon deregulation of SPDEF. SPDEF knockdown cells showed decreased biosynthesis of specific fatty acids, however, they restored their cellular fatty acid pool by increased uptake of exogenous fatty acids. Abolishing CD36 expression in AR+ breast cancer cells suggested that fatty acid uptake is critically required for cell growth of SPDEF knockdown cells. Pharmacological inhibition of CD36 induced a significant cytostatic effect in SPDEF knockdown cells. These data suggest that CD36 mediates exogenous fatty acid uptake as compensatory pathway when de novo fatty acid biosynthesis is decreased by SPDEF downregulation. In agreement, SPDEF knockdown cells did not have a significant growth disadvantage in vitro under saturated culture conditions. Usual cell culture media contain saturated levels of carbon sources and nutrients which do not reflect the physiological conditions found in the patient, making it difficult to study the metabolic profiles of cancer cells in vitro. However, when cells were cultured under physiological conditions resembling the natural cellular environment found in the patient, cells showed a significantly decreased growth rate when SPDEF was downregulated. These findings suggest that lipid and energy metabolism are transcriptionally regulated by SPDEF facilitating cell survival in nutrient-depleted environments and hence, tumor and metastatic outgrowth of AR+ breast cancer cells. Since initial data suggested that pharmacological inhibition of AR mimics the effect of SPDEF downregulation, targeting AR and CD36 simultaneously may be a treatment strategy for AR+ breast cancer patients.