No abstract

Prostate cancer (PCa) is the most common cancer amongst males and becomes incurable once it advances to the secondary site. The most common site of metastasis from PCa is the adipocyte-rich bone marrow. Adipocytes are metabolically active cells capable of secreting a variety of adipokines and free fatty acids, which can be utilized by tumor cells to enhance proliferation and cause resistance to chemotherapies. Our lab has shown that adipocytes promote PCa progression and therapy evasion through modulation of tumor metabolism and activation of pro-survival signaling; however, the molecular mechanisms behind tumor-promoting effects of fat cells are not understood. It is well known that high levels of iron within the tumor microenvironment help the growth of PCa cells. However, since iron overload can cause an increase in reactive oxygen species (ROS), which are harmful to cell structure, tumor cells have developed defense mechanisms to protect them from this oxidative damage. Intriguingly, our data show that the expression of ferritin, an iron storing protein, is reduced in PC3 and ARCaP(M) cells in co-culture with adipocytes. Since low ferritin levels have been associated with induction of ferroptosis, an iron and oxidative stress-regulated cell death process, we examined protein levels of ferroptosis markers GPX4 and NCOA4 in PCa cells. Surprisingly, our data show that interaction with adipocytes increases GPX4 and reduces NCOA4 expression in PCa cells suggesting an adipocyte-mediated defense mechanism against ferroptosis. The aim of the present study was to establish that adipocytes promote a defense mechanism against ferroptosis in PCa cells by the upregulation of GPX4 activity via the mTOR pathway. GPX4 is known to be an essential antioxidant enzyme that utilizes glutathione (GSH) to reduce lipid-peroxidation-induced ROS. We demonstrate that PCa cells exposed to adipocytes have reduced lipid-peroxidation induced ROS, which can be reversed with ferroptosis inducers. We also show that exposure of PC3 and ARCaP(M) cells to adipocytes leads to depletion of GSH, which may be indicative of higher GPX4 activity. It has been suggested that an increase in GPX4 can result from an interplay between ferroptosis and mTOR pathways, specifically mTORC1, which may mediate cysteine-induced GPX4 protein synthesis. Surprisingly, treatment of PCa cells co-cultured with adipocytes with mTORC1 inhibitor Everolimus (EVO) increases the protein and gene levels of GPX4. This coincides with reduced mRNA and protein expression of SLC7A11, transporter involved in the cysteine/glutamate antiporter system, which typically fuels GPX4 activity by bringing in the GSH precursor cysteine. Understanding the mechanisms of fat cell contribution to dysregulation of the mTOR pathway and escape from ferroptosis may have therapeutic implications for metastatic PCa. Citation Format: Alexis Wilson, Shane Mecca, Mackenzie Herroon, Laimar Garmo, Izabela Podgorski. Defense mechanism against oxidative damage in metastatic prostate cancer: Adipocyte-mediated modulation of mTOR and ferroptosis pathways [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 6319.

Bone is a site of metastasis from several tumor types including prostate cancer (PCa). Metastatic PCa is lethal, largely due to the complex nature of bone microenvironment, and the functional cross-talk between bone-resident cells and tumor cells that favors therapy evasion. We have shown previously that age- and obesity-induced changes in the bone microenvironment, specifically increased marrow adiposity, promote tumor adaptation in bone. Numerous studies have also linked the disruption in bone homeostasis with aggressive tumor phenotype. Whether environmental toxicants with propensity to accumulate in bone can modulate bone microenvironment to support tumor growth and progression is, however, not known. Per- and polyfluoroalkyl substances (PFAS) are “forever” chemicals with very long half-lives and ability to accumulate in bone. Limited reports have linked PFAS exposure with adipocyte differentiation and osteoclast activation, but their specific effects on bone are not known. It is also unclear whether the exposure to PFAS can directly impact metastatic tumor cells. Here, we hypothesized that PFAS contribute to metastatic progression by impacting both the tumor and its microenvironment. Specifically, we aimed to establish that exposure to PFAS promotes adipogenesis and osteoclastogenesis, creating microenvironment conducive to tumor growth. We also sought to determine whether direct effects of PFAS exposure on tumor cells lead to activation of growth and survival signaling that drives aggressive phenotype in bone. Our data show that 12-week exposure of mice to a cocktail of 5 PFAS chemicals (PFOS, PFOA, PFNA, PFHxS, and GenX) increases the number of adipocytes in the tibia and augments expression of adipogenesis-associated genes, such as FABP4 and adiponectin. RNAseq analyses of tibia from mice exposed to PFAS chemicals for 3 and 6 weeks indicate changes in bone metabolism and turnover, results supported by in vitro osteoclastogenesis assays. Mass spectrometry analyses of bone extracts from PFAS cocktail exposed mice reveal that the compound with highest concentration in bone is PFHxS. Interestingly, our in vitro assays employing bone marrow derived mesenchymal cells show that PFHxS can activate peroxisome proliferator-activated receptor (PPAR) signaling, leading to an increase in adipogenesis. Studies examining the direct impact of long term PFHxS as well as PFAS cocktail exposure on in vitro PCa cultures and progression of intratibially implanted PCa tumors are currently ongoing. Our findings to date indicate that changes in bone marrow microenvironment induced by PFAS chemicals have a potential to disrupt the balance between osteoblasts and adipocytes, promote marrow adipogenesis and osteoclastogenesis, and activate specific signaling pathways in tumor cells that support growth and survival. Citation Format: Laimar C. Garmo, Mackenzie K. Herroon, Shane Mecca, Alexis Wilson, Michael C. Petriello, Izabela Podgorski. Effects of per- and polyfluoroalkyl substances on bone marrow adipose environment: Potential implications for bone metastatic cancers [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 6318.

Bone metastatic disease is correlated with increased morbidity and mortality in prostate cancer (PCa) patients. Current treatments for bone-metastatic PCa are palliative, and new targets for therapy are desperately needed for this presently incurable disease. Various studies have determined that the supportive nature of the bone marrow (BM) niche and crosstalk between the bone-resident cells and the tumor lead to increased tumor cell survival and escape from therapy. Specifically, studies from our lab highlighted the critical role of bone marrow adipose tissue and its expansion due to obesity or age in PCa progression and chemoresistance in bone. Bone marrow adipocytes are metabolically active cells that, through secretion of lipids and adipokines, have the potential to affect the neighboring cells, including the tumor cells that have disseminated into the BM niche. We have shown previously that interaction with marrow adipocytes promotes lipid uptake by PCa cells, modulates their metabolic phenotype, and activates pro-survival signaling and ER/oxidative stress pathways; however, the molecular mechanisms behind the tumor-promoting effects of adipocytes are not understood. The main objective of this study was to investigate the molecular mechanisms underlying lipid-induced stress during tumor cell-adipocyte crosstalk and its functional relationship to tumor growth and progression in bone. Our preliminary data showed that adipocyte-tumor cell crosstalk increases lipid peroxidation in PCa cells, which coincides with augmented expression of ER stress markers and activation of mTOR signaling. We also observed that PCa cells under adipocyte exposure had augmented gene expression of stearoyl-CoA desaturase (SCD), an ER-resident enzyme responsible for the conversion of saturated fatty acids (SFA) to monounsaturated fatty acids (MUFA). We, therefore, hypothesized that SCD is a metabolic regulator that protects metastatic PCa cells against lipid-induced toxicity and ER stress and promotes tumor survival via activation of mTOR signaling. Here, we show that the inhibition of mTORC1 by Everolimus (EVO) decreases the gene expression of ER stress markers in PCa cells exposed to adipocytes. We also demonstrate that adipocyte-mediated induction of ER stress coincides with an increase in active mTOR signaling in PCa cells, indicating possible bidirectional crosstalk between ER stress and mTOR pathways. Notably, inhibition of SCD in PCa cells grown in Transwell co-culture with adipocytes by either siRNA or the small molecule inhibitor CAY10566 leads to reduced mTOR signaling and an increase in lipid peroxidation in PCa cells. SCD inhibition also increases the gene and protein expression of ER stress markers, specifically XBP1 (S) and ATF4, suggesting apoptosis induction. Collectively, our results place SCD as a mediator of ER stress-mTOR crosstalk and a promoter of tumor survival and suggest that targeting SCD activity might be a viable approach to rewire tumor metabolism, and inhibit metastatic progression in bone and sensitize PCa tumors to therapy. Citation Format: Alexis Wilson, Mackenzie Herroon, Shane Mecca, Laimar Garmo, Izabela Podgorski. Adipocyte regulation of ER stress and mTOR signaling in bone-metastatic PCa: The role of stearoyl-CoA desaturase [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B039.