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Figure S1: LNCaP, VCaP and DuCaP cells were grown in charcoal-dextran stripped serum (CSS) for 48 h and treated with increasing concentrations of R1881 or DHT in the presence or absence of Enzalutamide (10 µM) or vehicle (Ctrl) for 48 h. Figure S2: Androgens increased lipid uptake of long-chain fatty acids. LAPC4 cells were grown in CSS for 48 h and treated with 1 nM R1881 or vehicle for 48 h. Figure S3: (A) LNCaP cells were synchronized in G0/G1 by androgen deprivation (CSS for 48 h) followed by treatment with Tunicamycin (1 mg/mL), Hydroxyurea (1 M), or Nocodazole (25 ug/mL) for another 24 h, placing cell cycle blocks in G0/G1, S phase and mitosis, respectively. Figure S4: Oncomine analysis of candidate lipid transporters in (A) Grasso [2], (B) Varambally [3] and (C) LaTulippe datasets [4] comparing gene expression of normal prostate gland versus localized, primary prostate cancer tumor samples. Figure S5: (A) DuCaP (top panel) and VCaP cells (bottom panel) were grown in CSS for 48 h and treated with 10 nM DHT in the absence or presence of Enz (10 µM) or vehicle (Ctrl) for 48 h. Table S1. Primer sequences

De novo lipogenesis is a well-described androgen receptor (AR)–regulated metabolic pathway that supports prostate cancer tumor growth by providing fuel, membrane material, and steroid hormone precursor. In contrast, our current understanding of lipid supply from uptake of exogenous lipids and its regulation by AR is limited, and exogenous lipids may play a much more significant role in prostate cancer and disease progression than previously thought. By applying advanced automated quantitative fluorescence microscopy, we provide the most comprehensive functional analysis of lipid uptake in cancer cells to date and demonstrate that treatment of AR-positive prostate cancer cell lines with androgens results in significantly increased cellular uptake of fatty acids, cholesterol, and low-density lipoprotein particles. Consistent with a direct, regulatory role of AR in this process, androgen-enhanced lipid uptake can be blocked by the AR-antagonist enzalutamide, but is independent of proliferation and cell-cycle progression. This work for the first time comprehensively delineates the lipid transporter landscape in prostate cancer cell lines and patient samples by analysis of transcriptomics and proteomics data, including the plasma membrane proteome. We show that androgen exposure or deprivation regulates the expression of multiple lipid transporters in prostate cancer cell lines and tumor xenografts and that mRNA and protein expression of lipid transporters is enhanced in bone metastatic disease when compared with primary, localized prostate cancer. Our findings provide a strong rationale to investigate lipid uptake as a therapeutic cotarget in the fight against advanced prostate cancer in combination with inhibitors of lipogenesis to delay disease progression and metastasis. Implications: Prostate cancer exhibits metabolic plasticity in acquiring lipids from uptake and lipogenesis at different disease stages, indicating potential therapeutic benefit by cotargeting lipid supply.

The processes regulating tumor metastasis are multivariate and complex. Redox regulation of the tumor phenotype by GPx2 knockdown (KD) in breast cancer led us to uncover dramatic effects on spontaneous metastasis. Analysis of single cell RNA sequencing (scRNAseq) data from GPx2 KD tumor and control tumor, revealed that both tumors were comprised of several luminal-like tumor cell clusters and one mesenchymal-like cell cluster (cluster 3). Notably, GPx2 KD promoted a significant increase in the size of mesenchymal cells (cluster 3) relative to control, which might be due to the stimulation of epithelial-to-mesenchymal transition (EMT) in response to GPx2 loss. In support of this view, GPx2 KD stimulated an increase in mRNA expression of basal/mesenchymal (KRT5, KRT14, KRT17, Vimentin, Twist1, Twist2, CDH2) genes and a decrease in mRNA expression of epithelial/luminal (Cldn7 and Epcam) genes, especially in cluster 3. Moreover, GPx2 KD upregulated mRNA expression of basal/mesenchymal (Twist2, CDH2, and KRT14) genes in most luminal-like clusters expressing epithelial/luminal (Epcam, Cldn3/7, CDH1, KRT8/18) genes, implying these clusters may be undergoing EMT transition in a hybrid epithelial/mesenchymal state in response to GPx2 loss. Validation of these data in cell lines and tumors showed that GPx2 KD dramatically enhanced EMT via activation of ROS/HIF1α-mediated signaling. Importantly, these effects were reversed by GPx2 re-expression or HIF1α inhibition, which was capable of suppressing EMT and metastasis. Collectively, these results indicate that GPx2 loss promotes breast cancer metastasis by stimulating EMT due to HIF1α signaling, highlighting the impact of GPx2 and HIF1 on therapeutic intervention in metastasis. Citation Format: Zuen Ren, Huizhi Liang, Malindrie Dharmaratne, Miriam Morales-Gallego, Atefeh Taherian Fard, Jessica Mar, Kimita Suyama, Outhiriaradjou Benard, Michael B. Prystowsky, Larry Norton, Rachel B. Hazan. Loss of glutathione peroxidase 2 promotes epithelial to mesenchymal transition and breast cancer metastasis [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 968.

Tumor heterogeneity is known to drive breast cancer progression and metastasis. Current therapies target mostly inter-tumor heterogeneity (different patients) without taking into consideration intratumor heterogeneity (individual tumor). Hypoxia is a hallmark of aggressive tumors that is known to select for intratumor cells that are prone for metastasis. Here we show that knockdown of a ROS scavenging enzyme, Glutathione Peroxidase 2, in the PyMT mammary tumor model results in highly hypoxic and metastatic tumors. Single cell RNA-seq on both GPx2 knockdown and control tumors revealed a high degree of intratumor heterogeneity resulting in several clusters that were either of luminal/epithelial or basal-like/mesenchymal type shared by both tumors. Interestingly, GPx2 loss caused increases in a mesenchymal-like/quiescent and a highly proliferative luminal population, implying that GPx2 loss might drive a cancer stem-like population that is conducive of metastasis. Metascape pathway analysis of differentially expressed genes identified pathways that were significantly overrepresented in GPx2 knockdown tumors involving mitochondrial oxidative phosphorylation and response to hypoxia, consistent with the high levels of reactive oxygen species in these tumors. Finally, differential gene expression analysis on mesenchymal and luminal subpopulations predicts novel candidate gene signatures stimulated by GPx2 loss, that might be used to identify or target metastatic cells in tumors. In sum, our data imply that oxidative stress conveys a metastatic phenotype via effects on tumor cell heterogeneity. Citation Format: Zuen Ren, Malindrie Dharmaratne, Ameya Kulkarni, Atefeh Taherian Fard, Jessica Mar, Phillip M. Galbo, Yang Liu, Deyou Zheng, Huizhi Liang, Outhiriaradjou Benard, Kimita Suyama, Michael B. Prystowsky, Larry Norton, Rachel B. Hazan. Redox regulation of tumor cell heterogeneity in the PyMT mammary tumor model revealed by single cell RNA sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4426.