*2.6. Increased Lipid Catabolism and Decreased Androgen Response Is Associated with Poorer Progression-Free Survival*

To further validate the role of CPT1A and lipid catabolism in advanced PCa, we turned to publicly accessible TCGA (The Cancer Genome Atlas) Firehose Legacy dataset (492 samples). Pathways were scored in each patient using GSEA and grouped according by median split of each pathway. Examination of the same GSEA pathways identified in our RNAseq data (Figure 2C) showed that the lipid catabolic process (Figure 6A), and the androgen response hallmark (Figure 6B), were significantly associated with progression free survival (PFS) but in opposite directions. The increase in lipid catabolism genes and the decrease in androgen response genes shortened the PFS. We did not observe significant PFS changes with the GO serine metabolism and the unfolded protein response hallmark pathways (Figure S6).

**Figure 5.** Lipid and serine metabolism genes are associated with less androgen signaling and a neuroendocrine phenotype. (**A**) Gene set enrichment analysis (GSEA) plot of the OE versus KD analysis showing the decrease in the androgen response hallmark in OE cells. (**B**) Gene expression analysis of OE versus KD cells for neuronal-like markers like ENO2 *(p* = 6.1 <sup>×</sup> 10−10*),* SYP (*p* = ns), NCAM2 (*<sup>p</sup>* <sup>=</sup> 4.14 <sup>×</sup> <sup>10</sup><sup>−</sup>5), NXPH4 (*<sup>p</sup>* <sup>=</sup> 1.6 <sup>×</sup> <sup>10</sup><sup>−</sup>17). (**C**) GSEA plots of the public GSE32967 dataset showing the comparison between small cell carcinoma (*n* = 3) versus adenocarcinoma (*n* = 3) patient xenografts for the Hallmark Androgen Response; GO\_Serine and GO\_Tetrahydrofolate pathways. Normalized enrichment scores (NES) and statistical significance are indicated in the plots. (**D**) Heatmap of the leading-edge genes from the serine and tetrahydrofolate functional GSEA plots shown in panel C, which are also genes increased in the OE cells compared to KD cells. CPT1A and AR are also included and show an inverse correlation in one of the samples of each subtype of PCa studied.

**Figure 6.** Increased lipid catabolism and decreased androgen response is associated with less progression-free survival. The TCGA prostate adenocarcinoma (PRAD) Firehose legacy dataset (*n* = 492) was divided by median split into high and low pathway scores. Pathways from our OE versus KD RNAseq analyses (Figure 2C) were then studied for progression free survival (PFS) in the TCGA PRAD dataset. Kaplan–Meier (KM) plots for Lipid Catabolism Process (**A**) and Androgen Response Hallmark (**B**), with the number at risk and *p*-value (logrank test) are shown.
