*2.5. Lipid and Serine Metabolism Genes Are Associated with Less Androgen Signaling and a More Neuroendocrine Phenotype*

Gene set enrichment analysis of our RNAseq showed decreased expression of androgen response genes CPT1A OE cells (Figure 5A). Since decreased AR signaling is associated with changes to a neuroendoendocrine phenotype [7], we next looked for markers of neuronal-like differentiation and identified Enolase 2 (ENO2), Synaptophysin (SYP), Neural Cell Adhesion Molecule 2 (NCAM2), and Neurexophilin 4 (NXPH4) genes significantly upregulated in OE versus KD cells. The ENO2 and SYP genes are markers associated with neuroendocrine PCa (NEPC). To investigate the possibility that CPT1A overexpression (OE) is associated with more aggressive disease, we searched previous transcriptome analysis in the public databases. Particularly, we searched for studies that compared adenocarcinoma with aggressive disease like small cell NEPC and focused on serine and one carbon metabolism, CPT1A and AR expression in clinical data.

Using the dataset from GSE32967 [37], we compared gene expression between small cell carcinoma (a subset of NEPC) and adenocarcinoma patient derived PCa xenografts. GSE Analysis showed that the androgen response hallmark was significantly decreased in the NEPC samples, while significant increases were observed in the serine metabolism and one carbon (tetrahydrofolate) metabolism pathways (Figure 5C). Several of serine and tetrahydrofolate leading-edge genes increased in our RNAseq analysis were also increased in the GSE32967 dataset (Figure 5D). Addition of CPT1A and AR gene expression to the heatmap showed that one NEPC sample had low AR as expected, but modest increased CPT1A associated with increased SHMT2, MTHFD2 and PSPH expression. Conversely, one sample with adenocarcinoma features showed less CPT1A associated with less SHMT2, MTHFD2, and PSAT1 expression (Figure 5D). We further investigated the direction of the relationship between CPT1A and AR expression in metastatic disease (Figure S5), using the Taylor et al. dataset [38]. A positive correlation was observed in the primary tumors, as expected from the role of androgens in regulating lipid metabolism [39]. However, this positive correlation was lost in the metastatic samples (R = −0.28, *p* = 0.25), where AR expression was significantly increased compared to primary tumors (Wilcoxon *p* = 1.1 <sup>×</sup> 10−7). The possibility that a strong anti-androgen blockade in these metastatic samples could reverse the correlation to less AR and more CPT1A expression, as it happens in NEPC, warrants further investigation.
