Differential regulation of metabolic pathways by androgen receptor (AR) and its constitutively active splice variant, AR-V7, in prostate cancer cells

Abstract

Metastatic prostate cancer (PCa) is primarily an androgen-dependent disease, which is treated with androgen deprivation therapy (ADT). Tumors usually develop resistance (castration-resistant PCa [CRPC]), but remain androgen receptor (AR) dependent. Numerous mechanisms for AR-dependent resistance have been identified including expression of constitutively active AR splice variants lacking the hormone-binding domain. Recent clinical studies show that expression of the best-characterized AR variant, AR-V7, correlates with resistance to ADT and poor outcome. Whether AR-V7 is simply a constitutively active substitute for AR or has novel gene targets that cause unique downstream changes is unresolved. Several studies have shown that AR activation alters cell metabolism. Using LNCaP cells with inducible expression of AR-V7 as a model system, we found that AR-V7 stimulated growth, migration, and glycolysis measured by ECAR (extracellular acidification rate) similar to AR. However, further analyses using metabolomics and metabolic flux assays revealed several differences. Whereas AR increased citrate levels, AR-V7 reduced citrate mirroring metabolic shifts observed in CRPC patients. Flux analyses indicate that the low citrate is a result of enhanced utilization rather than a failure to synthesize citrate. Moreover, flux assays suggested that compared to AR, AR-V7 exhibits increased dependence on glutaminolysis and reductive carboxylation to produce some of the TCA (tricarboxylic acid cycle) metabolites. These findings suggest that these unique actions represent potential therapeutic targets.

Emerging data on androgen receptor splice variants in prostate cancer

“…Interestingly, we found that the promoter of the canonical AR target PSA is co-occupied by AR-V7 and AR-FL whereas the promoter of the UBE2C gene is bound by AR-V7 only (Cao et al 2014), suggesting that the AR-V/AR-FL dimers and the AR-V/AR-V dimers may regulate different sets of target genes. This is supported by transcriptome and metabolome data showing that AR-Vs can regulate some canonical AR targets as well as a distinct set of genes/pathways (Chan, et al 2015; Guo et al 2009; Hu, et al 2012; Li et al 2013; Lu, et al 2014; Shafi, et al 2015). Identifying the respective binding sites for AR-V/AR-FL dimers and AR-V/AR-V dimers across the genome and elucidating whether the genes and pathways regulated by the AR-V/AR-FL dimers fully overlap with those regulated by AR-FL homodimer could be vital in understanding how AR-Vs contribute to castration resistance.…”

Targeting AR Variant–Coactivator Interactions to Exploit Prostate Cancer Vulnerabilities

“…The extent to which AR-Vs regulate unique genes (compared to full length AR) to drive PC progression is under active investigation (27-30). Since AR-V activity is critical for CRPC cell survival and resistance to even the newest generation of AR-targeted therapies, these variants are attractive targets for CRPC treatment (31).…”

Androgen receptor variation affects prostate cancer progression and drug resistance

“…In normal cells, the AR consists of four domains: a transactivation domain (encoded by exon 1), a DNA-binding domain (exons 2–3), a hinge region (encoded by the 5′ portion of exon 4), and a ligand-binding domain (exons 4–8), as shown in Figure 1 (2). …”

“…AR-V7 results from the contiguous splicing of AR exons 1/2/3/CE3, the latter of which is 16 amino acids from exon 3b (see Figure 1) (2, 11, 25). Because this alternative splicing results in the exclusion of the ligand-binding domain, AR-V7 can remain constitutively active in the absence of androgens (25, 27).…”

“…Not only does AR-V7 increase citrate production by increasing expression of MDH1 (forming citrate from malate) and reducing OGDH overexpression (shunting α-ketoglutarate to citrate instead of succinate), but it also promotes citrate formation through GLUD1 overexpression by reductive carboxylation (forming α-ketoglutarate from glutamate). Increased citrate formation results in an increased formation of amino acids and steroids in AR-V7-expressing cells, ultimately causing lower intracellular citrate concentrations than would otherwise be present in cells expressing wt-AR (2). These findings, in part, explain why patients with AR-V7-positive tumors have reduced intratumoral citrate concentrations when compared to those tumors expressing wt-AR (2).…”