AR-V7 in Prostate Cancer Cells Introduces Potential Therapeutic Target for CRPC

A recent study showed that the androgen receptor (AR) splice variant AR-V7 activated full-length AR through an alternative biosynthesis pathway and increased citrate utilization through altered metabolic activities, which may partially explain its resistance to conventional androgen deprivation therapy (ADT).

Ayesha A. Shafi, BA, from Baylor College of Medicine in Houston, TX and lead researcher of the study, presented these findings on March 7, 2015 at The Endocrine Society’s 97th Annual Meeting (ENDO 2015) in San Diego, California.

Prostate cancer is an androgen-dependent disease, and treatment for castration-resistant prostate cancer typically involves ADT. Although these tumors are AR-dependent, they usually become resistant to ADT and recur within 2 years.

Although several mechanisms have been proposed for this aberrant reactivation of AR, Shafi and her colleagues focused on the expression of constitutively active AR splice variants and local synthesis of androgens within the tumor. AR-V7 is the most well-characterized splice variant implicated in resistant prostate cancer, but the mechanisms by which it initiates reactivation of AR have not been clarified.

Shafi et al developed an LNCaP human cell line (an androgen-sensitive human prostate adenocarcinoma cell line) that express AR-V7 in response to doxycycline. AR-V7 expression in the cells induced mRNA expression of the classical AR target genesFK506binding protein 5 (FKBP5) and prostate-specific antigen (PSA) to a similar level as the androgen-treated cells, confirming that the splice variant activated AR.

Using RNA sequencing, the researchers revealed that expression of steroid 5-alpha reductase (SRD5A1), a key enzyme involved in an alternative pathway of androgen synthesis that is commonly used in prostate cancer cells, increased in AR-V7-expressing cells, but not in those that express the full-length AR. According to Shafi, these results suggest that AR-V7 induces this alternative steroid biosynthesis pathway to activate full-length AR.

To test the biological significance of this activation, Shafi and colleagues used mRNA transcription of serine/threonine protein kinase 1 (SGK1) as an indicator of AR activation. A 24-hour incubation with androstenedione, a key intermediate in the synthesis of androgens from cholesterol, increased SGK1 in AR-V7-expressing cells to a similar level as the androgen-treated cells. This increase did not occur in the cells that did not express AR-V7 and was blocked by the AR antagonist MDV3100, supporting the notion that the increase in SGK1 expression in the presence of androstenedione occurred through AR and was stimulated by AR-V7.

“These findings show a unique role for AR-V7 in cell metabolism,” said Shafi.

To further investigate this role, Shafi and colleagues performed a metabolomics array using a metabolomics core developed at Baylor College of Medicine. Using liquid chromatography-mass spectrometry, they found differences in expression of metabolites in the glycolysis, Krebs cycle, and glutaminolysis pathways. Upon further investigation of the Krebs cycle pathway, Shafi et al found that relative to androgen-treated cells, presence of AR-V7 dramatically reduced steady-state levels of citrate.

Citrate levels within normal prostate cells are high relative to the surrounding tissue, although this elevation is notably absent in castration-resistant prostate cancer. Shafi demonstrated these trends in the AR-V7-expressing cells mirrored those in castration-resistant prostate tumors (but not other prostate cancer tumors), suggesting dysregulation in citrate metabolism may be unique to the resistant tumors.

To clarify the mechanism by which AR-V7 alters citrate metabolism in the LNCaP cell line, Shafi et al showed that although both AR-V7-expressing and androgen-treated cells increased glycolytic activity to a similar extent, AR-V7-expressing cells had greater citrate production.

Reductive carboxylation, which involves conversion of Krebs cycle intermediates to citrate, is a characteristic metabolic pathway used by many types of cancer cells to fuel cell growth. Using end-labeled [1-¹³C] glutamine, Shafi et al showed that AR-V7 enhanced citrate production through reductive carboxylation of glutamine in the cells, suggesting that this metabolic pathway could be a useful therapeutic target for prostate cancer that does not respond to conventional therapies.

“This [pathway] is a potential therapeutic target for prostate cancer [that could be targeted through] glutaminase inhibitors,” said Shafi.

Shafi indicated that because no targeted treatments are currently available for tumors that express AR-V7, recognizing this alternative metabolic pathway will likely be important for developing a more targeted treatment for castration-resistant prostate cancer.

Shafi AA, Arnold JM, Bingman III WE, et al. Androgen Receptor (AR) Isoform-Specific Effects on Cell Metabolism in Prostate Cancer Cells. Presented at: The Endocrine Society’s 97th Annual Meeting (ENDO 2015); March 5-8, 2015; San Diego, CA. Abstract OR 25-3.