Science Spotlight

Overcoming androgen therapy resistance in prostate cancer

From the Peter Nelson Lab, Human Biology Division

The vast majority of prostate carcinomas are highly dependent on androgen receptor (AR) signaling. Testosterone is the most well known androgen hormone, but there are several other androgens that bind to the AR as well. Because of high reliance on AR signaling, first line therapies for prostate cancers are AR pathway inhibitors such as enzalutamide and abiraterone. The widespread use of AR-repressing chemotherapies has impacted AR status of prostate metastases. In a recent paper, Dr. Peter Nelson and first author Dr. Eric Bluemn from the Human Biology Division, as well as colleagues from centers around the country, show that AR-directed therapies are resulting in the emergence of prostate metastases devoid of AR signaling and discover an effective pathway to target in these AR-null cancers.

Prostate cancers resistant to AR deprivation are termed castration-resistant prostate cancer (CRPC). Dr. Bluemn and colleagues characterized metastatic CRPCs from 84 patients for AR status and neuroendocrine status (NE). Neuroendocrine tumors express markers of neuroendocrine differentiation and are a subtype of prostate carcinoma separate from the more common epithelial type. The CRPCs collected from patients between 1997-2011 were 85% AR-positive, 10% NE-positive, and 5% negative for both. However, in samples collected between 2012-2016, when AR-targeting therapies became available, 69% were AR-positive, 10% were NE-positive, and 21% were double-negative, demonstrating that double-negative metastases were four times as prevalent in recent cases.

RNA sequencing analysis of double-negative CRPCs and AR-positive CRPCs did not reveal a genetic driver of AR signaling loss. Therefore, the authors developed a cellular model of AR resistant prostate cancer that reproduced the transition from a prostate tumor dependent upon AR signaling to one capable of AR-independent growth. They began with the LNCaP cell line, a commonly studied prostate cancer cell line that is androgen-sensitive, and modified it with an inducible anti-AR short hairpin RNA (shRNA) that silences the AR gene when doxycycline is added to the medium. For assurance of total AR ablation, an androgen response element-driven suicide gene was integrated into the genome, which induces cell death when AR is active. These cells were slowly deprived of androgen and selected for AR independent clones until AR expression was 45-fold lower than in normal LNCaP cells and the cells grew robustly without androgen.

Because the cells that survived the androgen deprivation must have developed reliance on other pathways for survival, the authors attempted to identify these pathways . First, they performed RNA sequencing and copy number analysis on the androgen-independent LnCAP cells and wild-type LnCAP cells. They found 548 differently expressed transcripts, but few copy number changes, and those few could not account for the striking changes in gene expression.

However, androgen independent LNCaP cells had higher levels of phosphorylated MEK and dual phosphorylated ERK. MAPK signaling is activated through a number of stimuli, including phosphorylation of MEK and dual phosphorylation of ERK, and MAPK signaling has been previously implicated in CRPC metastasis. Furthermore, fibroblast growth factor 8 (FGF8) expression was substantially up-regulated in AR independent cells, and a number of transcripts shown to reflect FGF receptor (FGFR) signaling were up-regulated as well. Because FGF8 is an activator of the MAPK pathway and known to be oncogenic, the authors hypothesized that FGF signaling is activated in the androgen independent LNCaP cells, and cell growth and survival is maintained through MAPK.

Targeting the FGF signaling pathway in LNCaP cells demonstrated that FGF signaling is necessary and sufficient to bypass AR activity. For example, adding FGF8 to the media increased the growth of wild type LNCaP cells in androgen deprivation conditions. By contrast, silencing FGF8 expression reduced androgen independent LNCaP cell growth by 80%.  FGFR inhibitors also blocked growth of androgen independent LNCaP cells, as did an inhibitor of MEK1/2, confirming that MAPK activity is required for FGF-mediated CRPC proliferation. A xenograft mouse model of the androgen independent LNCaP cells was made by injecting the cells into the flank of an immune-compromised mouse and allowing a tumor to form. The mice were treated with an FGFR inhibitor, and treatment significantly reduced tumor growth rates. 

Returning to patient CRPC tumor data, Bluemn et al. confirmed that gene sets reflecting the activity of FGF signaling, MAPK signaling, and MEK/ERK signaling are significantly enriched in AR-negative CRPC tumors. Across all of the CRPC metastases, AR activity was inversely associated with FGF8/9 expression and FGFR activity. The authors made a patient-derived xenograft (PDX) mouse model by injecting cells from an androgen-negative CRPC tumor into the flank of an immune-compromised mouse. The PDX tumor demonstrated high FGFR activity and low AR levels. Treatment with an FGFR inhibitor slowed the tumor growth significantly.

The downstream mediator of FGF/MAPK signaling ID1 (inhibitor of differentiation 1) was upregulated in androgen independent LNCaP cells in comparison to wild-type LNCaP cells. Stimulation of wild-type LNCaP cells with FGF8 in androgen-deprived conditions led to an increase in ID1 mRNA and protein levels, indicating that FGF8 activation is sufficient to trigger ID1 expression. While ID1 knockdown did not impair wild type LNCaP cell growth, it significantly impaired androgen independent LNCaP cell growth, ID1 transcripts were diminished in xenograft mouse models after treatment with an FGFR inhibitor. Together, these data support the hypothesis that ID1 mediates the FGFR dependent survival of AR-negative CRPCs (Figure 1). 

Diagram of FGF MAPK signaling
Figure 1: FGF8 triggers the MAPK pathway in androgen-negative CRPC cells leading to cell growth and proliferation. Image by H. Richards

As CRPCs become increasingly resistant to AR therapy, the need for alternative treatments is dire. The authors conclude that targeting FGFR may be a useful therapeutic strategy in CRPCs that are AR-negative. An ongoing clinical trial of the FGFR antagonist dovitinib in men with metastatic CRPC reported a 26% response rate in bone and tissue lesions. The authors hypothesize that the population responding to this treatment may have AR-negative cancers. In the future, they suggest conducting a clinical trial of FGFR or MAPK antagonists in patients selected for AR activity status to conclusively determine if targeting these pathways will help patients with AR-negative prostate cancer. 

Bluemn EG, Coleman IM, Lucas JM, Coleman RT, Hernandez-Lopez S, Tharakan R, Bianchi-Frias D, Dumpit RF, Kaipainen A, Corella AN, Yang YC, Nyquist MD, Mostaghel E, Hsieh AC, Zhang X, Corey E, Brown LG, Nguyen HM, Pienta K, Ittmann M, Schweizer M, True LD, Wise D, Rennie PS, Vessella RL, Morrissey C, Nelson PS. Androgen Receptor Pathway-Independent Prostate Cancer Is Sustained Through FGF Signaling. Cancer Cell. 2017 Oct 9;32(4):474-489. 

Funding for this work was provided by the National Institutes of Health, the Institute for Prostate Cancer Research, the Richard M. Lucas Foundation, and the Prostate Cancer Foundation.