Role of androgen receptor splice variant-7 (AR-V7) in prostate cancer resistance to 2nd-generation androgen receptor signaling inhibitors

Abstract

The role of truncated androgen receptor splice variant-7 (AR-V7) in prostate cancer biology is an unresolved question. Is it simply a marker of resistance to 2nd-generation androgen receptor signaling inhibitors (ARSi) like abiraterone acetate (Abi) and enzalutamide (Enza) or a functional driver of lethal resistance via its ligand-independent transcriptional activity? To resolve this question, the correlation between resistance to ARSi and genetic chances and expression of full length AR (AR-FL) vs. AR-V7 were evaluated in a series of independent patient-derived xenografts (PDXs). While all PDXs lack PTEN expression, there is no consistent requirement for mutation in TP53, RB1, BRCA2, PIK3CA, or MSH2, or expression of SOX2 or ERG and ARSi resistance. Elevated expression of AR-FL alone is sufficient for Abi but not Enza resistance, even if AR-FL is gain-of-function (GOF) mutated. Enza resistance is consistently correlated with enhanced AR-V7 expression. In vitro and in vivo growth responses of Abi-/Enza-resistant LNCaP-95 cells in which CRISPR-Cas9 was used to knockout AR-FL or AR-V7 alone or in combination were evaluated. Combining these growth responses with RNAseq analysis demonstrates that both AR-FL- and AR-V7-dependent transcriptional complementation are needed for Abi/Enza resistance.

Figure 1:. Characterization of CWR22-RH.

a) H & E histology (200x) of CWR22-RH xenografts. IHC staining (200x) for b) PTEN and c) AR. d) Western blot documenting AR expression in PC-82 relative to normal prostate, CWR22-RH, and LNCaP cells. e) m) Abi resistance of CWR22-RH xenografts in vivo (n = 3 each). f) Enzalutamide sensitivity of CWR22-RH xenografts in vivo (n = 5 each).

Figure 2:

RNA-seq based expression analysis of a subset of genes across PDX models expressed as Log2 FPKM.

Figure 3:. Characterization of LvCaP-2 and LvCaP-2R.

a) H & E histology (200x) of LvCaP-2 (inset, 400x). IHC (200x) for b) AR (inset, AR immunoblot), c) c-Myc, d) Ki67, e) HoxB13, f) Nkx3.1, g) cytokeratin-18, and h) PSA. i) Growth rate of LvCaP-2 in intact (i.e. ADT-equivalent) mice with subsequent regression and relapse in castrate (i.e. ARSi-equivalent) male NSG mice (n = 5 each). j) Growth rate of LvCaP-2R in intact vs. castrate hosts (n = 5 each). k) Abi resistance of LvCaP-2R xenografts in vivo (n = 3). i) H & E histology (200x) of LvCaP-2R (inset, 400x). IHC (200x) for m) Nkx3.1 and n) AR in LvCaP-2R PDX in castrate hosts. o) AR immunoblot of LvCaP-2 vs. LvCaP-2R and quantification based on densitometry. p) LvCaP-2R resistance to daily oral Enzalutamide treatment.

Figure 4:. Characterization of SkCaP-1 and SkCaP-1R.

a) H & E histology (200x) of SkCaP-1. IHC (200x) of SKCaP-1 for b) AR, c) Nkx3.1, and d) PSMA. e) Growth rate of SkCaP-1 in intact (i.e. ADT-equivalent) mice with subsequent regression and relapse in castrate (i.e. ARSi-equivalent) male NSG mice (n = 5 each). AR-FL and AR-V7 immunoblots of SkCaP-1 vs. SkCaP-1R (inset). f) IHC (200x) of SkCaP-1 for Ki67. g) Abi and Enza resistance of SkCaP-1R in vivo (n = 3 each). h) H & E histology (200x) of SkCaP-1R. IHC (200x) of i) AR, j) PSA, k) c-Myc, and i) Ki67 in SkCaP-1R PDX.

Figure 5:. Characterization of LNCaP variant under long-term ARSi-equivalent conditions (i.e. LN-95 cells).

a) AR-FL and AR-V7 immunoblot of LNCaP vs. LN-95 variant and quantification via densitometry. b) Cell number after 5 days of in vitro growth of LN-95 in 10% FBS media, 10% FBS media containing 10 μM enzalutamide, or 10% CS-FBS media vs. LNCaP growth under the same conditions with asterisks denoting significant difference at p < 0.05. c) Growth rate of LN-95 in castrated (i.e. ARSi-equivalent) vs. LNCaP in intact (i.e. ADT-equivalent) mice. d) Abi resistance of LN-95 xenografts in vivo (n = 3 each). e) In vivo growth response of LN-95 growing in castrated (i.e. ARSi-equivalent) male NSG mice given daily oral dosing with 25 mg of enzalutamide/kg/d vs. vehicle controls (n = 5 each).

Figure 6:. Characterization of AR-FL, AR-V7, vs. Total AR Knockout in LN-95 cells in vitro.

a) Overview of the CRISPR-Cas9 approach used to knockout AR-FL and/or AR-V7 in LN-95 cells. b) Western blot documenting knockout of AR-FL, AR-V7, or both in multiple LN-95 clones. c) IHC (200X) staining of parental LN-5 cells expressing both AR-FL and AR-V7 vs. AR-negative PC-3 cells and the relevant AR-knockout clones. d) Immunoblot documenting nuclear localization of LN-95 cell clones expressing only AR-V7 expressing (i.e. AR-FL KO) clones. e) RNAseq-based analysis of AR-target genes in parental, AR-FL, AR-V7, and total AR KO clones. f) In vitro growth after 6 days of the parental LN-95 cells vs. AR-FL, AR-V7, and total AR KO clones in 10% CS-FBS media.

Figure 7:. Characterization of AR-FL, AR-V7, vs. Total AR Knockout in LN-95 cells in vivo.

a) H & E histology and IHC for AR (200x) in parental LN-95 vs. AR-FL and total AR KO cells. b) Growth rate of parental LN-95 vs. total AR-KO clones in castrated hosts in vivo. c) Growth rate of parental LN-95 vs. AR-FL KO clones in castrated hosts in vivo. d) Growth rate of parental LN-95 s. AR-V7 KO clones in castrated hosts in vivo.