A co-clinical approach identifies mechanisms and potential therapies for androgen deprivation resistance in prostate cancer

Abstract

Here we report an integrated analysis that leverages data from treatment of genetic mouse models of prostate cancer along with clinical data from patients to elucidate new mechanisms of castration resistance. We show that castration counteracts tumor progression in a Pten loss-driven mouse model of prostate cancer through the induction of apoptosis and proliferation block. Conversely, this response is bypassed with deletion of either Trp53 or Zbtb7a together with Pten, leading to the development of castration-resistant prostate cancer (CRPC). Mechanistically, the integrated acquisition of data from mouse models and patients identifies the expression patterns of XAF1, XIAP and SRD5A1 as a predictive and actionable signature for CRPC. Notably, we show that combined inhibition of XIAP, SRD5A1 and AR pathways overcomes castration resistance. Thus, our co-clinical approach facilitates the stratification of patients and the development of tailored and innovative therapeutic treatments.

Figure 1. Prostate tumors characterized by distinct genetic alterations differentially respond to castration

(a) Timeline of analysis of Pten^flox/flox;Probasin(Pb)-Cre, Pten^flox/flox;Lrf^flox/flox;Probasin(Pb)-Cre, and Pten^flox/flox;p53^flox/flox;Probasin(Pb)-Cre noncastrated (n=15) and castrated (n=15) cohorts of mice. (b-c) Quantification of percentage area of normal epithelium in the prostate of Pten^flox/flox;Probasin(Pb)-Cre (left), Pten^flox/flox;Lrf^flox/flox;Probasin(Pb)-Cre (middle), and Pten^flox/flox;p53^flox/flox;Probasin(Pb)-Cre (right) castrated and non-castrated mice at 60 (b) and 90 (c) days post-castration. Representative images of H&E staining of DLP from castrated and non-castrated mice of the indicated genotypes are shown. (d) Representative MRI images of prostate cancers (Anterior Lobe AP) in castrated and non-castrated mice of indicated genotype 4 days before and 60 days post-castration. Tumor volume (area outlined in red) was quantified as described in the experimental procedures. Asterisk represents the location of the bladder.

Figure 2. Analysis of human prostate cancer unveils the relevance of LRF loss in the response to androgen deprivation

(a) Cross-species integrated genetic screenings (b) Histogram showing the percentage of PSA-reduction following treatment used to stratify patients as good responders (yellow) or poor responders (blue) to ADT. Mean value of percentage of PSA reduction distribution is shown (dashed line). (c-d) PTEN and LRF IHC in human prostate cancer TMAs. The percentage of tumor cells with loss of PTEN and LRF were measured. Patients were considered good responders (0) or poor responders (1) to ADT according to the percentage of PSA reduction. Arrows highlight PTEN and LRF null cells. (e) Percentage of patients that experienced combined loss of PTEN and LRF in the category of good (8.7%) and poor responders (30.5%). This distribution resulted statistically significant p=0.0132 in a Fisher test. (f) Schematic representation of the deletion of the p arm on chromosome 19 (green) in human CRPC. ZBTB7a genetic locus is indicated (arrow). (g) Diagram showing the distribution of PTEN-loss (orange) and LRF-loss (white) genetic alterations in castrated (blue) and noncastrated (green) metastasis. (h) CGH array data for 58 castration resistant prostate cancer metastatic samples shows genetic loss of the telomeric region of chromosome 19 containing the Zbtb7a locus. Data were processed by Partek Genomic Suite 6.4 Segmentation Algorithm (deletions are shown in green on the left side, amplification are showed in red on the right side). (i) Expression profile analysis of human castration resistant metastases shows statistically significant down-regulation of LRF expression.

Figure 3. Differential responses to castration in different genetic backgrounds

(a-b) IHC staining of DLP and AP from castrated and non-castrated mice at 30 days post-castration. Upper panel: Ki-67 staining on prostate sections from castrated and noncastrated mice of indicated genotype. Lower panel: quantification of Ki-67 staining. Quantification has been performed as described in the methods. For each genotype, castrated (n=3, grey bar) and non-castrated (n=3, black bar) mice were analyzed. Percentage of Ki67 positive cells in DLP and AP prostate lobes was evaluated on a total of 15,000 cells per lobe. Data are presented as mean ± standard deviation. (c) Western blot analysis on VP, DLP and AP from castrated Pten^flox/flox;Probasin(Pb)-Cre, Pten^flox/flox;Lrf^flox/flox;Probasin(Pb)-Cre and Pten^flox/flox;p53^flox/flox;Probasin(Pb)-Cre mice. Western blot analyses were performed on sample lysates collected from mice sacrificed 4 days after castration. (d) IHC staining of AR on DLP and AP from castrated and noncastrated mice sacrificed 30 days after castration. (e) Quantification of cells with nuclear AR localization in castrated Pten^flox/flox;Probasin(Pb)-Cre (light grey bar), Pten^flox/flox;Lrf^flox/flox;Probasin(Pb)-Cre (dark grey bar) and Pten^flox/flox;p53^flox/flox;Probasin(Pb)-Cre (black bar) prostate tumors. Number of cells with nuclear AR staining in DLP and AP prostate lobes was evaluated in n=3 castrated mice/genotype on a total of 2,500 cells for each prostate lobe. Data are presented as mean ± standard deviation.

Figure 4. Deregulation of XAF1 and SRD5A1 levels dictates prostate cancer progression and castration resistance in mouse and human

(a) Analysis of the expression profiles of Pten^flox/flox;Lrf^flox/flox;Probasin(Pb)-Cre versus Pten^flox/flox;Probasin(Pb)-Cre prostates shows a significant down-regulation of Xaf1 and a concomitant up-regulation of Srd5a1. (b) Quantitative RT-PCR of Xaf1 in VP (n=3, black bars), DLP (n=3, dark grey bars) and AP (n=3, light grey bars) samples collected from Pten^flox/flox;Probasin(Pb)-Cre, Pten^flox/flox;Lrf^flox/flox;Probasin(Pb)-Cre, and Pten^flox/flox;p53^flox/flox;Probasin(Pb)-Cre mice. Data are presented as mean ± standard deviation. (c) IHC with anti-Xaf1 of the same samples in b. (d) Western blot analysis on the same samples in b. (e) Quantitative RT-PCR for Srd5a1 in VP (n=3, black bars), DLP (n=3, dark grey bars) and AP (n=3, light grey bars) samples collected from WT, Pten^flox/flox;Probasin(Pb)-Cre, Pten^flox/flox;Lrf^flox/flox;Probasin(Pb)-Cre and Pten^flox/flox;p53^flox/flox;Probasin(Pb)-Cre mouse prostates. Data are presented as mean ± standard deviation. (f) XAF1 staining and relative quantification in human prostate cancer TMA. (g) LRF and XAF1 down regulation and SRD5A1 induction in castration resistant prostate cancer xenografts (orange bars) versus castration sensitive (black bars). (h-l) XAF1, XIAP, and SRD5A1 expression levels in primary and metastatic prostate cancer compared to normal prostatic epithelium.

Figure 5. Synergistic effect of ADT and embelin, in mouse CRPC

(a) Weight of VP, DLP, and AP of Pten^flox/flox;Lrf^flox/flox;Probasin(Pb)-Cre and Pten^flox/flox;p53^flox/flox;Probasin(Pb)-Cre mice treated with bicalutamide (Casodex) (n=8) (black bars) or bicalutamide plus embelin (n=8) (grey bars) for 4 weeks. Data are presented as mean ± standard deviation. (b-e) Proliferation rate analysis as percentage of Ki67 positive cells in VP, DLP, and AP of the samples described in a. Data are presented as mean ± standard deviation. (f-g) H&E and apoptosis analysis through Western blot quantification of Parp cleavage versus total Parp in VP, DLP, and AP of the Pten^flox/flox;Lrf^flox/flox;Probasin(Pb)-Cre prostate cancers described in a.

Figure 6. Genetic and molecular assessments in mice and patients dictate new experimental treatment to overcome CRPC

(a-d) Apoptosis analysis in human prostate cancer cell line (VCaP) treated with ADT, XIAP inhibitor (embelin), or SRD5A1 inhibitor (dutasteride), as single agent or in combination. Quantifications of PARP cleavage versus total PARP after 2 days of treatment are shown. (e) Weight of VP, DLP, and AP of castration resistant Pten^flox/flox;Lrf^flox/flox;Probasin(Pb)-Cre and Pten^flox/flox;p53^flox/flox;Probasin(Pb)-Cre mice treated with bicalutamide (Casodex) (n=4/genotype, black bars), bicalutamide plus embelin (n=4/genotype, dark grey bars), or bicalutamide plus embelin plus dutasteride (n=4/genotype, light grey bars) for 4 weeks. Data are presented as mean ± standard deviation.