Antagonizing CD105 and androgen receptor to target stromal-epithelial interactions for clinical benefit

Abstract

Androgen receptor signaling inhibitors (ARSIs) are standard of care for advanced prostate cancer (PCa) patients. Eventual resistance to ARSIs can include the expression of androgen receptor (AR) splice variant, AR-V7, expression as a recognized means of ligand-independent androgen signaling. We demonstrated that interleukin (IL)-6-mediated AR-V7 expression requires bone morphogenic protein (BMP) and CD105 receptor activity in both PCa and associated fibroblasts. Chromatin immunoprecipitation supported CD105-dependent ID1- and E2F-mediated expression of RBM38. Further, RNA immune precipitation demonstrated RBM38 binds the AR-cryptic exon 3 to enable AR-V7 generation. The forced expression of AR-V7 by primary prostatic fibroblasts diminished PCa sensitivity to ARSI. Conversely, downregulation of AR-V7 expression in cancer epithelia and associated fibroblasts was achieved by a CD105-neutralizing antibody, carotuximab. These compelling pre-clinical findings initiated an interventional study in PCa patients developing ARSI resistance. The combination of carotuximab and ARSI (i.e., enzalutamide or abiraterone) provided disease stabilization in four of nine assessable ARSI-refractory patients. Circulating tumor cell evaluation showed AR-V7 downregulation in the responsive subjects on combination treatment and revealed a three-gene panel that was predictive of response. The systemic antagonism of BMP/CD105 signaling can support ARSI re-sensitization in pre-clinical models and subjects that have otherwise developed resistance due to AR-V7 expression.

Keywords: CD105; PCa; androgen receptor splice variants; cancer-associated fibroblasts; endoglin.

Graphical abstract

Figure 1

AR-variant expression in PCa epithelia and prostate fibroblasts (A and B) Full-length AR (AR-FL) and splice variant mRNA expression in human PCa cell lines (22Rv1, LNCaP) and primary human prostatic fibroblasts (NAF and CAF) were evaluated by RT-PCR. β-actin or GAPDH were used as a loading control for RT-PCR gels and qPCR analyses (n ≥ 6). (C) mRNA expression in NAF and CAF were measured following culturing with dihydroxytestosterone (Tst; 10, 5, 2.5, 0 nM) for 14 days. AR-FL, AR-V2, and ARv567es mRNA expression in Tgfbr2^flox/flox (control) and Tgfbr2^fspKO (Tgfbr2-KO) mouse prostatic fibroblasts treated with R1881 (R, 10 nM), bicalutamide (B, 10 μM), or DMSO (C, control) for 48 h. (D) Hematoxylin and eosin (H&E) histological staining, immunohistochemical localization of AR, and AR-V7 expression was localized in PDX from mice treated with vehicle (V) or enzalutamide (E; 40 mg/kg) for 4 days (n ≥ 4). Nuclei were counterstained with hematoxylin and scale bar represents 30 μm. Data were analyzed using one-tailed t test, where statistical significance was indicated: ∗p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Figure 2

CD105 antagonism regulates AR-variant expression in PCa cells (A) Heatmap of TGF-β and BMP family ligand expression by 22Rv1 and C4-2B PCa cell lines treated with enzalutamide or vehicle for 72 h (two-way ANOVA, p < 0.0001). (B) mRNA expression of AR-FL and AR-V7 was measured in prostatic fibroblasts and PCa cells treated with BMP-2 for 0.5 or 6 h. (C) AR-FL, AR-V7, and UBE2C mRNA expression was measured in 22Rv1 cells treated with enzalutamide, carotuximab, and its combination, or vehicle for 72 h under 2% O₂. (D) Correlation between AR and CD105 mRNA expression was determined in three indicated datasets (R²). Enzalutamide was used at 5 μM; BMP-2 10 ng/mL; carotuximab 10 μg/mL; DMSO used as vehicle control. For qPCR data, GAPDH was used as loading control and data analyzed using two-tailed t test, where statistical significance was indicated: ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.

Figure 3

CD105 and RBM38 signaling in PCa cells (A) RNA-binding motif expression by primary human prostatic fibroblast RNA sequencing analysis from Kato et al. was analyzed. Data represents expression of genes as a ratio of CAF/NAF. (B) RBM38 mRNA expression was measured in prostatic CAF and 22Rv1 treated with enzalutamide (5 μM), carotuximab (10 μg/mL), or the combination, or vehicle for 72 h under 2% O₂. (C) AR-FL, AR-V1, AR-V7, and AR-v567es mRNA expression was measured in 22Rv1 following RBM38 siRNA for 48 h. (D) RBM38 promoter ChIP analysis examined the enrichment of RNA polymerase II (Pol II), ID1, and E2F1 in 22Rv1 cells treated with carotuximab or IgG control. A representative gel image is shown with densitometric analysis of enrichment as a percentage of input. Model of the role of ID1 at RBM38 promoter. ID1 recruitment at RBM30 promoter enables RNA polymerase II loading, whereas CD105 inhibition by carotuximab blocks both ID1 and RNA polymerase II. E2F recruitment to the RBM38 promoter is not dependent on ID1 loading. (E) RNA immunoprecipitation (RIP) assay was used to determine RBM38 enrichment at the AR-cryptic exon 3 loci hnRNA (heteronuclear RNA) in 22Rv1 PCa cells at the following loci: 66,831,239-66,840,000 (green), 66,831,239-66,843,000 (red), and 66,917,500-66920140 (blue). Cells were treated with vehicle, enzalutamide (5 μM/mL), or the combination of enzalutamide and carotuximab (10 μg/mL) for 48 h under 2% O₂. Data were analyzed using two-tailed t test, where statistical significance was indicated: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Figure 4

Androgen and CD105 signaling regulates fibroblastic AR-variant expression (A) The Jenkins dataset of PCa patients developing metastatic disease progression demonstrated a correlation between the expression of CD105 and RBM38 (p = 2.4 × 10⁻⁵⁰). (B) The Alumkal cohort of metastatic CRPC patients demonstrated a correlation between the expression of CD105 and RBM38 as well as a correlation between RBM38 and AR. The normalized expression values were centered by the mean across all the samples. The x and y axis are the mean centered log₂ transcripts per million relative expression values computed by using Pearson’s correlation method. (C) Flow cytometry for cell-surface CD105 was analyzed in 22Rv1 or C4-2B cells co-cultured in Transwells with CAF with indicated treatment for 72 h in 2% O₂. (D) AR-FL and AR-V2 mRNA expression was measured in wild-type (WT) mouse prostatic fibroblasts following the indicated treatments for 72 h. (E) AR-FL and AR-V7 mRNA expression in human CAFs were measured following 48-h incubation with LNCaP-conditioned media in the presence or absence of MAB-206. Western blot for ID1 expression was visualized in 22Rv1 cells following enzalutamide (Enza) and MAB-206 after 48-h treatment under 2% O₂. ID1 mRNA expression in both NAF and LNCaP was measured following co-culture with IgG or MAB-206. (F) Cell-surface CD105 was measured in LNCaP and CAFs following treatment with IgG or MAB-206 (MAB) for 48 h at 2% O₂. (G) RBM38 expression was measured in CAF cells treated with enzalutamide in the presence or absence of MAB-206 for 48 h at 2% O₂. (H) The data support a mechanism where AR antagonism promotes IL-6 for downstream CD105 signaling in RBM38-mediated AR-V7 generation. Enzalutamide was used at 5 μM, carotuximab 10 μg/mL, M1043 10 μg/mL, MAB-206 150 ng/mL, IgG 10 μg/mL, and DMSO was used as vehicle control. GAPDH was used as loading control for qPCR. Data were analyzed using two-tailed t test, where statistical significance was indicated: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001.

Figure 5

Fibroblastic AR-V7 expression is consequential to PCa enzalutamide sensitivity (A) Overexpression of AR-V7 (AR-V7^OE) in WT mouse prostatic fibroblasts was validated by RT-PCR and characterized for the expression of CAF marker expression in the presence or absence of enzalutamide and/or M1043 for 72 h. (B) Proliferation of 22Rv1 cells WS measured by flow cytometry for Ki67 following three-dimensional co-cultures with mouse prostatic fibroblasts (WT, AR-V7^OE, or CD105 knockout [CD105^KO]) under indicated treatments for 72 h (n = 3). Under the same conditions, apoptosis was measured by annexin V (n = 3). (C) Tissue recombinants of 22Rv1 and mouse prostatic fibroblasts (WT or AR-V7^OE) were orthotopically grafted and subsequently treated with enzalutamide for 4 days prior to sacrifice. The tumors were evaluated histologically for H&E, and immunohistochemistry (IHC) was used for phosphorylated-histone H3 and TUNEL staining (arrows indicate positive staining). Enzalutamide was used at 5 μM in culture or 1 mg/kg in mice, carotuximab at 10 μg/mL, M1043 at 10 μg/mL, and DMSO used as vehicle control. Data were analyzed using two-tailed t test, where statistical significance was indicated: ∗p < 0.05, ∗∗p < 0.01.

Figure 6

Targeted androgen and CD105 inhibition combination therapy were evaluated in patients (A) The time on combination therapy is indicated with relation to the number of clinical interventions prior to accrual to the phase 2 clinical trial for the individual patients. (B) CTC enrichment in patients revealed differentially expressed pre-treatment markers, ARG2, CSRP1, and KIAA1324; when combined, they distinguish non-responsive and responsive subjects. (C) 22Rv1 cells treated with enzalutamide (Enza), carotuximab (Carotux), or the combination for 72 h at 2% O₂ were evaluated by RT-PCR. Enzalutamide was used at 5 μM, carotuximab 10 μg/mL, and DMSO used as vehicle control. Data were analyzed using two-ANOVA, where statistical significance was indicated.