Tissue injury and hypoxia promote malignant progression of prostate cancer by inducing CXCL13 expression in tumor myofibroblasts

Abstract

Prostate cancer (PC) is a slowly progressing malignancy that often responds to androgen ablation or chemotherapy by becoming more aggressive, acquiring a neuroendocrine phenotype, and undergoing metastatic spread. We found that B lymphocytes recruited into regressing androgen-deprived tumors by C-X-C motif chemokine 13 (CXCL13), a chemokine whose expression correlates with clinical severity, play an important role in malignant progression and metastatic dissemination of PC. We now describe how androgen ablation induces CXCL13 expression. In both allografted and spontaneous mouse PC, CXCL13 is expressed by tumor-associated myofibroblasts that are activated on androgen ablation through a hypoxia-dependent mechanism. The same cells produce CXCL13 after chemotherapy. Myofibroblast activation and CXCL13 expression also occur in the normal prostate after androgen deprivation, and CXCL13 is expressed by myofibroblasts in human PC. Hypoxia activates hypoxia-inducible factor 1 (HIF-1) and induces autocrine TGF-β signaling that promotes myofibroblast activation and CXCL13 induction. In addition to TGF-β receptor kinase inhibitors, myofibroblast activation and CXCL13 induction are blocked by phosphodiesterase 5 (PDE5) inhibitors. Both inhibitor types and myofibroblast immunodepletion block the emergence of castration-resistant PC in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model of spontaneous metastatic PC with neuroendocrine differentiation.

Keywords: cancer; cancer-associated fibroblasts; inflammation; tumor microenvironment.

Fig. 1.

Androgen ablation induces CXCL13 in myofibroblasts. (A) Six-week-old FVB/N males (n = 10) were s.c. inoculated with Myc-CaP cells. When the tumors reached 1,000 mm³, mice were castrated or sham operated. Tumors were examined 1 wk later for SMA by immunohistochemistry (IHC) and CXCL13 mRNA by in situ hybridization (ISH). (Magnification: 200×.) Areas occupied by positive cells were determined by image analysis (n = 2). (B) FVB/N mice (n = 10 per group) bearing Myc-CaP tumors were castrated or sham operated and killed 1 wk later. Tumors were removed and digested, and the fibroblast (Fib) and epithelial (Epi) cell fractions were isolated, total RNA was extracted, and CXCL13 and FAP mRNAs were quantitated by quantitative PCR (Q-PCR) and normalized to cyclophilin A mRNA. Results are averages ± SD. (C) FVB/N mice (n = 9) bearing Myc-CaP tumors were castrated or sham operated, and tumors were collected 1 wk later. CD45, CXCL13, SMA, and GP38 expression was analyzed by flow cytometry. (D) Six-week-old male FVB/N mice (n = 10 per group) were vaccinated three times with 10⁸ cfu of TOPO or FAP vaccines every 5 d. Myc-CaP tumors were established as described earlier. Three days before castration, the mice were vaccinated again. One week after castration, tumors were collected, paraffin-embedded, sectioned, counterstained with DAPI, and analyzed by immunofluorescence (IF) for SMA and CXCL13 expression. (Magnification: 400×.) (E) FVB/N males (n = 10 per group) were vaccinated and Myc-CaP tumors were established as described earlier. Tumor volume was measured every 2–3 d after castration. Results are expressed as means ± SEM.

Fig. 2.

Castration-induced myofibroblast activation depends on TGF-β signaling. (A) Myc-CaP tumors (n = 10) were established as in Fig. 1A and were collected after sham operation (7 d) or at the indicated days after castration (C2, C4, C6, C7 refer to 2, 4, 6, and 7 d postcastration). (B–E) FVB/N mice bearing Myc-CaP tumors were castrated. Starting 1 d before castration, mice (n = 10 per group) were injected daily with vehicle or SB-431542 (0.2 mg/mouse; 10 mg/kg), a TGF-βR1 inhibitor. Tumors were collected 1 wk after castration and analyzed by IHC for SMA and nuclear SMAD2/3, SMA and CXCL13, and IKKα or subjected to RNA extraction and Q-PCR analysis of the indicated mRNAs normalized to the amount of cyclophilin A mRNA (C results are averages ± SD). (Magnification: B, 400×; D, 200×; E, 200×.) The area occupied by nuclear IKKα-expressing cells was determined as defined earlier. (F) Tumors were established and mice were treated with SB-431542 or vehicle, as described earlier. Tumor volume was measured every 2–3 d. Results are expressed as means ± SEM. P values >0.05 were considered nonsignificant, 0.01–0.05 was considered significant (*), 0.001–0.01 was considered very significant (**), and <0.001 was considered highly significant (***).

Fig. 3.

Hypoxia caused by androgen ablation induces TGF-β, CTGF, and IGF1 expression. (A) Fibroblasts were purified from Myc-CaP tumors 1 wk after sham operation or castration (C) (n = 10 per group), plated, and stimulated for 24 h with TGF-β1 (10 ng/mL), CTGF (50 ng/mL), or TGF-β1 plus CTGF. RNA was isolated, and expression of the indicated genes was analyzed as described earlier. Results are averages ± SD. (B) Fibroblasts were isolated from Myc-CaP tumors 1 wk after castration (C) or sham operation and incubated either in a standard incubator or a hypoxic chamber (1% O₂) for 24 h. Total RNA was isolated, and the indicated mRNAs were quantitated. Results are averages ± SD (C) FVB/N mice bearing Myc-CaP tumors were castrated, and tumors were collected at indicated times after castration. For hypoxia analysis, mice were i.p. injected 90 min before sacrifice with 60 mg/kg of pimonidazole hydrochloride (Hypoxyprobe-1). Tumors were removed, fixed, paraffin-embedded, sectioned, and subjected to IHC with Hypoxyprobe or HIF-1α antibodies. (Magnification: 200×.) The areas occupied by positive cells were determined as described earlier. Arrows indicate hypoxic areas and cells with nuclear HIF-1α.

Fig. 4.

PDE5 inhibition attenuates CRPC by interfering with myofibroblast activation. (A and B) Tumors were established in 6-wk-old FVB/N males (n = 10), as described earlier. Sildenafil was either added or not to the drinking water (0.7 g/L). (A) Tumor volume was measured every 2–3 d. Results are averages ± SEM. (B) Tumors were collected 1 wk after castration and analyzed for SMA by IHC. The areas occupied by SMA⁺ cells were determined as described earlier (n = 2). (C) Tumor-associated fibroblasts were purified from mice bearing Myc-CaP tumors and incubated in either a standard incubator or an hypoxic chamber (1% O₂) for 24 h in the absence or presence of indicated inhibitors. CT, control; H, hypoxia; PTX, pentoxyfidine; SILD, sildenafil. Expression of the indicated mRNAs was analyzed by Q-PCR as described earlier (n = 5). Results are averages ± SD.

Fig. 5.

Myofibroblasts are required for CXCL13 expression and control TRAMP tumor malignant progression. (A and B) Six-week-old FVB/N males (n = 10 per group) were castrated or sham operated, and their prostates were collected 1 wk after surgery. The indicated inflammatory cytokines and chemokines and cell marker mRNAs were quantitated by Q-PCR, as described earlier. Results are averages ± SD. (C and D) Twelve-week-old TRAMP males (n = 10) were castrated or sham operated, and their tumors were collected at the indicated times after castration. Expression of indicated mRNAs was quantified by Q-PCR, as described earlier. Results are averages ± SD (C). Tumors were fixed, paraffin embedded, sectioned, counterstained with DAPI, and analyzed for SMA and CXCL13 by IF (D). (Magnification: 200×.) (E) Six-week-old TRAMP males (n = 10 per group) were vaccinated three times with 10⁸ cfu of TOPO or FAP vaccines every 5 d, followed by a booster shot every 30 d. Mice were castrated when 12 wk old, and tumor weight was measured after 7 wk. Results are averages ± SD.

Fig. 6.

SMA, CXCL13, and HIF-1α are highly expressed in malignant human PC tissue. (A–C) Paraffin-embedded human prostate tissues [normal (n = 5), benign hyperplasia (n = 4), and malignant (n = 10)] were sectioned and analyzed for SMA, CXCL13, and HIF-1α by IHC. Shown are typical examples of each case. (A) The areas occupied by positive cells were calculated as described earlier. (Magnification: 400×.) The human prostate samples were also stained for SMA and CXCL13 (B) or CD20 and CXCL13 (C) and analyzed by IF. (Magnification: 400×.)