CXCL14 is an autocrine growth factor for fibroblasts and acts as a multi-modal stimulator of prostate tumor growth

Abstract

This study explored the role of secreted fibroblast-derived factors in prostate cancer growth. Analyses of matched normal and tumor tissue revealed up-regulation of CXCL14 in cancer-associated fibroblasts of a majority of prostate cancer. Fibroblasts over-expressing CXCL14 promoted the growth of prostate cancer xenografts, and increased tumor angiogenesis and macrophage infiltration. Mechanistic studies demonstrated that autocrine CXCL14-stimulation of fibroblasts stimulate migration and ERK-dependent proliferation of fibroblasts. CXCL14-stimulation of monocyte migration was also demonstrated. Furthermore, CXCL14-producing fibroblasts, but not recombinant CXCL14, enhanced in vitro proliferation and migration of prostate cancer cells and in vivo angiogenesis. These studies thus identify CXCL14 as a novel autocrine stimulator of fibroblast growth and migration, with multi-modal tumor-stimulatory activities. In more general terms, our findings suggest autocrine stimulation of fibroblasts as a previously unrecognized mechanism for chemokine-mediated stimulation of tumor growth, and suggest a novel mechanism whereby cancer-associated fibroblasts achieve their pro-tumorigenic phenotype.

Fig. 1.

CXCL14 is up-regulated in fibroblast-enriched prostate cancer stroma. (A) Stroma from normal prostate (open bars) and prostate cancer tissue (filled bars) was microdissected from 8 different patients and the expression of CXCL14 was analyzed with qRT-PCR. (B) Examples of CXCL14 immunohistochemistry analyses of paired tissues with up-regulation of CXCL14 in tumor stroma, together with unchanged (Top), up-regulated (Middle), or down-regulated (Bottom) expression in the epithelial cells. (Scale bar, 100 μm.)

Fig. 2.

NIH-CXCL14 cells accelerate growth of prostate cancer xenografts. (A) The tumor growth of injected LNCaP cells (filled triangles) or LNCaP cells together with NIH-ctr (open squares) or NIH-CXCL14 cells (filled squares) was followed over time after s.c. injection into SCID mice. (# n = 6; ^§ n = 5 remaining animals) (B) Tumor incidence at days 28 and 49, corresponding to the time when the first animal of the LNCaP/NIH-CXCL14 and LNCaP/NIH-ctr groups were killed, was calculated. Tumors from both groups were collected and analyzed for cell density (C), cell proliferation (D), and the abundance of vessels (E) and macrophages (G), using H&E staining or immunohistochemistry as indicated. Spearman correlation analysis was performed to analyze correlations between intratumor CXCL14 expression and CD31 (F) or CSF1R (H) expression. Error bars in B–D indicate SEM. (Scale bar, 100 μm.) *, P < 0.05, by unpaired t test.

Fig. 3.

CXCL14 promotes in vitro growth and migration of fibroblasts. (A) The growth of NIH-ctr (open squares) and NIH-CXCL14 cells (filled squares) in 1% FCS culture medium was determined by cell counting. (B) Western blot analysis of CXCL14-induced ERK1/2 phosphorylation in NIH-ctr cells. (C) The cell density of NIH-ctr and NIH-CXCL14 cells was determined by crystal violet staining after culture in low serum in the presence of the MEK inhibitor UO126. (D) For migration studies, fibroblasts were seeded in the upper compartment of a 2-compartment chamber and allowed to migrate for 20 h, after which migrated cells in the lower compartment were collected and counted using a hemocytometer. Error bars indicate SEM. *, P < 0.05, by unpaired t test (A), ANOVA (C), and paired t test (D).

Fig. 4.

Conditioned medium from NIH-CXCL14 cells, but not recombinant CXCL14, promote LNCaP proliferation and migration in vitro. (A) The effects of NIH-ctr or NIH-CXCL14 fibroblasts on LNCaP cell growth was determined in a co-culture assay by Rhodanile staining after 10 days. (B) LNCaP cells were allowed to migrate toward control medium alone or supplemented with 100 ng/mL CXCL14 or medium conditioned by NIH-ctr or NIH-CXCL14 cells for 20 h. The number of migrated cells was determined using a hemocytometer. (C) Effects on ERK1/2 phosphorylation in LNCaP cells and MCF cells treated with 100 ng/mL CXCL14 were analyzed by immunoblotting of total cell lysates. Error bars indicate SEM. *, P < 0.05, by paired t test (A) and ANOVA (B).

Fig. 5.

NIH-CXCL14 cells promote monocyte migration in vitro and angiogenesis in vivo. (A) Freshly isolated monocytes were allowed to migrate toward 10 ng/mL CXCL14, or conditioned medium derived from NIH-ctr or NIH-CXCL14 fibroblasts for 4 h. The number of migrated monocytes present in the lower compartment was determined by FACS analysis of CD14⁺ cells. (B) Effects on in vivo angiogenesis were analyzed by monitoring vessel in-growth in Matrigel plugs mixed with 1% FCS culture medium alone, or medium containing 100 ng/mL CXCL14, NIH-ctr or NIH-CXCL14 cells. Error bars indicate SEM. *, P < 0.05, by ANOVA.