miR-205 hinders the malignant interplay between prostate cancer cells and associated fibroblasts

Abstract

Aims: Tumor microenvironment is a strong determinant for the acquisition of metastatic potential of cancer cells. We have recently demonstrated that cancer-associated fibroblasts (CAFs) elicit a redox-dependent epithelial-mesenchymal transition (EMT) in prostate cancer (PCa) cells, driven by cycloxygenase-2/hypoxia-inducible factor-1 (HIF-1)/nuclear factor-κB pathway and enhancing tumor aggressiveness. Here, we investigated the involvement of microRNAs (miRNAs) in tumor-stroma interplay to identify possible tools to counteract oxidative stress and metastasis dissemination.

Results: We found that miR-205 is the most downmodulated miRNA in PCa cells upon CAF stimulation, due to direct transcriptional repression by HIF-1, a known redox-sensitive transcription factor. Rescue experiments demonstrated that ectopic miR-205 overexpression in PCa cells counteracts CAF-induced EMT, thus impairing enhancement of cell invasion, acquisition of stem cell traits, tumorigenicity, and metastatic dissemination. In addition, miR-205 blocks tumor-driven activation of surrounding fibroblasts by reducing pro-inflammatory cytokine secretion.

Innovation: Overall, such findings suggest miR-205 as a brake against PCa metastasis by blocking both the afferent and efferent arms of the circuit between tumor cells and associated fibroblasts, thus interrupting the pro-oxidant and pro-inflammatory circuitries engaged by reactive stroma.

Conclusion: The evidence that miR-205 replacement in PCa cells is able not only to prevent but also to revert the oxidative/pro-inflammatory axis leading to EMT induced by CAFs sets the rationale for developing miRNA-based approaches to prevent and treat metastatic disease.

FIG. 1.

miR-205 is downregulated in PCa cells stimulated with activated fibroblasts. (A) Unsupervised hierarchical clustering analysis based on miRNA expression data of PC-3 cells subjected to a variety of stimuli. miRNA expression profiles successfully distinguished cells that underwent EMT following stimulation with activated fibroblasts (here referred to as “mesenchymal”) from cells that did not (“epithelial”). A cluster of miRNAs downregulated in PC-3 cells undergoing CAF-induced EMT (including miR-200 family members and miR-205) is highlighted on the right. (B) Quantitative reverse transcriptase-polymerase chain reaction measurement of miR-205, miR-200b, and E-cadherin (CDH1) expression levels in PC-3 cells subjected to different stimuli. Data are reported as log₂-transformed relative quantity with respect to unstimulated cells and represent the mean±standard deviation (SD) of three experiments. miRNA, microRNA; PCa, prostate cancer; EMT, epithelial-mesenchymal transition; CAF, cancer-associated fibroblast.

FIG. 2.

miR-205 loss is associated with prostate cancer progression and metastasis. (A) miR-205 expression pattern was studied in normal prostate tissues (n=28), primary tumors (n=96), and metastatic lesions (n=12) from Taylor's dataset (GSE21032). Expression (here reported as log₂-transformed value of microarray intensity, I) is significantly lower in primary tumors than in normal prostate (P-value=0.0007, modified t test) and further reduced in metastatic lesions (P-value=7×10⁻¹⁵, modified t test). (B) A skewed distribution in miR-205 expression levels was observed in primary tumors, and “miR-low” and “miR-high” tumors were identified using the plotted threshold (dashed red line). (C) Kaplan–Meier curves showing a significant association between high or low miR-205 expression and time to biochemical relapse (BCR). (D) Representative gene sets significantly enriched among genes positively or negatively correlated with miR-205 expression in Taylor's dataset. Details and references of gene sets can be found at www.broadinstitute.org/gsea/msigdb/. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

CAF-induced proinflammatory signature correlates with miR-205 transcriptional repression. (A) The expression of pri-miR-205, mature miR-205, ZEB1, and ZEB2 was measured by quantitative reverse transcriptase-polymerase chain reaction in CAF-stimulated PC-3 cells at different time intervals. Data are reported as log₂-transformed relative expression with respect to serum-starved cells (named Starv). (B) The levels of E-cadherin, vimentin, HIF-1α, COX-2, and IκB-α (the phosphorylation of which is inversely proportional to NF-κB activation) were assessed by immunoblotting in CAF-stimulated PC-3 cells. (C) PC-3 cells were either incubated with CM from CAFs (obtained from fibroblasts treated with serum-free medium for 48 h) or serum-starved for 48 h and subsequently (i) harvested (lanes 1, 2), (ii) serum-starved for additional 48 h (lanes 3, 4), or (iii) serum-starved for 48 h, then treated with starvation medium or CM from CAFs for additional 48 h (lanes 5, 6). E-cadherin and vimentin levels were evaluated on cell lysates. HIF-1α, hypoxia-inducible factor-1α; COX-2, cycloxygenase-2; NF-κB, nuclear factor-κB; CM, conditioned medium. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

HIF-1α directly represses miR-205 transcription. (A) Representation of miR-205 promoter, recently identified as the 5 kb sequence upstream of miR-205 transcriptional start site (TSS) (3). Green ovals represent in silico-predicted HIF-1 binding sites. Light blue lines identify the regions amplified in chromatin immunoprecipitation experiments. (B) Chromatin from fibroblast-stimulated PC-3 cells was precipitated with anti-HIF-1α antibody, and then the regions putatively bound by HIF-1 were amplified by RT-PCR (top). Normal mouse preimmuned IgG was used as a negative control. Densitometric analysis of PCR signals in immunoprecipitants, reported as normalized to input DNA from each sample (bottom). Data are reported as mean±SD of three experiments. (C) HIF-1α immunoblotting on PC-3 cells either cultivated in hypoxia (1% O₂) or normoxia (20% O₂) for the indicated time intervals (either continuously or with a hypoxia pulse followed by recovery in 20% oxygen atmosphere). (D) qRT-PCR assessment of pri-miR-205 and mature miR-205 expression in PC-3 cells cultured as in (C). Data are reported as log₂-transformed relative expression with respect to cells cultured in normoxic conditions and harvested at each time point (24, 48, and 72 h). qRT-PCR, quantitative reverse transcriptase-polymerase chain reaction. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 5.

miR-205 prevents CAF-induced EMT in PCa cells in vitro. (A) PC-3 cells were transfected at low cell density (1×10⁵/p100) with miR-205 precursor or with miR-Neg and, after 48 h, treated or not with the CM from CAFs for an additional 48 h. The levels of E-cadherin were assessed by immunoblotting. Vehicle-transfected cells were used as a control. (B) Invasion of PC-3 cells, treated as in (A), was analyzed (24 h of invasion toward complete growth medium). Photographs are representative of six randomly chosen fields. (C) PC-3 cells treated as in (A) were detached and maintained in suspension for 48 h. Anoikis was quantified by FACS analysis as the percentage of annexin V/propidium-iodide-positive cells. After detachment, PC-3 cells were also analyzed for their clonogenic potential. First-passage individual prostaspheres were photographed (D), and the percentage of CD44^high/CD24^low cells (E) and CD133-positive cells (F) was analyzed by FACS analysis (data are reported as mean±standard deviation of four experiments). FACS, fluorescence activated cell sorter. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

miR-205 prevents CAF activation by PCa cells. (A) PC-3 cells were transfected at low cell density (1×10⁵/p100) with miR-205 precursor or with pre-miR-Neg as a control and, after 48 h, treated or not with CM from CAFs for an additional 48 h. Secreted IL-6 was evaluated by immunoblotting (top) and quantified by enzyme-linked immunosorbent assay (IL-6 concentration was normalized on protein content) (bottom) in the CM of the above treated cells. (B) HPFs were incubated with CM from PC-3 cells (treated as in A) for 48 h. Fibroblast activation protein (FAP) expression was assessed by immunoblotting on cell lysates. (C) Invasion of PC-3 cells, treated with CMs derived from the differently treated HPFs (see B), was analyzed (24 h of invasion toward complete growth medium). Photographs are representative of four randomly chosen fields. (D) Representative gene sets significantly enriched among genes downregulated after miR-205 restoring in DU145 PCa cells (dataset GSE11701), including gene sets related to cytokine activity, are reported. (E) qRT-PCR assessment of PKCɛ (PRKCE) and IL6 mRNA expression levels in PC-3 cells transfected with siRNA against PKCɛ (siPRKCE). Data are reported as log₂-relative quantity compared to cells transfected with control siRNA (siCTR). IL-6, interleukin-6; PKCɛ, protein kinase C epsilon. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 7.

miR-205 prevents CAF-induced enhancement of PCa cell tumorigenicity and metastatic dissemination in vivo. (A) PC-3 xenograft growth in SCID bg/bg mice. A mixture of 1×10⁶ PC-3 cells transfected either with miR-205 precursor or miR-Neg and 0.5×10⁶ CAFs was subcutaneously injected (n=8 mice per group). The onset and volume of the primary tumor are reported in the growth curve. P-values of the difference between tumor volumes in the two groups at each time point (miR-205 vs. miR-Neg) are reported. (B) PC-3 lung colonization in SCID bg/bg mice. PC-3 cells were transfected either with miR-205 precursor or miR-Neg and after 48 h were treated with CM from CAFs. After 72 h, cells were injected into the lateral tail vein of mice (n=8 per group). Animals were monitored at 3-day intervals and were sacrificed after 8 weeks. Lungs were inspected with the aid of a microscope and micrometastases were counted. (C) Paraffin-embedded tissue sections from lung micrometastases (original magnification 10×and 40×) obtained by miR-205- or miR-Neg-transfected PC-3 cells injected in SCID bg/bg mice were stained with hematoxylin and eosin.

FIG. 8.

miR-205 reverts CAF-induced EMT. PC-3 cells were treated with the CM from in vitro activated HPFs (10 ng/ml TGF-β1 or 50 ng/ml IL-6) or from CAFs for 48 h to induce EMT and then transfected with miR-205 precursor or silenced with siRNAs against COX-2 or HIF-1α. After 48 h, cell morphology (A) and the levels of E-cadherin (B) were assessed. Photographs are representative of randomly chosen fields. (C) Schematic representation of the central role played by miR-205 in the crosstalk between CAFs and PCa cells. CAFs secrete MMP-2 and MMP-9, which activate EMT and stemness in PCa cells, by promoting the COX-2 pro-inflammatory pathway. ROS produced by COX-2 activity favor a redox-dependent stabilization of HIF-1 (18,22), which, in turn, induces miR-205 transcriptional repression. Downregulation of the miRNA leads to derepression of its targets ZEB1/2 and PKC-ɛ (19), thus allowing respectively EMT and IL-6 secretion by PCa cells. By these two pathways miR-205 controls motility and stemness of PCa cells on one hand, and CAFs reactivity on the other, thereby closing the feed-forward loop between cancer and stromal cells. TGF-β1, tumor growth factor-β1; MMP, matrix metalloprotease; ROS, reactive oxygen species. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars