The online version of this article (doi:10.1186/1476-4598-11-3) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
QW and AW participated in the design of the study. QW performed experiments, analyzed data, and drafted the manuscript. RJ provided tissue samples. All authors read and approved the final manuscript.
Carcinoma cells must circumvent the normally suppressive signals to disseminate. While often considered 'stop' signals for adherent cells, CXCR3-binding chemokines have recently been correlated positively with cancer progression though the molecular basis remains unclear.
Here, we examined the expression and function of two CXCR3 variants in human prostate cancer biopsies and cell lines. Globally, both CXCR3 mRNA and protein were elevated in localized and metastatic human cancer biopsies compared to normal. Additionally, CXCR3A mRNA level was upregulated while CXCR3B mRNA was downregulated in these prostate cancer specimens. In contrast to normal prostate epithelial cells (RWPE-1), CXCR3A was up to half the receptor in the invasive and metastatic DU-145 and PC-3 prostate cancer cells, but not in the localized LNCaP cells. Instead of inhibiting cell migration as in RWPE-1 cells, the CXCR3 ligands CXCL4/PF4 and CXCL10/IP10 promoted cell motility and invasiveness in both DU-145 and PC-3 cells via PLCβ3 and μ-calpain activation. CXCR3-mediated diminution of cell motility in RWPE-1 cells is likely a result of cAMP upregulation and m-calpain inhibition via CXCR3B signal transduction. Interestingly, overexpression of CXCR3B in DU-145 cells decreased cell movement and invasion.
These data suggest that the aberrant expression of CXCR3A and down-regulation of CXCR3B may switch a progression "stop" to a "go" signal to promote prostate tumor metastasis via stimulating cell migration and invasion.
Additional file 1: CXCR3 expression levels in prostate cancer metastases were not organ specific (*P > 0.5). The analyses were based on data shown in Figure 1B. Metastatic prostate cancer localized in different organs was grouped and graphed with percentages of CXCR3-positive cells, including 5 lymph node metastases, 4 liver metastases, 2 lung metastases and 1 adrenal metastasis. Statistical analyses were not available in comparison to adrenal group due to a small sample size. (PPT 112 KB)
Additional file 2: CXCR3 chemokine expression in normal and prostate cancer cells. CXCR3 chemokine expression in normal and prostate cancer cells. (A) Chemokine mRNA expression in normal and prostate cancer cells. Ligand mRNA expression was normalized to GAPDH mRNA expression. Histogram represents mean values (+/-s.d.) of three separate experiments (*P < 0.05 compared to RWPE-1 cells). (B) Chemokine protein expression in normal and prostate cancer cells. (PPT 228 KB)12943_2011_994_MOESM2_ESM.PPT
Additional file 3: CXCR3 localization in normal and prostate cancer cells detected by flow cytometry. (A) Cells were collected and treated with or without PBST (PBS with 0.5% Tween-20) to permeablize cell membrane, then further stained with CXCR3 or CXCR3B antibody for flow cytomery. Black: IgG only; Red: surface CXCR3; Green: surface and intracellular CXCR3. The graphs are representative results from more than three experiments. (B) Quantitative analysis of CXCR3 and CXCR3B localization in prostate cells based on graphs showing in (A). Histogram represents mean values (+/-s.d.) of three separate experiments (*P < 0.05 compared to RWPE-1 cells). (PPT 162 KB)12943_2011_994_MOESM3_ESM.PPT
Additional file 4: CXCR3-chemokine-induced cell migration was blocked by CXCR3 antibody in DU-145 cells. Cells were treated with chemokine with or without CXCR3 blocking antibody. Cell migration was measured by the distance change (quantified by pixel) in 16 hrs. Histogram represents mean values (+/-s.d.) of three separate experiments (*P < 0.05). (PPT 110 KB)12943_2011_994_MOESM4_ESM.PPT
Additional file 5: CXCR3B downregulation did not change DU-145 cell migration. CXCR3B was knocked down by siRNA and evaluated at (A)mRNA and (B) protein expression levels. Histogram represents mean values (+/-s.d.) of three separate experiments (*P < 0.05).(C) No change of cell migration was observed after CXCR3B downregulation. Cell migration was evaluated by the distance change (quantified by pixel) in 16 hrs. Histogram represents mean values (+/-s.d.) of three separate experiments. (PPT 224 KB)12943_2011_994_MOESM5_ESM.PPT
Additional file 6: m-calpain expression in prostate normal and cancer cells. No changes of m-calpain expression were observed after chemokine treatments (A) in normal and prostate cancer cells and (B) in DU-145 and DU-145 CXCR3 overexpressing cells. Each experiment was repeated with similar results. (PPT 201 KB)12943_2011_994_MOESM6_ESM.PPT
Additional file 7: PKA activity in prostate normal and cancer cells. PKA activity in prostate cancer cells was assessed by using a commercially available PepTag Assay kit (Promega, WI). Higher PKA activity was found in prostate cancer cells than normal prostate epithelial cells. Graph represents mean values (+/-s.d.) of three separate experiments (*P < 0.05). (PPT 78 KB)12943_2011_994_MOESM7_ESM.PPT
Additional file 8: MMP mRNA expression in prostate normal and cancer cells. (A) MMP2 and MMP9 mRNA expression in prostate normal and cancer cells (*P < 0.05 compared to RWPE-1 cells). MMP mRNA expression was normalized to GAPDH mRNA expression in each cell lines. (B) MMP2 and MMP9 mRNA expression after CXCR3 chemokine treatment in prostate normal and cancer cells. The mRNA expression levels in untreated RWPE-1 cells were set as 1. Graphs represent mean values (+/-s.e.m.) of three separate experiments each in triplicate (**P < 0.05 compared to untreated within the group). (PPT 141 KB)12943_2011_994_MOESM8_ESM.PPT
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- Altered CXCR3 isoform expression regulates prostate cancer cell migration and invasion
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