JC virus mediates invasion and migration in colorectal metastasis

Abstract

Introduction: JC Virus (JCV), a human polyomavirus, is frequently present in colorectal cancers (CRCs). JCV large T-Ag (T-Ag) expressed in approximately half of all CRC's, however, its functional role in CRC is poorly understood. We hypothesized that JCV T-Ag may mediate metastasis in CRC cells through increased migration and invasion.

Material and methods: CRC cell lines (HCT116 and SW837) were stably transfected with JCV early transcript sequences cloned into pCR3 or empty vectors. Migration and invasion assays were performed using Boyden chambers. Global gene expression analysis was performed to identify genetic targets and pathways altered by T-Ag expression. Microarray results were validated by qRT-PCR, protein expression analyses and immunohistochemistry. Matching primary CRCs and liver metastases from 33 patients were analyzed for T-Ag expression by immunohistochemistry.

Results: T-Ag expressing cell lines showed 2 to 3-fold increase in migration and invasion compared to controls. JCV T-Ag expression resulted in differential expression of several genetic targets, including genes that mediate cell migration and invasion. Pathway analysis suggested a significant involvement of these genes with AKT and MAPK signaling. Treatment with selective PI3K/AKT and MAPK pathway inhibitors resulted in reduced migration and invasion. In support of our in-vitro results, immunohistochemical staining of the advanced stage tumors revealed frequent JCV T-Ag expression in metastatic primary tumors (92%) as well as in their matching liver metastasis (73%).

Conclusion: These data suggest that JCV T-Ag expression in CRC associates with a metastatic phenotype, which may partly be mediated through the AKT/MAPK signaling pathway. Frequent expression of JCV T-Ag in CRC liver metastasis provides further clues supporting a mechanistic role for JCV as a possible mediator of cellular motility and invasion in CRC.

Figure 1. JCV T-Ag expression in transfected colorectal cancer cells.

(A) An illustration of the JCV early transcript region that codes for 5 early transforming proteins, T-Ag, t-Ag and the 3 splice variants T'₁₆₅, T'₁₃₆, T'₁₃₅. T-Ag is predominant protein expressed after transfection (marked in red). (B) RT-PCR and Western immunoblotting gel images depicting JCV T-Ag specific mRNA and protein expression in stably transfected cells (indicated as T-Ag), while no T-Ag expression was observed in control cell lines (V, vector transfected). GAPDH (RT-PCR) and β-actin (WB) were used as loading controls. (C) Immunofluorescence staining with JCV T-Ag antibody shows nuclear expression of JCV T-Ag in transfected HCT116 and SW837 cells lines. The images were taken at a final magnification of 630×.

Figure 2. JCV T-Ag increases migration and invasion in vitro.

Migration and invasion assays were performed in Boyden chambers without and with Matrigel respectively. Representative images from one of the three independent experiments showing migration (A) and invasion (B) in control and T-Ag transfected HCT116 and SW837 cell lines (200× magnification). The bar graphs in panels B and D are quantitative determinations of data obtained using ImageJ cell counter software from 3 independent experiments. As indicated, T-Ag transfection (red and blue bars) showed significantly increased migration and invasion (p<0.05) in both cell lines.

Figure 3. Global gene expression analysis of JCV T-Ag transfected cells.

(A) The flow chart represents the strategy used for gene expression analysis. Using unsupervised analysis we selected 5559 genes. Dendograms in panels B and C illustrate clustering analysis of JCV-T-Ag transfected cells compared to vector controls. Gene trees are represented on the horizontal axis, while condition trees are represented on the vertical axis. The color conventions for all maps are as follows: red indicates over-expressed transcripts, blue are under-expressed transcripts, and yellow indicates transcripts that did not deviate from the controls. Clustering analysis in panel B illustrates Ingenuity pathway analysis which revealed 529 genes that are involved in regulation of cell motility. Of these, a subset of 43 genes that are specifically involved in migration or invasion and were up- or down-regulated in both cell lines are shown in panels C (as dendogram) and D (Venn diagram). Of this group of 43 genes, 20 genes that are directly or indirectly involved in AKT and MAPK pathways were shared between both HCT116 and SW837 cells as shown in panel E (Venn diagram) and panel F (as a schematic interaction of individual genes with each other). Red indicates up-regulated and green indicates down-regulated genes following T-Ag transfection (panel F).

Figure 4. Gene expression changes following T-Ag transfection in HCT116 cells.

(A) Microarray global gene expression data were validated in a randomly selected subset of genes by quantitative RT-PCR. Data are represented as the mean fold change normalized to vector cells. As indicated, there was a tight correlation between microarray and qRT-PCR in each instance. (B) A subset of cancer-metastasis related genes including MMP-9, uPA, CCL5 and NOS3 showed increased expression following T-Ag transfection in HCT116 cells. (C) Western blotting data indicated increased phosphorylation of AKT as well as ERK1/2 in T-Ag transfected cells in comparison to control cell lines.

Figure 5. PI3K and MAPK pathway inhibitors reduced migration and invasion of JCV T-Ag transfected cells.

Migration (panels A and B) and invasion (panels C and D) were performed using Boyden chambers without or with Matrigel. LY294002 (25 µM) and U0126 (10 µM) were added to both upper and lower chambers. Cells that invaded through Matrigel, and migrated through the 8 µm pores in the membrane were stained with DAPI. Cell counting was performed with ImageJ software. Data in bar graphs demonstrate fold changes normalized to vector cells (means±SE) obtained from 3 independent experiments. Significant inhibition in both migration and invasion was observed in both HCT116 and SW837 cells with LY294002 and U0126 individually and in combination.

Figure 6. JCV T-Ag expression in primary CRC tumors and in liver metastasis.

Staining was performed with Pab416 antibody against SV40 T-Ag with cross reactivity to JCV T-Ag. Images were obtained at 100× and 400× magnification (as shown in the insets within each photomicrograph) (A) Positive control staining from hamster brain tissue infected with JC virus shows strong nuclear T-Ag expression. Normal colonic mucosa (B) and liver hepatocytes (C) showed no JCV T-Ag expression and were used as negative controls with each staining. (D) A representative example of a CRC and colonic mucosa with no T-Ag expression. (E) A photomicrograph indicating strong nuclear T-Ag expression in invasive primary colonic tumor (F) The image illustrates T-Ag-specific expression in liver metastasis in cancer cells but no expression in surrounding normal liver hepatocytes.