Human cytomegalovirus infection enhances cell proliferation, migration and upregulation of EMT markers in colorectal cancer-derived stem cell-like cells

Abstract

Increasing evidence suggests a link between persistent human cytomegalovirus (HCMV) infection and cancer. Although the role of HCMV in cancer is still elusive, recent studies revealed the presence of HCMV nucleic acids and proteins in different cancer types such as glioblastoma, colorectal, breast, and prostate cancers, and neuroblastoma. Although HCMV may not be directly associated with the neoplastic transformation, the presence of HCMV DNA in the tumorous tissue has been associated with altered clinical outcomes in cancer patients. However, the mechanisms involved in the association between colorectal cancer (CRC) and HCMV are unclear. In this study, we investigated the influence of HCMV infection on CRC or their derived cells. Proliferation and migration assays revealed a high infection efficiency in CRC-derived HT29 and SW480 'stem‑like' cells. After 24, 48 and 72 h of HCMV infection, both HT29 and SW480 parental and stem‑like cells showed a significant increase in cell proliferation and viability (p<0.0001). Moreover, HCMV infection promoted cell migration. These results demonstrate a significant phenotypic alteration in the CRC cell line upon HCMV infection. Using epithelial to mesenchymal transition (EMT) assays, we demonstrated that the EMT markers and driver genes were upregulated during the virus infection. The WNT signaling pathway, which is associated with the proliferation and migration of CRC cells, was upregulated (6-fold) in HCMV-infected cells as compared to the non‑infected cells at day 7 from infection.

Figure 1

Generation of sphere from HT29 parental. (A) HT29 parental and stem-like cells. (B) Determination of the stemness marker CD44-FITC.

Figure 2

The infection of HCMV laboratory strain AD169 in colorectal cancer-derived cells. (A) HCMV AD169 was used to infect 10³ parental and stem-like HT29 and MRC-5 (control) cells at MOI of 5 on coverslips, followed by IE and (B) pp65 immunofluorescence. (C) IE-positive cells were observed after counter staining using a fluorescence microscope fitted with a camera. Five low magnification fields were counted for each condition. (D) The percentage of positive cells was calculated by dividing the number of IE-positive cells with the total number of nuclei, followed by multiplication with 100. ^****p<0.0001 by two-way ANOVA and Tukey's multiple comparisons test.

Figure 3

HCMV gene expression. (A) RT-PCR detection of IE1, US28, and β 2.7 kb transcripts in HACMV AD169-infected HT29 stem-like cells. M, 100 bp DNA ladder; P, positive control; and N, negative control. (B) Semi-quantitative SYBR Green-based RT-qPCR of IE1, US28, and β 2.7 kb at different time-points following HCMV AD169 infection of HT29 stem-like cells. ^****p<0.0001 by two-way ANOVA and Tukey's multiple comparisons test. (C) Determination of HCMV AD169 long-term infection in HT29 stem-like cells (4 weeks).

Figure 4

Cell viability and proliferation of AD169-infected and non-infected colorectal cancer-derived cells. HCMV AD169 was used to infect 10² parental and stem-like (A) HT29 cells and (B) SW480 cells at MOI of 5. Cells were harvested at 6, 12 24, 48 and 72 h following infection. Cells were subjected to WST-1 assays. AD169-infected cells showed significant proliferation compared to the non-infected cells. ^**p<0.01, ^**p<0.001 and ^***p<0.0001 by Student's t-test. For direct cell proliferation evaluation, 10⁴ parental and stem-like (C) HT29 cells and (D) SW480 cells were infected with AD169 at MOI of 5. Cells were harvested at 3, 6, 12 24, 48 and 72 h following infection and stained with 0.4% trypan blue. Cells were counted using the Countess Automated Cell Counter. ^*p<0.05, ^**p<0.01, and ^****p<0.0001 by two-way ANOVA and Tukey's multiple comparisons test.

Figure 5

Migration assay of AD169-infected and non-infected cells. Parental and stem-like (A) HT29 cells and (B) SW480 cells were treated with or without HCMV AD169 at MOI of 5. After 72 h of infection, 10⁴ cells were translocated in a 24-Transwell chamber with non-coated membrane and incubated for 6, 12 24, 48 and 72 h. The cells under the membrane were stained with crystal violet and counted under the microscope. ^***p<0.001 and ^****p<0.0001 by two-way ANOVA and Tukey's multiple comparisons test.

Figure 6

Human EMT RT² Profiler PCR Array of AD169-infected and non-infected cells. (A) HT29 stem-like cells were treated with or without HCMV AD169 at MOI of 5. After different time-points, cells were harvested and the total RNA was extracted and converted to cDNA. The hierarchical clustering of gene signatures was determined using RT² Profiler PCR Array of EMT and illustrated as heat maps (cut off value >2). (B) The expression of EMT markers and drivers gene of HT29 stem-like cells infected with AD169. (C) Expression of genes related to the WNT signaling pathway in HT29 stem-like cells infected with AD169. (D) Confirmation of WNT11/FZD7 expression in HT29 stem-like cells infected with AD169 and non-infected cells. ^*p<0.05, ^**p<0.01, and ^****p<0.0001 by two-way ANOVA and Tukey's multiple comparisons test.