HPV-transformed cells exhibit altered HMGB1-TLR4/MyD88-SARM1 signaling axis

Abstract

Cervical cancer is one of the leading causes of cancer death in women worldwide. Persistent infection with high-risk human papillomavirus (HPV) types is the main risk factor for the development of cervical cancer precursor lesions. HPV persistence and tumor development is usually characterized by innate immune system evasion. Alterations in Toll-like receptors (TLR) expression and activation may be important for the control of HPV infections and could play a role in the progression of lesions and tumors. In the present study, we analyzed the mRNA expression of 84 genes involved in TLR signaling pathways. We observed that 80% of the differentially expressed genes were downregulated in cervical cancer cell lines relative to normal keratinocytes. Major alterations were detected in genes coding for several proteins of the TLR signaling axis, including TLR adaptor molecules and genes associated with MAPK pathway, NFκB activation and antiviral immune response. In particular, we observed major alterations in the HMGB1-TLR4 signaling axis. Functional analysis also showed that HMGB1 expression is important for the proliferative and tumorigenic potential of cervical cancer cell lines. Taken together, these data indicate that alterations in TLR signaling pathways may play a role in the oncogenic potential of cells expressing HPV oncogenes.

Figure 1

Cervical cancer cell lines exhibit altered mRNA expression of genes involved TLR-signaling pathway. (A) Pathway network analysis of 47 TLR pathway genes differentially expressed in HPV positive tumor cell lines (HeLa and SiHa) relative to HPV negative tumor cells (C33A). Genes clustered by protein function and delimited by boxes (>2-fold or < −2-fold change and p value ≤ 0.05). Genes upregulated are red colored, genes downregulated are blue colored, and yellow-colored genes had opposite results in HeLa and SiHa cell lines (STRING: functional protein association networks, string-db.org/). (B) Biological processes distribution of genes differentially expressed in HPV positive tumor cell lines relative to HPV negative tumor cells. (WEB-based GEne SeT AnaLysis Toolkit, www.webgestalt.org/).

Figure 2

Cervical cancer cell lines and PHK expressing HPV16 oncogenes exhibit alterations in the expression of TLRs related proteins. (A) Tumor-derived cell lines C33A, SiHa, and HeLa, (B) PHK expressing HPV16 oncogenes E6 and/or E7. Protein levels were determined by Western blot analysis from cell culture extracts. Signals obtained were quantified using ImageJ software and are reported as expression relative to normal keratinocytes (PHK). Full-length blots are presented in a Supplementary Figure.

Figure 3

HMGB1 is upregulated in epithelial rafts expressing HPV16 E7 and in squamous cell carcinomas. The expression of HMGB1 was determined by immunohistochemistry in organotypic cultures sections of (A) control; (B) HPV16 E6 expressing; (C) HPV16 E7 expressing and (D) with HPV16 E6E7 expressing keratinocytes and in squamous cell carcinomas (E,F) sample 1 and (G,H) sample 2. Note limit area between normal epithelium and high-grade lesion (E,G).

Figure 4

HMGB1 silencing selectively inhibits cell viability and proliferation of SiHa and HeLa cell lines. (A) Viability of silenced cell lines was determined by AlamarBlue reduction. Cells were seeded in 96-well plates (2000 cells/well) and infected with lentiviral particles expressing specific shRNAs. After five days, AlamarBlue was added (10 µl/well) and reagent reduction was determined by absorbance measurement after four hours. Viability inhibition values for each cell line are reported as relative to those observed in cells transduced with a scrambled shRNA. (B,C) HMGB1 silencing has no effect on normal keratinocytes proliferation (B), but inhibits SiHa and HeLa growth (C), as well as, PHK expressing HPV16 E6E7 or only E7 (D), as determined by proliferation curves. Cells previously transduced with lentivirus vectors for silencing HMGB1 were seeded (1000 cells/well in 24-well plates) and the number of cells was counted in triplicate for eight days. (Student’s t test, p ≤ 0.05).

Figure 5

HMGB1 silencing affects the clonogenic potential of cervical cancer-derived cells. A lentiviral shRNA (MISSION®) was used specifically to silence HMGB1 in cervical cancer cell lines. After antibiotic selection, cells were seeded for colony formation assays. (A) Inhibition of colony formation in low-density assay. Cells were seeded in 6-well plates (100 cells/well); after 14 days, cells were fixed and the number of colonies was counted. (B) Inhibition of colony formation on soft agar in anchorage-independent growth assay. Cells were seeded in 24-well plates coated with agarose (500 cells/well). After 30 days, colonies were stained with MTT and counted. (Student’s t test, p ≤ 0.05).