Human Papillomavirus 16 E6 and E7 Oncoproteins Alter the Abundance of Proteins Associated with DNA Damage Response, Immune Signaling and Epidermal Differentiation

Abstract

The high-risk human papillomaviruses are oncogenic viruses associated with almost all cases of cervical carcinomas, and increasing numbers of anal, and oral cancers. Two oncogenic HPV proteins, E6 and E7, are capable of immortalizing keratinocytes and are required for HPV associated cell transformation. Currently, the influence of these oncoproteins on the global regulation of the host proteome is not well defined. Liquid chromatography coupled with quantitative tandem mass spectrometry using isobaric-tagged peptides was used to investigate the effects of the HPV16 oncoproteins E6 and E7 on protein levels in human neonatal keratinocytes (HEKn). Pathway and gene ontology enrichment analyses revealed that the cells expressing the HPV oncoproteins have elevated levels of proteins related to interferon response, inflammation and DNA damage response, while the proteins related to cell organization and epithelial development are downregulated. This study identifies dysregulated pathways and potential biomarkers associated with HPV oncoproteins in primary keratinocytes which may have therapeutic implications. Most notably, DNA damage response pathways, DNA replication, and interferon signaling pathways were affected in cells transduced with HPV16 E6 and E7 lentiviruses. Moreover, proteins associated with cell organization and differentiation were significantly downregulated in keratinocytes expressing HPV16 E6 + E7. High-risk HPV E6 and E7 oncoproteins are necessary for the HPV-associated transformation of keratinocytes. However their influence on the global dysregulation of keratinocyte proteome is not well documented. Here shotgun proteomics using TMT-labeling detected over 2500 significantly dysregulated proteins associated with E6 and E7 expression. Networks of proteins related to interferon response, inflammation and DNA damage repair pathways were altered.

Keywords: E6 protein; E7 protein; human papillomavirus; keratinocytes; proteomics; transformation.

Figure 1

(a) Western blot experiments showed that hTERT levels were elevated in cells transduced with HPV16 E6 and E7 lentiviruses, compared to control cells (LV-HEKn). (b) Bar charts indicate the average level of hTERT normalized to GAPDH for three replicates of each cell line.

Figure 2

Protein levels in HPV16 E6 + E7 cells and LV-HEKn control cells displayed similar trends in Western blots and LC-MS/MS experiments. (a) Western blots of UCHL1 and BST2 in HPV16 E6 + E7 cells and LV-HEKn control cells. (b) The grey bars represent the abundance of protein measured by Western blot relative to the abundance of load control protein. The red line represents the LC-MS/MS measurements in the two cell lines. Bar charts indicate the average of three replicates of each cell line.

Figure 3

(a) Western blot experiments showed that p53 levels were reduced in cells transduced with HPV16 E6 and E7 lentiviruses compared to the control cells (LV-HEKn). (b) Bar charts indicate the average level of p53 normalized to GAPDH for three replicates of each cell line.

Figure 4

Biological GO terms related to defense immune responses were associated with the proteins with significantly increased expression (adjusted p-value ≤ 0.02) by ≥±1.5-fold change in keratinocytes transduced with HPV16 E6 and E7. FDR (False discovery rate).

Figure 5

Biological GO terms related to DNA damage and apoptosis were associated with the proteins with significantly increased expression (adjusted p-value ≤ 0.02) by ≥±1.5-fold change in keratinocytes transduced with HPV16 E6 and E7. FDR (False discovery rate).

Figure 6

Thirty two- biological GO terms associated with epidermal differentiation, adhesion and cytoskeletal organization were predicted to be related to proteins with a reduced expression by ≥±1.5-fold change (adjusted p-value ≤ 0.02) in keratinocytes transduced with HPV16 E6 and E7 FDR (False discovery rate).

Figure 7

Canonical pathways predicted to be affected in HEKn cells expressing HPV16 E6 and E7 oncoproteins. Included in this analysis were proteins significantly differentially expressed (adjusted p-value ≤ 0.02) by ≥±1.5-fold change. Black shading indicates a predicted increase in activity and grey shading indicates a predicted inhibition of canonical pathway, based on current evidence in the Qiagen Ingenuity Pathway database.