Systematic Analysis of IL-1 Cytokine Signaling Suppression by HPV16 Oncoproteins

Abstract

The human papillomavirus (HPV) E6 and E7 oncogenes are expressed at all stages of HPV-mediated carcinogenesis and are essential drivers of cancers caused by high-risk HPV. Some of the activities of HPV E6 and E7, such as their interactions with host cellular tumor suppressors, have been characterized extensively. There is less information about how high-risk HPV E6 and E7 alter cellular responses to cytokines that are present in HPV-infected tissues and are an important component of the tumor microenvironment. We used several models of HPV oncoprotein activity to assess how HPV16 E6 and E7 alter the cellular response to the proinflammatory cytokine IL-1β. Models of early stage HPV infection and of established HPV-positive head and neck cancers exhibited similar dysregulation of IL-1 pathway genes and suppressed transcriptional responses to IL-1β treatment. Such overlap in cell responses supports that changes induced by HPV16 E6 and E7 early in infection could persist and contribute to a dysregulated immune environment throughout carcinogenesis. HPV16 E6 and E7 also drove the upregulation of several suppressors of IL-1 cytokine signaling, including SIGIRR, both in primary keratinocytes and in cancer cells. SIGIRR knockout was insufficient to increase IL-1β-dependent gene expression in the presence of HPV16 E6 and E7, suggesting that multiple suppressors of IL-1 signaling contribute to dampened IL-1 responses in HPV16-positive cells. IMPORTANCE Human papillomavirus (HPV) infection is responsible for nearly 5% of the worldwide cancer burden. HPV-positive tumors develop over years to decades in tissues that are subject to frequent stimulation by proinflammatory cytokines. However, the effects of HPV oncoproteins on the cellular response to cytokine stimulation are not well defined. We analyzed IL-1 cytokine signaling in several models of HPV biology and disease. We found that HPV16 E6 and E7 oncoproteins mediate a broad and potent suppression of cellular responses to IL-1β in models of both early and late stages of carcinogenesis. Our data provide a resource for future investigation of IL-1 signaling in HPV-positive cells and cancers.

Keywords: HPV; IL-1; carcinogenesis; cell growth; cytokine.

FIG 1

HPV16 oncoproteins dysregulate expression of genes involved in IL-1 signaling. (A) Primary human foreskin keratinocytes (HFK) were transduced with retroviruses encoding HPV16 E6 and HPV16 E7 or with matched GFP control retroviruses and selected with appropriate antibiotics. RNA-seq and bioinformatic analysis was performed on polyA-selected RNA from triplicate cell lines. Heat map and fold change values indicate differential expression of IL-1 pathway cytokines and regulatory proteins. Fold change values are indicated for genes that were significantly differentially expressed (fold change > 1.5, adjusted P value ≤ 0.05) in the RNA-seq analysis. (B) RNA from the same cell lines was analyzed by qRT-PCR using primers specific for HPV16 E6, HPV16 E7, or SIGIRR. Graphs display mean ± SD of the relative RNA level (versus GAPDH for HPV16 E6 and HPV16 E7, versus GAPDH for SIGIRR), and dots represent individual cell lines. Statistical significance was assessed using an unpaired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Protein lysates from four of the six cell lines in panels A and B were separated by SDS-PAGE and analyzed in Western blots with antibodies to SIGIRR and GAPDH.

FIG 2

Genes related to IL-1 signaling are differentially expressed in HPV-positive versus HPV-negative oropharyngeal squamous cell carcinomas (OPSCC). Gene expression data from 28 HPV-negative and 53 HPV-positive OPSCC in The Cancer Genome Atlas (TCGA) was accessed using cBioPortal. (A) Heat map displays summary mRNA expression data for selected genes encoding IL-1-related cytokines and IL-1 regulatory proteins. (B and C) violin plots display individual sample data from the same OPSCC for selected differentially expressed cytokines (B) and selected differentially expressed negative regulators (C). Solid horizontal bar indicates median, and dashed horizontal bars indicate top and bottom quartiles. Statistical significance was assessed using an unpaired t test with Welch's correction. **, P < 0.01; ****, P < 0.0001.

FIG 3

Differential expression of IL-1 regulatory genes in HPV-negative and HPV-positive patient-derived xenografts. Total RNA was purified from 11 HPV-negative and 8 HPV-positive patient-derived xenografts (PDX) and qRT-PCR was used to assess gene expression of IL1A and IL1B (A) or negative regulators of IL-1 signaling SIGIRR and IL1R2 (B). Data are expressed relative to G6PD expression. Statistical significance was determined using unpaired t test with Welch’s correction. P values of <0.1 are indicated; ns, not significant.

FIG 4

Differential expression of IL-1 family cytokines and IL-1 regulators in HPV-negative and HPV-positive HNSCC cell lines. (A and B) Total RNA was purified from four passages each of two HPV-negative (SCC4, SCC15) and four HPV-positive (SCC47, SCC90, SCC152, VU147T) HNSCC cell lines. qRT-PCR was used to assess gene expression of IL1A and IL1B (A) or selected negative regulators of IL-1 signaling (B). Data are expressed relative to G6PD expression. Statistical significance was determined using ordinary one-way ANOVA with Sidak’s multiple-comparison test. *, P < 0.05; **, P < 0.01. (C) Protein lysates from the cell lines in panels A and B were separated by SDS-PAGE and analyzed in Western blots with antibodies to SIGIRR and GAPDH.

FIG 5

HPV16 oncoproteins suppress the transcriptional response to IL-1β. Primary HFK stably expressing HPV16 E6 and HPV16 E7 or matched GFP control cells were treated with 0.5 ng/mL IL-1β for 3 h or left untreated. RNA-seq and bioinformatic analysis was performed on polyA-selected RNA from triplicate cell populations. (A) Heat map displays expression values for genes that are differentially expressed by ≥2.5-fold in GFP cells treated with IL-1β compared to untreated GFP cells. Gene Ontology (GO) analysis was performed on clusters of differentially expressed genes using DAVID. (B) Venn diagram indicates overlap between genes upregulated ≥1.5-fold upon IL-1β treatment in GFP cells or HPV16E6/E7 cells. (C) Heat maps display expression data for genes associated with selected GO terms.

FIG 6

HPV16 E6/E7 alleviate the growth-suppressive effect of IL-1β on primary keratinocytes. Primary HFK were transduced with retroviruses encoding HPV16 E6 and E7 or matched GFP controls. Each cell population was cultured with or without 0.5 ng/mL IL-1β for 30 days and population doublings were tracked with each passage. Graph displays the mean ± SD of three replicate cell populations per condition. Statistical significance was determined by two-way ANOVA with Tukey’s multiple-comparison test (ns, not significant; *, P < 0.05; **, P < 0.01).

FIG 7

HPV-negative HNSCC cell lines are more responsive to IL-1β treatment than HPV-positive HNSCC cell lines. (A to C) Two HPV-negative (SCC4, SCC15) and four HPV-positive (SCC47, SCC90, SCC152, VU147T) HNSCC cell lines were treated with 0.5 ng/mL IL-1β for 3 h or left untreated. qRT-PCR was used to assess gene expression of CXCL8 (A), CXCL1 (B), or SDC4 (C). Data are expressed relative to G6PD (for CXCL8) or GAPDH (for CXCL1 and SDC4). Statistical significance was determined by unpaired t test (*, P < 0.05 or as indicated).

FIG 8

SIGIRR is not the primary driver of the suppressed response to IL-1β in cells expressing HPV16 E6/E7. (A) HFK expressing HPV16 E6 and E7 were transduced with LentiCRISPRv2 vectors encoding a nontargeting (NT-2) sgRNA or an sgRNA targeting SIGIRR. SIGIRR depletion was confirmed by Western blotting. (B) Cells were treated with 0.5 ng/mL IL-1β for 3 h or left untreated. qRT-PCR was used to assess gene expression of CXCL8 or CXCL1. Data are expressed relative to GAPDH expression. (C) HPV-positive SCC47 cells were transduced with LentiCRISPRv2 vectors encoding nontargeting (NT) sgRNAs or sgRNAs targeting SIGIRR. SIGIRR depletion was confirmed by Western blotting. (D) Cells were treated with 0.5 ng/mL IL-1β for 3 h or left untreated. qRT-PCR was used to assess gene expression of CXCL8 or CXCL1. Data are expressed relative to GAPDH expression.