Downregulation of SMG-1 in HPV-positive head and neck squamous cell carcinoma due to promoter hypermethylation correlates with improved survival

Abstract

Purpose: Human papillomavirus (HPV) is linked with a subset of head and neck squamous cell carcinomas (HNSCC). HPV-positive HNSCCs show a better prognosis than HPV-negative HNSCCs, which may be explained by sensitivity of the HPV-positive HNSCCs to ionizing radiation (IR). Although the molecular mechanism behind sensitivity to IR in HPV-positive HNSCCs is unresolved, DNA damage response (DDR) might be a significant determinant of IR sensitivity. An important player in the DDR, SMG-1 (suppressor with morphogenetic effect on genitalia), is a potential tumor suppressor and may therefore be deregulated in cancer. No studies have yet been conducted linking defects in SMG-1 expression with cancer. We investigated whether deregulation of SMG-1 could be responsible for defects in the DDR in oropharyngeal HNSCC.

Experimental design: Expression and promoter methylation status of SMG-1 were investigated in HNSCCs. To identify a functional link between HPV infection and SMG-1, we transfected the HPV-negative cells with an E6/E7 expression construct. SMG-1 short hairpin RNAs were expressed in HPV-negative cells to estimate survival upon IR.

Results: Forced E6/E7 expression in HPV-negative cells resulted in SMG-1 promoter hypermethylation and decreased SMG-1 expression. Due to promoter hypermethylation, HPV-positive HNSCC cells and tumors express SMG-1 at lower levels than HPV-negative SCCs. Depletion of SMG-1 in HPV-negative HNSCC cells resulted in increased radiation sensitivity, whereas SMG-1 overexpression protected HPV-positive tumor cells from irradiation.

Conclusions: Levels of SMG-1 expression negatively correlated with HPV status in cancer cell lines and tumors. Diminished SMG-1 expression may contribute to the enhanced response to therapy exhibited by HPV-positive HNSCCs.

Figure 1. SMG-1, but not ATM, is expressed at lower levels in HPV-positive primary oropharyngeal HNSCC

(A) IHC analysis of tissue microarray (TMA) of primary HNSCC using antibodies against SMG-1. Representative images of TMA cores are presented, showing negative, weak, moderate and strong staining intensities. (B) Percentage of specimens with different SMG-1 staining intensities. Chi-square analysis was used to calculate p-value. (C) Western blot showing SMG-1 levels in lysates from HPV-positive and HPV-negative HNSCC tumors. (D) Scatter plots of expression levels of SMG-1 mRNA (left) and ATM mRNA (right) relative to normal uvula in HPV-positive and HPV-negative HNSCC tumors as assessed by qRT-PCR analysis.

Figure 2. Regulation of SMG-1 expression by HPV-16 E6/E7

(A) SCC61 cells were transiently transfected with empty vector or construct expressing HPV-16 E6/E7 (SCC61 E6/E7) and SMG-1 mRNA levels relative to normal human keratinocytes were analyzed by qRT-PCR. (B) Western blot showing SMG-1 protein levels in keratinocytes transfected with vector expressing HPV-16 E6/E7 or empty vector. (C) UMSCC47 cells were transduced with two individual E6 shRNAs (sh1 and sh2) or empty vector and analyzed by Western blotting (left panel). Downregulation of E6 was confirmed by qRT-PCR (right panel). (D) Left panel shows expression of E6 in SCC61 cells stably transfected with E6/E7 expression vector (SCC61 E6/E7) compared to cells harboring empty vector (SCC61v) as confirmed by qRT-PCR. Middle panel shows relative to parental SCC61 cells SMG-1 mRNA levels in SCC61 E6/E7 and SCC61v as assessed by qRT-PCR analysis. Right panel depicts Western blot showing SMG-1 protein levels in SCC61 E6/E7, SCC61v, and parental SCC61 cells. Error bars represent SD.

Figure 3. Expression of HPV-16 E6/E7 leads to SMG-1 promoter methylation

(A) SCC61 cells expressing HPV-16 E6/E7 (SCC61 E6/E7) or empty vector (SCC61v) were treated with azacitidine (aza) and analyzed by qRT-PCR for SMG1 expression. Data was normalized to value obtained for SCC61v cells. Error bars represent SD. (B) Percentage of hypermethylation in the SMG-1 promoter in SCC61v and SCC61 E6/E7 cells measured by the EpiTect Methyl DNA Restriction Kit. (C) Percentage of hypermethylation in the SMG1 promoter in HPV-positive and –negative oropharyngeal HNSCC patients measured using the same method as in B (left panel). SMG-1 mRNA expression in these patients was determined by qRT-PCR (right panel).

Figure 4. HPV correlates with improved survival and low SMG-1 expression levels

Kaplan-Meier curves showing disease-free survival (A) and overall survival (B) of patients with HPV-positive and HPV-negative HNSCC (left panel), as well as patients with low and moderate-high SMG-1 expression (right panel).

Figure 5. SMG-1 downregulation in HNSCC cells results in increased sensitivity to IR

(A) Clonogenic survival assay following treatment with increasing doses of ionizing radiation of HPV-positive (UMSCC47 and SCC090) and HPV-negative (JHU012, SCC61 and SCC25) HNSCC cell lines. Error bars represent SD. (B) Western blot showing SMG-1 depletion by two individual shRNAs (sh1 and sh2) in JHU012 and SCC61 cells compared to cells transduced with empty vector. (C) Clonogenic survival assay following treatment with increasing doses of ionizing radiation of JHU012 cells described in B. (D) Clonogenic survival assay following treatment with increasing doses of ionizing radiation of SCC61 cells described in B. Error bars represent SD.