# Title of Dataset: HPV upregulates MARCHF8 ubiquitin ligase and inhibits apoptosis by degrading the death receptors in head and neck cancer Since host cell survival is essential for viruses to replicate persistently, many viruses have evolved to prevent host cell apoptosis. The human papillomavirus (HPV) oncoproteins are known to dysregulate proapoptotic proteins. However, our understanding of detailed mechanisms for HPV to inhibit apoptosis is limited. Here, we report that HPV induces expression of the membrane-associated ubiquitin ligase MARCHF8, which is upregulated in HPV-positive head and neck cancer. MARCHF8 ubiquitinates the tumor necrosis factor receptor superfamily (TNFRSF) death receptors, FAS, TRAIL-R1, and TRAIL-R2 for degradation. We further revealed that downregulation of the death receptors by MARCHF8 prevents cancer cell apoptosis and that knockout of MARCHF8 expression significantly inhibits in vivo tumor growth and enhances tumor-free survival of mice transplanted with mouse oral cancer cells expressing HPV16 E6 and E7. These results suggest that virus-induced degradation of death receptors leads to cancer cell survival in HPV-positive head and neck cancer. ## Description of the Data and file structure FIG 1 Title: MARCHF8 expression is upregulated in HPV+ HNC. FIG 1A. MARCHF8 mRNA expression levels in microdissected human tissue samples from HPV+ (n = 16) and HPV- (n = 26) HNC patients, and normal individuals (n = 12) were analyzed using our previous gene expression data (GSE6791) and shown as fluorescence intensity (log2). FIG 1B. The MARCHF8 mRNA expression was validated in normal (N/Tert-1), HPV+ HNC (SCC2, SCC90, and SCC152), and HPV- HNC (SCC1, SCC9, and SCC19) cells by RT-qPCR. The data shown are ∆∆CT values normalized by the GAPDH mRNA level as an internal control. FIG 1C. MARCHF8 mRNA levels were determined in N/Tert-1 cells expressing E6 and/or E7 by RT-qPCR. The data shown are ∆∆CT values normalized by the GAPDH mRNA level as an internal control. FIG 1E. MARCHF8 protein levels (band density from western blotting imaging) were determined in HPV+ and HPV- HNC cells by western blotting. β-actin was used as a loading control. FIG 1G. MARCHF8 protein levels (band density from western blotting imaging) were determined in N/Tert-1 cells expressing HPV16 E6, E7, or E6 and E7 by western blotting. β-actin was used as a loading control. FIG 2 Title: The HPV oncoprotein E6 induces the MARCHF8 promoter activity mediated by the MYC/MAX complex. FIG 2B. The promoter-reporter constructs were transfected into HPV+ (SCC152) and HPV- (SCC1) cells. Luciferase activity was measured 48 h post-transfection. Data are shown as a fold change relative to the empty pGL4.2 vector (Basic). FIG 2D. The promoter-reporter constructs were cotransfected into SCC1 cells with HPV16 E6, E7, or E6 and E7 expression plasmids. Luciferase activity was measured 48 h post-transfection. Data are shown as a fold change relative to the empty pGL4.2 vector (Basic). FIG 2E. SCC152 cells were transfected with the 0.25 kb promoter (-90 to +160) reporter constructs of wildtype (WT) and E-box mutants containing single or double CG deletion in E-box1, E-box-2, and E-box1/2 (B and E). Luciferase activity was measured 48 h post-transfection. Data are shown as a fold change relative to the empty pGL4.2 vector (Basic). FIG 3 Title: Expression of FAS, TRAIL-R1, and TRAIL-R2 is downregulated in HPV+ HNC cells. FIG 3B. Total protein expression of FAS (FIG 3B1), TRAIL-R1 (FIG 3B2), and TRAIL-R2 (FIG 3B3) in HPV- (SCC1, SCC9, and SCC19) and HPV+ (SCC2, SCC90, and SCC152) HNC cells were determined by western blotting. FIG 3D. Cell surface expression of FAS proteins on HPV+ (SCC2, SCC90, and SCC152) and HPV- (SCC1, SCC9, and SCC19) HNC cells was analyzed by flow cytometry. FIG 3F. Cell surface expression of TRAIL-R1 proteins on HPV+ (SCC2, SCC90, and SCC152) and HPV- (SCC1, SCC9, and SCC19) HNC cells was analyzed by flow cytometry. FIG 3H. Cell surface expression of TRAIL-R2 proteins on HPV+ (SCC2, SCC90, and SCC152) and HPV- (SCC1, SCC9, and SCC19) HNC cells was analyzed by flow cytometry. FIG 3J. Cell surface expression of FAS proteins on N/Tert-1 cells expressing HPV16 E6, E7, and E6E7 was analyzed by flow cytometry. The values are Mean fluorescence intensities (MFI). FIG 3L. Cell surface expression of TRAIL-R1 proteins on N/Tert-1 cells expressing HPV16 E6, E7, and E6E7 was analyzed by flow cytometry. The values are Mean fluorescence intensities (MFI). FIG 3N. Cell surface expression of TRAIL-R2 proteins on N/Tert-1 cells expressing HPV16 E6, E7, and E6E7 was analyzed by flow cytometry. The values are Mean fluorescence intensities (MFI). FIG 4 Title: Knockdown of MARCHF8 expression increases FAS, TRAIL-R1, and TRAIL-R2 protein expression in HPV+ HNC cells. FIG 4B. HPV+ HNC (SCC152) cells were transduced with one of five lentiviral shRNAs against MARCHF8 (shR-MARCHF8 clones 1-5) or scrambled shRNA (shR-scr) as a control. Protein expression of MARCHF8 (FIG 4B1), FAS (FIG 4B2), TRAIL-R1 (FIG 4B3), and TRAIL-R2 (FIG 4B4) was determined by western blotting. FIG 4D. Cell surface expression of FAS proteins was analyzed by flow cytometry. The values are Mean fluorescence intensities (MFI). FIG 4F. Cell surface expression of TRAIL-R1 proteins was analyzed by flow cytometry. The values are Mean fluorescence intensities (MFI). FIG 4H. Cell surface expression of TRAIL-R2 proteins was analyzed by flow cytometry. The values are Mean fluorescence intensities (MFI). FIG 5 Title: MARCHF8 protein interacts with and ubiquitinates FAS, TRAIL-R1, and TRAIL-R2 proteins. FIG 5F. Ubiquitinated proteins were pulled down from the cell lysate of HPV+ HNC (SCC152) cells with scrambled shRNA (shR-scr) or shRNA against MARCHF8 (shR-MARCHF8 clone 3) treated with a proteasome inhibitor MG132 using an anti-ubiquitin antibody. FAS proteins were detected in the immunoprecipitated proteins by western blotting. FIG 5G. Ubiquitinated proteins were pulled down from the cell lysate of HPV+ HNC (SCC152) cells with scrambled shRNA (shR-scr) or shRNA against MARCHF8 (shR-MARCHF8 clone 3) treated with a proteasome inhibitor MG132 using an anti-ubiquitin antibody. TRAIL-R1 proteins were detected in the immunoprecipitated proteins by western blotting. FIG 5H. Ubiquitinated proteins were pulled down from the cell lysate of HPV+ HNC (SCC152) cells with scrambled shRNA (shR-scr) or shRNA against MARCHF8 (shR-MARCHF8 clone 3) treated with a proteasome inhibitor MG132 using an anti-ubiquitin antibody. TRAIL-R2 proteins were detected in the immunoprecipitated proteins by western blotting. FIG 6 Title: Knockdown of MARCHF8 enhances apoptosis of HPV+ HNC cells. FIG 6B. SCC152 cells with scrambled shRNA (shR-scr) or two shRNAs against MARCHF8 (shR-MARCHF8), were treated with an anti-human FAS antibody (Anti-FAS, clone EOS9.1, eBioscience) or the recombinant FAS ligand (rFAS-L, BioLegend #585404). The cells were stained with an anti-annexin V antibody and 7-AAD and analyzed by flow cytometry. FIG 6D. SCC2 cells with scrambled shRNA (shR-scr) or two shRNAs against MARCHF8 (shR-MARCHF8), were treated with an anti-human FAS antibody (Anti-FAS, clone EOS9.1, eBioscience) or the recombinant FAS ligand (rFAS-L, BioLegend #585404). The cells were stained with an anti-annexin V antibody and 7-AAD and analyzed by flow cytometry. FIG 7 Title: MARCHF8 is upregulated, and death receptor expression is downregulated in HPV+ mouse oral cancer cells. FIG 7B. Mouse MARCHF8 (FIG 7B1), FAS (FIG 7B2), TRAIL-R1 (FIG 7B3), and TRAIL-R2 (FIG 7B4) protein levels in mouse normal immortalized (NiMOE), HPV- transformed (HPV- MOE), and HPV+ transformed (HPV+ MOE) oral epithelial cells were determined by western blotting. β-actin was used as a loading control. FIG 7D. Cell surface expression of FAS proteins on NiMOE , HPV- MOE , and HPV+ MOE cells was analyzed by flow cytometry. FIG 7F. Cell surface expression of TRAIL-R2 proteins on NiMOE , HPV- MOE , and HPV+ MOE cells was analyzed by flow cytometry. FIG 8 Title: Knockout of Marchf8 expression increases FAS, TRAIL-R1, and TRAIL-R2 protein levels and enhances apoptosis of HPV+ mouse oral cancer cells. FIG 8B. mEERL cells were transduced with lentiviral Cas9 and one of two sgRNAs against Marchf8 (sgR-Marchf8-2 and sgR-Marchf8-3) or scrambled sgRNA (sgR-scr). Protein levels of MARCHF8 (FIG 8B1), FAS (FIG 8B2), TRAIL-R1 (FIG 8B3), and TRAIL-R2 (FIG 8B4) were determined by western blotting. FIG 8D. Cell surface expression of FAS proteins were analyzed by flow cytometry. FIG 8F. Cell surface expression of TRAIL-R2 proteins were analyzed by flow cytometry. FIG 8H. Untreated and rFAS-L-treated mEERL cells with sgR-Marchf8-2, sgR-Marchf8-3, or sgR-scr were stained with an anti-annexin V antibody and 7-AAD and analyzed by flow cytometry. FIG 9 Title: Knockout of Marchf8 expression suppresses HPV+ HNC tumor growth in vivo. Marchf8-knockout mEERL cells (mEERL/Marchf8-/-) were generated by lentiviral Cas9, and two sgRNAs targeting Marchf8 (sgR-Marchf8-2 and sgR-Marchf8-3) or scrambled sgRNA (sgR-scr). FIG 9A. mEERL/scr cells were injected into the rear right flank of C57BL/6J mice (n = 10 per group). Tumor volume was measured twice a week. FIG 9B and 9C. mEERL/Marchf8-/- (B and C) cells were injected into the rear right flank of C57BL/6J mice (n = 10 per group). Tumor volume was measured twice a week. FIG 9D. The data combined FIG 9A, 9B, and 9C. FIG 9E. Survival rates of mice were analyzed using a Kaplan-Meier estimator. FIG S1 Title: Protein and mRNA expression levels of HPV16 E6 and E7 in N/Tert-1 cells. FIG S1B. Total RNA was extracted from N/Tert-1 containing an empty vector and N/Tert-1 cells expressing HPV16 E6, E7, or E6 and E7 (E6E7). The HPV16 E6 mRNA expression levels were quantified by RT-qPCR. The data shown are normalized by the GAPDH mRNA level as an internal control. FIG S1C. Total RNA was extracted from N/Tert-1 containing an empty vector and N/Tert-1 cells expressing HPV16 E6, E7, or E6 and E7 (E6E7). The HPV16 E7 mRNA expression levels were quantified by RT-qPCR. The data shown are normalized by the GAPDH mRNA level as an internal control. FIG S2 Title: MARCHF8 expression is independent of p53 and E6AP and pRb binding domains of HPV16 E6 and E7, respectively. FIG S2A. MARCHF8 mRNA levels were determined in N/Tert-1 cells and N/Tert-1 cells expressing HPV16 E6, E6 8S9A10T, and E6 I128T or E7 and E7 ∆DLYC using RT-qPCR. FIG S2D. MARCHF8protein were determined in N/Tert-1 cells and N/Tert-1 cells expressing HPV16 E6, E6 8S9A10T, and E6 I128T or E7 and E7 ∆DLYC using western blotting. FIG S3 Title: The HPV oncoprotein E6 induces the MARCHF8 promoter activity independent of the p53 and E6AP binding domains. FIG S3B. The promoter-reporter construct (-1340 to +160) was transfected into HPV- (SCC1) cells and cotransfected with plasmids expressing wildtype E6, E6 Y54D, E6 I128T deficient in E6AP binding, wildtype E7, or E7 ∆DLYC deficient in pRb binding. Luciferase activity was measured 48 h post-transfection. The values are fold-changes relative to the empty pGL4.2 vector (Basic). FIG S4 Title: mRNA expression levels of FAS, TRAIL-R1, and TRAIL-R2 in HPV+ and HPV- cells. FIG S4A. The FAS mRNA expression levels in normal (N/Tert-1), HPV+ HNC (SCC2, SCC90, and SCC152), and HPV- HNC (SCC1, SCC9, and SCC19) cells were quantified by RT-qPCR. The data shown are normalized by the GAPDH mRNA level as an internal control. FIG S4B. The TRAIL-R1 mRNA expression levels in normal (N/Tert-1), HPV+ HNC (SCC2, SCC90, and SCC152), and HPV- HNC (SCC1, SCC9, and SCC19) cells were quantified by RT-qPCR. The data shown are normalized by the GAPDH mRNA level as an internal control. FIG S4C. The TRAIL-R2 mRNA expression levels in normal (N/Tert-1), HPV+ HNC (SCC2, SCC90, and SCC152), and HPV- HNC (SCC1, SCC9, and SCC19) cells were quantified by RT-qPCR. The data shown are normalized by the GAPDH mRNA level as an internal control. FIG S4D. The FAS mRNA expression levels in normal (N/Tert-1) and N/Tert-1 cells expressing HPV16 E6, E7, or E6 and E7 were quantified by RT-qPCR. The data shown are normalized by the GAPDH mRNA level as an internal control. FIG S4E. The TRAIL-R1 mRNA expression levels in normal (N/Tert-1) and N/Tert-1 cells expressing HPV16 E6, E7, or E6 and E7 were quantified by RT-qPCR. The data shown are normalized by the GAPDH mRNA level as an internal control. FIG S4F. The TRAIL-R2 mRNA expression levels in normal (N/Tert-1) and N/Tert-1 cells expressing HPV16 E6, E7, or E6 and E7 were quantified by RT-qPCR. The data shown are normalized by the GAPDH mRNA level as an internal control. FIG S5 Title: Knockdown of MARCHF8 expression increases FAS, TRAIL-R1, and TRAIL-R2 protein expression in HPV+ HNC cells. FIG S5B. HPV+ HNC (SCC2) cells were transduced with three lentiviral shRNAs against MARCHF8 (shR-MARCHF8 clones 3-5) along with scrambled shRNA (shR-scr). Protein expression of MARCHF8 (FIG S5B1), FAS (FIG S5B2), TRAIL-R1 (FIG S5B3), and TRAIL-R2 (FIG S5B4) was determined by western blotting. FIG S5D. Cell surface expression of FAS proteins was analyzed by flow cytometry. FIG S5F. Cell surface expression of TRAIL-R1 proteins was analyzed by flow cytometry. FIG S5H. Cell surface expression of TRAIL-R2 proteins was analyzed by flow cytometry. FIG S6 Title: mRNA expression levels of FAS, TRAIL-R1, and TRAIL-R2 by knockdown of MARCHF8 expression. FIG S6A. SCC152 cells were transduced with five and three lentiviral shRNAs against MARCHF8 (shR-MARCHF8), respectively, or scrambled shRNA (shR-scr). The mRNA levels of FAS were quantified by RT-qPCR. The data shown are normalized by the GAPDH mRNA level as an internal control. FIG S6B. SCC152 cells were transduced with five and three lentiviral shRNAs against MARCHF8 (shR-MARCHF8), respectively, or scrambled shRNA (shR-scr). The mRNA levels of TRAIL-R1 were quantified by RT-qPCR. The data shown are normalized by the GAPDH mRNA level as an internal control. FIG S6C. SCC152 cells were transduced with five and three lentiviral shRNAs against MARCHF8 (shR-MARCHF8), respectively, or scrambled shRNA (shR-scr). The mRNA levels of TRAIL-R2 were quantified by RT-qPCR. The data shown are normalized by the GAPDH mRNA level as an internal control. FIG S6D. SCC2 cells were transduced with five and three lentiviral shRNAs against MARCHF8 (shR-MARCHF8), respectively, or scrambled shRNA (shR-scr). The mRNA levels of FAS were quantified by RT-qPCR. The data shown are normalized by the GAPDH mRNA level as an internal control. FIG S6E. SCC2 cells were transduced with five and three lentiviral shRNAs against MARCHF8 (shR-MARCHF8), respectively, or scrambled shRNA (shR-scr). The mRNA levels of TRAIL-R1 were quantified by RT-qPCR. The data shown are normalized by the GAPDH mRNA level as an internal control. FIG S6F. SCC2 cells were transduced with five and three lentiviral shRNAs against MARCHF8 (shR-MARCHF8), respectively, or scrambled shRNA (shR-scr). The mRNA levels of TRAIL-R2 were quantified by RT-qPCR. The data shown are normalized by the GAPDH mRNA level as an internal control. ## Sharing/access Information None