MAL expression downregulation through suppressive H3K27me3 marks at the promoter in HPV16-related cervical cancers is prognostically relevant and manifested by the interplay of novel MAL antisense long noncoding RNA AC103563.8, E7 oncoprotein and EZH2.

BACKGROUND: MAL (T-lymphocyte maturation-associated protein) is highly downregulated in most cancers, including cervical cancer (CaCx), attributable to promoter hypermethylation. Long noncoding RNA genes (lncGs) play pivotal roles in CaCx pathogenesis, by interacting with human papillomavirus (HPV)-encoded oncoproteins, and epigenetically regulating coding gene expression. Hence, we attempted to decipher the impact and underlying mechanisms of MAL downregulation in HPV16-related CaCx pathogenesis, by interrogating the interactive roles of MAL antisense lncRNA AC103563.8, E7 oncoprotein and PRC2 complex protein, EZH2. RESULTS: Employing strand-specific RNA-sequencing, we confirmed the downregulated expression of MAL in association with poor overall survival of CaCx patients bearing HPV16, along with its antisense long noncoding RNA (lncRNA) AC103563.8. The strength of positive correlation between MAL and AC103563.8 was significantly high among patients compared to normal individuals. While downregulated expression of MAL was significantly associated with poor overall survival of CaCx patients bearing HPV16, AC103563.8 did not reveal any such association. We confirmed the enrichment of chromatin suppressive mark, H3K27me3 at MAL promoter, using ChIP-qPCR in HPV16-positive SiHa cells. Subsequent E7 knockdown in such cells significantly increased MAL expression, concomitant with decreased EZH2 expression and H3K27me3 marks at MAL promoter. In silico analysis revealed that both E7 and EZH2 bear the potential of interacting with AC103563.8, at the same binding domain. RNA immunoprecipitation with anti-EZH2 and anti-E7 antibodies, respectively, and subsequent quantitative PCR analysis in E7-silenced and unperturbed SiHa cells confirmed the interaction of AC103563.8 with EZH2 and E7, respectively. Apparently, AC103563.8 seems to preclude EZH2 and bind with E7, failing to block EZH2 function in patients. Thereby, enhanced EZH2 expression in the presence of E7 could potentially inactivate the MAL promoter through H3K27me3 marks, corroborating our previous results of MAL expression downregulation in patients. CONCLUSION: AC103563.8-E7-EZH2 axis, therefore, appears to crucially regulate the expression of MAL, through chromatin inactivation in HPV16-CaCx pathogenesis, warranting therapeutic strategy development.

Unfurling the functional association between long intergenic noncoding RNAs (lincRNAs) and HPV16-related cervical cancer pathogenesis through weighted gene co-expression network analysis of differentially expressed lincRNAs and coding genes.

Long intergenic noncoding RNAs (lincRNAs) do not overlap annotated coding genes and are located in intergenic regions, as opposed to antisense and sense-intronic lncRNAs, located in genic regions. LincRNAs influence gene expression profiles and are thereby key to disease pathogenesis. In this study, we assessed the association between lincRNAs and HPV16-positive cervical cancer (CaCx) pathogenesis using weighted gene co-expression network analysis (WGCNA) with coding genes, comparing differentially expressed lincRNA and coding genes (DElincGs and DEcGs, respectively) in HPV16-positive patients with CaCx (n = 44) with those in HPV-negative healthy individuals (n = 34). Our analysis revealed five DElincG modules, co-expressing and correlating with DEcGs. We validated a substantial number of such module-specific correlations in the HPV16-positive cancer TCGA-CESC dataset. Four such modules, displayed significant correlations with patient traits, such as HPV16 physical status, lymph node involvement, and overall survival (OS), highlighting a collaborative effect of all genes within specific modules on traits. Using the DAVID bioinformatics knowledgebase, we identified the underlying biological processes associated with these modules as cancer development and progression-associated pathways. Next, we identified the top 10 DElincGs with the highest connectivity within each functional module. Focusing on the prognostic module hub genes, downregulated CTD-2619J13.13 expression was associated with poor patient OS. This lincRNA gene interacted with 25 coding genes of its module and was associated with such biological processes as keratinization loss and keratinocyte differentiation, reflecting severe disease phenotypes. This study has translational relevance in fighting various cancers with high mortality rates in underdeveloped countries.

Identification of HPV16 positive cervical cancer subsets characterized by divergent immune and oncogenic phenotypes with potential implications for immunotherapy.

BACKGROUND: Cervical cancers (CaCx), like many other cancer types, portray high molecular heterogeneity that affects response to therapy, including immunotherapy. In India and other developing countries, CaCx mortality rates are very high because women report to the clinics with advanced cancers in absence of organized screening programs. This calls for implementation of newer therapeutic regimens for CaCx, like immunotherapy, which is again not used commonly in such countries. OBJECTIVE: Therefore, we focused on dissecting tumour immune heterogeneity, if any, identify immune gene-based biomarkers of heterogeneity and subsets of such cancers with the potential for immunotherapy. We also attempted to characterize the cancer-associated phenotypes of such subsets, including viral load, to decipher the relationship of tumour immunogenicity with oncogenicity. METHODS: Employing RNA-seq analysis of 44 HPV16 positive CaCx patients, immune subtypes were identified by unsupervised hierarchical clustering of global immune-gene expression profiles. Proportions of tumor infiltrating immune cells in the tumor milieu were estimated, employing Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT), using gene expression data from RNA-seq. The oncogenic phenotypes of the immune subtypes of CaCx were deciphered through differential gene expression (DEGs) and pathway enrichment analysis. Viral load was estimated through TaqMan-based qRT-PCR analysis. RESULTS: Analysis revealed the presence of two immune subtypes of CaCx, A (26/44; 59.09%) and B (18/44; 40.90%). Compared to Subtype-A, Subtype-B portrayed overexpression of immune genes and high infiltration of immune cells, specifically CD8+ T cells (p < 0.0001). Besides, a significant correlation between PD-1 and PD-L1 co-expression among Subtype-B, as opposed to Subtype-A, confirmed the interactive roles of these immune checkpoint molecules in Subtype B. Stepwise discriminant analysis pin-pointed ten immune-genes that could classify 100% of the patients significantly (p < 0.0001) into the two immune subtypes and serve as potential biomarkers of CaCx immunity. Differential gene expression analysis between the subtypes unveiled that Subtype-B was more biologically aggressive than Subtype-A, reflecting loss of structural integrity and promotion of cancer progression. The viral load was significantly lower in Subtype-B (average viral load = 10.74/100 ng of genomic DNA) compared to Subtype-A (average viral load = 14.29/100 ng of genomic DNA). Thus viral load and the ten-gene panel underscore their association with immunogenicity and oncogenicity. CONCLUSION: Our study provides strong evidence that only a subset, about 41% of HPV16 positive CaCx patients in India, portray immune enrichment of the tumor milieu coupled with aggressive phenotypes. Such subtypes are therefore likely to benefit through checkpoint molecule-based or tumor infiltrating lymphocyte-based immunotherapy, which could be a leap forward in tackling aggressive forms of such CaCx in India and other developing countries.