Noncanonical HPV carcinogenesis drives radiosensitization of head and neck tumors.

We analyzed transcriptional data from 104 HPV+ (Human papillomavirus) HNSCC (head and neck squamous cell carcinoma) tumors together with two publicly available sources to identify highly robust transcriptional programs (modules) which could be detected consistently despite heterogeneous sequencing and quantification methodologies. Among 22 modules identified, we found a single module that naturally subclassifies HPV+ HNSCC tumors based on a bimodal pattern of gene expression, clusters all atypical features of HPV+ HNSCC biology into a single subclass, and predicts patient outcome in four independent cohorts. The subclass-defining gene set was strongly correlated with Nuclear factor kappa B (NF-κB) target expression. Tumors with high expression of this NF-κB module were rarely associated with activating PIK3CA alterations or viral integration, and also expressed higher levels of HPHPV E2 and had decreased APOBEC mutagenesis. Alternatively, they harbored inactivating alterations of key regulators of NF-κB, TNF receptor associated factor 3 (TRAF3), and cylindromatosis (CYLD), as well as retinoblastoma protein (RB1). HPV+ HNSCC cells in culture with experimental depletion of TRAF3 or CYLD displayed increased expression of the subclass-defining genes, as well as robust radio-sensitization, thus recapitulating both the tumor transcriptional state and improved treatment response observed in patient data. Across all gene sets investigated, methylation to expression correlations were the strongest for the subclass-defining, NF-κB-related genes. Increased tumor-infiltrating CD4+ T cells and increased Estrogen receptors alpha (ERα) expression were identified in NF-κB active tumors. Based on the relatively high rates of cure in HPV+ HNSCC, deintensification of therapy to reduce treatment-related morbidity is being studied at many institutions. Tumor subclassification based on oncogenic subtypes may help guide the selection of therapeutic intensity or modality for patients with HPV+ HNSCC.

Direct Comparison of HPV16 Viral Genomic Integration, Copy Loss, and Structural Variants in Oropharyngeal and Uterine Cervical Cancers Reveal Distinct Relationships to E2 Disruption and Somatic Alteration.

Squamous cell carcinoma of the oropharynx caused by HPV type 16 (HPV16+ OPSCC) is the most common HPV-associated malignancy in the USA and has many molecular differences from uterine cervical squamous cell carcinoma (UCSCC). Our understanding of HPV oncogenesis relied on studies of UCSCC revealing a consensus model reliant on HPV integration with a loss of E2. Here, we compare patterns of HPV integration in UCSCC and OPSCC by analysis of affinity capture sequencing of the HPV16 genome in 104 OPSCC and 44 UCSCC tumors. These cohorts were contemporaneously sequenced using an identical strategy. Integration was identified using discordant read pair clustering and assembly-based approaches. Viral integration sites, structural variants, and copy losses were examined. While large-scale deep losses of HPV16 genes were common in UCSCC and were associated with E2 loss, deep copy losses of the HPV16 genome were infrequent in HPV16+ OPSCC. Similarly, structural variants within HPV16 favored E2 loss in UCSCC but not OPSCC. HPV16 integration sites were non-random, with recurrent integration hot-spots identified. OPSCC tumors had many more integration sites per tumor when compared to UCSCC and had more integration sites in genomic regions with high gene density. These data show that viral integration and E2 disruption are distinct in UCSCC and OPSCC. Our findings also add to growing literature suggesting that HPV tumorigenesis in OPSCC does not follow the model developed based on UCSCC.

Comprehensive Viral Genotyping Reveals Prognostic Viral Phylogenetic Groups in HPV16-Associated Squamous Cell Carcinoma of the Oropharynx.

Human papillomavirus-positive (HPV+) squamous cell carcinoma of the oropharynx (OPSCC) is the most prevalent HPV-associated malignancy in the United States and is primarily caused by HPV subtype 16 (HPV16). Favorable treatment outcomes have led to increasing interest in treatment deescalation to reduce treatment-related morbidity. Prognostic biomarkers are needed to identify appropriately low-risk patients for reduced treatment intensity. Targeted DNA sequencing including all HPV16 open reading frames was performed on tumors from 104 patients with HPV16+ OPSCC treated at a single center. Genotypes closely related to the HPV16-A1 reference were associated with increased numbers of somatic copy-number variants in the human genome and poor recurrence-free survival (RFS). Genotypes divergent from HPV16-A1 were associated with favorable RFS. These findings were independent of tobacco smoke exposure. Total RNA sequencing was performed on a second independent cohort of 89 HPV16+ OPSCC cases. HPV16 genotypes divergent from HPV16-A1 were again validated in this independent cohort, to be prognostic of improved RFS in patients with moderate (less than 30 pack-years) or low (no more than 10 pack-years) of tobacco smoke exposure. In summary, we show in two independent cohorts that viral sequence divergence from the HPV16-A1 reference is correlated with improved RFS in patients with moderate or low tobacco smoke exposure. IMPLICATIONS: HPV16 genotype is a potential biomarker that could be easily adopted to guide therapeutic decision-making related to deescalation therapy.

Addiction to protein kinase CÉ© due to PRKCI gene amplification can be exploited for an aptamer-based targeted therapy in ovarian cancer.

PRKCI, the gene for protein kinase Cι (PKCι), is frequently amplified in ovarian cancer and recent studies have shown that PKCι participates in ovary tumorigenesis. However, it is unknown whether PKCι is differentially involved in the growth/survival between PRKCI-amplified and non-amplified ovarian cancer cells. In this study, we analyzed ovarian cancer patient dataset and revealed that PRKCI is the only PKC family member significantly amplified in ovarian cancer and PRKCI amplification is associated with higher PKCι expression. Using a panel of ovarian cancer cell lines, we found that abundance of PKCι is generally associated with PRKCI amplification. Interestingly, silencing PKCι led to apoptosis in PRKCI-amplified ovarian cancer cells but not in those without PRKCI amplification, thus indicating an oncogenic addiction to PKCɩ in PRKCI-amplified cells. Since small-molecule inhibitors characterized to selectively block atypical PKCs did not offer selectivity nor sensitivity in PRKCI-amplified ovarian cancer cells and were even cytotoxic to non-cancerous ovary surface or fallopian tube epithelial cells, we designed an EpCAM aptamer-PKCι siRNA chimera (EpCAM-siPKCι aptamer). EpCAM-siPKCι aptamer not only effectively induced apoptosis of PRKCI-amplified ovarian cancer cells but also greatly deterred intraperitoneal tumor development in xenograft mouse model. This study has demonstrated a precision medicine-based strategy to target a subset of ovarian cancer that contains PRKCI amplification and shown that the EpCAM aptamer-delivered PKCι siRNA may be used to suppress such tumors.

SHOX2 is a direct miR-375 target and a novel epithelial-to-mesenchymal transition inducer in breast cancer cells.

MicroRNAs have added a new dimension to our understanding of tumorigenesis and associated processes like epithelial-to-mesenchymal transition (EMT). Here, we show that miR-375 is elevated in epithelial-like breast cancer cells, and ectopic miR-375 expression suppresses EMT in mesenchymal-like breast cancer cells. We identified short stature homeobox 2 (SHOX2) as a miR-375 target, and miR-375-mediated suppression in EMT was reversed by forced SHOX2 expression. Ectopic SHOX2 expression can induce EMT in epithelial-like breast cancer cells, whereas SHOX2 knockdown diminishes EMT traits in mesenchymal-like breast cancer cells, demonstrating SHOX2 as an EMT inducer. We show that SHOX2 acts as a transcription factor to upregulate transforming growth factor ß receptor I (TßR-I) expression, and TßR-I inhibitor LY364947 abolishes EMT elicited by ectopic SHOX2 expression, suggesting that transforming growth factor ß signaling is essential for SHOX2-induced EMT. Manipulating SHOX2 abundance in breast cancer cells impact in vitro invasion and in vivo dissemination. Analysis of breast tumor microarray database revealed that high SHOX2 expression significantly correlates with poor patient survival. Our study supports a critical role of SHOX2 in breast tumorigenicity.