cGAS-STING responses are dampened in high-risk HPV type 16 positive head and neck squamous cell carcinoma cells.

Head and neck cancers (HNCs) are a major health problem and a leading cause of morbidity and mortality worldwide. More than 90% of these tumours are head and neck squamous cell carcinomas (HNSCCs). Amongst the common risk factors for HNCs (tobacco and alcohol use), there is a strong association of human papillomavirus (HPV) with HNSCCs. HPV type 16 (HPV 16), the major high-risk HPV type, is most commonly associated with HPV-driven HNSCCs. The promiscuous nature of the major HPV oncogene, E7, allows its interaction with a myriad of host proteins including STING, a component of the viral DNA-sensing cyclic GMP-AMP synthase (cGAS) - stimulator of interferon genes (STING) machinery. Sensing of viral DNA by the cGAS-STING machinery results in a type I interferon (IFN)-mediated anti-viral response. Amelioration of IFN responses resulting from the direct blockade of STING by E7 was first demonstrated in high-risk HPV type 18 (HPV 18) positive (+) cervical squamous cell carcinoma (CESC) cells. However, the role of E7 from HPV 16 (HPV 16E7) in antagonising cGAS-STING responses have not been investigated, let alone in the context of HNSCCs. Here, we show that HPV 16E7+, but not HPV 16E7 negative (-), HNSCC cells respond poorly to cGAS-STING activation stimulus. We further confirm that this inhibition occurred via the highly conserved LXCXE motif in 16E7. This finding contributes to the better understanding of role of high-risk HPV E7 in blocking cGAS-STING pathway, especially in the context of HNSCCs.

Prevalence and types of high-risk human papillomaviruses in head and neck cancers from Bangladesh.

BACKGROUND: There is a dramatic rise in the incidence of Human papillomavirus (HPV) - associated head and neck squamous cell carcinoma (HNSCC) in the world, with considerable variation by geography, gender and ethnicity. Little is known about the situation in Bangladesh, where tobacco- and areca nut-related head and neck cancers (HNCs) are the most common cancers in men. We aimed to determine the prevalence of HPV in HNSCC in Bangladesh and to explore the possible value of cell cycle markers in clinical diagnostic settings. METHODS: One hundred and ninety six archival HNSCC tissue samples were analysed for the presence of HPV DNA. The DNA quality was assured, and then amplified using a nested PCR approach. The typing of HPV was performed by automated DNA sequencing. Cellular markers p53, Cyclin D1 and pRb were tested on all samples by immunohistochemistry (IHC), as well as p16 as a putative surrogate for the detection of HPV. RESULTS: HPV DNA was detected in 36/174 (~21%) samples: 36% of cancers from the oropharynx; 31% of oral cancers, and 22% from the larynx. HPV-16 was most common, being present in 33 samples, followed by HPV-33 (2 samples) and HPV-31 (1 sample). Twenty-eight out of 174 samples were positive for p16, predominantly in HPV-positive tissues (p < 0.001). No statistically significant association was observed between the cellular markers and HPV DNA positive cases. However, p16 positivity had excellent predictive value for the presence of HPV by PCR. CONCLUSION: There is a significant burden of HPV-associated HNSCC in Bangladesh, particularly in the oropharynx but also in oral and laryngeal cancers. Whilst a combination of PCR-based DNA detection and p16 IHC is useful, the latter has excellent specificity, acceptable sensitivity and good predictive value for carriage of HPV in this population and should be used for prognostic evaluation and treatment planning of all HNSCC patients in South Asia, as in the Western world.

Exploring Importance and Regulation of Autophagy in Cancer Stem Cells and Stem Cell-Based Therapies.

Autophagy is a globally conserved cellular activity that plays a critical role in maintaining cellular homeostasis through the breakdown and recycling of cellular constituents. In recent years, there has been much emphasis given to its complex role in cancer stem cells (CSCs) and stem cell treatment. This study examines the molecular processes that support autophagy and how it is regulated in the context of CSCs and stem cell treatment. Although autophagy plays a dual role in the management of CSCs, affecting their removal as well as their maintenance, the intricate interaction between the several signaling channels that control cellular survival and death as part of the molecular mechanism of autophagy has not been well elucidated. Given that CSCs have a role in the development, progression, and resistance to treatment of tumors, it is imperative to comprehend their biological activities. CSCs are important for cancer biology because they also show a tissue regeneration model that helps with organoid regeneration. In other words, the manipulation of autophagy is a viable therapeutic approach in the treatment of cancer and stem cell therapy. Both synthetic and natural substances that target autophagy pathways have demonstrated promise in improving stem cell-based therapies and eliminating CSCs. Nevertheless, there are difficulties associated with the limitations of autophagy in CSC regulation, including resistance mechanisms and off-target effects. Thus, the regulation of autophagy offers a versatile strategy for focusing on CSCs and enhancing the results of stem cell therapy. Therefore, understanding the complex interactions between autophagy and CSC biology would be essential for creating therapeutic treatments that work in both regenerative medicine and cancer treatment.

HPV-negative head and neck cancers with adverse pathological features carry specific molecular changes that are associated with survival.

BACKGROUND: Adverse pathological features following surgery in head and neck squamous cell carcinoma (HNSCC) are strongly associated with survival and guide adjuvant therapy. We investigated molecular changes associated with these features. METHODS: We downloaded data from the Cancer Genome Atlas and Cancer Proteome Atlas HNSCC cohorts. We compared tumors positive versus negative for perineural invasion (PNI), lymphovascular invasion (LVI), extracapsular spread (ECS), and positive margins (PSM), with multivariable analysis. RESULTS: All pathological features were associated with poor survival, as were the following molecular changes: low cyclin E1 (HR = 1.7) and high PKC-alpha (HR = 1.8) in tumors with PNI; six of 13 protein abundance changes with LVI; greater tumor hypoxia and high Raptor (HR = 2.0) and Rictor (HR = 1.6) with ECS; and low p38 (HR = 2.3), high fibronectin (HR = 1.6), low annexin A1 (HR = 3.1), and high caspase-9 (HR = 1.6) abundances with PSM. CONCLUSIONS: Pathological features in HNSCC carry specific molecular changes that may explain their poor prognostic associations.

Human papillomavirus and Epstein-Barr virus co-infection in oral and oropharyngeal squamous cell carcinomas: A systematic review and meta-analysis.

Squamous cell carcinoma of the oral cavity (OSCC) is the most common head-and-neck malignancy. Importantly, we are experiencing an alarming rise in the incidence of oropharyngeal squamous cell carcinoma (OPSCC) globally. Oncogenic viruses, human papillomavirus (HPV) and Epstein-Barr virus (EBV), are known to be co-associated with OSCC and OPSCC cases. However, the reported incidence of HPV and EBV co-infection in OSCCs and OPSCCs globally is unknown. To address this, we performed a formal meta-analysis and systematic review on published studies that report the detection of both EBV and HPV in OSCCs and OPSCCs. Our analysis revealed 18 relevant studies out of a total of 1820 cases (1181 from the oral cavity and 639 from the oropharynx). Overall, HPV and EBV co-infection was found in 11.9% of OSCC and OPSCC cases combined (95% CI: 8%-14.1%). Based on anatomical subsite, dual positivity estimates were 10.5% (95% CI: 6.7%-15.1%) for OSCC and 14.2% (95% CI: 9.1%-21.3%) for OPSCC. The highest dual positivity rates described were in European countries: for OSCC 34.7% (95% CI: 25.9%-44.6%) in Sweden and for OPSCC, 23.4% (95% CI: 16.9%-31.5%) in Poland. Given these substantive prevalence rates, the value of detecting dual infection in the diagnosis and prognosis of these cancers deserves careful longitudinal studies, as do implications for cancer prevention and therapy. We further proposed molecular mechanisms that could explain how HPV and EBV could co-contribute to the aetiology of OSCCs and OPSCCs.

Loss of LRP1B expression drives acquired chemo and radio-resistance in HPV-positive head and neck cancer.

OBJECTIVES: Although human papillomavirus positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients typically experience excellent survival, 15-20 % of patients recur after treatment with chemotherapy and radiation. Therefore, there is a need for biomarkers of treatment failure to guide treatment intensity. MATERIALS AND METHODS: Whole genome sequencing was carried out on HPV+OPSCC patients who were primarily treated with concurrent chemotherapy (cisplatin) and radiation. We then explored whether the loss of LRP1Bwas sufficient to drive an aggressive phenotype, and promote a resistance to cisplatin and radiation therapy both in vitro using HPV+ cell lines (93VU147T, UMSCC47, UWO37 and UWO23) and in vivo. RESULTS: Through integrative genomic analysis of three HPV+OPSCC tumour datasets, we identified that deletion of LRP1B was enriched in samples that recurred following chemo-radiation. Knockdown using siRNA in four HPV+ cell lines (UWO23, UWO37, UMSCC47 and 93VU147T) resulted in increased proliferation of all cases. CRISPR/Cas9 deletion of LRP1B in the same cell line panel demonstrated increased proliferation, clonogenic growth and migration, as well as resistance to both cisplatin and radiation in LRP1B deleted cells compared to their respective non-targeting control cells. Cell line derived xenograft studies indicated that the LRP1B knockout tumours were more resistant to cisplatin and radiation therapy compared to their controls invivo. CONCLUSION: Taken together, our work implicates LRP1B deletion as a potential biomarker for identifying treatment resistant HPV+ OPSCC cases.

Introduction and expression of PIK3CAE545K in a papillary thyroid cancer BRAFV600E cell line leads to a dedifferentiated aggressive phenotype.

Anaplastic thyroid cancer (ATC) is a rare, aggressive form of undifferentiated thyroid cancer, which exhibits rapid progression and is almost universally fatal. At least a subset of ATC is thought to arise from pre-existing well-differentiated thyroid cancer, most frequently papillary thyroid cancer (PTC). While PIK3CA mutations are rare in PTC, they are common in ATC and tend to co-occur with BRAF mutations. This provided the rationale for our study to identify the potential role of PIK3CA mutations in the progression from well-differentiated to undifferentiated thyroid cancer. We introduced PIK3CAE545K into the LAM1 PTC cell line, which carries a BRAFV600E mutation. In culture, the engineered cell line (LAM1:PIK3CAE545K) proliferated faster and demonstrated increased clonogenic potential relative to the parental line carrying an empty vector (LAM1EV). Both the LAM1EV and LAM1:PIK3CAE545K edited lines were implanted into hind flanks of athymic nude mice for in vivo determination of disease progression. While tumour weights and volumes were not significantly higher in LAM1:PIK3CAE545K mice, there was a decrease in expression of thyroid differentiation markers TTF-1, thyroglobulin, PAX8 and B-catenin, suggesting that introduction of PIK3CAE545K led to dedifferentiation in vivo. Collectively, this study provides evidence of a role for PIK3CAE545K in driving disease progression from a well-differentiated to an undifferentiated thyroid cancer; however, over-expression was not a determinant of an accelerated growth phenotype in ATC.

Tumor molecular differences associated with outcome disparities of Black patients with head and neck cancer.

BACKGROUND: Numerous studies of head and neck squamous cell carcinoma (HNSCC) have demonstrated disparate outcomes by race and ethnicity. Beyond known associations with socioeconomic variables, whether these are also associated with differences in tumor molecular composition has thus far been poorly explored. METHODS: We downloaded clinical and multiplatform molecular data from The Cancer Genome Atlas and other published studies. These were compared between non-Hispanic Black (n = 43) and White (n = 354) patients with non-HPV-related tumors, using multivariable models. Publicly available validation cohorts were used. RESULTS: Black patients had poorer progression-free survival than White patients. Tumors of Black patients had greater copy number aberrations, and increased SFRP1 methylation and miRNA-mediated PRG4 silencing associated with poor survival. PI3K/AkT/mTOR pathway proteins were differentially expressed. CONCLUSIONS: There are molecular differences between tumors of Black and White patients that may partially account for differences in survival. These may inform targeted treatment decisions to achieve equitable outcomes.

Immune-based classification of HPV-associated oropharyngeal cancer with implications for biomarker-driven treatment de-intensification.

BACKGROUND: There is significant interest in treatment de-escalation for human papillomavirus-associated (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients given the generally favourable prognosis. However, 15-30% of patients recur after primary treatment, reflecting a need for improved risk-stratification tools. We sought to develop a molecular test to risk stratify HPV+ OPSCC patients. METHODS: We created an immune score (UWO3) associated with survival outcomes in six independent cohorts comprising 906 patients, including blinded retrospective and prospective external validations. Two aggressive radiation de-escalation cohorts were used to assess the ability of UWO3 to identify patients who recur. Multivariate Cox models were used to assess the associations between the UWO3 immune class and outcomes. FINDINGS: A three-gene immune score classified patients into three immune classes (immune rich, mixed, or immune desert) and was strongly associated with disease-free survival in six datasets, including large retrospective and prospective datasets. Pooled analysis demonstrated that the immune rich group had superior disease-free survival compared to the immune desert (HR = 9.0, 95% CI: 3.2-25.5, P = 3.6 × 10-5) and mixed (HR = 6.4, 95% CI: 2.2-18.7, P = 0.006) groups after adjusting for age, sex, smoking status, and AJCC8 clinical stage. Finally, UWO3 was able to identify patients from two small treatment de-escalation cohorts who remain disease-free after aggressive de-escalation to 30 Gy radiation. INTERPRETATION: With additional prospective validation, the UWO3 score could enable biomarker-driven clinical decision-making for patients with HPV+ OPSCC based on robust outcome prediction across six independent cohorts. Prospective de-escalation and intensification clinical trials are currently being planned. FUNDING: CIHR, European Union, and the NIH.

3p Arm Loss and Survival in Head and Neck Cancer: An Analysis of TCGA Dataset.

Loss of the 3p chromosome arm has previously been reported to be a biomarker of poorer outcome in both human papillomavirus (HPV)-positive and HPV-negative head and neck cancer. However, the precise operational measurement of 3p arm loss is unclear and the mutational profile associated with the event has not been thoroughly characterized. We downloaded the clinical, single nucleotide variation (SNV), copy number aberration (CNA), RNA sequencing, and reverse phase protein assay (RPPA) data from The Cancer Genome Atlas (TCGA) and The Cancer Proteome Atlas HNSCC cohorts. Survival data and hypoxia scores were downloaded from published studies. In addition, we report the inclusion of an independent Memorial Sloan Kettering cohort. We assessed the frequency of loci deletions across the 3p arm separately in HPV-positive and -negative disease. We found that deletions on chromosome 3p were almost exclusively an all or none event in the HPV-negative cohort; patients either had <1% or >97% of the arm deleted. 3p arm loss, defined as >97% deletion in HPV-positive patients and >50% in HPV-negative patients, had no impact on survival (p > 0.05). However, HPV-negative tumors with 3p arm loss presented at a higher N-category and overall stage and developed more distant metastases (p < 0.05). They were enriched for SNVs in TP53, and depleted for point mutations in CASP8, HRAS, HLA-A, HUWE1, HLA-B, and COL22A1 (false discovery rate, FDR < 0.05). 3p arm loss was associated with CNAs across the whole genome (FDR < 0.1), and pathway analysis revealed low lymphoid-non-lymphoid cell interactions and cytokine signaling (FDR < 0.1). In the tumor microenvironment, 3p arm lost tumors had low immune cell infiltration (FDR < 0.1) and elevated hypoxia (FDR < 0.1). 3p arm lost tumors had lower abundance of proteins phospho-HER3 and ANXA1, and higher abundance of miRNAs hsa-miR-548k and hsa-miR-421, which were all associated with survival. There were no molecular differences by 3p arm status in HPV-positive patients, at least at our statistical power level. 3p arm loss is largely an all or none phenomenon in HPV-negative disease and does not predict poorer survival from the time of diagnosis in TCGA cohort. However, it produces tumors with distinct molecular characteristics and may represent a clinically useful biomarker to guide treatment decisions for HPV-negative patients.

All HPV-negative head and neck cancers are not the same: Analysis of the TCGA dataset reveals that anatomical sites have distinct mutation, transcriptome, hypoxia, and tumor microenvironment profiles.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) affects various anatomical sites, which often dictates whether the cancer is managed with primary surgery or radiation. This study aimed to assess differences in single nucleotide variation (SNV), copy number, mRNA abundance, methylation, and tumor microenvironment (TME) between HPV-negative oral cavity (OC), oropharyngeal (OPC), hypopharyngeal (HPC), and laryngeal (LC) cancers within The Cancer Genome Atlas (TCGA). METHODS: We downloaded the clinical information and molecular data for the TCGA HNSCC cohort from the data portal and published literature. The TME was estimated using mRNA abundance data. We conducted our analyses within the Bioconductor statistical framework in the R environment. CNA and mRNA abundance results were correlated and grouped with SNV results for downstream pathway analysis. RESULTS: LC had a higher mutational burden than OC and OPC (p <10-4). LC tumors were enriched in CSMD3, NSD1, DCHS2 and ANK2 SNVs, while OC tumors were enriched in CASP8 SNVs (FDR < 0.1). LCs were enriched for neuronal and glycosylation pathways, while OCs were enriched for extracellular matrix pathways. B cells and endothelial cells were more abundant in LC while monocytes were more abundant in OC (FDR < 0.1). OPC was the most hypoxic, followed by OC then LC (FDR < 0.05). OC had greater methylation of Hox genes than LC. Subsite analysis revealed that oral tongue cancers had fewer CASP8 and FBN2 mutations and higher dendritic cell abundance than other oral cavity cancers. CONCLUSIONS: We identified significant genomic, transcriptional, and microenvironmental differences between HPV-negative HNSCC. Further study is warranted to determine if these findings portend differential response to specific treatment modalities.

Chromosome 3p loss in the progression and prognosis of head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is characterized by aggressive behavior with a tendency for recurrence and metastasis. Analyses of The Cancer Genome Atlas (TCGA) data and other cohort studies suggest that the loss of the chromosomal 3p arm is a frequent genetic event observed in both human papillomavirus positive and negative HNSCC. Early molecular analyses (i.e. RFLP, CGH) identified three common regions (3p14.2, 3p21.3 and 3p25) that frequently exhibited loss of genetic material on one arm of the 3p chromosome. More recently, next generation sequencing has revealed the loss of larger regions of this arm. Here we review the role of chromosomal 3p arm loss in early initiation and progression of HNSCC, and its relationship with poor patient prognosis.

Analysis of the TCGA Dataset Reveals that Subsites of Laryngeal Squamous Cell Carcinoma are Molecularly Distinct.

Laryngeal squamous cell carcinoma (LSCC) from different subsites have distinct presentations and prognosis. In this study, we carried out a multiomic comparison of LSCC subsites. The Cancer Genome Atlas (TCGA) LSCC cohort was analyzed in the R statistical environment for differences between supraglottic and glottic cancers in single nucleotide variations (SNVs), copy number alterations (CNAs), mRNA abundance, protein abundance, pathway overrepresentation, tumor microenvironment (TME), hypoxia status, and patient outcome. Supraglottic cancers had significantly higher overall and smoking-associated SNV mutational load. Pathway analysis revealed upregulation of muscle related pathways in glottic cancer and neural pathways in supraglottic cancer. Proteins involved in cancer relevant signaling pathways including PI3K/Akt/mTOR, the cell cycle, and PDL1 were differentially abundant between subsites. Glottic and supraglottic tumors have different molecular profiles, which may partially account for differences in presentation and response to therapy.

Sex disparities in head & neck cancer driver genes: An analysis of the TCGA dataset.

OBJECTIVES: Survival in head and neck squamous cell carcinoma (HNSCC) has been associated with patient sex, typically with males experiencing poorer outcomes. It is unclear if this disparity is based in divergent tumor biology. We analyzed the TCGA HNSCC cohort to uncover disparities in the somatic single nucleotide variation (SNV), copy number alteration (CNA) and mRNA abundance profiles between males and females. Critically, we stratified our results by tumor HPV status to control for this significant confounder. METHODS: SNV, CNA and mRNA abundance differences between males and females were compared separately for the HPV-positive (n = 67) and negative (n = 431) TCGA HNSCC cohorts. Overall survival outcomes were compared in males and females in both HPV-positive and HPV-negative subsets of patients. RESULTS: Females were found to have poorer overall survival than males (p = 0.048), largely due to higher rates of HPV-positive disease among men. SNV analysis revealed that in HPV-positive disease, there were no differences by sex after accounting for the false discovery rate (FDR). In HPV-negative tumors, BRWD3 mutations occurred more frequently in the tumors of female patients compared to males after adjusting for the FDR (p = 0.02). Further, HPV-negative BRWD3 mutant tumors were found to have significantly worse 5-year overall survival compared to wildtype on multivariate analysis (p = 0.02). There were 88 heterozygous deletions and 14 amplifications that were differentially altered between male and female HPV-negative tumors and associated with expression changes. Pathway analysis of these genes revealed that tumors from males were enriched in five pathways including chemokine and phosphophatidylinositol signaling. CONCLUSIONS: Reanalysis of the TCGA HNSCC dataset stratified by sex revealed that males in this cohort had a significant survival advantage, due to a higher proportion of HPV-positive disease. Mutations in BRWD3 were more frequent in HPV-negative tumors of females and were associated with poorer overall survival. BRWD3 may represent a novel biomarker of patient outcomes, but will require additional validation.

Spleen tyrosine kinase expression is correlated with human papillomavirus in head and neck cancer.

OBJECTIVES: Spleen tyrosine kinase (SYK) is a promoter of cell survival in a variety of cell types, including normal and cancerous epithelial cells. We hypothesized that SYK would an important therapeutic target to inhibit for the treatment of HNSCC. MATERIALS AND METHODS: SYK protein abundance in patient tumours was evaluated. SYK protein and mRNA abundance was used to examine patient survival and human papillomavirus (HPV) status. Small-interfering RNAs and gene editing with CRISPR/Cas9 were used to evaluate SYK expression on proliferation in HNSCC cell lines. The potency of SYK inhibitor ER27319 maleate on cellular proliferation was tested using a panel of 28 HNSCC cell lines and in vivo in HNSCC patient-derived xenograft (PDX) models. RESULTS: Moderate to high protein expression of SYK was observed in 24% of patient tumors and high SYK expression was exclusively observed in HPV-positive samples (p < 0.001). SYK inhibition with RNA interference, gene editing or a SYK inhibitor (ER27319) decreased cell proliferation and migration. Treatment of PDXs with ER27319 maleate was observed to reduce tumour burden in vivo in two of three models. CONCLUSIONS: HPV-positive HNSCC harbours high SYK protein levels. We demonstrate that proliferation, migration and overall burden of these tumours can be reduced by genetic or pharmacologic inhibition of SYK. Taken together, these data establish SYK as a therapeutic target for HNSCC.

Aurora kinases are a novel therapeutic target for HPV-positive head and neck cancers.

OBJECTIVES: Human papilloma virus (HPV) is the main culprit in cancers of the cervix, penis, anus, skin, eye and head and neck. Current treatments for HPV cancers have not altered survival outcomes for 30 years and there is a significant lack of targeted therapeutic agents in the management of advanced HPV-related HNSCC. Here we show that survival and maintenance of HPV-positive HNC cells relies on the continuous expression of the major HPV oncogene, E7, and that Aurora kinases are critical for survival of high-risk HPV-positive HNC cells. MATERIALS AND METHODS: To assess the role of HPV E7 on HNC cell survival, RNA interference (RNAi) of the E7 gene was initially performed. Using an Aurora kinase inhibitor, Alisertib, the role of Aurora kinases in the carcinogenesis of HPV E7 positive HNC tumour lines was then investigated. An in vivo HNC xenograft model was also utilised to assess loss of tumour volume in response to RNAi E7 gene silencing and Alisertib treatment. RESULTS: RNAi silencing of the HPV E7 gene inhibited the growth of HPV-positive HNC cells and in vivo tumour load. We show that HPV E7 oncogene expression confers sensitivity to Alisertib on HNC cells where Alisertib-mediated loss in in vitro cell viability and in vivo tumour load is dependent on E7 expression. Moreover, Aurora kinase inhibition induced degradation of MCL-1 in HPV E7-expressing HNC cells. CONCLUSION: Overall, we show that Aurora kinases are a novel therapeutic target for HPV-positive HNCs. It might be feasible to combine Aurora kinase and MCL-1 inhibitors for future HNC therapies.

Can gene editing and silencing technologies play a role in the treatment of head and neck cancer?

Conventional treatment strategies have done little to improve the prognosis or disease-free survival in head and neck cancer (HNC) patients. Recent progress in our understanding of molecular aspects of head and neck squamous cell carcinoma (HNSCC) has provided insights into the potential use of molecular targeted therapies in combination with current treatment strategies. Here we review the current understanding of treatment modalities for both HPV-positive and HPV-negative HNSCCs with the potential to use gene editing and silencing technologies therapeutically. The development of sequence-specific RNA interference (RNAi) with its strong gene-specific silencing ability, high target specificity, greater potency and reduced side effects, has shown it to be a promising therapeutic candidate for treating cancers. CRISPR/Cas gene editing is the newest technology with the ability to delete, mutate or replace genes of interest and has great potential for treating HNSCCs. We also discuss the major challenge in using these approaches in HNSCC; that being the choice of target and the ability to deliver the payload. Finally, we highlight the potential combination of RNAi or CRIPSR/Cas with current treatment strategies and outline the possible path to the clinic.

HPV-associated head and neck cancers in the Asia Pacific: A critical literature review & meta-analysis.

BACKGROUND: Malignancies of the upper aero-digestive tract are a major public health problem, especially in the Asia Pacific. Certain Human papillomaviruses (HPVs) are well-established risk factors for carcinoma of the uterine cervix and for a subset of head and neck carcinomata: however their true importance in different populations and anatomical subsites remains unclear. The major risk factors in Asia Pacific remain smoked/smokeless tobacco, areca nut, alcohol abuse and poor diet, with limited evidence for HPVs. We review published studies of association of HPV with anatomical site-specific Head & Neck Squamous Cell Carcinoma (HNSCC) in these populations and attempt a meta-analysis. MATERIALS AND METHODS: From MEDLINE/PubMed/WEB-of SCIENCE/EMBASE/Scopus databases we found 67 relevant studies with a total of 7280 cases: 15 case-control studies met our inclusion criteria for meta-analysis, totaling 1106 cases & 638 controls. HPV detection rates, sample site and size, and methods of tissue preservation and HPV detection were tabulated for each study. RESULTS: Studies were heterogeneous in terms of sample selection and method of detection of HPVs. Most were of limited quality. Averaging data from 67 studies of HNSCC, the prevalence of HPV of any subtype is approximately 36%. PCR (polymerase chain reaction) was the most used detection method and HPV16 the most common genotype reported. Meta-analyses of case-control studies from this region reveal significant heterogeneity but suggest higher HPV prevalence in oropharyngeal cancer (OR: 14.66; 95%CI: 6.09-35.26) compared to oral cavity cancer and laryngeal cancer; (OR: 4.06; 95%CI: 3.05-5.39 & OR: 3.23; 95%CI: 1.37-7.61) respectively. CONCLUSION: In view of the significant association of HPV with HNSCC, studies with accurate subsite classification and more sensitive detection methods are necessary. Accurate data from this geographical region are essential to inform public health policies and treatment decisions, especially as studies from Europe and North America reveal HPV-driven cancers to be less aggressive, permitting treatment de-intensification.

Aurora A Is Critical for Survival in HPV-Transformed Cervical Cancer.

Human papillomavirus (HPV) is the causative agent in cervical cancer. HPV oncogenes are major drivers of the transformed phenotype, and the cancers remain addicted to these oncogenes. A screen of the human kinome has identified inhibition of Aurora kinase A (AURKA) as being synthetically lethal on the background of HPV E7 expression. The investigational AURKA inhibitor MLN8237/Alisertib selectively promoted apoptosis in the HPV cancers. The apoptosis was driven by an extended mitotic delay in the Alisertib-treated HPV E7-expressing cells. This had the effect of reducing Mcl-1 levels, which is destabilized in mitosis, and increasing BIM levels, normally destabilized by Aurora A in mitosis. Overexpression of Mcl-1 reduced sensitivity to the drug. The level of HPV E7 expression influenced the extent of Alisertib-induced mitotic delay and Mcl-1 reduction. Xenograft experiments with three cervical cancer cell lines showed Alisertib inhibited growth of HPV and non-HPV xenografts during treatment. Growth of non-HPV tumors was delayed, but in two separate HPV cancer cell lines, regression with no resumption of growth was detected, even at 50 days after treatment. A transgenic model of premalignant disease driven solely by HPV E7 also demonstrated sensitivity to drug treatment. Here, we show for the first time that targeting of the Aurora A kinase in mice using drugs such as Alisertib results in a curative sterilizing therapy that may be useful in treating HPV-driven cancers.

The induction of apoptosis in pre-malignant keratinocytes by omega-3 polyunsaturated fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) is inhibited by albumin.

The long chain omega-3 polyunsaturated fatty acids (PUFA) have been reported to exert anti-cancer effects. At this study we tested the effect of the omega-3 PUFA, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), on pre-malignant keratinocytes growth in the well-characterised human pre-malignant epidermal cell line, HaCaT and attempted to identify a PUFA serum antagonist. Both EPA and DHA inhibited HaCaT growth and induced apoptosis. At the 10% (v/v) foetal bovine serum (FBS) medium, limited growth inhibition (3-20% for 50µM DHA and EPA respectively) and negligible apoptosis were observed with PUFA use. However, at 3% (v/v) FBS medium, 30-50µM of PUFA caused impressive levels of growth inhibition (82-83% for 50µM DHA and EPA respectively) and increase of apoptosis (8-19% increase in 72h). None of the numerous serum growth factors present in FBS or the antioxidant n-tert-butyl-α-phenylnitrone could inhibit the PUFA-induced cytotoxicity. In contrast, bovine and human albumin (0.1-0.3%, w/v) significantly antagonized the growth inhibitory and apoptosis-inducing effects of PUFA. In conclusion, we have shown for the first time that omega-3 PUFA inhibit the growth and induce apoptosis of pre-malignant keratinocytes and identified albumin as a major antagonistic factor in serum that could limit their effectiveness at pharmacologically-achievable doses.