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.

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.

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.

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.

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.

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.

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.

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.

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.