Therapeutic Advances and Challenges for the Management of HPV-Associated Oropharyngeal Cancer.

The use of conventional chemotherapy in conjunction with targeted and immunotherapy drugs has emerged as an option to limit the severity of side effects in patients diagnosed with head and neck cancer (HNC), particularly oropharyngeal cancer (OPC). OPC prevalence has increased exponentially in the past 30 years due to the prevalence of human papillomavirus (HPV) infection. This study reports a comprehensive review of clinical trials registered in public databases and reported in the literature (PubMed/Medline, Scopus, and ISI web of science databases). Of the 55 clinical trials identified, the majority (83.3%) were conducted after 2015, of which 77.7% were performed in the United States alone. Eight drugs have been approved by the FDA for HNC, including both generic and commercial forms: bleomycin sulfate, cetuximab (Erbitux), docetaxel (Taxotere), hydroxyurea (Hydrea), pembrolizumab (Keytruda), loqtorzi (Toripalimab-tpzi), methotrexate sodium (Trexall), and nivolumab (Opdivo). The most common drugs to treat HPV-associated OPC under these clinical trials and implemented as well for HPV-negative HNC include cisplatin, nivolumab, cetuximab, paclitaxel, pembrolizumab, 5-fluorouracil, and docetaxel. Few studies have highlighted the necessity for new drugs specifically tailored to patients with HPV-associated OPC, where molecular mechanisms and clinical prognosis are distinct from HPV-negative tumors. In this context, we identified most mutated genes found in HPV-associated OPC that can represent potential targets for drug development. These include TP53, PIK3CA, PTEN, NOTCH1, RB1, FAT1, FBXW7, HRAS, KRAS, and CDKN2A.

Rethinking treatment paradigms: Neoadjuvant therapy and de-escalation strategies in HPV-positive head and neck cancer.

Head and neck cancer (HNC) is the 6th most common cancer across the world, with a particular increase in HNC associated with human papilloma virus (HPV) among younger populations. Historically, the standard treatment for this disease consisted of combined surgery and radiotherapy or curative platinum-based concurrent chemoradiotherapy, with associated long term and late toxicities. However, HPV-positive HNC is recognized as a unique cancer subtype, typically with improved clinical outcomes. As such, treatment de-escalation strategies have been widely researched to mitigate the adverse effects associated with the current standard of care without compromising efficacy. These strategies include treatment de-escalation, such as novel surgical techniques, alternative radiation technologies, radiation dose and volume reduction, as well as neoadjuvant chemotherapies, immunotherapies, and combined therapies. Although these therapies show great promise, many of them are still under investigation due to hesitation surrounding their widespread implementation. The objective of this review is to summarize the most recent progress in de-escalation strategies and neoadjuvant therapies designed for HPV-positive HNC. While specific treatments may require additional research before being widely adopted, encouraging results from recent studies have highlighted the advantages of neoadjuvant chemotherapy and immunotherapy, as well as radiation and surgical de-escalation approaches in managing HPV-positive HNC.

RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells.

Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops.

Discovery of a Small-Molecule Inhibitor Targeting the Androgen Receptor N-Terminal Domain for Castration-Resistant Prostate Cancer.

The current mainstay therapeutic strategy for advanced prostate cancer is to suppress androgen receptor (AR) signaling. However, castration-resistant prostate cancer (CRPC) invariably arises with restored AR signaling activity. To date, the AR ligand-binding domain (LBD) is the only targeted region for all clinically available AR signaling antagonists, such as enzalutamide (ENZ). Major resistance mechanisms have been uncovered to sustain the AR signaling in CRPC despite these treatments, including AR amplification, AR LBD mutants, and the emergence of AR splice variants (AR-Vs) such as AR-V7. AR-V7 is a constitutively active truncated form of AR that lacks the LBD; thus, it can not be inhibited by AR LBD-targeting drugs. Therefore, an approach to inhibit AR through the regions outside of LBD is urgently needed. In this study, we have discovered a novel small molecule SC428, which directly binds to the AR N-terminal domain (NTD) and exhibits pan-AR inhibitory effect. SC428 potently decreased the transactivation of AR-V7, ARv567es, as well as full-length AR (AR-FL) and its LBD mutants. SC428 substantially suppressed androgen-stimulated AR-FL nuclear translocation, chromatin binding, and AR-regulated gene transcription. Moreover, SC428 also significantly attenuated AR-V7-mediated AR signaling that does not rely on androgen, hampered AR-V7 nuclear localization, and disrupted AR-V7 homodimerization. SC428 inhibited in vitro proliferation and in vivo tumor growth of cells that expressed a high level of AR-V7 and were unresponsive to ENZ treatment. Together, these results indicated the potential therapeutic benefits of AR-NTD targeting for overcoming drug resistance in CRPC.

E-liquid alters oral epithelial cell function to promote epithelial to mesenchymal transition and invasiveness in preclinical oral squamous cell carcinoma.

The gaining popularity of tobacco and nicotine delivery products, such as electronic cigarettes (e-cigarettes) being perceived as relatively safe is of a medical concern. The long-term safety of these new products remains uncertain for oral health. In this study, in vitro effects of e-liquid were assessed in a panel of normal oral epithelium cell lines (NOE and HMK), oral squamous cell carcinoma (OSCC) human cell lines (CAL27 and HSC3), and a mouse oral cancer cell line (AT84) using cell proliferation, survival/cell death, and cell invasion assays. In addition, signaling pathways underlying the pro-invasive activity of e-cigarettes were evaluated by gene and protein expression analysis. We demonstrated that e-liquid promotes proliferation and anchorage-independent growth of OSCC and induces morphological changes associated with enhanced motility and invasive phenotypes. Furthermore, e-liquid-exposed cells express significantly reduced cell viability, regardless of e-cigarette flavour content. At the gene expression level, e-liquid induces changes in gene expression consistent with epithelial to mesenchymal transition (EMT) revealed by reduced expression of cell epithelial markers such as E-cadherin and enhanced expression of mesenchymal proteins like vimentin and B-catenin seen both in OSCC cell lines and normal oral epithelium cells. In summary, the ability of e-liquid to induce proliferative and invasive properties along the activation of the EMT process can contribute to the development of tumorigenesis in normal epithelial cells and promote aggressive phenotype in pre-existing oral malignant cells.

Genetic Mutations Associated with Inflammatory Response Caused by HPV Integration in Oropharyngeal Squamous Cell Carcinoma.

(1) Background: Head and neck cancer (HNC) ranks as the sixth most prevalent cancer in the world. In addition to the traditional risk factors such as alcohol and tobacco consumption, the implication of the human papillomavirus (HPV) is becoming increasingly significant, particularly in oropharyngeal cancer (OPC). (2) Methods: This study is based on a review analysis of different articles and repositories investigating the mutation profile of HPV-related OPC and its impact on patient outcomes. (3) Results: By compiling data from 38 datasets involving 8311 patients from 12 countries, we identified 330 genes that were further analyzed. These genes were enriched for regulation of the inflammatory response (RB1, JAK2, FANCA, CYLD, SYK, ABCC1, SYK, BCL6, CEBPA, SRC, BAP1, FOXP1, FGR, BCR, LRRK2, RICTOR, IGF1, and ATM), among other biological processes. Hierarchical cluster analysis showed the most relevant biological processes were linked with the regulation of mast cell cytokine production, neutrophil activation and degranulation, and leukocyte activation (FDR < 0.001; p-value < 0.05), suggesting that neutrophils may be involved in the development and progression of HPV-related OPC. (4) Conclusions: The neutrophil infiltration and HPV status emerge as a potential prognostic factor for OPC. HPV-infected HNC cells could potentially lead to a decrease in neutrophil infiltration. By gaining a better molecular understanding of HPV-mediated neutrophil immunosuppression activity, it is possible to identify a meaningful target to boost antitumor immune response in HNC and hence to improve the survival of patients with HNC.

Implications and Emerging Therapeutic Avenues of Inflammatory Response in HPV+ Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinomas (HNSCC) are a heterogeneous group of malignancies which have shown exponential incidence in the last two decades especially due to human papillomavirus (HPV) infection. The HPV family comprises more than 100 types of viruses with HPV16 and HPV18 being the most prevalent strains in HNSCC. Literature data reveal that the mutation profile as well as the response to chemotherapy and radiotherapy are distinct among HPV+ versus HPV-negative tumors. Furthermore, the presence of the virus induces activation of an immune response, in particular the recruitment of specific antiviral T lymphocytes to tumor sites. These T cells when activated produce soluble factors including cytokines and chemokines capable of modifying the local immune tumor microenvironment and impact on tumor response to the treatment. In this comprehensive review we investigated current knowledge on how the presence of an HPV can modify the inflammatory response systemically and within the tumor microenvironment's immunological responses, thereby impacting on disease prognosis and survival. We highlighted the research gaps and emerging approaches necessary to discover novel immunotherapeutic targets for HPV-associated HNSCC.

Therapeutic Vaccines for HPV-Associated Oropharyngeal and Cervical Cancer: The Next De-Intensification Strategy?

The rise in human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) has prompted a quest for further understanding of the role of high-risk HPV in tumor initiation and progression. Patients with HPV-positive OPSCC (HPV+ OPSCC) have better prognoses than their HPV-negative counterparts; however, current therapeutic strategies for HPV+ OPSCC are overly aggressive and leave patients with life-long sequalae and poor quality of life. This highlights a need for customized treatment. Several clinical trials of treatment de-intensification to reduce acute and late toxicity without compromising efficacy have been conducted. This article reviews the differences and similarities in the pathogenesis and progression of HPV-related OPSCC compared to cervical cancer, with emphasis on the role of prophylactic and therapeutic vaccines as a potential de-intensification treatment strategy. Overall, the future development of novel and effective therapeutic agents for HPV-associated head and neck tumors promises to meet the challenges posed by this growing epidemic.

NEDD9 links anaplastic thyroid cancer stemness to chromosomal instability through integrated centrosome asymmetry and DNA sensing regulation.

Stemness and chromosomal instability (CIN) are two common contributors to intratumor heterogeneity and therapy relapse in advanced cancer, but their interplays are poorly defined. Here, in anaplastic thyroid cancer (ATC), we show that ALDH+ stem-like cancer cells possess increased CIN-tolerance owing to transcriptional upregulation of the scaffolding protein NEDD9. Thyroid patient tissues and transcriptomic data reveals NEDD9/ALDH1A3 to be co-expressed and co-upregulated in ATC. Compared to bulk ALDH- cells, ALDH+ cells were highly efficient at propagating CIN due to their intrinsic tolerance of both centrosome amplification and micronuclei. ALDH+ cells mitigated the fitness-impairing effects of centrosome amplification by partially inactivating supernumerary centrosomes. Meanwhile, ALDH+ cells also mitigated cell death caused by micronuclei-mediated type 1 interferon secretion by suppressing the expression of the DNA-sensor protein STING. Both mechanisms of CIN-tolerance were lost upon RNAi-mediated NEDD9 silencing. Both in vitro and in vivo, NEDD9-depletion attenuated stemness, CIN, cell/tumor growth, while enhancing paclitaxel effectiveness. Collectively, these findings reveal that ATC progression can involve an ALDH1A3/NEDD9-regulated program linking their stemness to CIN-tolerance that could be leveraged for ATC treatment.

Novel Aurora A and Protein Kinase C (α, ß1, ß2, and θ) Multitarget Inhibitors: Impact of Selenium Atoms on the Potency and Selectivity.

Aurora kinases and protein kinase C (PKC) have been shown to be involved in different aspects of cancer progression. To date, no dual Aurora/PKC inhibitor with clinical efficacy and low toxicity is available. Here, we report the identification of compound 2e as a potent small molecule capable of selectively inhibiting Aurora A kinase and PKC isoforms α, ß1, ß2 and θ. Compound 2e demonstrated significant inhibition of the colony forming ability of metastatic breast cancer cells in vitro and metastasis development in vivo. In vitro kinase screening and molecular modeling studies revealed the critical role of the selenium-containing side chains within 2e, where selenium atoms were shown to significantly improve its selectivity and potency by forming additional interactions and modulating the protein dynamics. In comparison to other H-bonding heteroatoms such as sulfur, our studies suggested that these selenium atoms also confer more favorable PK properties.

Molecular prognostic indicators in HPV-positive oropharyngeal cancer: an updated review.

Infection with HPV virus and exposure to extrinsic carcinogens are the main causative factors for oropharyngeal squamous cell carcinoma (OPSCC). While HPV-related OPSCC typically shows a better prognosis and may be a candidate for de-intensification therapy, there is a subset of HPV-related cancers that show aggressive phenotype with frequent metastatic spread. The identification and refinement of molecular markers can better serve for prediction of prognosis and thus improve treatment decisions and outcome. We conducted a systematic review according to the PRISMA guidelines of all relevant studies addressing novel biomarkers in publications prior to July 2021. We identified studies that evaluated the association between molecular markers and prognosis in HPV-positive OPSCC. Full-text publications were entirely reviewed, classified, and selected if a clear predictive/prognostic value was seen in patients with HPV-positive OPSCC. Furthermore, a functional analysis of the target genes was conducted to understand biological processes and molecular pathways impacting on HPV-positive OPSCC outcomes. The systematic review yielded a total of 14 studies that matched the inclusion and exclusion criteria. Differential expression was identified for 31 different biomarkers. The first common pattern identified was the association of HPV-related circulating antibodies to activated immune function. Second, gene-gene interaction analysis further identified interacting gene networks tightly implicated in hypoxia tumor metabolism including the Warburg effect. Survival in HPV-positive OPSCC can be predicted by distinct selective biomarkers mainly indicative of immune host response and oxidative metabolism. Among these markers, some were identified to be unsuitable for HPV-positive de-escalation trials aimed at improving patients' quality of life.

Identification of R-Spondin Gene Signature Predictive of Metastatic Progression in BRAFV600E-Positive Papillary Thyroid Cancer.

Papillary thyroid carcinoma (PTC) is the most common malignancy of the thyroid gland and early stages are curable. However, a subset of PTCs shows an unusually aggressive phenotype with extensive lymph node metastasis and higher incidence of locoregional recurrence. In this study, we investigated a large cohort of PTC cases with an unusual aggressive phenotype using a high-throughput RNA sequencing (RNA-Seq) to identify differentially regulated genes associated with metastatic PTC. All metastatic PTC with mutated BRAF (V600E) but not BRAF wild-type expressed an up-regulation of R-Spondin Protein 4 (RSPO4) concomitant with an upregulation of genes involved in focal adhesion and cell-extracellular matrix signaling. Further immunohistochemistry validation confirmed the upregulation of these target genes in metastatic PTC cases. Preclinical studies using established PTC cell lines support that RSPO4 overexpression is associated with BRAF V600E mutation and is a critical upstream event that promote activation of kinases of focal adhesion signaling known to drive cancer cell locomotion and invasion. This finding opens up the potential of co-targeting B-Raf, RSPO and focal adhesion proteins as a pharmacological approach for aggressive BRAF V600E PTC.

Portrait of DNA methylated genes predictive of poor prognosis in head and neck cancer and the implication for targeted therapy.

In addition to chronic infection with human papilloma virus (HPV) and exposure to environmental carcinogens, genetic and epigenetic factors act as major risk factors for head and neck cancer (HNC) development and progression. Here, we conducted a systematic review in order to assess whether DNA hypermethylated genes are predictive of high risk of developing HNC and/or impact on survival and outcomes in non-HPV/non-tobacco/non-alcohol associated HNC. We identified 85 studies covering 32,187 subjects where the relationship between DNA methylation, risk factors and survival outcomes were addressed. Changes in DNA hypermethylation were identified for 120 genes. Interactome analysis revealed enrichment in complex regulatory pathways that coordinate cell cycle progression (CCNA1, SFN, ATM, GADD45A, CDK2NA, TP53, RB1 and RASSF1). However, not all these genes showed significant statistical association with alcohol consumption, tobacco and/or HPV infection in the multivariate analysis. Genes with the most robust HNC risk association included TIMP3, DCC, DAPK, CDH1, CCNA1, MGMT, P16, MINT31, CD44, RARß. From these candidates, we further validated CD44 at translational level in an independent cohort of 100 patients with tongue cancer followed-up beyond 10 years. CD44 expression was associated with high-risk of tumor recurrence and metastasis (P = 0.01) in HPV-cases. In summary, genes regulated by methylation play a modulatory function in HNC susceptibility and it represent a critical therapeutic target to manage patients with advanced disease.

Chitosan/PCL nanoparticles can improve anti-neoplastic activity of 5-fluorouracil in head and neck cancer through autophagy activation.

Head and neck squamous cell carcinoma (HNSCC), a prevalent cancer worldwide, has a high incidence of loco-regional dissemination, frequent recurrence, and lower 5-year survival rates. Current gold standard treatments for advanced HNSCC rely primarily on radiotherapy and chemotherapy but with limited efficacy and significant side effects. In this study, we characterized a novel 5-fluorouracil (5-FU) carrier composed of chitosan solution (CS) and polycaprolactone (PCL) microparticles (MPs) in HNSCC preclinical models. The designed MPs were evaluated for their size, morphology, drug entrapment efficiency (EE%) and in vitro drug release profile. The anti-cancer activity of 5-FU-loaded particles was assessed in HNSCC human cell lines (CAL27 and HSC3) and in a preclinical mouse model (AT84) utilizing cell proliferation and survival, cell motility, and autophagy endpoints. The results demonstrated a 38.57 % in 5-FU entrapment efficiency associated with reduced 5-FU in vitro release up to 96 h post-exposure. Furthermore, CS-decorated PCL MPs were able to promote a significant inhibition of cancer cell proliferation based on the metabolic and colony formation assays, in comparison to controls. In contrast, CS-decorated PCL MPs did not influence the pharmacological efficacy of 5-FU to inhibit in vitro cancer cell migration. Last, cell protein analysis revealed a significant increase of autophagy and cell death evaluated by LC3-II expression and PARP1 cleavage, respectively. In summary, these results support the potential utility of CS-decorated PCL MPs as an effective 5-FU-delivery carrier to improve HNSCC therapeutic management.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Co-Overexpression of TWIST1-CSF1 Is a Common Event in Metastatic Oral Cancer and Drives Biologically Aggressive Phenotype.

Invasive oral squamous cell carcinoma (OSCC) is often ulcerated and heavily infiltrated by pro-inflammatory cells. We conducted a genome-wide profiling of tissues from OSCC patients (early versus advanced stages) with 10 years follow-up. Co-amplification and co-overexpression of TWIST1, a transcriptional activator of epithelial-mesenchymal-transition (EMT), and colony-stimulating factor-1 (CSF1), a major chemotactic agent for tumor-associated macrophages (TAMs), were observed in metastatic OSCC cases. The overexpression of these markers strongly predicted poor patient survival (log-rank test, p = 0.0035 and p = 0.0219). Protein analysis confirmed the enhanced expression of TWIST1 and CSF1 in metastatic tissues. In preclinical models using OSCC cell lines, macrophages, and an in vivo matrigel plug assay, we demonstrated that TWIST1 gene overexpression induces the activation of CSF1 while TWIST1 gene silencing down-regulates CSF1 preventing OSCC invasion. Furthermore, excessive macrophage activation and polarization was observed in co-culture system involving OSCC cells overexpressing TWIST1. In summary, this study provides insight into the cooperation between TWIST1 transcription factor and CSF1 to promote OSCC invasiveness and opens up the potential therapeutic utility of currently developed antibodies and small molecules targeting cancer-associated macrophages.