Human Papillomavirus Negative Cell Research Articles

Urdamycin V from Streptomyces sp induces p53 independent apoptosis in cervical cancer cells inconsiderate of HPV status and inhibited growth of gram-positive human pathogens

In cervical cancer, loss of p53 or mutant non-functional p53 and hyperactivated mTOR/Akt pathway positively correlates to cancer progression. Urdamycin V isolated from Streptomyces OA293 is a recently isolated novel angucycline derivative that in the present study showcased induction of p53 independent apoptosis in both HPV (Human papillomavirus) positive and negative cervical cancer cell lines. Apoptosis induction was via phosphorylation modulation in the cell growth regulating proteins along mTORC2/Akt/p38/Erk pathway. The compound was also tested against human pathogens and selectively inhibited gram-positive strains, Streptococcus pyogenes and Staphylococcus aureus. The present study put forward urdamycins as a potential therapeutic that places promise for further research.

EGCG attenuate EGF triggered matrix abundance and migration in HPV positive and HPV negative cervical cancer cells.

Our previous laboratory findings suggested the beneficial effects of epigallocatechin gallate (EGCG) against cervical cancer (CC) cells survival. The present study is aimed at identifying the effects of EGCG in preventing the actions of epidermal growth factor (EGF) in human papilloma virus (HPV) 68 positive ME180 and HPV negative C33A CC cells. An elevated level of EGF in tumor micro-environment (TME) is linked to the metastasis of several cancers including CC. We hypothesized that EGCG has the ability to block the actions of EGF. To test this, survival assay was performed in cells treated with or without EGF and EGCG. The mitochondrial activity of cells was ascertained using MTT assay and mitored staining. Protein and non-protein components in the extracellular matrix such as collagen and sulphated glycosaminoglycans (GAGs) were evaluated using sirius red and alcian blue staining, respectively. Matrix metalloproteinase-2 (MMP-2) gene expression and enzymatic activity were assessed using real time-reverse transcriptase-polymerase chain reaction (RT-PCR) and gelatin zymography. Wound healing assay was performed to assess the EGF induced migratory ability and its inhibition by EGCG pre-treatment. Clonogenic assay showed thatEGCG pre-treatment blocked the EGF driven colony formation. In silico analysis performed identified the efficacy of EGCG in binding with different domains of EGF receptor (EGFR). EGCG pre-treatment prevented the epithelial-mesenchymal transition (EMT) and metabolic activity induced by EGF, this is associated with concomitant reduction in the gene expression and enzyme activity of MMP-2. Further, reduced migration and ability to form colonies were observed in EGCG pre-treated cells when stimulated with EGF. HPV positive ME180 cells showed increased migratory and clonogenic ability upon EGF stimulation, whose effects were not much significant in HPV negative C33A cells. EGCG effectively blocked the actions of EGF in both HPV positive and HPV negative conditions and can be advocated as supplementary therapy for the management of EGF driven CC. However, further studies using cell line-derived xenograft(CDX)/patient-derived xenograft(PDX) model system is warranted to validate the therapeutic utility of EGCG.

Identification and validation of a prognostic signature of autophagy, apoptosis and pyroptosis-related genes for head and neck squamous cell carcinoma: to imply therapeutic choices of HPV negative patients.

An effective tool is needed to predict the prognosis of head and neck squamous cell carcinoma (HNSCC). Human papillomavirus (HPV) positive HNSCC patients generally have a favorable survival and a promising responsiveness to radiotherapy, chemoradiotherapy and checkpoint blockades. However, HPV negative patients, the majority of HNSCC patients, have been largely overlooked. Cell death has been involved in the therapeutic resistance of cancers. To this end, we aimed to identify the association of autophagy, apoptosis and pyroptosis-related genes with the prognosis of HNSCC, and construct a prognostic signature to predict the prognosis for HNSCC, especially for HPV negative HNSCC. Autophagy and apoptosis-related genes were obtained from Gene Set Enrichment Analysis (GSEA) website, and pyroptosis-related genes were obtained from GSEA and Gene Ontology (GO) database. We established the cell death index (CDI) based on RNA sequencing (RNA-seq) data and clinicopathological information from The Cancer Genome Atlas (TCGA) dataset. The prognostic value of CDI was verified by Kaplan-Meier, receiver operating characteristic (ROC) and univariate and multivariate Cox regression analyses in TCGA dataset, and validated with the datasets from Gene Expression Omnibus (GEO) and Qilu Hospital of Shandong University. We further assessed the immune microenvironment of patients with high and low CDI scores. Moreover, the expression of the signature genes in HNSCC cell lines were explored. We found that CDI was an independent prognostic indicator for overall survival (hazard ratio 3.80, 95% confidential interval: 2.70-5.40, P < 0.001). Furthermore, HNSCC patients with high CDI scores obtained increased overall survival post radiation indicating benefits from radiotherapy of this subgroup. On the other hand, HPV negative HNSCC patients with low CDI exhibited increased checkpoint gene expressions, an inflamed tumor microenvironment and an enriched immune response-related functions, suggesting the potential benefits from checkpoint immunotherapies of this subgroup. Moreover, we validated the baseline and induced expressions of above 16 genes in two HPV negative HNSCC cell lines, CAL27 and SCC-15. We established a prognostic signature and emphasized its implements in the therapeutic choices of HPV negative HNSCC patients, the majority and the poor outcome population of HNSCC.

Accuracy of Response Assessment FDG PET-CT Post (Chemo)Radiotherapy in HPV Negative Oropharynx Squamous Cell Carcinoma.

Simple SummaryOropharyngeal squamous cell carcinoma is often treated with (chemo)radiotherapy with curative intent. Human papilloma virus (HPV) is a key risk factor for the development of a majority of oropharynx cancers in many parts of the world. PET-CT is widely used as an accurate method of assessing response following (chemo)radiotherapy and most of the data supporting this is based upon HPV-related disease. Oropharynx squamous cell carcinoma that is not related to human papilloma virus has an inferior prognosis and there is little data regarding the accuracy of response assessment PET-CT after chemoradiotherapy. This study shows that a negative PET-CT after treatment for patients with HPV-negative oropharynx cancer has a high negative predictive value with treatment having been successsful; however if the PET-CT is equivocal there is a significant chance of disease being persistent.Background: Data on the accuracy of response assessment 2-[fluorine-18]-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography-computed tomography (PET-CT) following (chemo)radiotherapy in patients with oropharynx squamous cell carcinoma (OPSCC) is predominantly based on HPV-positive disease. There is a paucity of data for HPV-negative disease, which has a less favourable prognosis. Methods: 96 patients treated with (chemo)radiotherapy for HPV-negative OPSCC with baseline and response assessment FDG PET-CT between 2013–2020, were analysed. PET-CT response was classified as negative, equivocal, or positive based on qualitative reporting. PET-CT response categories were analysed with reference to clinicopathological outcomes. Test characteristics were evaluated, comparing negative results to equivocal and positive results together. Post-test probabilities were calculated separately for positive and equivocal or negative results. Results: Median follow-up was 26 months. The negative predictive value of a negative scan was 93.7 and 93.2%, respectively, for primary tumour and nodal disease. For a negative scan, the post-test probability was 0.06 for primary and 0.07 for nodal disease. The post-test probability of an equivocal scan was 0.51 and 0.72 for primary and lymph node, respectively. The post-test probability of a positive scan approached 1. For patients with/without a negative scan, two-year overall survival and progression-free survival were 83% versus 30% and 79% versus 17% (p < 0.001), respectively. Conclusion: The NPV of a negative response assessment PET-CT in HPV-negative OPSCC is high, supporting a strategy of clinical monitoring. Contrasting with the published literature for HPV-positive OPSCC, an equivocal response scan was associated with a moderate rate of residual disease.

4NQO enhances differential activation of DNA repair proteins in HPV positive and HPV negative HNSCC cells

Tobacco exposure and human papillomavirus (HPV) infection are among the main risk factors for the development of head and neck squamous cell carcinoma (HNSCC). Interestingly, recent studies show that tumors from HPV positive (HPV+) smokers and non-smokers have similar mutational profiles, which suggests that HPV could prevent mutation induction or accumulation in the intermediate risk group composed of HPV+ smokers. Hence, we tested this observation by analyzing the effects of 4-Nitroquinoline N-oxide (4NQO), a mutagen and smoking mimetic, in NOK (normal oral keratinocytes), NOKE6.E7 (NOK cells transfected with E6.E7 oncogenes of HPV), HPV+ and HPV negative (HPV−) HNSCC cells. Oxidative DNA damage, γH2AX foci formation, DNA repair protein activation, cell cycle phase analysis, apoptotic cell death, cell viability and clonogenic cell survival were analyzed after 4NQO treatment in NOK, NOKE6.E7, HPV+ and HPV− HNSCC cells. 4NQO increased oxidative base damage and γH2AX foci formation in NOKE6.E7, HPV+ and HPV− HNSCC cells. Phosphorylation of homologous recombination (HR) repair proteins was higher in NOKE6.E7 and HPV+ HNSCC cells compared to NOK and HPV− HNSCC cells respectively. HPV+ and HPV− HNSCC cells showed differential activation of cell cycle regulatory proteins, increased apoptosis, and decreased cell viability upon 4NQO-induced DNA damage. Taken together, 4NQO (a smoking mimetic), induced higher activation of HR repair in HPV+ HNSCC cells compared to HPV− HNSCC cells. This may allow for increased mutational resistance and help explain why HPV+ smokers have a worse prognosis than HPV+ non-smokers.

CD68+ Macrophage Infiltration Associates With Poor Outcome of HPV Negative Oral Squamous Carcinoma Patients Receiving Radiation: Poly(I:C) Enhances Radiosensitivity of CAL-27 Cells but Promotes Macrophage Recruitment Through HMGB1.

Patients with human papillomavirus (HPV) negative oral squamous cell carcinoma (OSCC) generally have poor clinical outcomes and worse responses to radiotherapy. It is urgent to explore the underlining mechanisms of the distinct prognoses between HPV negative and HPV positive OSCC and to develop effective therapy strategy to increase the survival rate of HPV negative OSCC patients. We conducted a retrospective cohort of 99 resected OSCC patients to evaluate the prognosis of HPV negative and HPV positive OSCC patients receiving radiation or not. We further addressed the association of CD68+ macrophage infiltration with HPV status and the effects on survival of OSCC patients. We also used the TCGA-OSCC cohort for further verification. Based on the cohort study, we applied a synthetic dsRNA polymer, polyriboinosinic-polyribocytidylic acid (poly(I:C)), on CAL-27 (HPV negative OSCC cells). We co-cultured its condition medium with THP-1 derived macrophage and examined the cytokines and macrophage migration. We found that high CD68+ macrophage infiltration associated with poor overall survival in HPV negative OSCC patients receiving radiation. In vitro, poly(I:C) could induce apoptosis and enhance the radiosensitivity, but increase macrophage recruitment. Targeting HMGB1 could inhibit IL-6 induction and macrophage recruitment. Our findings indicated that CD68+ macrophage might play an important role in the outcomes of HPV negative OSCC patients receiving radiation. Our findings also suggested that radiation combined poly(I:C) might be a potential therapy strategy to increase the radiation response and prognosis of HPV negative OSCC. Notably, HMGB1 should be targeted to inhibit macrophage recruitment and enhance overall therapy effects.

Hypofractionated Radiotherapy Upregulates Several Immune Checkpoint Molecules in Head and Neck Squamous Cell Carcinoma Cells Independently of the HPV Status While ICOS-L Is Upregulated Only on HPV-Positive Cells.

While the treatment of squamous cell carcinoma of the head and neck (HNSCC) with radiotherapy (RT) is complemented more and more by immunotherapy in clinical trials, little is known about the impact of the human papillomavirus (HPV) status or the applied RT scheme on the immune phenotype of the tumor cells. Therefore, we aimed to examine the impact of the HPV status of four human HNSCC cell lines on cell death and the expression of immune checkpoint molecules (ICMs) after RT with either hypofractionation irradiation (5x3.0Gy) or a high single dose (1x19.3Gy) via multicolor flow cytometry and quantitative PCR at an early time point after therapy. In our study, 5x3.0Gy RT induced high numbers of early and late apoptotic cells independent of the HPV status, but necrosis was only increased in the HPV-positive UM-Scc-47 cells. Generally, the immune stimulatory ICMs (CD70, CD137-L, ICOS-L) were less affected by RT compared to the immune suppressive ones (PD-L1, PD-L2, and the herpesvirus entry mediator (HVEM)). A significant higher surface expression of the analyzed ICMs was found after hypofractionated RT compared to a single high dose; however, regardless of the HPV status, with the exception of ICOS-L. Here, HPV-positive HNSCC tumor cells showed a stronger response to 5x3.0Gy than HPV-negative ones. On the RNA level, only minor alterations of ICMs were observed following RT, with the exception of the HPV negative cell line CAL33 treated with 5x3.0Gy, where PD-L2, HVEM and CD70 were significantly increased. We conclude that the HPV status may not distinctly predict immunological responses following RT, and thus cannot be used as a single predictive marker for therapy responses in HNSCC. In contrast, the patient-specific individual expression of ICMs following RT is preferable for the targeted patient selection for immune therapy directed against distinct ICM.

Abstract 272: Glycomics: Protein glycosylation changes in the pathogenesis of head and neck cancer

Abstract Background: Glycosylation is the most common post-translational modification of proteins, and glycosylation changes at cell surfaces are frequently associated with malignant epithelia. These affect many functions including proliferation, motility and invasiveness, increasing the propensity to metastasise. Many head &amp; neck squamous cell carcinomata (HNSCC), especially those originating in the lymphoid mucosa of the oropharynx, are driven by so-called high risk genotypes of Human Papillomavirus (HPV). Five-year survival remains poor, averaging around 50% globally: this is partly related to late diagnosis. Specific protein glycosylation signatures on malignant keratinocytes have promise as diagnostic and prognostic markers and as therapeutic targets. Here we begin creation of a glyco-fingerprint library mapping the glyco-space of HNSCC. Materials and methods: Six established HNSCC cell lines were used to capture the qualitative and semi-quantitative profile of the N- and O-glycome using the well-established porous graphitized carbon (PGC) glycomics approach. Furthermore, we attempted to evaluate the effect sialylation has on HNSCC cell migration by enzymatically removing sialic acid residues from cell surface glycoconjugates. Therefore, we used the enzyme neuraminidase to remove sialic acids from HNSCC cells and measured cell proliferation rates by using incucyte. Results: In Human Papillomavirus (HPV) negative HNSCC cell lines, 37-41% of cells carried oligomannoses on their surfaces compared to 45% in HPV+ cell lines. Sialylated N-glycan were the most abundant type of N-glycan, contributing to &amp;gt;40% of the N-glycome in the HPV negative cell lines: but just 32-38% in HPV+ cell lines; di- and tri-sialylated forms were most frequent. The majority of complex type N-glycans (33%) carried core-fucose residues with just minor levels (&amp;lt;3%) of Lewis-type fucosylation identified. We also observed unusual types of oligomannose N-glycans carrying core-fucose residues. Enzymatic sialic acid removal resulted in 40% reduction in cell doubling numbers. Conclusion: This first systematic mapping reveals diverse glycosylation of head and neck cancer cell lines with high levels of core fucosylation and sialylation. Differences between HPV positive and negative cells may partly explain the comparatively good prognosis of HPV-driven HNC. This mapping forms the basis of histopathological profiling of real tumours which might have prognostic potential and suggest targets for immunotherapy. Removal of sialic acids resulted in decreased cell proliferation in vitro, indicating the fundamental role of protein glycosylation in the pathogenesis of HNC. Citation Format: Mohammad Rasheduzzaman, Xi Zhang, Riccardo Dolcetti, Liz Kenny, Newell W. Johnson, Daniel Kolarich, Chamindie Punyadeera. Glycomics: Protein glycosylation changes in the pathogenesis of head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 272.

Abstract 1930: 4-nitroquinoline-1-oxide enhances induction of apoptosis preferentially in HPV positive head and neck squamous cell carcinoma cells

Abstract Background: Tobacco use and human papillomavirus (HPV) infection are both strong contributors to the oncogenesis of head and neck squamous cell carcinoma (HNSCC). Despite HPV's causative role in HNSCC, it confers no statistically significant difference in mutational profile between HPV-associated HNSCC smokers and non-smokers. Our earlier preliminary data suggested that HPV positive (+) HNSCC cells have better activation of DNA repair proteins compared to HPV negative (-) HNSCC cells when treated with 4NQO, a mutagen and carcinogen which serves as a surrogate for tobacco exposure. This may partially explain HPV+ HNSCC's higher mutational resistance. In addition to increased DNA repair, HPV+ cells may also eliminate mutations through higher rates of apoptosis compared to HPV- cells. Hence, we wanted to test this hypothesis by analyzing the effects of 4NQO on apoptosis in HPV+ and HPV- HNSCC cells. Methods: NOK, NOK E6.E7, SCC40 (HPV-), SCC104 and UMSCC47 (HPV+) cells were treated with different concentrations of 4NQO (0, 0.1, and 0.2 ug/ml) for 24 and 48 hours. Untreated (control) and 4NQO treated cells were assessed for cell viability using MTT assay. Apoptotic cells were determined using Annexin-V FITC flow cytometry analysis, and Western blot was performed to check the apoptotic proteins for the afore-mentioned control and 4NQO treatments. Results: MTT assay showed decreased cell viability of NOK E6.E7, SCC47 and SCC104 cells in response to 4NQO treatment compared to relatively higher cell viability of SCC40 cells. Flow cytometry confirmed that HPV+ HNSCC cells are relatively more apoptotic compared to HPV- cells following 4NQO treatment at 24 and 48 hours. This is further supported by activation of pro-apoptotic proteins including p53, cleaved caspase 3, cleaved PARP, cleaved caspase 9, and p21 to a greater extent in HPV+ cells at different time points of 4NQO treatment using Western blot analysis. Conclusion: Our in-vitro data suggest HPV may stimulate a protective measure against smoking-induced mutations by increasing apoptosis before cells can contribute to the HNSCC mutational profile. Taken together, these data explain why HPV associated HNSCC have a better prognosis than HPV negative irrespective of smoking history. Citation Format: Gauri Shishodia, Alice Lee, Rhodee Ric Toledo, Emily Zimmerman, Xiaohua Rong, Adam Xiao, Lynn Harrison, Cherie Ann Nathan. 4-nitroquinoline-1-oxide enhances induction of apoptosis preferentially in HPV positive head and neck squamous cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1930.

Effect of 4‐Nitroquinoline‐1‐Oxide on DNA damage and repair in head and neck squamous cell carcinoma cells

Background Tobacco exposure and human papillomavirus (HPV) infection are among the main factors for development of head and neck squamous cell carcinoma (HNSCC). Interestingly, recent studies show that HPV positive (+) smokers and non-smokers have similar mutational profiles, which suggests that mutations induced in smokers could be associated with the protective effects of HPV. Our preliminary data showed that 4NQO, a mutagen and carcinogen which serves as a surrogate for tobacco exposure induces oxidative DNA damage and DSB formation in HNSCC cells at 6 hrs. HPV+ HNSCC cells also showed a relative increase in activation of homologous recombination (HR) DNA repair proteins compared to HPV negative (-) HNSCC cells at 6 hrs. In this study, we wanted to investigate the effect of 4NQO on DNA damage and repair activation at 24 hrs in HPV+ and HPV- HNSCC cells. Methods NOK E6.E7 +/-, UPCI-SCC40 (HPV-), UM-SCC104 and UM-SCC47 (HPV+) cells were utilized to check effects of 4NQO on DNA repair. Oxidative DNA damage, double strand break formation (DSBs) and protein expression were analyzed by FPG-modified alkaline comet assay, γH2AX staining and Western analysis following treatment with 4NQO for 6 hr, replacement with fresh media, and sample collection at 24 hr. Results 4NQO increased oxidative DNA damage, single strand breaks, and yH2AX foci at 6 hrs; however, these lesions were no longer present at 24 hrs. This suggests DNA repair had probably occurred upon removal of 4NQO. We no longer observed an increase in phosphorylation of BRCA1, Rad51, H2AX, and p21 levels in HNSCC cells at 24 hr although it was present at 6 hrs. Conclusion Our in-vitro data suggests that HPV may have a role in the activation of DNA repair proteins by 4NQO at 6 hrs. This may have allowed for successful repair of DNA damage in both HPV+ and HPV- HNSCC cells by 24 hrs. Taken together, these data suggest that HPV may protect against smoking-induced mutations by preferentially increasing HR DNA repair protein activation.

Genotyping and Characterization of HPV Status, Hypoxia, and Radiosensitivity in 22 Head and Neck Cancer Cell Lines.

Simple SummaryCell lines are widely used for research, but even commonly used cell lines may not be well characterized, making validation of published data and generalizing the results to the clinic problematic. Here, we have established a head and neck squamous cell carcinomas (HNSCC) cell line database. We believe that these cell lines are suitable models to study HNSCC disease in vitro and in vivo and may provide a valuable tool for investigators wishing to study the relation between e.g., hypoxia and radiosensitivity in HNSCC.To study head and neck squamous cell carcinomas (HNSCC) in vitro, a large variety of HNSCC cell lines have been developed. Here, we characterize a panel of 22 HNSCC cell lines, thereby providing a tool for research into tumor-specific treatment options in HNSCC. Both human papillomavirus (HPV) positive and HPV negative tumor cell lines were collected from commercial and collaborative sources. Short tandem repeat profiling was used to confirm or characterize the identity of the cell lines. Targeted sequencing was performed using a standard pathology single molecule Molecular Inversion Probe panel to detect mutations for 23 tumor suppressors and oncogenes. HPV status, p16 status, radiosensitivity data, and hypoxia data are summarized from all cell lines. We detected HPV transcripts in five cell lines, all of which overexpressed p16. One HPV negative cell line was also p16 positive. We detected mutations in KIT (SCCNij185), PIK3CA (SCCNij185), and CDKN2A (UT-SCC-5 and UT-SCC-38). TP53 mutations were the most frequent, occurring in 16/22 cell lines. HPV infection and TP53 mutations were almost mutually exclusive, with the exception of 93-VU-147T. The cell lines exhibited a wide range of sensitivities towards hypoxia and irradiation. Here, we provide a description of a set of frequently used HNSCC cell lines with diverse characteristics as found in HNSCC patients.

Abstract PO-033: Metabolic differences between human papillomavirus-related and smoking-related squamous cell carcinoma of the head and neck independently predict overall patient survival

Abstract Background: Human papillomavirus (HPV) status and tobacco use are the strongest prognostic factors of survival for squamous cell carcinoma of the head and neck (SCCHN). Patients with HPV-related SCCHN live more than twice as long as patients with tobacco-related SCCHN. The underlying metabolic dysregulation characterizing these two clinically distinct groups may pose exciting translational targets that are yet unknown. Using a metabolome-wide approach, we investigated the metabolic differences of HPV vs smoking-related SCCHN in a large patient population. We then linked those metabolic differences to patient survival. Methods: We recruited 137 patients from Emory University hospitals and clinics: 55 HPV-positive never smokers (i.e., HPV-related SCCHN) and 82 HPV-negative current or former smokers (i.e., smoking-related SCCHN). We performed untargeted high-resolution metabolomics using liquid chromatography-mass spectrometry on pre-treatment patient plasma at the Emory Clinical Biomarkers Laboratory. Our statistical approach has three steps. 1) Metabolite feature selection: We aggregated the results from two selection algorithms (5-fold cross-validated partial least squares discriminant analysis and support vector machine) to determine the metabolites that differentiated HPV vs smoking-related SCCHN. 2) Metabolic pathway analysis (Mummichog): We identified statistically significant (P&amp;lt;0.05) metabolic pathways stemming from the step 1 metabolites. 3) Metabolic profile and survival analysis: We created a metabolite-based linear principal component using the significant pathway metabolites in step 2 and estimated its association with overall survival using adjusted Cox models. Results: Of the 9,640 extracted metabolites, the two models found 229 that differentiated HPV vs smoking-related SCCHN. Metabolic pathway analysis found fatty acid and steroid metabolism was significantly upregulated in HPV patients: bile acid biosynthesis (P&amp;lt;0.0001), 21-carbon steroid hormone metabolism (P=&amp;lt;0.0001), fatty acid beta-oxidation (P=0.04). Pathways related to sugar metabolism were significantly upregulated in smoking patients: galactose metabolism (P=0.003), starch and sucrose metabolism (P=0.007), hexose phosphorylation (P=0.03), sialic acid metabolism (P=0.03). A principal component comprised of the 16 pathway metabolites was significantly associated with overall survival (HR=1.27 per standard deviation, P=0.01, 95% CI: 1.06-1.51) independent of HPV, smoking, tumor site, age, sex, race, and body mass index. Conclusions: Metabolites corresponding to fatty acid, steroid, and sugar metabolism differentiated HPV-related SCCHN from smoking-related SCCHN. They may be clinically informative as prognostic markers of survival. Our novel findings in patient plasma parallel those from HPV positive vs negative SCCHN cell lines, suggesting that metabolites in blood may be putative biomarkers of tumor metabolic activity and thus potentially useful for drug discovery. Citation Format: Ronald C. Eldridge, Karan Uppal, Jonathan Beitler, Kristin Higgins, Evanthia C. Wommack, Dong M. Shin, Nabil F. Saba, Andrew Miller, Deborah W. Bruner, Canhua Xiao. Metabolic differences between human papillomavirus-related and smoking-related squamous cell carcinoma of the head and neck independently predict overall patient survival [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-033.

Abstract 6185: In vivo screening in HPV positive and HPV negative head and neck squamous cell carcinoma

Abstract Background: The pathogenesis of head and neck squamous cell carcinoma (HNSCC) largely occurs through two pathways, human papillomavirus (HPV) mediated or chemical carcinogenesis secondary to alcohol and tobacco use. HPV status is an important prognostic factor and marker for sensitivity to radiation therapy in HNSCC patients. HPV negative HNSCC has been shown to have increased radioresistance compared to HPV positive HNSCC leading to poorer prognosis. The goal of this work is to utilize in vivo screening technology to identify key differential targets between HPV positive and negative tumors for both therapeutic purposes as well as to identify differential pathways between these two malignancies. Methods: Pooled, barcoded shRNA libraries focused on DNA damage repair (DDR) and currently targetable genes were transduced into three HPV positive (UM-SCC-147, UD-SCC-2, UPCI:SCC152) and three HPV negative (UM-SCC-22A, Cal-27, HN31) cell lines. Each cell line was injected into the mouse flank and allowed to grow to ~100mm3. Tumors were divided into control and radiation groups; the latter being treated with 6-10 Gy at 2 Gy/treatment. For each tumor type radiation was titrated to generate an approximate 20% reduction in tumor size. Tumors were then allowed to grow for an additional 2 weeks and then harvested. Barcodes from each tumor were sequenced and compared to both the reference input cells as well as untreated control tumors. Barcodes observed at decreased frequency in treated tumors indicated a potential target for therapeutic sensitization. Both p-values as well as percentile median fold change for each gene were calculated. These values were averaged for the three HPV negative and three HPV positive cell lines and used for analysis. Results: Multiple genes in the DDR and FDA libraries were identified from the HPV negative and HPV positive groups that may be associated with radioresistance in vivo. Example genes that were identified primarily in the HPV negative group include BTK, TXN, TTK, while those in the HPV positive group include RNF168, RAD50, ESR1. Inhibition of these genes may lead to increased cell death following radiation therapy through diminished DNA repair of radiation-induced damage. Conclusion: Our study identifies targetable genes that may contribute to radioresistance in both HPV negative and HPV positive head and neck squamous cell carcinoma and warrants further analysis. Citation Format: Mohamed K. Abdelhakiem, Liangpeng Yang, Tongxin Xie, Manish Kumar, David Molkentine, Aakash Sheth, Mitchell Frederick, Jeffrey Myers, Curtis R. Pickering, Heath D. Skinner. In vivo screening in HPV positive and HPV negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6185.

Human myoma tissue-based extracellular matrix models for testing the effects of irradiation on the HPV positive cells

BackgroundThis study was designed to investigate the invasion of human papillomavirus (HPV) positive human cervical carcinoma cell lines in human leiomyoma-based extracellular matrices in vitro, and to test the suitability of the model for studying the irradiation effects on the cancer cell invasion.MethodsHPV positive cervical carcinoma cell lines SiHa and CaSki, and HPV negative squamous cell carcinoma cell line HSC-3 were used. CaSki cells contain around 600 copies of HPV 16 virus in the genome, whereas SiHa have only 1–2 copies per cell. Cells were analyzed using two different human tumor derived extracellular matrix methods (3D myoma disc model, and Myogel Transwell invasion assay). Cultures were irradiated with 4 Gy. Myoma invasion area and the depth of invasion were measured with ImageJ 1.51j8 software. Statistical analyses were performed with SPSS Statistics (IBM SPSS® Statistics 25).ResultsAll cells invaded through Myogel coated Transwell membranes and within myoma discs. In myoma discs, a difference in the invasion depth (p = 0.0001) but not in invasion area (p = 0.310) between the HPV positive cell lines was seen, since SiHa (less HPV) invaded slightly better than CaSki (more HPV). HSC-3 cells (HPV negative) invaded deepest (p = 0.048) than either of the HPV positive cell line cells. No difference was detected in the invasion area (p = 0.892) between HPV positive and HPV negative cells. The ionized radiation significantly reduced the invasion depth of HSC-3 (p = 0.008), SiHa (p = 0.0001) and CaSki (p = 0.005). No significant effect on the invasion area was detected in any of the cell lines. However, a significant difference was observed between SiHa and CaSki in the reduction of the invasion depth after radiation (p = 0.013) as the reduction was greater with SiHa than CaSki.ConclusionsBoth solid and gelatinous human leiomyoma-based extracellular matrix models were suitable platforms to study the invasion of HPV positive cervical carcinoma cells in vitro. SiHa cells with less HPV copy number cells invaded slightly better and were slightly more sensitive to irradiation than CaSki cells with high HPV copy number. However, there was no drastic differences between the invasion properties of these carcinoma cells.

HPV Infection Significantly Accelerates Glycogen Metabolism in Cervical Cells with Large Nuclei: Raman Microscopic Study with Subcellular Resolution.

Using Raman microscopy, we investigated epithelial cervical cells collected from 96 women with squamous cell carcinoma (SCC) or belonging to groups I, IIa, IIID-1 and IIID-2 according to Munich III classification (IIID-1 and IIID-2 corresponding to Bethesda LSIL and HSIL groups, respectively). All women were tested for human papillomavirus (HPV) infection using PCR. Subcellular resolution of Raman microscopy enabled to understand phenotypic differences in a heterogeneous population of cervical cells in the following groups: I/HPV−, IIa/HPV−, IIa/HPV−, LSIL/HPV−, LSIL/HPV+, HSIL/HPV−, HSIL/HPV+ and cancer cells (SCC/HPV+). We showed for the first time that the glycogen content in the cytoplasm decreased with the nucleus size of cervical cells in all studied groups apart from the cancer group. For the subpopulation of large-nucleus cells HPV infection resulted in considerable glycogen depletion compared to HPV negative cells in IIa, LSIL (for both statistical significance, ca. 45%) and HSIL (trend, 37%) groups. We hypothesize that accelerated glycogenolysis in large-nucleus cells may be associated with the increased protein metabolism for HPV positive cells. Our work underlines unique capabilities of Raman microscopy in single cell studies and demonstrate potential of Raman-based methods in HPV diagnostics.

IFI16 promotes cervical cancer progression by upregulating PD-L1 in immunomicroenvironment through STING-TBK1-NF-kB pathway

Cervical cancer remains one of the leading causes of cancer death worldwide. Immunotherapy is the most promising cancer therapeutics in recent years and has gain positive results in several cancers in the clinic. This study was aimed to investigate the roles and mechanism of IFI16 in cervical cancer immunotherapy. We observed an abnormally high expression of Programmed cell death 1 ligand 1 (PD-L1) and Interferon‐inducible 16 (IFI16) in Human papillomavirus (HPV) positive cervical cancer cells compared with HPV negative cervical cancer cells. Moreover, IFI16 promotes cervical cancer development in vitro and in vivo as the oncogenic role of PD-L1. In the subsequent mechanism investigation, we found that IFI16 activated STING-TBK1-mediated immunoregulation, and subsequently activated downstream NF-kB pathway, which interacted with the proximal region of PD-L1 promoter to facilitate PD-L1 expression. In conclusion, we found that IFI16 positively regulate PD-L1 through STING-TBK1-NF-kB pathway, thus promoting cervical cancer progression. The roles of IFI16 in cervical cancer progression deserve further investigation and hold the promise of being developed as a novel immunotherapy target in the future.

Single Nucleotide Polymorphism rs6942067 Is a Risk Factor in Young and in Non-Smoking Patients with HPV Negative Head and Neck Squamous Cell Carcinoma.

Genetic factors behind the increasing incidence of human papillomavirus (HPV) negative head and neck squamous cell carcinoma (HNSCC) in young non-smokers are suspected, but have not been identified. Recently, rs6942067, a single nucleotide polymorphism (SNP) located upstream of the DCBLD1 gene, was found associated with non-smoking lung adenocarcinoma. To validate if this SNP is also implicated in HNSCC, participants of The Cancer Genome Atlas HNSCC cohort were investigated for rs6942067 status, associated DCBLD1 expression, and clinical characteristics. Occurrence of the rs6942067 GG genotype is significantly higher in young and in HPV negative non-smoking HNSCC than in other HNSCC. Additionally, rs6942067 GG is associated with higher DCBLD1 expression in HNSCC and patients with high DCBLD1 expression have a worse overall survival at three years, both in univariate and multivariate analysis. Furthermore, high DCBLD1 expression is associated with activation of the integrin signaling pathway and its phosphorylation with EGFR and MET. Collectively, these findings suggest that DCBLD1 plays a critical role in HNSCC and demonstrate an association between rs6942067 and clinical characteristics of young age and HPV negative non-smoking status in HNSCC patients.

Abstract 1401: Sensitive detection of integrated HPV in carcinoma cells

Abstract It is estimated that 79 million people in the US are currently infected with human papillomavirus (HPV). There are more than 200 different HPV types that are common worldwide. HPV types are categorized as either high risk (HR) or low risk (LR). LR types, such as HPV 6 and HPV 11, can cause genital warts or squamous papillomas of the oral cavity, while persistent infection with HR HPV types can induce cervical, anal, or head and neck cancers. Episomal HPV DNA can be detected in advanced stages of cancer. HPV integration in the host genome drives oncogenesis by the expression of the two major viral oncogenes, E6 and E7, which abrogate cell cycle checkpoints, promote cellular proliferation, and cause progressive genetic instability. Hence, screening for the presence of E6/E7 RNA transcripts expression is considered the gold standard for confirming active HPV infection. Early detection of active HPV is critical for better understanding of prognosis and therapeutics. So far, detection of integrated HPV DNA in tissue specimens has been challenging. A novel, cost-effective, in situ hybridization (ISH) detection assay has been developed using two sets of HPV probes with enhanced signaling, termed hyper probes. The Hyper HPV D probe detects HR HPV DNA and the Hyper HPV DR probe can simultaneously detect DNA and E6/E7 RNA of HR HPV subtypes in cancer cell lines and formalin fixed paraffin embedded (FFPE) biopsy specimens. The probes were tested on cervical carcinoma SiHa cells containing 1-3 copies of integrated DNA of HPV 16 and HPV negative cervical cancer C33A cell line. The Hyper HPV DR probe resulted in 1-3 puncta in more than 90% of SiHa cell nuclei, indicating integrated copies of HPV DNA into the host genome. There was also the additional presence of a diffuse signal which was abolished by pretreating SiHa cells with RNase. These data suggest the diffuse signal corresponds to E6/E7 mRNA expression. No signals were detected in HPV negative C33A cells. When cells were stained with Hyper HPV D probes, only punctate signals in nuclei of SiHa cells was present and no signal was evident in C33A control cells. Additionally, human cervical and anal FFPE biopsies were analyzed. Simultaneous presence of both punctate signals and diffuse signals were detected when using Hyper HPV DR probes in HR HPV infected tissues, while only punctate signal was detected when using HPV D hyper probes. The signal from the hyper probes was significantly stronger when compared to other commercially available HPV detection kits. Moreover, other commercially available kits failed to detect low copy of integrated HPV. In conclusion, these data demonstrate the Hyper HPV probes can reliably detect low copies of HPV DNA integrated in the host genome and the presence of HPV mRNA using ISH in HPV infected cells. Citation Format: Maurizio Mauro, Richard Delle Bovi, Jack Coleman. Sensitive detection of integrated HPV in carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1401.

Autocrine STAT3 activation in HPV positive cervical cancer through a virus-driven Rac1-NFκB-IL-6 signalling axis.

Persistent human papillomavirus (HPV) infection is the leading cause of cervical cancer. Although the fundamental link between HPV infection and oncogenesis is established, the specific mechanisms of virus-mediated transformation are not fully understood. We previously demonstrated that the HPV encoded E6 protein increases the activity of the proto-oncogenic transcription factor STAT3 in primary human keratinocytes; however, the molecular basis for STAT3 activation in cervical cancer remains unclear. Here, we show that STAT3 phosphorylation in HPV positive cervical cancer cells is mediated primarily via autocrine activation by the pro-inflammatory cytokine Interleukin 6 (IL-6). Antibody-mediated blockade of IL-6 signalling in HPV positive cells inhibits STAT3 phosphorylation, whereas both recombinant IL-6 and conditioned media from HPV positive cells leads to increased STAT3 phosphorylation within HPV negative cervical cancer cells. Interestingly, we demonstrate that activation of the transcription factor NFκB, involving the small GTPase Rac1, is required for IL-6 production and subsequent STAT3 activation. Our data provides new insights into the molecular re-wiring of cancer cells by HPV E6. We reveal that activation of an IL-6 signalling axis drives the autocrine and paracrine phosphorylation of STAT3 within HPV positive cervical cancers cells and that activation of this pathway is essential for cervical cancer cell proliferation and survival. Greater understanding of this pathway provides a potential opportunity for the use of existing clinically approved drugs for the treatment of HPV-mediated cervical cancer.

Sex differences in patients with high risk HPV-associated and HPV negative oropharyngeal and oral cavity squamous cell carcinomas

Background Human papilloma virus (HPV)-associated head and neck cancer is now recognized as a distinct clinical entity from HPV-negative tumors, which are primarily associated with tobacco and alcohol exposure.Little is known, however, about the behavior of HPV-associated oropharynx (OP) and oral cavity (OC) SCCs as two distinct cancers and how sex affects the overall survival (OS) in these two cancers. The objective of our study is to determine if sex is associated with overall survival (OS) in patients with high-risk human papillomavirus (HPV)-positive and HPV-negative squamous cell carcinomas (SCC) in the oropharynx and oral cavity sites.MethodsThis is a retrospective cohort study using a national database. Data were extracted from the National Cancer Database (NCDB) of patients diagnosed with OP or OC SCC from 2010 to 2014. Univariate and multivariate survival analyses were conducted with chi-square tests, Kaplan-Meier estimates, log-rank tests, and Cox proportional hazards multivariable modeling.ResultsA total of 30,707 patients (13,694 OP HPV-associated, 7933 OP HPV-, 1220 OC HPV-associated, 7860 OC HPV-) were identified. In all four groups, women tended to be older and have lower T and N clinical classification than men. Though there were no significant differences in OS between the sexes in OP HPV-associated cancers, female sex was associated with worse OS in OP HPV- cancers (HR: 1.15; 95% CI 1.04–1.28, p = 0.004), whereas it was associated with improved OS in OC HPV-associated and HPV- cancers (HPV-associated: HR: 0.71; 95% CI 0.50–0.99, p = 0.048; HPV-: HR: 0.87; 95% CI 0.78–0.95, p = 0.004).ConclusionThe effect of sex on OS in OC and OP SCC appears to vary based on tumor location and HPV status. While the source of this difference in prognostic association is unclear, it may be related to an emerging difference in the biology of HPV carcinogenesis in these locations.