Effect of ATR Inhibition in RT Response of HPV-Negative and HPV-Positive Head and Neck Cancers.
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ABSTRACT:
SUBMITTER: Dok R
PROVIDER: S-EPMC7913134 | biostudies-literature | 2021 Feb
REPOSITORIES: biostudies-literature
Publications
Effect of ATR Inhibition in RT Response of HPV-Negative and HPV-Positive Head and Neck Cancers.
Dok Rüveyda R Glorieux Mary M Bamps Marieke M Nuyts Sandra S
International journal of molecular sciences 20210203 4
Radiotherapy (RT) has a central role in head and neck squamous cell carcinoma (HNSCC) treatment. Targeted therapies modulating DNA damage response (DDR) and more specific cell cycle checkpoints can improve the radiotherapeutic response. Here, we assessed the influence of ataxia-telangiectasia mutated and Rad3-related (ATR) inhibition with the ATR inhibitor AZD6738 on RT response in both human papillomavirus (HPV)-negative and HPV-positive HNSCC. We found that ATR inhibition enhanced RT response ...[more]
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Project description:The DNA cytosine deaminase APOBEC3B (A3B) is a newly recognized endogenous source of mutations in a range of human tumors, including head/neck cancer. A3B inflicts C-to-T and C-to-G base substitutions in 5'-TCA/T trinucleotide motifs, contributes to accelerated rates of tumor development, and affects clinical outcomes in a variety of cancer types. High-risk human papillomavirus (HPV) infection causes A3B overexpression, and HPV-positive cervical and head/neck cancers are among tumor types with the highest degree of APOBEC signature mutations. A3B overexpression in HPV-positive tumor types is caused by the viral E6/E7 oncoproteins and may be an early off-to-on switch in tumorigenesis. In comparison, less is known about the molecular mechanisms responsible for A3B overexpression in HPV-negative head/neck cancers. Here, we utilize an immunohistochemical approach to determine whether A3B is turned from off-to-on or if it undergoes a more gradual transition to overexpression in HPV-negative head/neck cancers. As positive controls, almost all HPV-positive oral epithelial dysplasias and oropharyngeal cancers showed high levels of nuclear A3B staining regardless of diagnosis. As negative controls, A3B levels were low in phenotypically normal epithelium adjacent to cancer and oral epithelial hyperplasias. Interestingly, HPV-negative and low-grade oral epithelial dysplasias showed intermediate A3B levels, while high-grade oral dysplasias showed high A3B levels similar to oral squamous cell carcinomas. A3B levels were highest in grade 2 and grade 3 oral squamous cell carcinomas. In addition, a strong positive association was found between nuclear A3B and Ki67 scores suggesting a linkage to the cell cycle. Overall, these results support a model in which gradual activation of A3B expression occurs during HPV-negative tumor development and suggest that A3B overexpression may provide a marker for advanced grade oral dysplasia and cancer.
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Project description:Human papillomaviruses (HPVs) are associated with nearly all cervical cancers (CCs), 20-30% of head and neck cancers (HNCs), and other cancers. Because HNCs also arise in HPV-negative patients, this type of cancer provides unique opportunities to define similarities and differences of HPV-positive versus HPV-negative cancers arising in the same tissue. Here, we describe genome-wide expression profiling of 84 HNCs, CCs and site-matched normal epithelial samples in which we used laser capture microdissection to enrich samples for tumor-derived versus normal epithelial cells. This analysis revealed that HPV+HNCs and CCs differed in their patterns of gene expression yet shared many changes compared to HPV-HNCs. Some of these shared changes were predicted, but many others were not. Notably, HPV+HNCs and CCs were found to be upregulated in their expression of a distinct and larger subset of cell cycle genes than observed in HPV-HNC. Moreover, HPV+ cancers over-expressed testis-specific genes that are normally expressed only in meiotic cells. Many, though not all, of the hallmark differences between HPV+HNC and HPV-HNC were a direct consequence of HPV and in particular the viral E6 and E7 oncogenes. This included a novel association of HPV oncogenes with testes specific gene expression. These findings in primary human tumors provide novel biomarkers for early detection of HPV+ and HPV- cancers, and emphasize the potential value of targeting E6 and E7 function, alone or combined with radiation and/or traditional chemotherapy, in the treatment of HPV+ cancers. Experiment Overall Design: All tissue samples were fresh frozen in liquid nitrogen and collected with patientsâ?? consent under approval of the Institutional Review Boards from all participating institutions. Each tissue sample was cryosectioned, and selected sections stained with hematoxylin/eosin, and reviewed to determine tumor content, pathological status, and freedom from necrosis and freezing artifacts. Epithelial cells from all normal samples and tumor cells from HNC or CC samples with less than 80% tumor were laser capture microdissected from adjacent sections using a PixCell II LCM system. For guidance, an adjacent section was briefly stained with hematoxylin to visualize tissue structure. Total RNA was extracted from sectioned and/or microdissected samples as follows: 1 ml of TRIzol (Invitrogen, Carlsbad, CA) was added to each tissue sample, homogenized by passing through a 20 gauge needle, and added to 0.2 ml chloroform. After centrifugation at 20,000 xg for 20 min at 4oC, RNA in the aqueous phase was precipitated with an equal volume of isopropanol for 30 min at 4oC, pelleted, and washed twice with cold 70% ethanol. Double strand (ds) cDNA was synthesized from this RNA using a SuperScript ds cDNA synthesis kit (Invitrogen) and T7 promoter-linked oligo (dT)24. Complementary RNA (cRNA) was synthesized from T7 promoter-linked ds cDNA using a MEGAscript high transcription kit (Ambion, Austin, TX). To obtain a sufficient cRNA for 2 microarray hybridizations, this amplification process was repeated. Second round cRNA was biotin labeled using a BioArray High Yield RNA Transcript Labeling Kit (Enzo Life Sciences, Farmingdale, NY) and stored at -80oC until hybridized. cRNA quality and quantity was determined by gel electrophoresis and UV spectrophotometry. Whole human gene expression was profiled using Affymetrix Human Genome U133 Plus 2.0 arrays.
2008-06-15 | E-GEOD-6791 | biostudies-arrayexpress
Project description:Human papillomaviruses (HPVs) are associated with nearly all cervical cancers (CCs), 20-30% of head and neck cancers (HNCs), and other cancers. Because HNCs also arise in HPV-negative patients, this type of cancer provides unique opportunities to define similarities and differences of HPV-positive versus HPV-negative cancers arising in the same tissue. Here, we describe genome-wide expression profiling of 84 HNCs, CCs and site-matched normal epithelial samples in which we used laser capture microdissection to enrich samples for tumor-derived versus normal epithelial cells. This analysis revealed that HPV+HNCs and CCs differed in their patterns of gene expression yet shared many changes compared to HPV-HNCs. Some of these shared changes were predicted, but many others were not. Notably, HPV+HNCs and CCs were found to be upregulated in their expression of a distinct and larger subset of cell cycle genes than observed in HPV-HNC. Moreover, HPV+ cancers over-expressed testis-specific genes that are normally expressed only in meiotic cells. Many, though not all, of the hallmark differences between HPV+HNC and HPV-HNC were a direct consequence of HPV and in particular the viral E6 and E7 oncogenes. This included a novel association of HPV oncogenes with testes specific gene expression. These findings in primary human tumors provide novel biomarkers for early detection of HPV+ and HPV- cancers, and emphasize the potential value of targeting E6 and E7 function, alone or combined with radiation and/or traditional chemotherapy, in the treatment of HPV+ cancers. Keywords: Gene expression ptofiles of primary cancers
2007-05-15 | GSE6791 | GEO
Project description:Human papillomavirus (HPV) was recently recognized as a new risk factor for head and neck squamous cell carcinoma. For oropharyngeal cancers, an HPV+ status is associated with better prognosis in a subgroup of nonsmokers and nondrinkers. However, HPV infection is also involved in the biology of head and neck carcinoma (HNC) in patients with a history of tobacco use and/or alcohol consumption. Thus, the involvement of HPV infection in HN carcinogenesis remains unclear, and further studies are needed to identify and analyze HPV-specific pathways that are involved in this process. Using a quantitative proteomics-based approach, we compared the protein expression profiles of two HPV+ HNC cell lines and one HPV- HNC cell line. We identified 155 proteins that are differentially expressed (P < 0.01) in these three lines. Among the identified proteins, prostate stem cell antigen (PSCA) was upregulated and eukaryotic elongation factor 1 alpha (EEF1?) was downregulated in the HPV+ cell lines. Immunofluorescence and western blotting analyses confirmed these results. Moreover, PSCA and EEF1 ? were differentially expressed in two clinical series of 50 HPV+ and 50 HPV- oral cavity carcinomas. Thus, our study reveals for the first time that PSCA and EEF1 ? are associated with the HPV-status, suggesting that these proteins could be involved in HPV-associated carcinogenesis.
| S-EPMC3955617 | biostudies-literature
Project description:Radiotherapy is one of the most used treatment approaches for head and neck squamous cell carcinoma (HNSCC). Targeted inhibition of DNA repair machinery has the potential to improve treatment response by tailoring treatment to cancer cells lacking specific DNA repair pathways. Human papillomavirus (HPV)-negative and HPV-positive HNSCCs respond differently to radiotherapy treatment, suggesting that different approaches of DNA repair inhibition should be employed for these HNSCC groups. Here, we searched for optimal radiosensitization approaches for HPV-positive and HPV-negative HNSCCs by performing a targeted CRISPR-Cas9 screen. We found that inhibition of base excision repair resulted in a better radiotherapy response in HPV-positive HNSCC, which is correlated with upregulation of genes involved in base excision repair. In contrast, inhibition of nonhomologous end-joining and mismatch repair showed strong effects in both HNSCC groups. We validated the screen results by combining radiotherapy with targeted inhibition of DNA repair in several preclinical models including primary and recurrent patient-derived HNSCC xenografts. These findings underline the importance of stratifying HNSCC patients for combination treatments.
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Project description:Head and neck squamous cell carcinomas (HNSCC) are a heterogeneous group of tumours that originate predominantly from the oral cavity, pharynx and larynx. Our aim was to determine whether salivary miRNA expression levels can diagnose these cancer subtypes. Saliva samples were collected from healthy controls (n=113, smoker and non-smokers), HPV-positive (n=54) and HPV-negative (n=47) HNSCC patients. The miRNA expression levels in saliva was quantified using qPCR. The potential of salivary miRNAs to discriminate these groups of patients was evaluated using multiple logistic regression with ROC analysis and a 10-fold cross-validation analysis. Salivary miRNA-9, -127, -134, -191, -222 and -455 were shown to discriminate a control group from a HPV-negative HNSCC patient group with a sensitivity of 60% and a specificity of 94%; whilst salivary miRNA-9,-134, -196b, -210, and -455 were the most parsimonious subset discriminating a control group from a HPV-positive HNSCC group, with a sensitivity of 65% and a specificity of 95%. Furthermore, miRNA-9, -134, -196b, -210 and -455 as a panel, was the most parsimonious subset to discriminate HPV-positive HNSCC patients from HPV-negative HNSCC patients. In addition, the expression levels of miRNA-9, -127, -196a, -196b, -210, -222 and -455 were significantly increased in the saliva collected from early stage HNSCC patients compared to controls. A future multi-centre confirmatory study is warranted to test the diagnostic performance of these salivary miRNA prior to clinical implementation.
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Project description:ObjectivesCisplatin is commonly used in the treatment of head and neck squamous cell carcinoma (HNSCC), and the repair of cisplatin-induced DNA damage involves activation of the DNA damage response protein ataxia telangiectasia and Rad3-related (ATR). Resistance to cisplatin therapy exacerbates adverse toxicities and is associated with poor outcomes. Since repair of cisplatin-induced DNA damage contributes to resistance, we hypothesized that inhibition of ATR using AZD6738, a well-tolerated and orally-bioavailable inhibitor, would enhance the sensitivity of HNSCC cells and tumors to cisplatin.Materials and methodsA panel of human papilloma virus-negative (HPV-) and HPV+ HNSCC cell lines were treated with cisplatin in the absence or presence of AZD6738, and effects on cell viability, colony formation, apoptosis signaling, and DNA damage were assessed. The impact of co-treatment with cisplatin plus AZD6738 on the growth of HPV- and HPV+ cell line- and patient-derived xenograft tumors was also examined.ResultsInhibition of ATR with AZD6738 enhanced cisplatin-induced growth inhibition of HNSCC cell lines and tumors, in association with increased apoptosis signaling and DNA damage. Both HPV- and HPV+ models were sensitized to cisplatin by ATR inhibition.ConclusionInhibition of ATR promotes sensitization to cisplatin in preclinical in vitro and in vivo models of HPV- and HVP+ HNSCC, supporting clinical evaluation of this strategy in this disease.
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Project description:PURPOSE:To develop a clinically relevant model system to study head and neck squamous cell carcinoma (HNSCC), we have established and characterized a direct-from-patient tumorgraft model of human papillomavirus (HPV)-positive and HPV-negative cancers. EXPERIMENTAL DESIGN:Patients with newly diagnosed or recurrent HNSCC were consented for donation of tumor specimens. Surgically obtained tissue was implanted subcutaneously into immunodeficient mice. During subsequent passages, both formalin-fixed/paraffin-embedded as well as flash-frozen tissues were harvested. Tumors were analyzed for a variety of relevant tumor markers. Tumor growth rates and response to radiation, cisplatin, or cetuximab were assessed and early passage cell strains were developed for rapid testing of drug sensitivity. RESULTS:Tumorgrafts have been established in 22 of 26 patients to date. Significant diversity in tumorgraft tumor differentiation was observed with good agreement in degree of differentiation between patient tumor and tumorgraft (Kappa 0.72). Six tumorgrafts were HPV-positive on the basis of p16 staining. A strong inverse correlation between tumorgraft p16 and p53 or Rb was identified (Spearman correlations P = 0.085 and P = 0.002, respectively). Significant growth inhibition of representative tumorgrafts was shown with cisplatin, cetuximab, or radiation treatment delivered over a two-week period. Early passage cell strains showed high consistency in response to cancer therapy between tumorgraft and cell strain. CONCLUSIONS:We have established a robust human tumorgraft model system for investigating HPV-positive and HPV-negative HNSCC. These tumorgrafts show strong correlation with the original tumor specimens and provide a powerful resource for investigating mechanisms of therapeutic response as well as preclinical testing.
| S-EPMC3581858 | biostudies-literature
Project description:PURPOSE:The genetic differences between human papilloma virus (HPV)-positive and -negative head and neck squamous cell carcinomas (HNSCC) remain largely unknown. To identify differential biology and novel therapeutic targets for both entities, we determined mutations and copy-number aberrations in a large cohort of locoregionally advanced HNSCC. EXPERIMENTAL DESIGN:We performed massively parallel sequencing of 617 cancer-associated genes in 120 matched tumor/normal samples (42.5% HPV-positive). Mutations and copy-number aberrations were determined and results validated with a secondary method. RESULTS:The overall mutational burden in HPV-negative and HPV-positive HNSCC was similar with an average of 15.2 versus 14.4 somatic exonic mutations in the targeted cancer-associated genes. HPV-negative tumors showed a mutational spectrum concordant with published lung squamous cell carcinoma analyses with enrichment for mutations in TP53, CDKN2A, MLL2, CUL3, NSD1, PIK3CA, and NOTCH genes. HPV-positive tumors showed unique mutations in DDX3X, FGFR2/3 and aberrations in PIK3CA, KRAS, MLL2/3, and NOTCH1 were enriched in HPV-positive tumors. Currently targetable genomic alterations were identified in FGFR1, DDR2, EGFR, FGFR2/3, EPHA2, and PIK3CA. EGFR, CCND1, and FGFR1 amplifications occurred in HPV-negative tumors, whereas 17.6% of HPV-positive tumors harbored mutations in fibroblast growth factor receptor genes (FGFR2/3), including six recurrent FGFR3 S249C mutations. HPV-positive tumors showed a 5.8% incidence of KRAS mutations, and DNA-repair gene aberrations, including 7.8% BRCA1/2 mutations, were identified. CONCLUSIONS:The mutational makeup of HPV-positive and HPV-negative HNSCC differs significantly, including targetable genes. HNSCC harbors multiple therapeutically important genetic aberrations, including frequent aberrations in the FGFR and PI3K pathway genes. See related commentary by Krigsfeld and Chung, p. 495.
| S-EPMC4305034 | biostudies-literature
Project description:Head and neck squamous cancers are a heterogeneous group of cancers that arise from the upper aerodigestive tract. Etiologically, these tumors are linked to alcohol/tobacco abuse and infections with high-risk human papillomavirus (HPV). HPV-positive HNSCCs are characterized by a different biology and also demonstrate better therapy response and survival compared to alcohol/tobacco-related HNSCCs. Despite this advantageous therapy response and the clear biological differences, all locally advanced HNSCCs are treated with the same chemo-radiotherapy schedules. Although we have a better understanding of the biology of both groups of HNSCC, the biological factors associated with the increased radiotherapy response are still unclear. Hypoxia, i.e., low oxygen levels because of an imbalance between oxygen demand and supply, is an important biological factor associated with radiotherapy response and has been linked with HPV infections. In this review, we discuss the effects of hypoxia on radiotherapy response, on the tumor biology, and the tumor microenvironment of HPV-positive and HPV-negative HNSCCs by pointing out the differences between these two tumor types. In addition, we provide an overview of the current strategies to detect and target hypoxia.
| S-EPMC8656584 | biostudies-literature