Human Papillomavirus Positive Cell Lines Research Articles

Abstract 2937: Accurate detection of human papillomavirus breakpoints in cervical cancers using a novel library preparation strategy for nanopore sequencing technology

Abstract Introduction: Integration of human papillomavirus (HPV) DNA into the human genome is considered as a key event in cervical carcinogenesis. Different methods have been employed in exploring HPV integration sites, such as APOT, DIPS-PCR, and NGS. However, these methods have their drawbacks. APOT heavily relies on the quality of the sample due to its dependence on RNA, while DIPS-PCR mostly identifies known sites and has limited capability in uncovering new integration sites. Although the detection sensitivity of virus infection status increased significantly through the Illumina sequencing platform, there were still disadvantages remain for further improvement, including the detection accuracy and the complex integrated genome structure identification, etc. Also, it requires large amounts of sequencing data, and thus is not applicable in clinical usage, which requires fast and accurate results. Therefore, the development of a new method for HPV integration detection with high accuracy and prompt reporting capacity is crucial for future clinical application. Methods: We established a novel library preparation strategy for the detection of HPV integration in the human genome using Oxford Nanopore sequencing. Using this novel technology, Nanopore libraries were prepared from HPV positive cell lines HeLa and SiHa followed by sequencing on the Oxford Nanopore. We also determined HPV integration sites in five patients with cervical cancer patients with HPV positive tumors using Ilumina WGS. HPV integrations and breakpoints in human genome from both Nanopore and Ilumina WGS were extracted using Minimap and ViFi tool respectively. All HPV integration sites from cell lines and patient samples were validated using Sanger sequencing. Results: The novel library preparation approach for detection of HPV integrations with Nanopore sequencing technology were sensitive and efficient in detection of commonly reported HPV breakpoints in HPV positive cell lines HeLa and SiHa. Additionally, we applied this method to clinical samples from five patients subjected to Whole Genome Sequencing, consistently yielding concordant results. Conclusion: Our Nanopore based assay for detecting HPV integrations without the need for WGS demonstrated high efficacy. These data highlight the sensitivity and efficiency of this methodology in detecting HPV integrations events, emphasizing its potential as a reliable diagnostic tool in understanding pathophysiology of HPV-related malignancies and its use as prognostic markers in clinical settings. Citation Format: Preetiparna Parida, Nivedita Mukherjee, Agastya Singh, Ashima Singh, Krishna Sharan, Mahadev Rao, Shirley Lewis, Sabarinathan Radhakrishnan, Rama Rao Damerla. Accurate detection of human papillomavirus breakpoints in cervical cancers using a novel library preparation strategy for nanopore sequencing technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2937.

Detection of Multiple HPV Types in Liquid Biopsies of Cervical Neoplasia.

More than 95% of cervical cancers and their precancerous lesions are caused by human papillomavirus (HPV). Cell-free (cf) HPV DNA detection in blood samples may serve as a monitoring tool for cervical cancer. In our methodological study, an HPV panel for simultaneous detection of 24 types using mass spectrometry-based analysis was developed for liquid biopsy approaches and tested on HPV positive cell lines, plasmid controls, and cervical high-grade squamous intraepithelial lesions (HSIL) in positive smear samples (n = 52). It was validated in cfDNA blood samples (n = 40) of cervical cancer patients. The HPV panel showed proficient results in cell lines and viral plasmids with a limit of detection of 1 IU (international units)/µL for HPV16/18 and 10GE/µL for HPV11/31/33/39/45/51/52/58/59 and a specificity of 100% for the tested HPV types. In cervical smear samples, HPV DNA was detected with a sensitivity of 98.14%. The overall agreement between the new HPV panel and clinical records was 97.2% (κ = 0.84). In cervical cancer cfDNA, 26/40 (65.0%) tested positive for any HPV type, with most infections due to hrHPV (24/26). HPV positive samples were found in all FIGO stages, with the highest positivity ratio in FIGO III and IV. Even the lowest stage, FIGO I, had 12/23 (52.2%) patients with a positive HPV plasma status. This proof-of-concept paper shows that the described assay produces reliable results for detecting HPV types in a multiplex mass spectrometry-based assay in cervical smear and cfDNA with high specificity and sensitivity in both cohorts. The assay shows potential for liquid biopsy-based applications in monitoring cervical cancer progression.

Development of a touchdown droplet digital PCR assay for the detection and quantitation of human papillomavirus 16 and 18 from self-collected anal samples

ABSTRACT Broad-spectrum droplet digital polymerase chain reaction (ddPCR) assays can be used to quantitate multiple human papillomavirus (HPV) types. However, the presence of nucleotide mismatches at the primer binding sites may result in different optimal annealing temperatures and, consequently, under detection of some HPV types. Touchdown PCR uses decreasing annealing temperature over the first PCR cycles allowing for specific and efficient amplification of the target sequence. Here, as a proof-of-concept, we assessed if touchdown ddPCR (TD-ddPCR) using broad-spectrum oligonucleotides and genotype-specific probes targeting HPV L1 can be used to quantitate HPV. We tested our hypothesis using HPV positive cell lines and anal samples which potentially contain PCR inhibitors. Using serial dilutions of HPV16 and HPV18 DNA, the TD-ddPCR assays were performed with linearity across the dilution series (Pearson’s correlation r = 0.9999) and detected one copy of both HPV types, with no cross-reactivity to 27 other low-risk and high-risk HPV types. The PCRs were highly reproducible, with inter- and intra-assay coefficient of variations ranging 2.0–6.3% for HPV16 and 4.9–11.6% for HPV18. Testing self-collected anal samples positive for HPV16 ( n = 29) or HPV18 ( n = 33) and those negative for both HPV types ( n = 22) with the ddPCR assay and Seegene Anyplex II HPV28 (Seegene, TD-ddPCR) showed high agreements between the two assays. In conclusion, HPV broad-spectrum primers containing mismatches at their target binding sites and a touchdown thermal cycling approach resulted in sensitive and specific amplification of HPV genome using ddPCR. The assay has potential clinical diagnostic applications for detection, quantitation, and monitoring of anal HPV infections. IMPORTANCE The quantity of the human papillomavirus (HPV) is associated with disease outcome. We designed an accurate and precise digital PCR assay for quantitating HPV in anal samples, a sample type that is typically problematic due to the presence of PCR inhibitors.

Hinokitiol Dysregulates Metabolism of Carcinoma Cell Lines and Induces Downregulation of HPV16E6 and E7 Oncogenes and p21 Upregulation in HPV Positive Cell Lines

Background: Hinokitiol (β-thujaplicin), isolated from the wood of Chamaecyparis taiwanensis, has a wide variety of biological properties including anti-inflammatory, anti-microbial, and anti-tumor effects. Therefore, hinokitiol has become a frequent additive in oral and other healthcare products. Objectives: Our goal was to determine the anti-tumor activity of hinokitiol on human papillomavirus (HPV) positive (n = 3) and negative (n = 2) cell lines derived from cervical or head and neck squamous cell carcinoma (HNSCC) and keratinocyte cell lines (n = 3) transformed spontaneously or with HPV16E6 and E7 oncogenes. Methods: The cell-lines were exposed to hinokitiol at different concentrations (0–200 µM) for 24 h. Cell metabolism, proliferation, and the cell cycle distribution were assessed by MTT- and 3H-thymidine incorporation and flow cytometry. Expressions of p21 and on HPV16E6 and E7 oncogenes were assessed by qPCR. Results: In all carcinoma cell lines, hinokitiol treatment declined the metabolic activity irrespective of the HPV status. This decline was statistically significant, however, only in HPV-positive cell lines CaSki and UD-SCC-2 when exposed to hinokitiol concentrations at 100 and 200 µM, respectively (p < 0.05). Immortalized cell lines, HMK and HPV-positive IHGK, were more sensitive as a similar metabolic effect was achieved at lower hinokitiol concentrations of 3.1, 6.25, and 50 µM, respectively. Hinokitiol blocked DNA synthesis of all carcinoma cell lines without evident association with HPV status. G1 cell cycle arrest and p21 upregulation was found in all cell lines after hinokitiol treatment at higher concentration. However, when the p21 results of all HPV-positive cells were pooled together, the increase in p21 expression was statistically significantly higher in HPV-positive than in HPV-negative cell lines (p = 0.03), but only at the highest hinokitiol concentration (200 µM). In HPV-positive cell lines hinokitiol declined the expression of HPV16E7 and E6 along the increase of p21 expression. The dose-dependent inverse correlation between p21 and E7 was statistically significant in SiHa cells (r = −0.975, p-value = 0.03) and borderline in UD-SCC-2 cells (r = −0.944, p-value = 0.06), in which p21 and E6 were also inversely correlated (r = −0.989). Conclusions: Our results indicate that hinokitiol might have potential in preventing the progress of immortalized cells toward malignancy and the growth of malignant lesions. Hinokitiol can also influence on the progression of HPV-associated lesions by downregulating the E6 and E7 expression.

Characteristic differences in radiation-induced DNA damage response in human papillomavirus-negative and human papillomavirus-positive head and neck cancers with accumulation of fractional radiation dose.

Superior treatment responses by patients with human papillomavirus (HPV) positive head and neck squamous cell carcinoma (HNSCC), compared to patients with HNSCC from other causes, drive biomarker research to optimize treatment. Most HNSCC patients receive radiation therapy delivered as a fractionated course. Changing HPV status in HNSCC from a positive prognostic marker to a predictive one requires biomarkers that capture cellular radiation response to cumulative dose. Nuclear enlargement, γH2AX expression and micronuclei count, were studied in six HNSCC cell lines after 4 Gy fractionated X-irradiation. All HNSCC cell lines displayed altered cellular responses, indicating increasing inability to repair radiation damage with subsequent radiation fractions. Increases in nuclear area were significantly greater among HPV positive cell lines (207% and 67% for the HPV positive and HPV negative groups, respectively). A different character of DNA repair dysfunction in the HPV positive group suggests greater chromosomal translocation with accumulated radiation dose.

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<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<0.0001), 21-carbon steroid hormone metabolism (P=<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.

Intrinsic Radiosensitivity Is Not the Determining Factor in Treatment Response Differences between HPV Negative and HPV Positive Head and Neck Cancers.

Head and neck squamous cell carcinomas (HNSCC) resulting from human papillomavirus (HPV) are increasing in incidence but demonstrate significantly better treatment response than HNSCC from other causes such as tobacco and alcohol. This study sought to identify differences in HNSCC, intrinsic to HPV status, in their response to radiation dose. Previously unexamined changes in radio-responsiveness following fractionated X-ray irradiation were compared between HPV positive and negative statuses of HNSCC. Six HNSCC cell lines, 3 of each HPV status, were investigated for radiosensitivity by clonogenic assay and modelled by response as a function of dose. Generational cultures of each cell line were developed to follow changes in radiosensitivity after repeated irradiations simulating fractionated radiation therapy. As a group, the HPV positive cell lines were more radiosensitive, but with changes following repeated fractions of dose, and modelling of response as a function of dose, both statuses displayed large radiobiological heterogeneity. These findings challenge current radiobiological assumptions of head and neck cancers as early responding tissue to radiation and may go some way in explaining difficulties reaching consensus in stratification of treatment by HPV status. Consequently, results from this study do not support stratifying radiation therapy by HPV status.

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.

Roscovitine strongly enhances the effect of olaparib on radiosensitivity for HPV neg. but not for HPV pos. HNSCC cell lines.

At present, advanced stage human Papillomavirus (HPV) negative and positive head and neck squamous cell carcinoma (HNSCC) are treated by intense multimodal therapy that includes radiochemotherapy, which are associated with relevant side effects. Patients with HPV positive tumors possess a far better prognosis than those with HPV negative cancers. Therefore, new therapeutic strategies are needed to improve the outcome especially of the latter one as well as quality of life for all HNSCC patients. Here we tested whether roscovitine, an inhibitor of cyclin-dependent kinases (CDKs), which hereby also blocks homologous recombination (HR), can be used to enhance the radiation sensitivity of HNSCC cell lines.In all five HPV negative and HPV positive cell lines tested, roscovitine caused inhibition of CDK1 and 2. Surprisingly, all HPV positive cell lines were found to be defective in HR. In contrast, HPV negative strains demonstrated efficient HR, which was completely suppressed by roscovitine. In line with this, for HPV negative but not for HPV positive cell lines, treatment with roscovitine resulted in a pronounced enhancement of the radiation-induced G2 arrest as well as a significant increase in radiosensitivity. Due to a defect in HR, all HPV positive cell lines were efficiently radiosensitized by the PARP-1 inhibitor olaparib. In contrast, in HPV negative cell lines a significant radiosensitization by olaparib was only achieved when combined with roscovitine.

Abstract 4102: Overcoming intrinsic and acquired cetuximab resistance: an in vitro study on the potential of afatinib in HNSCC cell lines

Abstract Aberrant signalling of the epidermal growth factor receptor (EGFR) plays an integral role in the tumorigenesis of many cancer types, including head and neck squamous cell carcinoma (HNSCC), making it a compelling drug target. After the initial promising results of EGFR-targeted therapies, the problem of therapeutic resistance is emerging and new treatment options are necessary. In contrast to the first generation EGFR inhibitors, afatinib is an irreversible ErbB family blocker that inhibits EGFR as well as HER2 and HER4. The objective of this study was to investigate whether afatinib is able to overcome intrinsic and acquired cetuximab resistance in human papillomavirus (HPV) positive and negative HNSCC cell lines under normal and reduced oxygen conditions and to identify the molecular mechanisms underlying afatinib’s cytotoxic effect. Sensitivity to cetuximab treatment was investigated in a panel of HPV positive and negative HNSCC cell lines using the colorimetric sulforhodamine B (SRB) assay (168h, 0-2μM). Cetuximab sensitive and intrinsically resistant cell lines were identified. Acquired cetuximab resistant cell lines were generated by chronically exposing initially sensitive cell lines to cetuximab. In parallel, control cell lines were established by exposing these cells to the vehicle control (PBS). Cytotoxicity of afatinib (72h, 0 - 5μM) was assessed under normoxic and hypoxic (1% O2) conditions. Cell cycle distribution and induction of apoptotic cell death were assessed flow cytometrically using the Vindelov method and Annexin V-FITC/PI assay, respectively. Data analysis was performed using WinNonlin and FlowJo software. We identified intrinsic cetuximab resistance in 5 out of 7 HNSCC cell lines (i.e. LICR-HN1, Cal-27, SQD9, 93-VU-147T and UM-SCC104) and generated acquired cetuximab resistant cell lines (i.e. SCC22b-R and SC263-R). Afatinib showed a clear concentration-dependent cytotoxic effect in both cetuximab sensitive and resistant cell lines with IC50 values ranging between 0.019 and 4.04 μM. Furthermore, afatinib maintained its cytotoxic effect under hypoxic conditions. Treatment with afatinib led to an increase of the proportion of cells in the G0/G1 phase of the cell cycle. Afatinib also induced an increase in the percentage of AnnV+/PI- and AnnV+/PI+ cells with a corresponding decrease of the percentage viable (AnnV-/PI-) cells. In conclusion, our results suggest that afatinib has the potential to overcome cetuximab resistance, as it was able to establish cytotoxicity in HPV positive and negative HNSCC cell lines that were intrinsically and acquired resistant to cetuximab. Furthermore, treatment with afatinib caused a G0/G1 cell cycle arrest and induced apoptotic cell death. These data support the hypothesis that afatinib might be a promising novel therapeutic strategy to treat HNSCC patients experiencing intrinsic or acquired cetuximab resistance. Citation Format: Ines De Pauw, An Wouters, Jolien Van den Bossche, Vanessa Deschoolmeester, Patrick Pauwels, Jan B. Vermorken, Marc Peeters, Filip Lardon. Overcoming intrinsic and acquired cetuximab resistance: an in vitro study on the potential of afatinib in HNSCC cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4102. doi:10.1158/1538-7445.AM2017-4102

103. High-Capacity Adenoviral Vectors (HCAdV) Armed with a High Risk Human Papillomavirus (HPV) Oncogene Specific CRISPR/Cas9 Machinery Specifically Kill Cervical Cancer Cells

Persistent high risk human Papillomavirus (HPV) infections are the main cause of cervical cancer and partially also head and neck cancer. Recent studies showed that plasmid transfection of HPV oncogene specific designer nucleases effectively induced in/del mutations in the early promotors or oncogenes of HPV genomes integrated into the genome of cervical cancer cells by double-strand DNA break mediated non homologous end joining (NHEJ). Destruction or suppression of HPV oncogenes lead to decreased suppression of HPV oncogenes mediated by tumor suppressor protein p53 and retinoblastoma protein pRB inducing recovery from HPV oncogene mediated inhibition of apoptosis induction and cell cycle control. However, only few attempts have been made to improve delivery of respective designer nucleases by means of viral delivery allowing for translating these promising findings towards in vivo applications. Here we aimed at arming gene deleted high-capacity adenoviral vectors (HCAdVs) with HPV-specific CRISPR/cas9 machineries. By using a new toolbox that facilitates customization, cloning and production of CRISPR-HCAdVs we assembled HCAdV genomes containing the Streptococcus pyogenes Cas9 (spCas9) gene including either one gRNA expression unit specific for HPV18-E6 or two gRNA expression units specific for HPV18 and HPV16-E6 that have been shown efficiency to destroy respective genes. HPV specific CRISPR-HCAdVs were amplified in medium scale using a shortened protocol yielding high virus titers. Hela and Caski cervical cancer cells containing HPV18 or HPV16 genomes integrated into their cellular genome, as well as HPV negative A549 cells were infected with HPV specific CRISPR-HCAdVs. Adenoviral delivery of HPV specific CRISPR/Cas9 resulted in strong cell death in HPV positive cervical cancer cell lines whereas HPV negative A549 cells were unaffected. Moreover, HPV-specific CRISPR-HCAdVs infected Hela and Caski cells showed decreased proliferation compared to untreated cells and HPV negative control cells and cytotoxicity assays revealed strongly decreased cell viability of cervical cancer cells. In sharp contrast to control groups, increased apoptosis in HPV specific CRISPR-HCAdV treated cervical cancer cells was measured after performing apoptosis detection assays. Our results suggest that also HCAdVs can serve as oncolytic agents when armed with target specific designer nucleases such as CRISPR/Cas9. We believe that our approach will pave the way towards in vivo applications of CRISPR/Cas9 mediated oncolysis of HPV induced cervical cancer. As our CRISPR/Cas9-HCAdV production pipeline is adaptable to incorporate other or even more gRNA expression units specific for further HPV types, it provides a valuable platform to develop personalized antiviral or oncolytic vectors for any specific HPV target.

In vitro 3-dimensional tumor model for radiosensitivity of HPV positive OSCC cell lines

The incidence of oropharyngeal squamous cell carcinoma (OSCC) is increasing due to the rising prevalence of human papillomavirus (HPV) positive OSCC. HPV positive OSCC is associated with better outcomes than HPV negative OSCC. Our aim was to explore the possibility that this favorable prognosis is due to the enhanced radiosensitivity of HPV positive OSCC. HPV positive OSCC cell lines were generated from the primary OSCCs of 2 patients, and corresponding HPV positive cell lines generated from nodal metastases following xenografting in nude mice. Monolayer and 3 dimensional (3D) culture techniques were used to compare the radiosensitivity of HPV positive lines with that of 2 HPV negative OSCC lines. Clonogenic and protein assays were used to measure survival post radiation. Radiation induced cell cycle changes were studied using flow cytometry. In both monolayer and 3D culture, HPV positive cells exhibited a heterogeneous appearance whereas HPV negative cells tended to be homogeneous. After irradiation, HPV positive cells had a lower survival in clonogenic assays and lower total protein levels in 3D cultures than HPV negative cells. Irradiated HPV positive cells showed a high proportion of cells in G1/S phase, increased apoptosis, an increased proliferation rate, and an inability to form 3D tumor clumps. In conclusion, HPV positive OSCC cells are more radiosensitive than HPV negative OSCC cells in vitro, supporting a more radiosensitive nature of HPV positive OSCC.

P27 The combined use of anti-EGFr and anti-JAK/STAT-3 agents as radiosensitizers in HPV negative and positive head and neck cancer cell lines

Human Papilloma Virus (HPV) is a common etiological factor in human head and neck squamous cell cancer (HHNSCC). Our group has reported that HPV-infected HHNSCC cell lines are more sensitive to radiation and deficient in DNA repair compared to uninfected lines (Cooper T, et al. Cancer Research, 73(8 Supplement 1), AACR, 2013). EGFr inhibitors (primarily cetuximab) have evolved as an important therapeutic modality for head and neck cancer since it was found that cetuximab sensitizes HHNSCC’s to radiation in laboratory studies, as well as in phase III clinical studies (Bonner JA, et al. Lancet Oncology; 11(1):21–28, Epub 2009, November 2010). Early on, it was recognized that cetuximab not only inhibits EGFr, but the downstream inhibitor of apoptosis STAT-3 (Bonner JA, et al. J Clin Oncol 2001;19:3234–3243). Therefore, we have previously assessed cetuximab in combination with the inhibition of JAK/STAT as a means of accentuating the targeting of this pathway and increasing radiosensitization (Bonner JA, et al. Radiother Oncol 2011;99:339–343). Herein, we report the radiosensitizing properties of dual EGFr and JAK/STAT inhibition in three HPV negative HHNSCC’s and two HPV positive HHNSCC’s. Three HPV negative HHNSCC’s (UM-SCC-1, UM-SCC-6, UM-SCC-5) and two HPV positive HHNSCC’s (SCC-47, SCC-90) cell lines were used for these studies. All five cell lines showed greater anti-proliferative effects for the combination of cetuximab (0.25 μg/ML) followed 1hr by the JAK2/STAT-3 inhibitor, BMS911543 (10 μM) than for either agent alone. This enhanced anti-proliferative effect, for the combination of inhibitors (EGFr and JAK1i/STAT3), correlated with greater inhibition of phosphorylated STAT-3 as measured by immunoblot. Additionally, there was a suggestion that greater radiosensitization (proliferation and colony formation) occurred when both agents were used prior to radiation compared to the use of either single agent in all five cell lines. The enhanced anti-proliferative effects of combining the agents (JAK2/STAT-3 and cetuximab) showed correlation with enhanced apoptosis. Additionally, there was a suggestion of enhanced apoptosis when both agents were given prior to radiation compared to either single agent treatment given prior to radiation. However, the apoptotic events were markedly pronounced (2-fold increase) in the HPV negative cell lines compared to the HPV positive cell lines even though the HPV positive cell lines showed 5–10-fold greater radiosensitivity compared to the HPV negative lines (colony formation). Additional studies are being performed to assess mechanisms associated with the regulation of cellular proliferation that may account for the discordant findings with respect to apoptosis and radiosensitivity relative to HPV status. The combined treatment of Cetuximab (anti-EGFr) and BMS911543 (anti-JAK-STAT-3) with or without radiation was studied in four HHNSCC cell lines. The combination of these two inhibitors increased the anti-proliferative effects of all cell lines compared to either individual treatment in HPV positive and negative human head and neck squamous cell cancers. The combination treatment also led to marked radiosensitization which correlated with apoptotic events. However, the treatment-induced apoptotic events were muted in the HPV positive cell lines compared to the HPV negative cell lines even though the HPV positive cell lines showed the greatest radiosensitivity. Further study of the effects of these inhibitors on the regulation of cellular proliferation in HPV positive and HPV negative tumors is warranted.

Oncolytic activity of reovirus in HPV positive and negative head and neck squamous cell carcinoma.

BackgroundThe management of patients with advanced stages of head and neck cancer requires a multidisciplinary and multimodality treatment approach which includes a combination of surgery, radiation, and chemotherapy. These toxic treatment protocols have significantly improved survival outcomes in a distinct population of human papillomavirus (HPV) associated oropharyngeal cancer. HPV negative head and neck squamous cell carcinoma (HNSCC) remains a challenge to treat because there is only a modest improvement in survival with the present treatment regimens, requiring innovative and new treatment approaches. Oncolytic viruses used as low toxicity adjunct cancer therapies are novel, potentially effective treatments for HNSCC. One such oncolytic virus is Respiratory Orphan Enteric virus or reovirus. Susceptibility of HNSCC cells towards reovirus infection and reovirus-induced cell death has been previously demonstrated but has not been compared in HPV positive and negative HNSCC cell lines.ObjectivesTo compare the infectivity and oncolytic activity of reovirus in HPV positive and negative HNSCC cell lines.MethodsSeven HNSCC cell lines were infected with serial dilutions of reovirus. Two cell lines (UM-SCC-47 and UM-SCC-104) were positive for type 16 HPV. Infectivity was measured using a cell-based ELISA assay 18 h after infection. Oncolytic activity was determined using an alamar blue viability assay 96 h after infection. Non-linear regression models were used to calculate the amounts of virus required to infect and to cause cell death in 50% of a given cell line (EC50). EC50 values were compared.ResultsHPV negative cells were more susceptible to viral infection and oncolysis compared to HPV positive cell lines. EC50 for infectivity at 18 h ranged from multiplicity of infection (MOI) values (PFU/cell) of 18.6 (SCC-9) to 3133 (UM-SCC 104). EC50 for cell death at 96 h ranged from a MOI (PFU/cell) of 1.02×102 (UM-SCC-14A) to 3.19×108 (UM-SCC-47). There was a 3×106 fold difference between the least susceptible cell line (UM-SCC-47) and the most susceptible line (UM-SCC 14A) EC50 for cell death at 96 h.ConclusionsHPV negative HNSCC cell lines appear to demonstrate greater reovirus infectivity and virus-mediated oncolysis compared to HPV positive HNSCC. Reovirus shows promise as a novel therapy in HNSCC, and may be of particular benefit in HPV negative patients.

Abstract 4896: Identification of the mechanisms of radiosensitization by human papillomavirus (HPV) in head and neck cancers cell lines

Abstract It has been shown in several clinical studies that HPV-positive head and neck squamous cell carcinoma (HNSCC) have a more favorable outcome and greater response to therapy. The reason for improved prognosis of HPV-related head and neck cancers remains speculative. One hypothesis is that HPV-positive cells are intrinsically more sensitive to standard therapies and thus, respond better to treatment. Clinical studies suggest indeed that HPV-related HNSCC actually display enhanced sensitivity to concurrent chemoradiation therapy. The goal of this project is to verify this hypothesis and elucidate the underlying mechanisms. For this purpose, we determined radiosensitivity by clonogenic survival of two HPV positive HNSCC cell lines (UPCI-SCC-154 & UPCI-SCC-90) compared to two HPV negative ones (SCC-61 & SQD9). Apoptosis was measured by Annexin V/PI staining. Cell cycle distribution and G2/M checkpoint were assessed by flow cytometry. DNA damage repair was evaluated by gamma-H2Ax assay. In addition, we investigated basal DNA damage repair efficiency by reporter gene assay for homologous recombination (HR) and non-homologous end-joining (NHEJ). The surviving fraction at 2Gy (SF2) for the two HPV+ cell lines was 0,13 and 0,15 for UPCI-SCC-90 and UPCI-SCC-154, respectively while SF2 for SQD9 was 0,49 and 0,16 for SCC-61 a HNSCC cell line described as radiosensitive. At 24h after irradiation (2Gy) there was no significant difference in apoptosis (less than 5%) between HPV- and HPV+ cell lines. However further time points still need to be analysed. Cell cycle distribution 24h after irradiation, indicated a significant increase in G2/M in HPV+ cells but also in SCC61. The kinetics of H2Ax phosphorylation after a single dose of 2 Gy correlated the SF2 data with a slower clearance of H2Ax for HPV+ cells, as well as for radiosensitive SCC61. However, analysis of remaining gH2AX depending on cell cycle phase indicated a slower clearance for cells in G2/M phase in HPV+ cell lines (p<0,001). Furthermore, HPV+ cells show decreased efficiency in NHEJ but also in HR which occurs mainly in G2/M phase. These results taken together, confirm an increased radiosensitivity of HPV+ cells and an impaired DNA damage repair mainly by HR although at this point further investigation is needed to elucidate the exact mechanisms. In order to investigate whether increased radiosensitivity is directly related to the presence of viral oncoproteins E6 and/or E7 we transfected HPV- HCT116 colon carcinoma cell line (p53 wt and -/-) with the oncoproteins and we plan to assess their radioresponse in terms of cell cycle arrest and DNA damage repair. Citation Format: Vanesa Bol, Vincent Grégoire. Identification of the mechanisms of radiosensitization by human papillomavirus (HPV) in head and neck cancers cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4896. doi:10.1158/1538-7445.AM2014-4896

Arsenic trioxide inhibits cell proliferation and human papillomavirus oncogene expression in cervical cancer cells

Arsenic trioxide (As2O3) has shown therapeutic effects in some leukemias and solid cancers. However, the molecular mechanisms of its anticancer efficacy have not been clearly elucidated, particularly in solid cancers. Our previous data showed that As2O3 induced apoptosis of human papillomavirus (HPV) 16 DNA-immortalized human cervical epithelial cells and cervical cancer cells and inhibited the expression of HPV oncogenes in these cells. In the present study, we systemically examined the effects of As2O3 on five human cervical cancer cell lines and explored the possible molecular mechanisms. MTT assay showed that HPV-negative C33A cells were more sensitive to growth inhibition induced by As2O3 than HPV-positive cervical cancer cells, and HPV 18-positive HeLa and C4-I cells were more sensitive to As2O3 than HPV 16-positive CaSki and SiHa cells. After As2O3 treatment, both mRNA and protein levels of HPV E6 and E7 obviously decreased in all HPV positive cell lines. In contrast, p53 and Rb protein levels increased in all tested cell lines. Transcription factor AP-1 protein expression decreased significantly in HeLa, CaSki and C33A cells with ELISA method. These results suggest that As2O3 is a potential anticancer drug for cervical cancer.

HPV associated tumor cells control tumor microenvironment and leukocytosis in experimental models.

Human papillomavirus (HPV) is the main etiological factor for cervical cancer development. HPV is also associated with other anogenital and oropharyngeal tumors. HPV associated tumors are frequent and constitute a public health problem, mainly in developing countries. Therapy against such tumors is usually excisional, causing iatrogenic morbidity. Therefore, development of strategies for new therapies is desirable. The tumor microenvironment is essential for tumor growth, where inflammation is an important component, displaying a central role in tumor progression. Inflammation may be a causal agent, suppressor of anti-tumor T cell responses, or may have a role in angiogenesis, drug resistance, and metastasis. The aim of this work was to investigate the role of HPV transformed cells in the tumor microenvironment and tumor effects on myeloid populations in lymphoid organs in the host. We used experimental models, where we injected cervical cancer derived cell lines in immunodeficient mice, comparing HPV positive, SiHa, and HeLa cells (HPV 16 and HPV18, respectively), with HPV negative cell line, C33A. Our data shows that HPV positive cell lines were more efficient than the HPV negative cell line in leukocyte recruitment to the tumor microenvironment and increase in myeloid cell proliferation in the bone marrow and spleen. We also observed that HPV positive cells lines expressed significantly higher levels of IL-6 and IL-8, while C33A expressed significantly higher levels of IL-16 and IL-17. Finally, in spite of cytokine secretion by tumor cells, leukocytes infiltrating SiHa and HeLa tumors displayed almost negligible STAT3 and no NFκB phosphorylation. Only the inflammatory infiltrate of C33A tumors had NFκB and STAT3 activated isoforms. Our results indicate that, although from the same anatomical site, the uterine cervix, these cell lines display important differences regarding inflammation. These results are important for the design of immunotherapies against cervical cancer, and possibly against HPV associated tumors in other anatomical sites.

Structure Based Identification and Characterization of Flavonoids That Disrupt Human Papillomavirus-16 E6 Function

Expression and function of the human papillomavirus (HPV) early protein 6 (E6) is necessary for viral replication and oncogenesis in cervical cancers. HPV E6 targets the tumor suppressor protein p53 for degradation. To achieve this, “high-risk” HPV E6 proteins bind to and modify the target specificity of the ubiquitin ligase E6AP (E6 associated protein). This E6-dependent loss of p53 enables the virus to bypass host cell defenses and facilitates virally induced activation of the cell cycle progression during viral replication. Disruption of the interaction between E6 and E6AP and stabilization of p53 should decrease viability and proliferation of HPV positive cells. A new in vitro high-throughput binding assay was developed to assay binding between HPV-16 E6 and E6AP and to identify compounds that inhibit this interaction. The compound luteolin emerged from the screen and a library of novel flavones based on its structure was synthesized and characterized using this in vitro binding assay. The compounds identified in this study disrupt the E6/E6AP interaction, increase the levels of p53 and p21Cip1/Waf1, and decrease proliferation of HPV positive cell lines. The new class of flavonoid E6 inhibitors displays a high degree of specificity for HPV positive cells. Docking analyses suggest that these compounds bind in a hydrophobic pocket at the interface between E6 and E6AP and mimic the leucines in the conserved α-helical motif of E6AP. The activity and specificity of these compounds represent a promising new lead for development as an antiviral therapy in the treatment of HPV infection and cervical cancer.

278 Epigenetic regulation to anti-cancer drugs in HPV positive cell lines

Infectious etiologies for certain human cancers have long been suggested by epidemiological studies and studies with experimental animals. Important support for this concept came from the discovery by Harald zur Hausen's group that human cervical carcinoma almost universally contains certain “high-risk” human papillomavirus (HPV) types. Over the years, much has been learned about the carcinogenic activities of high-risk HPVs. These studies have revealed that two viral proteins, E6 and E7, that are consistently expressed in HPV-associated carcinomas, are necessary for induction and maintenance of the transformed phenotype. Hence, HPV-associated tumors are unique amongst human solid tumors in that they are universally caused by exposure to the same, molecularly defined oncogenic agents, and the molecular signal transduction pathways subverted by these viral transforming agents are frequently disrupted in other, non-virus-associated human cancers.