Oropharyngeal Squamous Cell Carcinoma Cell Lines Research Articles

Curcumin Alone and Combined With PI3K Inhibitors Elicits Positive Effects on Oropharyngeal Cancer Cell Lines Regardless of HPV Status.

Human papillomavirus positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) is rising in incidence. Compared to HPV-negative (HPV-) OPSCC, HPV+ cases have a better 5-year survival. With its severe side-effects, today's chemoradiotherapy has not improved outcome compared to radiotherapy alone, so new therapies are needed. Mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), fibroblast growth factor receptor 3 (FGFR3) and cell division cycle 27 (CDC27) are found in HPV+ OPSCC, and in vitro targeted therapy combining PI3K and FGFR inhibitors showed synergistic effects. Here the effects of targeting CDC27 with curcumin with/without various inhibitors or cisplatin on OPSCC cell lines were examined. Curcumin was administered to HPV+ OPSCC cell lines CU-OP-2, CU-OP-3 and CU-OP-20, and HPV- CU-OP-17 with/without PI3K, cyclin-dependent kinase 4/6, FGFR, poly (ADP-ribose) polymerase or WEE1 inhibitors (BYL719, PD-0332991, JNJ-42756493, BMN-673 and MK-1775, respectively), or cisplatin. The cell lines were then assessed for 72 h after treatment for viability, proliferation and cytotoxicity. Curcumin led to dose-dependent responses with reduced viability and proliferation; upon combining it with BYL719, additional positive effects were found for most OPSCC lines grown as monolayers, and these effects were validated in CU-OP-2 cells grown as spheroids. Curcumin with MK-1775 or PD-0332991 also elicited some positive effects on CU-OP-2 and CU-OP-17 cells. Curcumin alone led to dose-dependent responses and when combined with BYL719, positive effects were revealed, as they were when it was combined with MK-1775 or PD-0332991, suggesting a potential use of some of these combinations for HPV+ OPSCC.

Abstract PO-097: Tumor-associated extracellular vesicles from HPV+ oropharyngeal cancer lymphatic fluid trigger an anti-tumor response through activation of the cGAS-STING pathway

Abstract Background: Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPC) is increasing rapidly in incidence and is now the most common HPV-associated malignancy in the U.S. Our group previously demonstrated that lymph-enriched fluid from surgical drains in HPV+ OPC patients is rich in tumor-derived cell-free DNA, with prognostic implications. In this study, we characterize extracellular vesicles (EVs) derived from cervical lymph in HPV+ OPC patients and explore the impact on innate immune response. The cyclic GMP-AMP synthase (cGAS) pathway plays a key role in immune response by detecting pathogenic or damaged host DNA. We propose that HPV DNA-containing EVs uptake by HPV-negative cells modulates their immune responses via activation of cGAS-STING signaling. Methods: Surgical lymphatic fluid samples were collected from 31 HPV(+) and 10 HPV(-) OPC patients 24 hours post neck dissection, along with supernatant from HPV(+) and HPV(-) OPC cell lines. EVs were isolated from both biofluids and characterized using immunoblotting, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). EVs were then treated with DNase to eliminate external DNA before extracting EV DNA. Lymph cell-free DNA (cfDNA) was also extracted. We used qPCR to quantify HPV16 levels in supernatant and lymph EVs. Next, we incubated HPV(-) OPC cells with EVs that were isolated from the supernatant from HPV (+) or HPV (-) cells. Finally, we compared cGAS-STING activation induced by HPV (+) EVs to the STING agonist 2,3-GAMP (Sigma) through IFNβ mRNA expression and phosphor-TBK1 immunoblotting. Results: Lymphatic EVs showed typical morphology with diameters ranging 70–150 nm, and were CD63 and TSG101 positive by immunoblot. 27 HPV (+) OPC patients (87%) had detectable HPV16 DNA in their postoperative lymph EVs, while all HPV(-) lymph EVs were negative for HPV16. Comparison of bulk lymph cfDNA and lymph EV DNA showed a significant proportion (39.5±24.4%) of lymph HPV16 DNA is contained by EVs. Further, an EV uptake assay showed HPV16 DNA was trafficked into HPV(-) cells through EVs, and blocking EV internalization with heparin reduced recipient cell HPV16 levels. Crucially, HPV (-) cells cultured with HPV (+) EVs for 8 hours increased IFNβ1 expression, while HPV(-) EVs did not increase IFNβ1 expression. Lastly, we observed TBK1 phosphorylation, a reporter of STING activation, was elevated in these cells treated with HPV(+) EVs. Conclusions: Our study is the first to characterize tumor-associated EVs in lymphatic fluid. We showed many of these tumor-associated lymph EVs in HPV(+) patients harbor HPV16. Additionally, we demonstrate lymph EVs containing HPV16 activate cellular cGAS-STING intermediates. Ongoing studies are investigating the impact of EVs on the tumor microenvironment. Citation Format: Zhongping Xu, Noah Earland, Peter K. Harris, Sophie P. Gerndt, Ricardo Ramirez, Nicholas P. Semenkovich, Aadel A. Chaudhuri, Jose P. Zevallos. Tumor-associated extracellular vesicles from HPV+ oropharyngeal cancer lymphatic fluid trigger an anti-tumor response through activation of the cGAS-STING pathway [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-097.

Abstract 4896: Transcriptomic alterations by HPV-human fusion transcripts in HPV+ HNSCC cell lines

Abstract Human papillomavirus (HPV) genomic integration is frequently seen in oropharyngeal squamous cell carcinoma (OPSCC). Integration into cellular genes can cause the generation of HPV-host fusion transcripts, which have been associated with altered gene expression of critical cellular pathways and worse survival in OPSCC patients. The mechanism of these transcriptomic alterations by fusion transcripts and how they contribute to poor outcome are unknown. We began our analysis by investigating whether our cell lines models of HPV+ OPSCC recapitulate these gene expression alterations using a qRT-PCR profiling system. We compared the expression of 128 genes in relevant pathways in four fusion transcript positive cell lines versus two fusion transcript negative cell lines and found a number of genes that were differentially expressed between the two groups. This suggests that these cell lines are appropriate models to study the mechanism of fusion transcript-dependent transcriptional regulation. To confirm our results in another system, we created fusion transcript positive/negative cell lines by cloning two pairs of either an HPV-human fusion transcript or an HPV only transcript into normal keratinocytes. These cloned cells were then run through the same qRT-PCR profiling system, and we saw differentially expressed genes between the two groups. There was an overlapping set of gene pathways between the HPV+ OPSCC cell lines and cloned keratinocytes, suggesting HPV-human fusion transcripts are sufficient to alter cellular gene expression. Subsequent in-depth molecular characterization and in vitro studies will help clarify the mechanism of these changes and their effects on cancer formation and metastasis. These studies will advance our understanding of how fusion transcripts lead to worse patient survival and help develop new therapies for these patients. Citation Format: Lisa M. Pinatti, Hana Sinha, Chad Brenner, Heather M. Walline, Thomas E. Carey. Transcriptomic alterations by HPV-human fusion transcripts in HPV+ HNSCC cell lines [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 4896.

Sensitivity of human papillomavirus‑positive and ‑negative oropharyngeal cancer cell lines to ionizing irradiation.

Human papillomavirus‑positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) has increased in incidence and has a much better prognosis than HPV‑negative (HPV‑) OPSCC with radiotherapy alone, but exactly why is unknown. The present study therefore aimed to further examine the sensitivity and possible changes in gene expression of several HPV+ and HPV‑ OPSCC, including various novel cell lines, upon ionizing irradiation (IR). Previously established HPV+ UM‑SCC‑47, UPCI‑SCC‑90, CU‑OP‑2, CU‑OP‑3 and HPV‑ UM‑SCC‑4, UM‑SCC‑6, UM‑SCC‑74a, UM‑SCC‑19 and newly established CU‑OP‑17 and CU‑OP‑20, characterised here, were subjected to 0‑6Gy. Surviving fractions of each cell line were tested by clonogenic assays, and irregularities in cell cycle responses were examined by flow cytometry, while changes in gene expression were followed by mRNA sequencing. HPV+ OPSCC cell lines showed greater variation in sensitivity to ionizing irradiation (IR) and tended to be more sensitive than HPV‑ OPSCC cell lines. However, their IR sensitivity was not correlated to the proportion of cells in G2 arrest, and HPV‑ cell lines generally showed lower increases in G2 after IR. Upon IR with 2Gy, mRNA sequencing revealed an increase in minor HPV integration sites in HPV+ cell lines, and some changes in gene expression in OPSCC cell lines, but not primarily those associated with DNA repair. To conclude, HPV+ OPSCC cell lines showed greater variation in their sensitivity to IR, with some that were radioresistant, but overall the HPV+ OPSCC group still tended to be more sensitive to IR than the HPV‑ OPSCC group. In addition, HPV+ OPSCC lines were more frequently in G2 as compared to HPV‑ cell lines, but the increase in G2 arrest upon IR in HPV+ OPSCC was not correlated to sensitivity to IR. Increases in minor HPV integration sites and changes in gene expression were also demonstrated after irradiation with 2Gy.

Elevated TLR5 expression in vivo and loss of NF-κΒ activation via TLR5 in vitro detected in HPV-negative oropharyngeal squamous cell carcinoma

In oropharyngeal squamous cell carcinoma (OPSCC), the expression pattern of toll-like receptors (TLRs), in comparison between human papillomavirus (HPV)-positive and -negative tumors differs. TLRs control innate immune responses by activating, among others, the nuclear factor-κΒ (NF-κΒ) signaling pathway. Elevated NF-κΒ activity is detectable in several cancers and regulates cancer development and progression. We studied TLR5 expression in 143 unselected consecutive OPSCC tumors, and its relation to HPV-DNA and p16 status, clinicopathological parameters, and patient outcome, and studied TLR5 stimulation and consecutive NF-κB cascade activation in vitro in two human OPSCC cell lines and immortalized human keratinocytes (HaCat).Clinicopathological data came from hospital registries, and TLR5 immunoexpression was evaluated by immunohistochemistry. Flagellin served to stimulate TLR5 in cultured cells, followed by analysis of the activity of the NF-κB signaling cascade with In-Cell Western for IκΒ and p-IκΒ.High TLR5 expression was associated with poor disease-specific survival in HPV-positive OPSCC, which typically shows low TLR5 immunoexpression. High TLR5 immunoexpression was more common in HPV-negative OPSCC, known for its less-favorable prognosis. In vitro, we detected NF-κΒ cascade activation in the HPV-positive OPSCC cell line and in HaCat cells, but not in the HPV-negative OPSCC cell line.Our results suggest that elevated TLR5 immunoexpression may be related to reduced NF-κΒ activity in HPV-negative OPSCC. The possible prognosis-worsening mechanisms among these high-risk OPSCC patients however, require further evaluation.

Sensitivity to inhibition of DNA repair by Olaparib in novel oropharyngeal cancer cell lines infected with Human Papillomavirus

The incidence of Human Papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is increasing rapidly in the UK. Patients with HPV-positive OPSCC generally show superior clinical responses relative to HPV-negative patients. We hypothesised that these superior responses could be associated with defective repair of DNA double strand breaks (DSB). The study aimed to determine whether defective DNA repair could be associated with sensitivity to inhibition of DNA repair using the PARP inhibitor Olaparib. Sensitivity to Olaparib, and induction and repair of DNA damage, were assessed in a panel of 8 OPSCC cell-lines, including 2 novel HPV-positive lines. Effects on cell cycle distribution and levels of PARP1 and p53 were quantified. RNA-sequencing was used to assess differences in activity of DNA repair pathways. Two HPV-positive OPSCC lines were sensitive to Olaparib at potentially therapeutic doses (0.1–0.5 μM). Two HPV-negative lines were sensitive at an intermediate dose. Four other lines, derived from HPV-positive and HPV-negative tumours, were resistant to PARP inhibition. Only one cell-line, UPCISCC90, showed results consistent with the original hypothesis i.e. that in HPV-positive cells, treatment with Olaparib would cause accumulation of DSB, resulting in cell cycle arrest. There was no evidence that HPV-positive tumours exhibit defective repair of DSB. However, the data suggest that a subset of OPSCC may be susceptible to PARP-inhibitor based therapy.

Extracellular vesicle microRNA cargo is correlated with HPV status in oropharyngeal carcinoma.

The incidence of human papilloma virus positive (HPV+ ) oropharyngeal squamous cell carcinoma (OPSCC) has increased rapidly in recent decades. These tumours have a favourable outcome compared to HPV-negative (HPV- ) OPSCC. However, HPV+ tumours are more likely to metastasise to distant sites, suggesting a difference in how these tumour subtypes interact with the metastatic niche. Extracellular vesicles (EVs) have emerged as important players in cell-to-cell communication and are a potential source of biomarkers for cancer diagnosis. This study aims to characterise the microRNA cargo of small EVs released by HPV+ and HPV- OPSCC cell lines. Extracellular vesicles produced by HPV+ (SCC2 and SCC90) and HPV- (SCC72 an SCC89) OPSCC cells were characterised by tunable resistive pulse sensing (TRPS) and western blotting. RNA was extracted from EVs and analysed by small RNA sequencing. A bioinformatics approach was used to identify EV miRNA signatures associated with HPV status. HPV- OPSCC cells produced more EVs than HPV+ OPSCC cells. EVs were positive for the common EV markers CD63, CD9 and TSG101. Unbiased hierarchical clustering analysis of EV miRNA cargo revealed that samples clustered based on HPV status. 14 miRNA were enriched in HPV+ cell-derived EVs, whereas 19 miRNA were enriched in EVs derived from HPV- cell lines. Here, we identify EV miRNA signatures indicative of the HPV status of the parent cell. This may provide a platform from which to validate salivary or blood-based biomarkers with utility for early detection and stratifying risk in OPSCC patients.

Altered Toll-like receptor expression and function in HPV-associated oropharyngeal carcinoma.

Toll-like receptors (TLRs) have been widely investigated due to their importance in the inflammatory response and possible links to tumor promotion/regression and prognosis. In cancers with an infective etiology, such as human papillomavirus (HPV)-associated Oropharyngeal Squamous Cell Carcinoma (OPSCC), TLR responses may be activated and play a role in tumorigenesis. Our aim was to assess the expression of all TLRs in OPSCC cell lines (both HPV+ and HPV–) by qPCR, Western Blot and flow cytometry and assess their response to TLR ligands lipopolysaccharide (LPS), LPS ultra-pure (LPS-UP) and peptidoglycan (PGN) by analyzing IL-8 and IL-6 production. We also immunostained 61 OPSCC tissue samples with anti-TLR4. Results showed lower TLR1 and TLR6 mRNA expression and higher TLR9 protein expression in HPV+ when compared to HPV–OPSCC cells. TLR4 expression did not vary by HPV status in OPSCC cells, but TLR4 expression was significantly lower in HPV+OPSCC tissues. After stimulation with PGN, only one cell line (HPV+) did not secrete IL-6 or IL-8. Furthermore, HPV+OPSCC lines showed no IL-6 or IL-8 production on treatment with LPS/LPS-UP. The data suggest changes in TLR4 signaling in HPV+OPSCC, since we have shown lower tissue expression of TLR4 and no pro-inflammatory response after stimulation with LPS and LPS-UP. Also, it suggests that OPSCC may respond to HPV infection by increased expression of TLR9. This study demonstrates differences in expression and function of TLRs in OPSCC, which are dependent on HPV status, and may indicate subversion of the innate immune response by HPV infection.

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.

Human papillomavirus DNA and p16 expression in Japanese patients with oropharyngeal squamous cell carcinoma

Human papillomavirus (HPV) is a major etiologic factor for oropharyngeal squamous cell carcinoma (OPSCC). However, little is known about HPV-related OPSCC in Japan. During the study, formalin-fixed, paraffin-embedded OPSCC specimens from Japanese patients were analyzed for HPV DNA by the polymerase chain reaction (PCR) and for the surrogate marker p16 by immuno-histochemistry. For HPV DNA-positive, p16-negative specimens, the methylation status of the p16 gene promoter was examined by methylation-specific PCR. Overall survival was calculated in relation to HPV DNA and p16 status and was subjected to multivariate analysis. OPSCC cell lines were examined for sensitivity to radiation or cisplatin in vitro. The study results showed that tumor specimens from 40 (38%) of the 104 study patients contained HPV DNA, with such positivity being associated with tumors of the tonsils, lymph node metastasis, and nonsmoking. Overall survival was better for OPSCC patients with HPV DNA than for those without it (hazard ratio, 0.214; 95% confidence interval, 0.074–0.614; P = 0.002). Multivariate analysis revealed HPV DNA to be an independent prognostic factor for overall survival (P = 0.015). Expression of p16 was associated with HPV DNA positivity. However, 20% of HPV DNA-positive tumors were negative for p16, with most of these tumors manifesting DNA methylation at the p16 gene promoter. Radiation or cisplatin sensitivity did not differ between OPSCC cell lines positive or negative for HPV DNA. Thus, positivity for HPV DNA identifies a distinct clinical subset of OPSCC with a more favorable outcome in Japanese.

Radiosensitization of Oropharyngeal Squamous Cell Carcinoma Cells by Human Papillomavirus 16 Oncoprotein E6∗I

Patients with oropharyngeal squamous cell carcinoma (OSCC) whose disease is associated with high-risk human papillomavirus (HPV) infection have a significantly better outcome than those with HPV-negative disease, but the reasons for the better outcome are not known. We postulated that they might relate to an ability of HPV proteins to confer a better response to radiotherapy, a commonly used treatment for OSCC. We stably expressed the specific splicing-derived isoforms, E6∗I and E6∗II, or the entire E6 open reading frame (E6total), which gives rise to both full length and E6∗I isoforms, in OSCC cell lines. Radiation resistance was measured in clonogenicity assays, p53 activity was measured using transfected reporter genes, and flow cytometry was used to analyze cell cycle and apoptosis. E6∗I and E6total sensitized the OSCC cells to irradiation, E6∗I giving the greatest degree of radiosensitization (approximately eightfold lower surviving cell fraction at 10 Gy), whereas E6∗II had no effect. In contrast to radiosensitivity, E6∗I was a weaker inhibitor than E6total of tumor suppressor p53 transactivator activity in the same cells. Flow cytometric analyses showed that irradiated E6∗I expressing cells had a much higher G2M:G1 ratio than control cells, indicating that, after G2, cells were diverted from the cell cycle to programmed cell death. This study supports a role for E6∗I in the enhanced responsiveness of HPV-positive oropharyngeal carcinomas to p53-independent radiation-induced death.

Poster 053: Rap1 Enhances Beta-Catenin-Dependent Transcription and Cell Invasion in Oral and Oropharyngeal Squamous Cell Carcinoma

Statement of the Problem: In the U.S., oral and oropharyngeal squamous cell carcinoma (OSCC) accounts for more deaths annually than cervical cancer, melanoma, or lymphoma. Despite the aggressive surgery and radiation therapy for this disease, the prognosis has not improved in four decades. Elucidation of signaling pathways that regulate proliferation in OSCC will facilitate the identification of novel treatment targets. Previously, we reported that active, GTP-bound Rap1 is strongly expressed in nuclei of OSCC. Importantly, because it has a canonical nuclear localization sequence that binds armadillo proteins, Rap1 is a candidate protein for the nuclear transport of armadillo proteins. One such protein is beta-catenin (beta-cat), which is a central molecule in the Wnt signal pathway. Beta-cat translocates from the cytoplasm to the nucleus, where as a co-factor with T-cell factor/ lymphoid enhancer factor (TCF/LEF), it triggers the expression of genes that control proliferation, migration, and invasion, and has been implicated in various cancers. However, there are conflicting reports on the nuclear translocation of beta-cat in SCC. Hence, the objective of this study was to investigate whether; a) beta-cat localizes in the nucleus of OSCC cells; b) Rap1 interacts with beta-cat; c) Rap1 induces nuclear translocation of beta-cat and enhances TCF-dependent transcription of beta-cat target genes; d) Rap1 promotes invasion of OSCC cells. Materials and Methods: Western blot analysis: Wholecell lysates were electrophoresed on 12% sodium dodecyl sulfate-polyacrylamide gels, transferred to nitrocellulose membranes and blotted with mouse anti-Rap1 monoclonal antibody or mouse anti-beta-cat monoclonal antibody. Rap1 activation assay: Active, GTP-bound Rap1 was detected by the ralGDS pull down assay followed by immunoblot analysis with the Rap1 antibody. Transfection and luciferase assay: To determine if Rap1 induces nuclear translocation and transcription of beta-cat target genes, cells were co transfected with TOPFlash or FOPFlash, renilla luciferase (internal control), beta-cat construct, and Rap1AG12 and luminescence was measured 24 h later. TOPFlash contains the TCF binding site for beta-cat, upstream of the luciferase gene. FOPFlash, which has a scrambled binding site, was used as a negative control. Immunostaining: Immunohistochemistry was performed on human OSCC tissue sections or human OSCC cells with mouse anti-beta-cat monoclonal antibody or rabbit anti-beta-cat polyclonal antibody, respectively. Double-labeling immunofluorescence on transfected cells was also performed to identify the nuclear localization of beta-cat. Cell invasion assay: Cell invasion was determined by Matrigel invasion chamber (BD Biosciences) according to the manufacturer’s instructions. Method of Data Analysis: Statistical analysis was performed using Student’s t-test. P value of 0.05 or less was considered statistically significant. Results: a) Immunohistochemical studies showed nuclear beta-cat expression in OSCC cell lines and human tissue. b) The ralGDS pull down assay indicated that beta-cat binds to active, GTP-bound Rap1. c) Immunofluorescence studies showed that Rap1A enhanced nuclear translocation of beta-cat. d) Consistent with these findings, Rap1A upregulates beta-cat induced transcription and enhances beta-cat-mediated invasion of OSCC cells. Conclusion: Rap1 binds to beta-cat and upregulates beta-cat-dependent transcription and invasion, consistent with the notion that Rap1 facilitates nuclear transport of beta-cat. Since free beta-cat translocates to the nucleus and induces the expression of genes that regulate proliferation and invasion, elucidating the nuclear transport mechanism in OSCC could identify an important therapeutic target.