Head And Neck Squamous Cell Cancer Cell Lines Research Articles

Circular RNA from Tyrosylprotein Sulfotransferase 2 Gene Inhibits Cisplatin Sensitivity in Head and Neck Squamous Cell Carcinoma by Sponging miR-770-5p and Interacting with Nucleolin.

Chemoresistance poses a significant challenge in the treatment of advanced head and neck squamous cell cancer (HNSCC). The role and mechanism of circular RNAs (circRNAs) in HNSCC chemoresistance remain understudied. We conducted circRNA microarray analysis to identify differentially expressed circRNAs in HNSCC. The expression of circRNAs from the tyrosylprotein sulfotransferase 2 (TPST2) gene and miRNAs was evaluated through qPCR, while the circular structure of circTPST2 was verified using Sanger sequencing and RNase R. Through Western blotting, biotin-labeled RNA pulldown, RNA immunoprecipitation, mass spectrometry, and rescue experiments, we discovered miR-770-5p and nucleolin as downstream targets of circTPST2. Functional tests, including CCK8 assays and flow cytometry, assessed the chemoresistance ability of circTPST2, miR-770-5p, and Nucleolin. Additionally, FISH assays determined the subcellular localization of circTPST2, miR-770-5p, and Nucleolin. IHC staining was employed to detect circTPST2 and Nucleolin expression in HNSCC patients. circTPST2 expression was inversely correlated with cisplatin sensitivity in HNSCC cell lines. Remarkably, high circTPST2 expression correlated with lower overall survival rates in chemotherapeutic HNSCC patients. Mechanistically, circTPST2 reduced chemosensitivity through sponge-like adsorption of miR-770-5p and upregulation of the downstream protein Nucleolin in HNSCC cells. The TCGA database revealed improved prognosis for patients with low circTPST2 expression after chemotherapy. Moreover, analysis of HNSCC cohorts demonstrated better prognosis for patients with low Nucleolin protein expression after chemotherapy. We unveil circTPST2 as a circRNA associated with chemoresistance in HNSCC, suggesting its potential as a marker for selecting chemotherapy regimens in HNSCC patients. Further exploration of the downstream targets of circTPST2 advanced our understanding and improved treatment strategies for HNSCC.

85 Detection of human angiotensin-converting enzyme 2 receptor (hACE2R) on human cancer cell lines

BackgroundSARS-CoV-2 infections have delayed administration of treatments for some patients with cancer, increasing the number of avoidable deaths. However, we hypothesized that infection of cancer cells with SARS-CoV-2 might increase the immunogenicity of those cancer cells. Here we sought to determine whether non-small cell lung cancer (NSCLC) and head and neck squamous cell cancer (HNSCC) cell lines could be a potential target of SARS-CoV-2, which binds and infects host cells via interactions between the viral spike glycoprotein and the human angiotensin-converting enzyme 2 receptor (hACE2) receptor. Through an institutional research tissue protocol, our lab has established a panel of cancer cell lines of various histologies. Here we set out to identify whether HNSCC and NSCLC cell lines expressed the hACE2R. We also investigated the expression of neuropilin-1, a molecule reported to facilitate SARS-CoV-2 cell entry.MethodsEstablished cell lines were phenotyped by flow cytometric analysis utilizing the anti-hACE2R antibody from Novus Biologicals. Cell lines were also stained with mIgG1 and anti-CD3 antibodies as negative staining controls.ResultsWe identified that three of eight NSCLC cell lines expressed the hACE2R and two of these had strong expression of neuropilin-1. Evaluation of HNSCC cell lines identified seven of seven cell lines expressed detectable levels of hACE2R but only one of seven HNSCC celllines expressed substantial levels of neuropilin-1. Preliminary evaluation of a renal cell carcinoma (RCC) cell line revealed strong staining for hACE2R.ConclusionsOur study found that a majority of HNSCC cell lines (100% n=7) and approximately a third of the NSCLC cell lines (37.5%, n=8) tested express the hACE2R. Some cell lines express both hACE2R and neuropilin-1, potentially increasing their susceptibility for infection with SARS-C0V-2. While these studies were performed with cultured cell lines that may have modulated expression of hACE2R, it is possible that in vivo these tumors express the ACE2R and could serve as a target and possible reservoir for SARS-CoV-2.AcknowledgementsSupportThe Chiles foundation, Nancy Lematta

Abstract 2412: Decursin inhibits tumor progression in head and neck squamous cell cancer by down regulating CXCR7 expression

Abstract Background: CX chemokine receptor 7 (CXCR7) is frequently overexpressed in a variety of tumors and plays a significant role in tumor growth and metastasis. Thus, inhibition of CXCR7 is an important potential anticancer treatment strategy. Decursin, a derivative from Angelica gigas Nakai has been reported to have anti-tumor activity in various cancers. However, little is known about its anti-tumor activity and underlying mechanisms of action in head and neck squamous cell cancer (HNSCC). Methods: CXCR7-overexpressing HNSCC cell lines were established in vitro. The effects of decursin on cell growth, motility, migration, and invasion were investigated using CCK-8, wound healing, and transwell assays. In addition, the mechanism by which decusin shows antitumor activity was investigated. Results: Overexpression of CXCR7 significantly increased cell proliferation, migration, invasion, and cell motility compared with control cells. Treatment with decursin decreased membrane expression of CXCR7 in a concentration dependent manner and significantly inhibited tumor progression by suppressing cell growth, migration, invasion, and motility. Decursin was found to induce G0/G1 cell cycle arrest and regulate cell cycle-related proteins such as Cyclin A and CDK2 in HNSCC cells. In addition, decursin inhibited CXCR7-induced STAT3/c-MYC activation. Conclusion: These results demonstrate that CXCR7 promotes cancer progression through the STAT3/c-Myc pathway in HNSCC and that decursin is a natural product that may target CXCR7 for HNSCC treatment. Citation Format: Mina Joo, Solbi Kim, Nayoung Kim, Myung-Won Lee, Heung Jin Jeon, Hyewon Ryu, Ik-Chan Song, Jin-Man Kim, Hyo Jin Lee. Decursin inhibits tumor progression in head and neck squamous cell cancer by down regulating CXCR7 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2412.

MiR-34a-5p functions as a tumor suppressor in head and neck squamous cell cancer progression by targeting Flotillin-2.

While a number of therapeutic advances have been made in recent years, the overall survival of patients with head and neck squamous cell cancer (HNSCC) remains poor. MicroRNAs (miRNAs) are key drivers of oncogenic progression, with miR-34a-5p downregulation having been observed in many different tumor types. Here, we assessed the link between miR-34a-5p and HNSCC progression and the mechanistic basis for this relationship. Levels of miR-34a-5p in HNSCC tumors and cell lines were assessed via qPCR, after which we explored the functional importance of this miRNA in this oncogenic setting. Through luciferase reporter assays, the ability of miR-34a-5p to regulate flotillin-2 (FLOT-2) was further clarified. Overall, these analyses revealed that HNSCC tumors and cells exhibited marked miR-34a-5p downregulation that was linked to the progression of this tumor type. At a functional level, miR-34a-5p constrained the proliferation, migratory/invasive activity, and epithelial-mesenchymal transition induction in HNSCC cells. At the mechanistic level, miR-34a-5p was found to suppress FLOT-2 expression and to activate the MEK/ERK1/2 pathway. Overall, these results suggest that miR-34a-5p can function as a tumor suppressor miRNA in HNSCC owing to its ability to target FLOT-2, highlighting the promise of targeting this regulatory axis to treat HNSCC.

HPRT promotes proliferation and metastasis in head and neck squamous cell carcinoma through direct interaction with STAT3

Increasing effort has been put into finding novel molecular pathways to improve the efficiency of EGFR inhibitors against head and neck squamous cell cancer (HNSCC). In this study, we performed data mining and bioinformatically analysed RNA-Seq data downloaded from TCGA and confirmed that higher expression of HPRT in HNSCC tissue was related to poor prognosis of patients. Then, we conducted in vitro and in vivo loss- and gain-of-function experiments to demonstrate the role of HPRT in HNSCC cell lines. Overexpression of HPRT increased the gene expression of epithelial mesenchymal transition markers via direct interaction with STAT3. Knocking down HPRT significantly decreased tumour growth and enhanced the anticancer effect of EGFR inhibitors against HNSCC xenografts. In conclusion, HPRT is a binding partner of STAT3 that promotes EMT and proliferation. Our findings support HPRT as a promising prognostic indicator and potential therapeutic target for HNSCC.

Silencing novel long non-coding RNA FKBP9P1 represses malignant progression and inhibits PI3K/AKT signaling of head and neck squamous cell carcinoma in vitro.

BackgroundLong non-coding RNAs (lncRNAs) play key roles in human cancers. In our previous study, we demonstrated that lncRNA FKBP prolyl isomerase 9 pseudogene 1 (FKBP9P1) was highly expressed in head and neck squamous cell cancer (HNSCC) tissues. However, its functional significance remains poorly understood. In the present study, we identify the role and potential molecular biologic mechanisms of FKBP9P1 in HNSCC.MethodsQuantitative real-time polymerase chain reaction was used to detect the expression of FKBP9P1 in HNSCC tissues, matched adjacent normal tissues, human HNSCC cells (FaDu, Cal-27, SCC4, and SCC9), and human immortalized keratinocytes cell HaCaT (normal control). Cal-27 and SCC9 cells were transfected with sh-FKBP9P1-1, sh-FKBP9P1-2, and normal control (sh-NC) lentivirus. Cell counting kit-8 assay, colony formation assay, wound healing assay, and trans-well assay were used to explore the biologic function of FKBP9P1 in HNSCC cells. Furthermore, western blotting was used to determine the mechanism of FKBP9P1 in HNSCC progression. Chi-squared test was performed to assess the clinical significance among FKBP9P1 high-expression and low-expression groups. Survival analyses were performed using the Kaplan-Meier method and assessed using the log-rank test. The comparison between two groups was analyzed by Student t test, and comparisons among multiple samples were performed by one-way analysis of variance and a Bonferroni post hoc test.ResultsFKBP9P1 expression was significantly up-regulated in HNSCC tissues (tumor vs. normal, 1.914 vs. 0.957, t = 7.746, P < 0.001) and cell lines (P < 0.01 in all HNSCC cell lines). Besides, the median FKBP9P1 expression of HNSCC tissues (1.677) was considered as the threshold. High FKBP9P1 level was correlated with advanced T stage (P = 0.022), advanced N stage (P = 0.036), advanced clinical stage (P = 0.018), and poor prognosis of HNSCC patients (overall survival, P = 0.002 and disease-free survival, P < 0.001). Knockdown of FKBP9P1 led to marked repression in proliferation, migration, and invasion of HNSCC cells in vitro (P all < 0.01). Mechanistically, silencing FKBP9P1 was observed to restrain the PI3K/AKT signaling pathway.ConclusionsSilencing lncRNA FKBP9P1 represses HNSCC progression and inhibits PI3K/AKT (phosphatidylinositol 3 kinase/AKT Serine/Threonine Kinase) signaling in vitro. Therefore, FKBP9P1 could be a potential new target for the diagnosis and treatment of HNSCC patients.

Prognostic Significance of Serum Free Amino Acids in Head and Neck Cancers.

Despite distinctive advances in the field of head and neck squamous cell cancer (HNSCC) biomarker discovery, the spectrum of clinically useful prognostic serum biomarkers is limited. As metabolic activities in highly proliferative transformed cells are fundamentally different from those in non-transformed cells, specific shifts in concentration of different metabolites may serve as diagnostic or prognostic markers. Blood amino acids have been identified as promising biomarkers in different cancers before, but little is known about this field in HNSCC. Blood amino acid profiles of 140 HNSCC patients were examined using high-performance liquid chromatography. Cox proportional hazards regression model was used to assess the prognostic value of amino acid concentrations in serum. Colony forming assay was used to identify the effect of amino acids that were significant in Cox proportional hazards regression models on colony forming ability of FaDu and Detroit 562 cell lines. In the multivariable Cox regression model for overall survival (OS), palliative treatment was associated with an unfavourable prognosis while high serum levels of methionine have had a positive prognostic impact. In the relapse-free survival (RFS) multivariable model, methionine was similarly identified as a positive prognostic factor, along with tumor localization in the oropharynx. Oral cavity localization and primary radio(chemo)therapy treatment strategy have been linked to poorer RFS. 1mM serine was shown to support the forming of colonies in both tested HNSCC cell lines. Effect of methionine was exactly the opposite.

Combination of Ionizing Radiation with Glutaminase Inhibition Improves Treatment Response in Head and Neck Squamous Cell Carcinoma

Head and neck cancer is the 6th most common cancer worldwide. Ionizing radiation (IR) is an important aspect of cancer treatment, however its efficacy is limited by radioresistance, resulting in locoregional failure rates as high as 50%. The pathways used by cells to evade the cellular damage caused by IR are poorly understood. Initial Reverse Phase Protein Array analyses in our laboratory demonstrated that IR induced metabolic pathways, including those involving the TCA cycle. Glutamate is a key substrate of the TCA cycle, and essential for the production of biomolecules necessary for tumor proliferation. Glutaminase is the enzyme that converts glutamine to glutamate. Analysis of The Cancer Genome Atlas's transcriptome database revealed that glutaminase overexpression (upper 25%) in patients with head and neck squamous cell cancer (HNSCC) was associated with significantly reduced patient survival (p&lt;0.03) . In this study, we aimed to identify whether glutaminase inhibition with CB‐839 (Caymen Chemical) could enhance the cellular response to IR. To do this, we used three representative human‐derived HNSCC cell lines: CAL‐27, FaDu, and HN5. We first established that proliferation of all three HNSCC cell lines was glutamine dependent by Dojindo CCK‐8 cell proliferation assay (20–50% increase, n=3 per line, p&lt;0.01). Moreover, a synergistic decrease in cell survival was observed in clonogenic assays with HNSCC cell lines treated with IR and CB‐839 relative to either treatment alone (n=3 per line, p&lt;0.05). In 3D cell culture, which better represents the host microenvironment, combinatorial treatment significantly reduced HNSCC spheroid size (11–26% decrease, n=3 per line, p&lt;0.0001). To further understand the mechanism behind this reduced cell growth and survival, we examined CB‐839's effect on cellular metabolism using Seahorse MitoStress test assays. CB‐839 significantly reduced spare respiratory capacity and OCR/ECAR rate in HNSCC cell lines examined (p&lt;0.001, p&lt; 2.0×10−6,n&gt;3). Mims et al. 2015 previously demonstrated that suppressing spare respiratory capacity enhances radiation sensitivity, which may explain the observed synergistic effects of CB‐839 in combination with IR. In summary, radiation resistance and locoregional failure is a major hurdle in the successful treatment of HNSCC. Combinatorial treatment with IR and the glutaminase inhibitor, CB‐839, led to an enhanced response over IR alone in HNSCC. These results support glutaminase as a possible target to overcome radiation related treatment resistance in patients with HNSCC.Support or Funding InformationPathways to Cancer Therapeutics Training Grant (CMP &amp; SW T32CA117846‐11A1)VA Career Development Award (V.T. IK2 BX004360‐01A1)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 1690: NKG2A immune checkpoint blockade potentiates cetuximab induced ADCC in head and neck cancer preclinical model

Abstract Monalizumab (IPH2201) is a first-in-class humanized IgG4 targeting NKG2A (Natural Killer Group 2A), which is expressed as a heterodimer with CD94 at the surface of subsets of NK (Natural Killer) cells, γδ T cells and tumor infiltrating CD8+ T cells. This inhibitory receptor binds to HLA-E (Human Leukocytes Antigen-E) molecules that are frequently up-regulated on human cancer cells, preventing from killing by NKG2A+ immune cells. HLA-E is expressed on most of the patients with Head and Neck Squamous Cell Cancer (HNSCC). Monalizumab blocks the binding of CD94/NKG2A to HLA-E, reducing inhibitory signaling thereby unleashing NK and T cell responses. High expression of EGFR occurs in most epithelial malignancies and particularly in HNSCC and is associated with poor prognosis. The anti-EGFR monoclonal antibody cetuximab (Ctx) is thought to act through blocking oncogenic signaling and by inducing Fcγ receptor-mediated antibody dependent cell cytotoxicity (ADCC) which involves human NK cells. We investigated ex vivo and in vitro the rationale of combining monalizumab with Ctx in the treatment of oral cancers. We first analyzed formalin-fixed paraffin-embedded samples of HNSCC patients by immunohistochemistry (n=65). We confirmed that HLA-E was expressed on carcinoma cells in all patients. We also observed that most of the tumors were highly infiltrated by CD8+ T cells both in the tumor beds and in the stroma and that the majority of tumors were also infiltrated by NKp46+ NK cells in higher numbers in the non-metastatic tumors. Interestingly, HNSCC was found as being one of the tumor types with the highest NKp46+ cell density as compared with renal cell carcinoma, non-small cell lung cancer, colorectal cancer or pancreatic tumors. Moreover CD94+ lymphocytes were detected in the stroma and tumor beds in close contact to tumor cells in about half of the patients. Using multicolor flow cytometry, NK cells and CD8+ T cells from tumor and non-involved adjacent tissues, metastatic lymph nodes and peripheral blood from patients relapsing post-chemotherapy were characterized for expression of activating and inhibitory receptors. Interestingly, NKG2A expression was higher on tumor infiltrating NK cells (CD56dim or CD56bright) and CD8+ T cell compared to the one observed in non-involved adjacent tissue, metastatic lymph node or blood. In vitro, monalizumab increased CD107 mobilization and CD137 upregulation on NKG2A+ NK cells in response to HNSCC cell lines with endogenous HLA-E expression and enhanced Ctx-mediated ADCC in a dose dependent manner. Altogether, these data support the rationale for investigating monalizumab in HNSCC patients and in combination with cetuximab in clinical trials (NCT02643550). Citation Format: Caroline Soulas, Ana Lalanne, Cécile Bonnafous, Caroline Hoffman, Elodie Bonnet, Arnaud Dujardin, Violette Breso, Mathieu Bléry, Olivier Lantz, Romain Remark, Eric Vivier, Pascale Andre. NKG2A immune checkpoint blockade potentiates cetuximab induced ADCC in head and neck cancer preclinical model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1690.

Inhibition of GSK3α/β impairs the progression of HNSCC.

BackgroundHead and neck squamous cell cancer (HNSCC) is one of the most common tumors worldwide and there is an enormous need for innovative therapy approaches. Several recent studies suggest tumor entity specific roles of glycogen synthase kinase 3 (GSK3) in different human cancers, acting as tumor suppressor or as tumor promoter. Here we describe the role of GSK3 with respect to different parameters within HNSCC progression.MethodsBase line expression and activity profiles of p-GSK3α/β (Ser21/9) and p-GSK3α/β (Tyr279/216) were analyzed by immunohistochemistry and western blotting. Four different permanent HNSCC cell lines were exposed to the potent GSK3α/β inhibitor SB 216763. Cell viability was controlled via the MTT test. Cell migration was quantified with the Real Time Cell Analyzer (RCTA) xCELLigence. Regulation of the epithelial-mesenchymal transition (EMT) was measured with the Human Epithelial to Mesenchymal Transition (EMT) RT2 Profiler™ PCR Array and scratch assays. Taqman probes were used to detect the specific gene expression profiles of inflammatory cytokines Interleukin IL1β, IL6, IL8, IL10, TNFα and IFNβ.ResultsExposure of permanent HNSCC cell lines to the specific GSK3α/β inhibitor SB 216763 leads to significant growth inhibition, inhibition of migration and decreased levels of active GSK3α/β in a dose dependent manner.Exposure of HNSCC lines to SB 216763 also resulted in a markable shift of EMT markers and functional EMT dysregulation. Functionally GSK3 differentially mediates the expression of TLR4- and TLR3-induced inflammatory cytokines in HNSCC, whereas no effect of SB 216763 on the NFkB activity was noticed.ConclusionGSK3α/β plays a crucial role in a variety of regulatory networks for HNSCC cancer progression as it drives proliferation or migration and thus GSK3 could serve as an interesting target for clinical drug development.

High NOTCH1 mRNA Expression Is Associated with Better Survival in HNSCC.

The clinical impact of the expression of NOTCH1 signaling components in squamous cell carcinoma of the pharynx and larynx has only been evaluated in subgroups. The aim of this study was therefore to evaluate NOTCH1 expression in head and neck squamous cell cancer (HNSCC) patient tissue and cell lines. We analyzed tissue from 195 HNSCCs and tissue from 30 normal patients for mRNA expression of NOTCH1, NOTCH3, HES1, HEY1, and JAG1 using quantitative real-time PCR. Association of expression results and clinical orpathological factors was examined with multivariate Cox regression. NOTCH1 expression was determined in three Human Papilloma Virus (HPV)-positive and nine HPV-negative HNSCC cell lines. High expression of NOTCH1 was associated with better overall survival (p = 0.013) and disease-free survival (p = 0.040). Multivariate Cox regression confirmed the significant influence of NOTCH1 expression on overall survival (p = 0.033) and disease-free survival (p = 0.029). A significant correlation was found between p16 staining and NOTCH1 mRNA expression (correlation coefficient 0.28; p = 0.01). NOTCH1 was expressed at higher levels in HPV-positive HNSCC cell lines compared with HPV-negative cell lines, which was not statistically significant (p = 0.068). We conclude that NOTCH1 expression is associated with overall survival, and that inhibition of NOTCH1 therefore seems less promising.

BAP1 Is a Novel Target in HPV-Negative Head and Neck Cancer.

Purpose: This study examined the potential role of the nuclear deubiquitinating enzyme BRCA1-associated protein-1 (BAP1) in radioresistance in head and neck squamous cell cancer (HNSCC).Experimental Design: We overexpressed, knocked down, and rescued BAP1 expression in six HNSCC cell lines, three human papillomavirus (HPV)-negative and three HPV-positive, and examined the effects on radiosensitivity in vitro and in an HNSCC mouse xenograft model. Radiosensitivity was assessed by clonogenic cell survival and tumor growth delay assays; changes in protein expression were analyzed by immunofluorescence staining and Western blotting. We also analyzed The Cancer Genome Atlas HNSCC database to test for associations between BAP1 expression and outcome in patients.Results: Overexpression of BAP1 induced radioresistance in both cell lines and xenograft models; conversely, BAP1 knockdown led to increased ubiquitination of histone H2A, which has been implicated in DNA repair. We further found that BAP1 depletion suppressed the assembly of constitutive BRCA1 foci, which are associated with homologous recombination (HR), but had minimal effect on γ-H2AX foci and did not affect proteins associated with nonhomologous end joining, suggesting that BAP1 affects radiosensitivity in HNSCC by modifying HR. Finally, in patients with HNSCC, overexpression of BAP1 was associated with higher failure rates after radiotherapy.Conclusions: BAP1 can induce radioresistance in HNSCC cells, possibly via deubiquitination of H2Aub and modulation of HR, and was associated with poor outcomes in patients with HNSCC. BAP1 may be a potential therapeutic target in HNSCC. Clin Cancer Res; 24(3); 600-7. ©2017 AACR.

Repurposing Albendazole: new potential as a chemotherapeutic agent with preferential activity against HPV-negative head and neck squamous cell cancer.

Albendazole is an anti-helminthic drug that has been shown to exhibit anti-cancer properties, however its activity in head and neck squamous cell cancer (HNSCC) was unknown. Using a series of in vitro assays, we assessed the ability of albendazole to inhibit proliferation in 20 HNSCC cell lines across a range of albendazole doses (1 nM–10 μM). Cell lines that responded to treatment were further examined for cell death, inhibition of migration and cell cycle arrest. Thirteen of fourteen human papillomavirus-negative HNSCC cell lines responded to albendazole, with an average IC50 of 152 nM. In contrast, only 3 of 6 human papillomavirus-positive HNSCC cell lines responded. Albendazole treatment resulted in apoptosis, inhibition of cell migration, cell cycle arrest in the G2/M phase and altered tubulin distribution. Normal control cells were not measurably affected by any dose tested. This study indicates that albendazole acts to inhibit the proliferation of human papillomavirus-negative HNSCC cell lines and thus warrants further study as a potential chemotherapeutic agent for patients suffering from head and neck cancer.

Abstract 372: Taselisib enhances the potency of ERBB inhibitors in biomarker-defined subsets of head and neck squamous carcinoma cell lines

Abstract The phosphoinositol-3-kinase (PI3K) signaling pathway is one of the most frequently activated pathways in oncogenesis, and controls critical cellular processes such as proliferation, transcription and survival. Taselisib (GDC 0032) is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30 fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. The gene that encodes the p110 alpha isoform of PI3K, PIK3CA, is frequently mutated in breast, colorectal and endometrial cancers. Previously published data demonstrated that taselisib has increased activity against PIK3CA mutant cancer cell lines (Ndubaku CO et al, J Med Chem, 2013). To determine if there are additional predictive biomarkers outside of PIK3CA mutations for sensitivity to taselisib, we profiled over 550 cell lines, encompassing 13 of the main tissue types, to increasing concentrations of taselisib. As expected, a small percentage of all tumor types responded to taselisib and correlated strongly with PIK3CA mutations. Intriguingly, the majority of head and neck squamous cell cancer (HNSCC) cell lines showed an IC50 concentration of less than 0.5uM, suggesting that HNSCC cell lines may be particularly susceptible to PI3K inhibition. Interestingly, the majority of HNSCC cell lines displayed an activated PI3K pathway as evidenced by phosphorylation of key proteins, such as AKT, S6 and PRAS40 implicating that downstream phospho-protein analysis of the PI3K pathway may not predict for sensitivity to taselisib. Mutational profiling identified that only 3 out of the tested 31 HNSCC cell lines harbored a mutation within PIK3CA, suggesting that additional genotypes may explain the sensitivity to taselisib. Additional molecular analysis assessing both gene expression and copy number levels of genes relevant to the PI3K pathway found that many of the taselisib sensitive cell lines had an activated ERBB signaling node through low level amplification of ERBB receptors (e.g. EGFR, FGFR1) or via a NRG1:ERBB3 autocrine mechanism. As many ERBB receptors heterodimerize with ERBB3, which contains p85 binding sites and potentially activates the PI3K pathway, we next tested whether taselisib would enhance the potency of ERBB receptor inhibitors in biomarker-defined subset of HNSCC cell lines. Combination screens were performed in cell lines with amplified EGFR, ERBB2, and NRG1:ERBB3 autocrine signaling with taselisib + tarceva or lapatinib. We found that combination effects assessed using the BLISS excess method showed a synergistic interaction. These results suggest that taselisib may have therapeutic potential for the treatment of HNSCC. Citation Format: Heidi M. Savage, Carol O’Brien, Heather Moore, Mark R. Lackner, Robert Yauch, Jeffrey Settleman, Timothy R. Wilson. Taselisib enhances the potency of ERBB inhibitors in biomarker-defined subsets of head and neck squamous carcinoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 372.

Abstract 4666: Phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitors demonstrate broad efficacy and synergy in head and neck cancer cell lines and patient-derived xenografts

Abstract Background: There is an urgent need for improved therapeutics in head and neck squamous cell cancer (HNSCC) to improve survival and decrease treatment morbidity. The Phosphatidylinositol 3-kinase (PI3K) pathway is frequently altered in HNSCC, particularly in human papillomavirus (HPV) positive disease. PI3K pathway inhibitors are under active investigation in preclinical models and clinical trials, however it is not clear which patient population will benefit most from these agents. Methods: Thirty unique HNSCC cell lines including five HPV positive lines were characterized with whole exome sequencing and copy number arrays. All lines were treated over a 10-point dose range with a PIK3CA specific inhibitor (BYL719), a pan PI3K inhibitor (GDC-0941), a combined pan-PI3k and mTOR inhibitor (BEZ-235), mTOR inhibitor (everolimus) and a combination of BYL719 and everolimus. Five unique patient derived xenografts (PDX) were treated with BYL719 and everolimus alone and in combination. Results: The selective PIK3CA inhibitor BYL719 was preferentially active in cell lines with PIK3CA activating mutations and amplifications (&amp;gt;100% relative PIK3CA amplification), however HRAS mutant lines were resistant. These genomic markers of sensitivity and resistance did not apply to the pan-PI3K inhibitor GDC-0941 or combined PI3K-mTOR inhibitor BEZ-235, however these agents were more active in HPV-positive cell lines (P&amp;lt;0.05). BYL719 and everolimus were individually effective at decreasing tumor growth in four PDX models including a model with an activating PIK3CA mutation, two PIK3CA amplified models and a PIK3CA wild-type model. Combined PIK3CA and mTOR inhibition appeared to be synergistic and evolved resistance never developed to this treatment, however the combination caused significant weight loss in the PDX models. Interpretation: PI3K inhibitors were broadly active in HNSCC cell lines and PDXs and their application should not be solely restricted to tumors with activating PIK3CA mutations or amplifications. Agents with broader isoform activity and combined PI3K and mTOR treatment were more potent, however combined inhibitor treatment appeared toxic in the PDX models. This trade off of toxicity and efficacy needs to balanced in the clinical setting. Citation Format: Anthony C. Nichols, Laurie Ailkes, Ren Sun, Morgan Black, Alessandro Datti, Frederick Vizeacoumar, Nicole Pinto, Kara Ruicci, John Yoo, Kevin Fung, Danielle MacNeil, Joe Mymryk, Paul C. Boutros, John W. Barrett. Phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitors demonstrate broad efficacy and synergy in head and neck cancer cell lines and patient-derived xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4666.

Cold Atmospheric Plasma: A Promising Complementary Therapy for Squamous Head and Neck Cancer.

Head and neck squamous cell cancer (HNSCC) is the 7th most common cancer worldwide. Despite the development of new therapeutic agents such as monoclonal antibodies, prognosis did not change for the last decades. Cold atmospheric plasma (CAP) presents the most promising new technology in cancer treatment. In this study the efficacy of a surface micro discharging (SMD) plasma device against two head and neck cancer cell lines was proved. Effects on the cell viability, DNA fragmentation and apoptosis induction were evaluated with the MTT assay, alkaline microgel electrophoresis (comet assay) and Annexin-V/PI staining. MTT assay revealed that the CAP treatment markedly decreases the cell viability for all tested treatment times (30, 60, 90, 120 and 180 s). IC 50 was reached within maximal 120 seconds of CAP treatment. Comet assay analysis showed a dose dependent high DNA fragmentation being one of the key players in anti-cancer activity of CAP. Annexin-V/PI staining revealed induction of apoptosis in CAP treated HNSCC cell lines but no significant dose dependency was seen. Thus, we confirmed that SMD Plasma technology is definitely a promising new approach on cancer treatment.

Exploiting high-throughput cell line drug screening studies to identify candidate therapeutic agents in head and neck cancer.

BackgroundThere is an urgent need for better therapeutics in head and neck squamous cell cancer (HNSCC) to improve survival and decrease treatment morbidity. Recent advances in high-throughput drug screening techniques and next-generation sequencing have identified new therapeutic targets in other cancer types, but an HNSCC-specific study has not yet been carried out. We have exploited data from two large-scale cell line projects to clearly describe the mutational and copy number status of HNSCC cell lines and identify candidate drugs with elevated efficacy in HNSCC.MethodsThe genetic landscape of 42 HNSCC cell lines including mutational and copy number data from studies by Garnett et al., and Barretina et al., were analyzed. Data from Garnett et al. was interrogated for relationships between HNSCC cells versus the entire cell line pool using one- and two-way analyses of variance (ANOVAs). As only seven HNSCC cell lines were tested with drugs by Barretina et al., a similar analysis was not carried out.ResultsRecurrent mutations in human papillomavirus (HPV)-negative patient tumors were confirmed in HNSCC cell lines, however additional, recurrent, cell line-specific mutations were identified. Four drugs, Bosutinib, Docetaxel, BIBW2992, and Gefitinib, were found via multiple-test corrected ANOVA to have lower IC50 values, suggesting higher drug sensitivity, in HNSCC lines versus non-HNSCC lines. Furthermore, the PI3K inhibitor AZD6482 demonstrated significantly higher activity (as measured by the IC50) in HNSCC cell lines harbouring PIK3CA mutations versus those that did not.ConclusionHNSCC-specific reanalysis of large-scale drug screening studies has identified candidate drugs that may be of therapeutic benefit and provided insights into strategies to target PIK3CA mutant tumors. PIK3CA mutations may represent a predictive biomarker for response to PI3K inhibitors. A large-scale study focused on HNSCC cell lines and including HPV-positive lines is necessary and has the potential to accelerate the development of improved therapeutics for patients suffering with head and neck cancer. This strategy can potentially be used as a template for drug discovery in any cancer type.Electronic supplementary materialThe online version of this article (doi:10.1186/2050-6511-15-66) contains supplementary material, which is available to authorized users.

Abstract 2631: Neuregulin-1 expression correlates with sensitivity to the PI3K inhibitor, GDC-0032, outside of PIK3CA mutations in head and neck cancer

Abstract The phosphoinositol-3-kinase (PI3K) signaling pathway is one of the most frequently activated pathways in oncogenesis, and controls critical cellular processes such as proliferation, transcription and survival. GDC 0032 is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30 fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. The gene that encodes the p110 alpha isoform of PI3K, PIK3CA, is frequently mutated in breast, colorectal and endometrial cancers. Previously published data demonstrated that GDC-0032 has increased activity against PIK3CA mutant cancer cell lines (Olivero A. et al., DDT02-01, AACR 2013). To determine if there are additional predictive biomarkers outside of PIK3CA mutations for sensitivity to GDC-0032, we profiled over 550 cell lines, encompassing 13 of the main cancer types, to increasing concentrations of GDC-0032. As expected, a small percentage of all tumor types responded to GDC-0032 and, were assessed, correlated strongly with PIK3CA mutations. Intriguingly, the majority of head and neck squamous cell cancer (HNSCC) cell lines showed an IC50 concentration of less than 1uM, suggesting that HNSCC cell lines may be particularly susceptible to PI3K inhibition. Molecular profiling identified that only 10% of the HNSCC cell lines had a mutation within PIK3CA, and no consistent copy number alterations were observed in receptor tyrosine kinases or PTEN. To investigate this observation further, we developed a ”PIK3CA activation” algorithm, by utilizing the mutant-selective property of GDC-0032, and calculated a ratio of GDC-0032 to GDC-0941, a pan PI3K inhibitor with reduced in vitro selectivity for PIK3CA mutations. Following a proof of principle experiment in breast cancer cells, a GDC-0941/GDC-0032 ratio of ≥4 enriched for breast cancer cells with mutant PIK3CA (11 out of 13 cell lines). Application of the algorithm, to our panel of HNSCC cell lines identified 10 out of 34 cell lines that had a GDC-0941/GDC-0032 ratio of ≥4, with only 1 of these cell lines being PIK3CA mutant. Further molecular characterization identified high mRNA expression of neuregulin-1, the ligand for HER3, in GDC-0032 cell lines with a GDC-0941/GDC-0032 ratio of ≥4, compared to cell lines with a ratio of &amp;lt;4. Pharmacodynamic analysis of sensitive and refractory HNSCC cell lines demonstrated that GDC-0032 inhibited the phospho-AKT pathway in both groups of cell lines, but GDC-0032 only suppressed downstream phospo-S6 signaling in the sensitive cell lines. In conclusion, high throughput cell line screening identified a “PIK3CA activated” subset of HNSCC cell lines that correlated with increased neuregulin-1 expression and corresponding sensitivity to GDC-0032, outside of PIK3CA mutations. Citation Format: Heidi M. Savage, Carol O'Brien, Bob Yauch, Jeff Settleman, Mark R. Lackner, Timothy R. Wilson. Neuregulin-1 expression correlates with sensitivity to the PI3K inhibitor, GDC-0032, outside of PIK3CA mutations in head and neck cancer. [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 2631. doi:10.1158/1538-7445.AM2014-2631

Abstract 4668: Antitumor efficacy of AZD1480 in head and neck squamous cell carcinoma.

Abstract Aberrant activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway has been implicated in cancer cell proliferation and survival. AZD1480, a pharmacologic inhibitor of JAK2, has been tested in several cancer models including glioblastoma, lung, renal, prostate, breast, and ovarian cancer. However JAK inhibitors, including AZD1480 has not been evaluated in head and neck squamous cell cancer (HNSCC). In the present study, the in vitro and in vivo efficacy of AZD1480 was evaluated in HNSCC preclinical models. AZD1480 decreased HNSCC proliferation in cell lines with varying levels of pJAK2 and pSTAT3 with an IC50 ranging from 0.9μM - 4μM. Further evaluation demonstrated downmodulation of pJAK2 and pSTAT3 in HNSCC cell lines by AZD1480. Furthermore, in vivo antitumor efficacy of AZD1480 was tested in heterotopic xenograft tumor models derived from 2 HNSCC patient tumors. AZD1480 at a dose of 30mg/kg, was given twice daily via oral gavage and compared with vehicle (0.5% Hypromellose) control. Tumors treated with AZD1480 exhibited significant growth inhibition relative to the tumors treated with vehicle (p=0.0037). In addition, significant decrease in pSTAT3Tyr705 expression was observed in both the patient tumors treated with AZD1480 compared to vehicle alone indicating that AZD1480 targets pSTAT3 and may be considered for study in the treatment of HNSCC patients. Citation Format: Malabika Sen, John Black, Sarah Wheeler, Maria L. Freilino, Sonali Joyce, Vivian Lui, Yan Zeng, Korey Marshall, Jennifer R. Grandis. Antitumor efficacy of AZD1480 in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4668. doi:10.1158/1538-7445.AM2013-4668

Abstract 5359: Gene expression characterization of different cancer stem cell isolation methods in head and neck cancer

Abstract Background: Head and neck squamous cell cancer (HNSCC) is a heterogeneous cancer that continues to pose major clinical problems. There is growing evidence that cancer stem cells (CSCs) may be an important factor in disease progression and resistance to current treatments. Establishing that stem cells are present in HNSCC and characterizing their global gene expression patterns provides the rationale for this study. Methods: A series of 5 HNSCC cell lines that have been maintained at low passage number such that they maintain phenotypic and morphological characteristics similar to the primary tumor were used in this study. Three different strategies were used to isolate stem cells by flow cytometry sorting: 1) Hoechst 33342 “side” population, 2) Aldefluor, and 3) CD44 bright cells. Global gene expression for each isolated CSC-enriched population and its associated CSC-depleted population was analyzed using Affymetrix Human Exon 1.0ST arrays. Gene set enrichment analysis was performed using Ariadne Pathway Studio. Results: The different methods of identifying CSCs produced widely different populations. In side population cells, there were 77 differentially expressed genes (p&amp;lt;0.05 and 1.5-fold cutoff) while only 59 and 17 genes were differentially expressed in the CD44 bright and Aldefluor bright populations, respectively. Not a single gene was commonly altered between any two of the isolation methods. Gene set enrichment analysis, however, was able to identify categories that differed between the CSC-enriched and CSC-depleted populations. Biological processes of cell cycle, cell division, mitosis, translational elongation, and spindle assembly were highly regulated. Microtubule binding/microtubule plus-end binding and structural constituent of ribosome were commonly altered molecular functions. Conclusions: This study suggests that current methods for isolating putative stem cells in HNSCC result in diverse populations with differing expression of specific genes. However, when examined for global expression patterns, several important categories of genes are commonly altered between the CSC-enriched and CSC-depleted populations. Some of the known characteristics of stem cells such as asymmetric division and unlimited growth potential are suggested by the highly regulated categories of cell division and spindle assembly. From these studies we hope to gain greater insight into the importance of stem cells in HNSCC as well as improve the capability of identifying these potentially important new targets for treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5359. doi:1538-7445.AM2012-5359