Head And Neck Squamous Cell Carcinoma Patient Samples Research Articles

Knockdown of Ubiquitin-Conjugating Enzyme E2 T Abolishes the Progression of Head and Neck Squamous Cell Carcinoma by Inhibiting NF-Κb Signaling and inducing Ferroptosis.

Ubiquitin-conjugating enzyme 2T (UBE2T) has been reported to be associated with uncontrolled cell growth and tumorigenesis in multiple cancer types. However, the understanding of its regulatory role in the carcinogenesis of Head And Neck Squamous Cell Carcinoma (HNSC) is limited. UBE2T expression in HNSC patient samples and the correlation between its expression and patients' survival rates were evaluated using The Cancer Genome Atlas (TCGA) database. Cell survival and proliferation were investigated in UM-SCC1 and UM-SCC15 cells infected with control and shUBE2T lentivirus. The xenograft mouse model was established using UM-SCC15 cells to examine HNSC tumorigenesis with or without UBE2T. Western blot, qRT-PCR, and ferroptosis assays were carried out to disclose the interaction between UBE2T and NF-κB signaling and ferroptosis. The increased expression of UBE2T was noted in tumor tissues of patients with HNSC, correlating with a significantly reduced overall survival time in this patient cohort. Knockdown of UBE2T inhibited HNSC tumorigenesis and tumor growth. Mechanistically, inhibition of UBE2T suppressed NF-κB signaling and induced ferroptosis in HNSC. Our study underscores the multifaceted role of UBE2T in HNSC, illuminating its potential as a biomarker and therapeutic target.

A pilot study of the relative number of circulating tumor cells and leukocytes containing actin-binding proteins in head and neck cancer patients.

Circulating tumor cells (CTCs) play an important role in tumor metastases, which is positively correlated with an increased risk of death. Actin-binding proteins, including cofilin (CFL1), profilin 1 (PFN1), and adenylate cyclase-associated protein 1 (CAP1), are thought to be involved in tumor cell motility and metastasis, specifically in head and neck squamous cell carcinoma (HNSCC). However, currently, there are no published studies on CFL1, PFN1, and CAP1 in CTCs and leukocytes in HNSCC patients. We assessed serum levels of CFL1, PFN1, and CAP1 and the number of CTCs and leukocytes containing these proteins in blood from 31 HNSCC patients (T1-4N0-2M0). The analysis used flow cytometry and an enzyme-linked immunosorbent assay kit. We found that CAP1 + CTCs and CAP1 + leukocyte subpopulations were prevalent in these HNSCC patient samples, while the prevalence rates of CFL1 + and PFN1 + CTCs were relatively low. Patients with stage T2-4N1-2M0 had CFL1 + and PFN1 + CTCs with an elevated PFN1 serum level, compared with the T1-3N0M0 group. In summary, the PFN1 serum level and the relative number of PFN1 +CD326 + CTCs could be valuable prognostic markers for HNSCC metastases. The current study is the first to obtain data regarding the contents of actin-binding proteins (ABPs) in CTCs, and leukocytes in blood from HNSCC patients. This is also the first to assess the relationship between the number of CTCs subgroups and disease characteristics.

IGF2BP2 serves as a core m6A regulator in head and neck squamous cell carcinoma.

Methylation of N6 adenosine (m6A) plays a crucial role in the development and progression of cancers. Its modification is regulated by three types of m6A-related regulators (methyltransferases (writers), demethylases (erasers), and RNA-binding proteins (readers)). Till now, the functions and roles of these regulators in head and neck squamous cell carcinoma (HNSC) remain largely unexplored. Therefore, we utilized the open HNSC dataset in The Cancer Genome Atlas (TCGA), four different cell lines, and our HNSC patient samples (n=40) to explore the clinical significance of 19 m6A regulators, and selected the most significant prognosis-related regulator. Authentic analyses based on online websites were also used in the study (Oncomine, UALCAN, Kaplan-Meier plotter, Human Protein Atlas (HPA), cBioPortal, LinkedOmics, String, etc.). From the results, general overexpression of m6A regulators was observed in pan-cancer, especially in HNSC. IGF2BP2 was recognized as the hub m6A regulator, which was an independent, unfavorable prognostic factor in HNSC. Its mRNA and protein expression in HNSC were significantly up-regulated. Gene mutation types of IGF2BP2 in HNSC (32%) were mainly mRNA High or Amplification, which represented the high expression of IGF2BP2. And these mutations were associated with a poor prognosis. In functional analysis, IGF2BP2 was negatively correlated to tumor immune infiltration in HNSC. Finally, HMGA2 might interact with the IGF2BP2 in HNSC. In conclusion, IGF2BP2 serves as a core m6A regulator among all regulators in HNSC, which has a high expression and predicts the poor prognosis of HNSC patients independently. IGF2BP2 might bring a new direction for HNSC treatment in the future.

A novel neutrophil extracellular trap signature to predict prognosis and immunotherapy response in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignant cancers, and patients with HNSCC possess early metastases and poor prognosis. Systematic therapies (including chemotherapy, targeted therapy, and immunotherapy) are generally applied in the advanced/late stages of HNSCC, but primary and acquired resistance eventually occurs. At present, reliable biomarkers to predict the prognosis of HNSCC have not been completely identified. Recent studies have shown that neutrophil extracellular traps (NETs) are implicated in cancer progression, metastasis and cancer immune response, and NET-related gene signatures are associated with the prognosis of patients with several human cancers. To explore whether NET-related genes play crucial roles in HNSCC, we have performed systematic analysis and reported several findings in the current study. Firstly, we identified seven novel NET-related genes and developed a NET-score signature, which was highly associated with the clinicopathological and immune traits of the HNSCC patients. Then, we, for the first time, found that NIFK was significantly upregulated in HNSCC patient samples, and its levels were significantly linked to tumor malignancy and immune status. Moreover, functional experiments confirmed that NIFK was required for HNSCC cell proliferation and metastasis. Altogether, this study has identified a novel NET-score signature based on seven novel NET-related genes to predict the prognosis of HNSCC and NIFK has also explored a new method for personalized chemo-/immuno-therapy of HNSCC.

Evaluation of correlation between CD44, radiotherapy response, and survival rate in patients with advanced stage of head and neck squamous cell carcinoma (HNSCC).

PurposeCancer stem cells (CSCs) constitute a distinctive subpopulation of cancer cells that are competent in tumor initiation, invasion, recurrence, and resistance to chemoradiotherapy. CD44, a hyaluronic acid (HA) receptor has been considered as a potential CSC marker in head and neck cancer. The purpose of this study is to evaluate the correlation between CD44 and clinicopathological parameters, treatment response, survival, and recurrence.MethodsThe CD44 expression was examined by immunohistochemistry (IHC) in 90 samples of head and neck squamous cell carcinoma (HNSCC) confirmed patients. The expression of CD44 and its association with clinicopathological parameters, treatment response, and survival was determined.ResultsIn all HNSCC patient samples, CD44 was expressed consistently at different intensities. Tumor size (p < 0.001), stage (p < 0.001), and treatment response (p < 0.001) showed statistically significant association with CD44 expression. Alcohol and CD44 were observed as independent predictors of response to radiotherapy using multivariate ordinal logistic regression analysis. Analysis of 2‐year overall survival (OS) showed that CD44 expression (p = 0.02), tumor size (p = 0.001), lymph node status (p < 0.001), stage (p < 0.001), and grade (p = 0.007) were significantly associated with OS. Using Cox regression analysis, lymph node status (p = 0.001), grade (p < 0.001), recurrence (p < 0.001), and CD44 expression (p = 0.003) were found to be potential independent predictors of OS.ConclusionOur findings suggest that CD44 contributes to resistance to radiotherapy and poor OS. The results also suggest that except for CD44 there could be other factors such as lymph node metastasis, grade, and alcohol which should be investigated as potential targets for therapy.

Abstract 2367: Role of miR-34a-MET axis in head and neck cancer squamous cell carcinoma

Abstract MicroRNAs (miR) are evolutionarily highly conserved non-coding RNAs that play roles in fundamental cellular functions by post-translational gene regulation. In cancer pathogenesis, miRs exert both anti-tumorigenic and pro-tumorigenic effects by virtue of miR-specific and context-dependent mechanisms. The role of chromosomal changes and mutation status on miR expression in cancer remains unknown. Here we provide evidence that chromosome arm 1p loss and P53 mutations are associated with lower levels of miR-34a, particularly in squamous cancers such as head and neck squamous cell carcinoma (HNSCC). miR-34a acts as a tumor suppressor and physically interacts with and functionally targets the proto-oncogene MET, and modulates cell proliferation. miR-34a expression is inversely correlated with MET in tumor tissue. We found that miR-34a suppresses HNSCC carcinogenesis, at least in part, by downregulating MET, consequently inhibiting HNSCC proliferation. Moreover, ectopic expression of miR-34a reduces HNSCC cell proliferation and tumor burden in vitro and in vivo, represses expression of genes and proteins involved in epithelial-mesenchymal transition, and negates the oncogenic effect of MET in mouse tumors. Our work demonstrates a role of miR-34a in maintaining tumor immunity, as overexpression of miR-34a resulted in a decease in percentage of anti-inflammatory (M2-like) TAMs and PDL1+TAMs, which are reported to have pro-tumorigenic activity. In HNSCC patient samples, higher levels of miR-34a are significantly associated with a higher frequency of Th1 cells, myeloid cells, and regulatory T cells. Our results demonstrate that miR-34a directly targets MET and maintains anti-tumor immune activity, and could potentially represent a new therapeutic approach for HNSCC. Citation Format: Xun Wu, Yi-Shing Lisa Cheng, Methew Matthen, Angela Yoon, Gary Schwartz, Shashi Bala, Alison M. Taylor, Fatemeh Momen-Heravi. Role of miR-34a-MET axis in head and neck cancer squamous cell carcinoma [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 2367.

The functional GRHL3-filaggrin axis maintains a tumor differentiation potential and influences drug sensitivity

Current therapies for treating heterogeneous cancers such as head and neck squamous cell carcinoma (HNSCC) are non-selective and are administered independent of response biomarkers. Therapy resistance subsequently emerges, resulting in increased cellular proliferation that is associated with loss of differentiation. Whether a cancer cell differentiation potential can dictate therapy responsiveness is still currently unknown. A multi-omic approach integrating whole-genome and whole-transcriptome sequencing with drug sensitivity was employed in a HNSCC mouse model, primary patients’ data, and human cell lines to assess the potential of functional differentiation in predicting therapy response. Interestingly, a subset of HNSCC with effective GRHL3-dependent differentiation was the most sensitive to inhibitors of PI3K/mTOR, c-Myc, and STAT3 signaling. Furthermore, we identified the GRHL3-differentiation target gene Filaggrin (FLG) as a response biomarker and more importantly, stratified HNSCC subsets as treatment resistant based on their FLG mutational profile. The loss of FLG in sensitive HNSCC resulted in a dramatic resistance to targeted therapies while the GRHL3-FLG signature predicted a favorable patient prognosis. This study provides evidence for a functional GRHL3-FLG tumor-specific differentiation axis that regulates targeted therapy response in HNSCC and establishes a rationale for clinical investigation of differentiation-paired targeted therapy in heterogeneous cancers.

Down-regulation of the tumor suppressor miR-34a contributes to head and neck cancer by up-regulating the MET oncogene and modulating tumor immune evasion

BackgroundMicroRNAs (miRs) have been shown to play an important role in tumorigenesis, including in head and neck squamous cell carcinoma (HNSCC). The miR-34 family is thought to play a role in tumor suppression, but the exact mechanism of their action in HNSCC is not well understood. Moreover, the impact of chromosomal changes and mutation status on miR-34a expression remains unknown.MethodsDifferential expression of miR-34a, MET, and genomic alterations were assessed in the Cancer Genome Atlas (TCGA) datasets as well as in primary HNSCC and adjacent normal tissue. The biological functions of miR-34a in HNSCC were investigated in samples derived from primary human tumors and HNSCC cell lines. The expression of MET was evaluated using immunohistochemistry, and the molecular interaction of miR-34a and MET were demonstrated by RNA pulldown, RNA immunoprecipitation, luciferase reporter assay, and rescue experiments. Lastly, locked nucleic acid (LNA) miRs in mouse xenograft models were used to evaluate the clinical relevance of miR-34a in HNSCC tumor growth and modulation of the tumor microenvironment in vivo.ResultsChromosome arm 1p loss and P53 mutations are both associated with lower levels of miR-34a. In HNSCC, miR-34a acts as a tumor suppressor and physically interacts with and functionally targets the proto-oncogene MET. Our studies found that miR-34a suppresses HNSCC carcinogenesis, at least in part, by downregulating MET, consequently inhibiting HNSCC proliferation. Consistent with these findings, administration of LNA-miR-34a in an in vivo model of HNSCC leads to diminished HNSCC cell proliferation and tumor burden in vitro and in vivo, represses expression of genes involved in epithelial-mesenchymal transition, and negates the oncogenic effect of MET in mouse tumors. Consistently, LNA-miR-34a induced a decreased number of immunosuppressive PDL1-expressing tumor-associated macrophages in the tumor microenvironment. In HNSCC patient samples, higher levels of miR-34a are significantly associated with a higher frequency of Th1 cells and CD8 naïve T cells.ConclusionsOur results demonstrate that miR-34a directly targets MET and maintains anti-tumor immune activity. We propose miR-34a as a potential new therapeutic approach for HNSCC.

Abstract A31: Deregulation of NOTCH 1/NR4A2 signaling axis in head and neck cancer pathogenesis

Abstract Background: Notch1 and its intracellular cytoplasmic domain (NICD) are dysregulated in many solid tumors, including head and neck squamous cell carcinoma (HNSCC), and associated with disease initiation and progression. NICD-mediated upregulation of nuclear receptor related 1 (NURR1/NR4A2) is linked to differentiation/development, homeostasis, and cellular metabolism. However, the role and molecular mechanism(s) of Notch1/NICD- NR4A2 axis in HNSCC pathogenesis are still unknown and need to be investigated. Methods: The clinicopathologic importance of Notch1 and NR4A2 was investigated using HNSCC patient samples and publicly available databases. NR4A2 was stably knocked out (KO) and Notch1 signaling was blocked using small-molecule inhibitor (PF3084014) in HNSCC cell lines and analyzed for tumorigenic and metastatic potential. We also examined the whole-genome NR4A2 target genes using ChIP sequencing. Results: Both Notch1 and NR4A2 were highly overexpressed in HNSCC patients. Inhibition of Notch1 signaling or NR4A2 KO significantly decreased the clonogenicity and migratory potential of HNSCC cells. Our ChIP analysis revealed that NR4A2 regulates genes involved in metabolism, hypoxia, cell cycle progression, integrin and Wnt signaling. Interestingly, we observed that NR4A2 regulates stem cell markers including Sox2, Nanog, and CD44 and their expression was drastically reduced upon Notch 1 inhibition. Conclusion: Targeting Notch1-NR4A2 signaling axis may be crucial for inhibition of cancer stem cell-mediated HNSCC pathogenesis. Citation Format: Sanjib Chaudhary, Ramesh Pothuraju, Zafar Sayed, Dwight T. Jones, Surinder K. Batra, Muzafar A. Macha. Deregulation of NOTCH 1/NR4A2 signaling axis in head and neck cancer pathogenesis [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A31.

Fibroblast Growth Factor Receptors as Targets for Radiosensitization in Head and Neck Squamous Cell Carcinomas

We examined the capacity of the pan-fibroblast growth factor receptor (FGFR) inhibitor AZD4547 to augment radiation response across a panel of head and neck squamous cell carcinoma (HNSCC) cell lines and xenografts. FGFR1, FGFR2, and FGFR3 RNA in situ hybridization expression was assessed in a cohort of HNSCC patient samples, cell lines, and patient-derived xenografts (PDXs). Invitro effects of AZD4547 and radiation on cell survival, FGFR signaling, apoptosis, autophagy, cell cycle, and DNA damage repair were evaluated. Reverse phase protein array was used to identify differentially phosphorylated proteins in cells treated with AZD4547. Invivo tumor responses were evaluated in cell lines and PDX models. FGFR1, FGFR2, and FGFR3 RNA in situ hybridization were expressed in 41%, 81%, and 89% of 107 oropharynx patient samples. Sensitivity to AZD4547 did not directly correlate with FGFR protein or RNA expression. In sensitive cell lines, AZD4547 inhibited p-MAPK in a time-dependent manner. Significant radiosensitization with AZD4547 was observed in cell lines that were sensitive to AZD4547. The mechanism underlying these effects appears to be multifactorial, involving inhibition of the MTOR pathway and subsequent enhancement of autophagy and activation of apoptotic pathways. Significant tumor growth delay was observed when AZD4547 was combined with radiation compared with radiation or drug alone in an FGFR-expressing HNSCC cell line xenograft and PDX. These findings suggest that AZD4547 can augment the response of radiation in FGFR-expressing HNSCC invivo model systems. FGFR1 and FGFR2 may prove worthy targets for radiosensitization in HNSCC clinical investigations.

In vitro humanized 3D microfluidic chip for testing personalized immunotherapeutics for head and neck cancer patients

ObjectivesImmunotherapy and personalized medicine therapeutics are emerging as promising approaches in the management of head and neck squamous cell carcinoma (HNSCC). In spite of that, there is yet no assay that could predict individual response to immunotherapy. MethodsWe manufactured an in vitro 3D microfluidic chip to test the efficacy of immunotherapy. The assay was first tested using a tongue cancer cell line (HSC-3) embedded in a human tumour-derived matrix “Myogel/fibrin” and immune cells from three healthy donors. Next, the chips were used with freshly isolated cancer cells, patients' serum and immune cells. Chips were loaded with different immune checkpoint inhibitors, PD-L1 antibody and IDO 1 inhibitor. Migration of immune cells towards cancer cells and the cancer cell proliferation rate were evaluated. ResultsImmune cell migration towards HSC-3 cells was cancer cell density dependent. IDO 1 inhibitor induced immune cells to migrate towards cancer cells both in HSC-3 and in two HNSCC patient samples. Efficacy of PD-L1 antibody and IDO 1 inhibitor was patient dependent. ConclusionWe introduced the first humanized in vitro microfluidic chip assay to test immunotherapeutic drugs against HNSCC patient samples. This assay could be used to predict the efficacy of immunotherapeutic drugs for individual patients.

Abstract 5862: 3,5-bis(2,4-difluorobenzylidene)-4-piperidone, a novel compound potently inhibits HNSCC through a DCLK1 mediated mechanism

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) affects 40,000 patients annually and is associated with &amp;lt;50% 5-year survival despite aggressive therapy. Cancer stem cells (CSC) have been reported to contribute to tumor aggressiveness and therapeutic resistance in several tumor types, including HNSCC. Very little is known about the molecular characterization of CSCs in HNSCC. High expression of Doublecortin-like Kinase 1 (DCLK1), a microtubule associated protein that has been previously reported to mark quiescent CSCs of the colon and pancreas, is correlated with poor prognostic outcomes according to TCGA databases. We hypothesized that DCLK1 expression confers stem cell properties and hence is a target for therapy in HNSCC. Methods: HNSCC tumor micro array was stained with DCLK1 to evaluate DCLK1 expression in HNSCC compared to normal mucosal tissue. Additionally, HNSCC cells were screened for expression of DCLK1 to assess expression. HN5 and FaDu cells were treated with DiFiD (0-10µM) and analyzed for cell viability in 2-dimensional and 3-dimensional cultures. Expression for markers of apoptosis, stemness, and cell cycle regulation were determined by western blotting. Furthermore, Caspase 3/7 assay and Annexin V/Pi staining were performed to confirm induction of apoptosis. Thermal shift binding assays (CETSA) were performed to confirm DiFiD:DCLK1 interaction. Lastly, HN5 tumor xenograft toxicity studies were performed in Balb c/ nude FOXN1 mice to determine the antitumor efficacy of DiFiD in vivo. Results: We identified expression of DCLK1 in HNSCC cell lines and patient samples. IHC demonstrated increased expression of DCLK1 in HNSCC compared to normal mucosal tissue. Furthermore, DCLK1 was upregulated in HNSCC cells lines. CETSA assay demonstrated increased thermal stability of DCLK1 following incubation with DiFiD, suggesting DiFiD binds to DCLK1. There was a dose and time dependent reduction in cell viability both in 2-dimensional and 3-dimensional culture models following treatment with DiFiD in both cell lines. DiFiD induced caspase mediated apoptosis following G2/M cell cycle arrest. DiFiD significantly inhibited tumor growth in vivo, as confirmed by reduction in tumor volume and weight. DiFiD also showed little off target toxicity, as mouse weights were not significantly altered following DiFiD treatment. Conclusions: Taken together, these data demonstrate that DiFiD has potent anti-cancer activity and therefore may serve as a potential novel therapy for the treatment of HNSCC. Citation Format: David Standing, Jake New, Prasad Dandawate, Dharmalingam Subramaniam, Vusala Snyder, Jonathan Enders, Afreen Sayed, Shrikant Anant, Sufi M. Thomas. 3,5-bis(2,4-difluorobenzylidene)-4-piperidone, a novel compound potently inhibits HNSCC through a DCLK1 mediated mechanism [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 5862.

PO-031 Sensitising P53 mutant HNSCC to oncolytic reovirus (RT3D) by targeting the unfolded protein response

Introduction Oncolytic viruses are able to enter and selectively replicate in cancer cells. These self-propagating agents kill through multiple mechanisms and have the potential to promote anti-tumour immune responses. Reovirus strain type 3 Dearing (RT3D) is an oncolytic virus actively under clinical investigation. RT3D has been shown to induce ER stress and trigger the downstream unfolded protein response (UPR) in a variety of tumour types. Recent evidence has indicated that proteasome inhibition in multiple myeloma, and BRAF inhibition in BRAF mutant melanoma, sensitise to RT3D through perturbation of the normal cellular response to ER stress. We sought to investigate if more direct modulation of UPR signalling could sensitise head and neck squamous cell carcinoma (HNSCC) to RT3D. Material and methods ER stress agents including novel agents targeting UPR signalling were screened for sensitisation to RT3D in a panel of HNSCC cell lines. Alterations to reovirus protein levels and viable virus particle production was assessed. A 3D tumour spheroid model was used as a more accurate method to study stress in vitro. Recombinant protein expression and shRNA techniques were utilised to study how the unfolded protein response impacts reovirus replication. ER stress measured by GRP78 expression was also assessed in HNSCC patient samples. Results and discussions The PERK inhibitor GSK2606414 and the canonical ER stress agent thapsigargin were shown to sensitise HNSCC to RT3D. PERK inhibition resulted in increased viral capsid protein levels and increased viable RT3D production from target cells. PERK inhibition enhanced the ability of RT3D to eradicate HNSCC 3D tumour spheroids. PERK inhibition increased RT3D spread in 3D spheroids while perturbing normal ER chaperone levels and inhibiting RT3D induced LC3A/B. In HNSCC patient samples, GRP78 was found to be elevated in tumour vs stroma with significant variation between patients. This highlights the importance in understanding the link between ER stress levels/UPR signalling and reovirus efficacy. Conclusion We demonstrate that modulators of the ER stress response represent a novel mechanism of sensitisation to RT3D in head and neck.

Upregulation of cell cycle genes in head and neck cancer patients may be antagonized by erufosine's down regulation of cell cycle processes in OSCC cells.

The TCGA database was analyzed to identify deregulation of cell cycle genes across 24 cancer types and ensuing effects on patient survival. Pan-cancer analysis showed that head and neck squamous cell carcinoma (HNSCC) ranks amongst the top four cancers showing deregulated cell cycle genes. Also, the median gene expression of all CDKs and cyclins in HNSCC patient samples was higher than that of the global gene expression. This was verified by IHC staining of CCND1 from HNSCC patients. When evaluating the quartiles with highest and lowest expression, increased CCND1/CDK6 levels had negative implication on patient survival. In search for a drug, which may antagonize this tumor profile, the potential of the alkylphosphocholine erufosine was evaluated against cell lines of the HNSCC subtype, oral squamous cell carcinoma (OSCC) using in-vitro and in-vivo assays. Erufosine inhibited growth of OSCC cell lines concentration dependently. Initial microarray findings revealed that cyclins and CDKs were down-regulated concentration dependently upon exposure to erufosine and participated in negative enrichment of cell cycle processes. These findings, indicating a pan-cdk/cyclin inhibition by erufosine, were verified at both, mRNA and protein levels. Erufosine caused a G2/M block and inhibition of colony formation. Significant tumor growth retardation was seen upon treatment with erufosine in a xenograft model. For the decreased cyclin D1 and CDK 4/6 levels found in tumor tissue, these proteins can serve as biomarker for erufosine intervention. The findings demonstrate the potential of erufosine as cell cycle inhibitor in HNSCC treatment, alone or in combination with current therapeutic agents.

ErbB activation signatures as potential biomarkers for anti-ErbB3 treatment in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) accounts for 3–5% of all tumor types and remains an unmet medical need with only two targeted therapies approved to date. ErbB3 (HER3), the kinase-impaired member of the EGFR/ErbB family, has been implicated as a disease driver in a number of solid tumors, including a subset of HNSCC. Here we show that the molecular components required for ErbB3 activation, including its ligand neuregulin-1 (NRG1), are highly prevalent in HNSCC and that HER2, but not EGFR, is the major activating ErbB3 kinase partner. We demonstrate that cetuximab treatment primarily inhibits the ERK signaling pathway and KTN3379, an anti-ErbB3 monoclonal antibody, inhibits the AKT signaling pathway, and that dual ErbB receptor inhibition results in enhanced anti-tumor activity in HNSCC models. Surprisingly, we found that while NRG1 is required for ErbB3 activation, it was not sufficient to fully predict for KTN3379 activity. An evaluation of HNSCC patient samples demonstrated that NRG1 expression was significantly associated with expression of the EGFR ligands amphiregulin (AREG) and transforming growth factor α (TGFα). Furthermore, NRG1-positive HNSCC cell lines that secreted high levels of AREG and TGFα or contained high levels of EGFR homodimers (H11D) demonstrated a better response to KTN3379. Although ErbB3 and EGFR activation are uncoupled at the receptor level, their respective signaling pathways are linked through co-expression of their respective ligands. We propose that NRG1 expression and EGFR activation signatures may enrich for improved efficacy of anti-ErbB3 therapeutic mAb approaches when combined with EGFR-targeting therapies in HNSCC.

Changes in programmed death-ligand 1 expression during cisplatin treatment in patients with head and neck squamous cell carcinoma.

Programmed death-ligand 1 (PD-L1) expression is regarded as a predictive marker for anti-PD-1/PD-L1 therapy. The purpose of study was to explore the changes in PD-L1 expression in head and neck squamous cell carcinoma (HNSCC) during treatment. Paired HNSCC tissues prior to and after cisplatin-based treatment were evaluated to determine PD-L1 protein expression by immunohistochemistry. Among the 35 HNSCC patient samples, PD-L1 expression status changed after treatment in 37.1% (13/35) of samples. Among the 13 patients whose baseline PD-L1 was negative, PD-L1 expression was increased in 9 cases (69.2%) and remained negative in 4 cases (30.8%, P = 0.003). Patients exposed to cisplatin generally showed PD-L1 up-regulation (83.3%, P = 0.037) compared to those not exposed to cisplatin (57.1%, P = 0.072). To validate these findings in vitro, changes in PD-L1 expression in HNSCC cell lines (Detroit-562, PCI-13, SNU-1041, SNU-1066, SNU-1076, and FaDu) were analyzed by western blotting and flow cytometry after treatment with cisplatin and interferon-gamma. In HNSCC cell lines, PD-L1 expression was significantly up-regulated after cisplatin, along with phosphor-MAPK/ERK kinase up-regulation. In conclusion, PD-L1 expression in HNSCC may be altered during cisplatin treatment, activating the MAPK/ERK kinase pathway.

Epidermal growth factor receptor variant III in head and neck squamous cell carcinoma is not relevant for targeted therapy and irradiation.

BackgroundThe epidermal growth factor receptor (EGFR) is an important regulator of cell growth and survival, and is highly variable in tumor cells. The most prevalent variation of the EGFR extracellular domain is the EGFR variant III (EGFRvIII). Some studies imply that EGFRvIII may be responsible for the poor response to the monoclonal EGFR-antibody Cetuximab, used therapeutically in head and neck squamous cell carcinoma (HNSCC). Due to inconsistent data in the literature regarding EGFRvIII prevalence and clinical relevance in HNSCC, especially its predictive value, we examined EGFRvIII-transfected cell lines and patient tissue samples.ResultsIn contrast to other recent publications, we were able to demonstrate EGFRvIII expression in HNSCC. However, we noted that the different detection methods yielded inconsistent results. Furthermore, our EGFRvIII transfected and EGFR wild type cell lines exhibited similar characteristics and response rates in the performed in vitro experiments.Materials and MethodsWe conducted various inhibition and combined irradiation experiments using three EGFRvIII-transfected cell lines. Moreover, a patient cohort of 149 cases consisting of formalin fixed and paraffin embedded (FFPE) and fresh-frozen specimens was assayed via reverse transcriptase PCR (rtPCR) with gel electrophoresis and sequencing for EGFRvIII prevalence. In the rtPCR assays, we used five previously published EGFRvIII primers and EGFRvIII-positive glioblastoma tissue as a positive control. In addition, immunohistochemical staining was conducted.ConclusionsEGFRvIII can be detected in HNSCC patient samples. Nevertheless, the low prevalence and similar response rates to targeted drugs and irradiation in vitro cast doubt regarding the clinical relevance of EGFRvIII in HNSCC.

Developing Robust Predictive Models for Head and Neck Cancer across Microarray and RNA-seq Data.

Increased understanding of the transcriptomic patterns underlying head and neck squamous cell carcinoma (HNSCC) can facilitate earlier diagnosis and better treatment outcomes. Integrating knowledge from multiple studies is necessary to identify fundamental, consistent gene expression signatures that distinguish HNSCC patient samples from disease-free samples, and particularly for detecting HNSCC at an early pathological stage. This study utilizes feature integration and heterogeneous ensemble modeling techniques to develop robust models for predicting HNSCC disease status in both microarray and RNAseq datasets. Several alternative models demonstrated good performance, with MCC and AUC values exceeding 0.8. These models were also applied to discriminate between early pathological stage HNSCC and normal RNA-seq samples, showing encouraging results. The predictive modeling workflow was integrated into a software tool with a graphical user interface. This tool enables HNSCC researchers to harness frequently observed transcriptomic features and ensembles of previously developed models when investigating new HNSCC gene expression datasets.

RacGAP1 Is a Novel Downstream Effector of E2F7-Dependent Resistance to Doxorubicin and Is Prognostic for Overall Survival in Squamous Cell Carcinoma

We have previously shown that E2F7 contributes to drug resistance in head and neck squamous cell carcinoma (HNSCC) cells. Considering that dysregulation of responses to chemotherapy-induced cytotoxicity is one of the major reasons for treatment failure in HNSCC, identifying the downstream effectors that regulate E2F7-dependent sensitivity to chemotherapeutic agents may have direct clinical impact. We used transcriptomic profiling to identify candidate pathways that contribute to E2F7-dependent resistance to doxorubicin. We then manipulated the expression of the candidate pathway using overexpression and knockdown in in vitro and in vivo models of SCC to demonstrate causality. In addition, we examined the expression of E2F7 and RacGAP1 in a custom tissue microarray (TMA) generated from HNSCC patient samples. Transcriptomic profiling identified RacGAP1 as a potential mediator of E2F7-dependent drug resistance. We validated E2F7-dependent upregulation of RacGAP1 in doxorubicin-insensitive SCC25 cells. Extending this, we found that selective upregulation of RacGAP1 induced doxorubicin resistance in previously sensitive KJDSV40. Similarly, stable knockdown of RacGAP1 in insensitive SCC25 cells induced sensitivity to doxorubicin in vitro and in vivo. RacGAP1 expression was validated in a TMA, and we showed that HNSCCs that overexpress RacGAP1 are associated with a poorer patient overall survival. Furthermore, E2F7-induced doxorubicin resistance was mediated via RacGAP1-dependent activation of AKT. Finally, we show that SCC cells deficient in RacGAP1 grow slower and are sensitized to the cytotoxic actions of doxorubicin in vivo. These findings identify RacGAP1 overexpression as a novel prognostic marker of survival and a potential target to sensitize SCC to doxorubicin.

Abstract 3934: Restoration of NOTCH signaling in head and neck squamous cell carcinoma inhibits tumor growth and cell migratory abilities

Abstract Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common malignancy worldwide, with roughly 300,000 cancer related deaths occurring globally each year. The survival of patients with HNSCC hasn't changed significantly over the past decade leading investigators to search for promising molecular targets. To identify new treatment targets and biomarkers that could better guide therapy, we previously characterized the genomic alterations from primary HNSCC patient samples. We were among the first to discover that NOTCH1 is one of the most frequently mutated genes in this cancer type. The spectrum of inactivating NOTCH1 mutations in HNSCC suggested a tumor suppressive role for this protein, however, the mechanism of its function is currently unknown. Procedure: We used Sanger sequencing and immunoblotting to characterize 50 well- established HNSCC cell lines as being wild-type or mutant for NOTCH1. We cloned the full length NOTCH1 receptor to restore the NOTCH signaling function and activated this pathway by culturing cells on immobilized NOTCH ligand, Jagged1, coated plates. Clonogenic assays and competitive cell proliferation assays was used to evaluate cell growth and proliferation. Transwell Boyden chambers was used to investigate migration/invasion. Tumorigenicty was evaluated in vivo in a mice orthotopic model of oral cancer. An unbiased gene expression analysis was performed to identify potential downstream targets of the NOTCH pathway. Results: We show here that restoration of NOTCH signaling in mutant HNSCC cell lines significantly decreases cell growth and migratory abilities. Activation of NOTCH1 led to decreased tumorigenicity in a mice orthotopic oral cancer model. Conversely, abrogation of the NOTCH pathway using the dominant negative form of the NOTCH co-activator, MAML1, enhanced tumor growth in a weakly tumorigenic HNSCC cell line harboring wild-type NOTCH1. To evaluate the mechanism of NOTCH induced inhibition of growth and migration, we performed an unbiased microarray analysis and observed suppression of two proto-oncogenes, AXL and CTNNAL1 in the presence of NOTCH1, but not in its absence. Restoration of NOTCH1 significantly decreases protein expression levels of AXL and CTNNAL1 which then results in decreased cell growth and migratory abilities. Furthermore, we show here a novel link between NOTCH1, AXL and CTNNAL1 via HES5, one of the canonical NOTCH downstream targets. We suggest that NOTCH1 activates HES5 which then downregulates AXL and CTNNAL1, thus inhibiting cell growth and migratory abilities. Conclusions: We have data linking NOTCH1 reactivation to suppression of proto-oncogenes, AXL and CTNNAL1 along with decreased growth and migration of HNSCC cells. This underscores the need to understand the clinical significance of the NOTCH pathway and its downstream targets which can then be exploited for potential therapeutic strategies. Citation Format: Shhyam Moorthy, Rami Saade, Curtis Pickering, David Neskey, Noriaki Tanaka, Patrick Zweidler-McKay, Mitchell Frederick, Jeffrey Myers. Restoration of NOTCH signaling in head and neck squamous cell carcinoma inhibits tumor growth and cell migratory abilities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3934. doi:10.1158/1538-7445.AM2015-3934