Head And Neck Squamous Cell Carcinoma Tumor Growth Research Articles

Sensory nerve release of CGRP increases tumor growth in HNSCC by suppressing TILs

Perineural invasion (PNI) and nerve density within the tumor microenvironment (TME) have long been associated with worse outcomes in head and neck squamous cell carcinoma (HNSCC). This prompted an investigation into how nerves within the tumor microenvironment affect the adaptive immune system and tumor growth. We used RNA sequencing analysis of human tumor tissue from a recent HNSCC clinical trial, proteomics of human nerves from HNSCC patients, and syngeneic orthotopic murine models of HPV-unrelated HNSCC to investigate how sensory nerves modulate the adaptive immune system. Calcitonin gene-related peptide (CGRP) directly inhibited CD8 Tcell activity invitro, and blocking sensory nerve function surgically, pharmacologically, or genetically increased CD8 and CD4 Tcell activity invivo. Our data support sensory nerves playing a role in accelerating tumor growth by directly acting on the adaptive immune system to decrease Th1 CD4 Tcells and activated CD8 Tcells in the TME. These data support further investigation into the role of sensory nerves in the TME of HNSCC and points toward the possible treatment efficacy of blocking sensory nerve function or specifically inhibiting CGRP release or activity within the TME to improve outcomes. 1R01DE028282-01, 1R01DE028529-01, 1P50CA261605-01 (to S.D.K.), 1R01CA284651-01 (to S.D.K.), and F31 DE029997 (to L.B.D.).

PROTAC Prodrug-Integrated Nanosensitizer for Potentiating Radiation Therapy of Cancer.

Radiation therapy (RT) is one of the primary options for clinical cancer therapy, in particular advanced head and neck squamous cell carcinoma (HNSCC). Herein, the crucial role of bromodomain-containing protein 4 (BRD4)-RAD51 associated protein 1 (RAD51AP1) axis in sensitizing RT of HNSCC is revealed. A versatile nanosensitizer (RPB7H) is thus innovatively engineered by integrating a PROteolysis TArgeting Chimeras (PROTAC) prodrug (BPA771) and hafnium dioxide (HfO2 ) nanoparticles to downregulate BRD4-RAD51AP1 pathway and sensitize HNSCC tumor to RT. Upon intravenous administration, the RPB7H nanoparticles selectively accumulate at the tumor tissue and internalize into tumor cells by recognizing neuropilin-1 overexpressed in the tumor mass. HfO2 nanoparticles enhance RT effectiveness by amplifying X-ray deposition, intensifying DNA damage, and boosting oxidative stress. Meanwhile, BPA771 can be activated by RT-induced H2 O2 secretion to degrade BRD4 and inactivate RAD51AP1, thus impeding RT-induced DNA damage repair. This versatile nanosensitizer, combined with X-ray irradiation, effectively regresses HNSCC tumor growth in a mouse model. The findings introduce a PROTAC prodrug-based radiosensitization strategy by targeting the BRD4-RAD51AP1 axis, may offer a promising avenue to augment RT and more effective HNSCC therapy.

Diallyl Trisulfide Induces ROS-Mediated Mitotic Arrest and Apoptosis and Inhibits HNSCC Tumor Growth and Cancer Stemness.

Despite advances in therapeutic approaches, the five-year survival rate for head and neck squamous cell carcinoma (HNSCC) patients is still less than fifty percent. Research has indicated that the consumption of Allium vegetables or processed garlic containing diallyl trisulfide (DATS) can lower the risk of multiple types of cancer. Nevertheless, the effectiveness and underlying mechanisms of DATS against HNSCC have not been thoroughly explored until the current study. In this research, it was found that DATS notably curtailed the growth and viability of HNSCC cells. Additionally, DATS triggered a significant G2/M cell cycle arrest in these cells, accumulating cyclin B1, Cip1/p21, and Ser-10 phospho-histone H3-this was indicative of mitotic arrest attenuated by NAC pretreatment, suggesting the role of reactive oxygen species (ROS) induction. The production of ROS induced by DATS led to DNA damage and apoptosis, a process associated with elevated levels of cleaved caspase-3 and cleaved PARP, along with reduced XIAP. When HNSCC cells were exposed to pharmacological concentrations of DATS, it resulted in the suppression of cancer stem cell (CSC) populations, as indicated by a decrease in the CD133high/CD44high cell fraction, reduced aldehyde dehydrogenase 1 (ALDH1) activity, inhibited spheroid formation and downregulated SOX2 and Oct4 expression. Furthermore, the administration of DATS to tumor xenografts demonstrated its in vivo capacity to hinder CSCs. Further, DATS treatment inhibited the growth of UMSCC-22B head and neck cancer tumor xenograft in immunocompromised mice. Overall, DATS inhibited cell proliferation; induced cell cycle mitotic arrest and apoptosis involving DNA damage through ROS generation; reduced the CSC fraction and spheroid formation; and downregulated SOX2 and Oct4 expression. More importantly, DATS inhibited HNSCC tumor growth and CSC fraction in vivo. Thus, DATS could be a potential anticancer agent that can be used against head and neck cancer.

Recombinant immunotoxin induces tumor intrinsic STING signaling against head and neck squamous cell carcinoma

The innate immune stimulator of interferon genes (STING) pathway is known to activate type I interferons (IFN-I) and participate in generating antitumor immunity. We previously produced hDT806, a recombinant diphtheria immunotoxin, and demonstrated its efficacy against head and neck squamous cell carcinoma (HNSCC). However, it’s unknown whether the tumor-intrinsic STING plays a role in the anti-HNSCC effects of hDT806. In this study, we investigated the innate immune modulation of hDT806 on HNSCC. hDT806 significantly upregulated the level of STING and the ratio of p-TBK1/TBK1 in the HNSCC cells. Moreover, intratumoral hDT806 treatment increased the expression of STING-IFN-I signaling proteins including IFNA1, IFNB, CXCL10 and MX1, a marker of IFN-I receptor activity, in the HNSCC xenografts. Overexpression of STING mimicked the hDT806-induced upregulation of the STING-IFN-I signaling and induced apoptosis in the HNSCC cells. In the mouse xenograft models of HNSCC with STING overexpression, we observed a significant suppression of tumor growth and reduced tumor weight with increased apoptosis compared to their control xenograft counterparts without STING overexpression. Collectively, our data revealed that hDT806 may act as a stimulator of tumor-intrinsic STING-IFN-I signaling to inhibit tumor growth in HNSCC.

Abstract 1834: Cholinergic nerve release of CGRP increases tumor growth in HNSCC by suppressing TILs

Abstract Perineural invasion (PNI) and nerve density within the tumor microenvironment (TME) have long been associated with worse outcomes in head and neck squamous cell carcinoma (HNSCC). We investigate how nerves within the tumor microenvironment impact the adaptive immune system and tumor growth. RNA sequencing and proteomics data led us to identify cholinergic nerves as the potential drivers of tumor growth in human and mouse HNSCC. Using three syngeneic orthotopic murine models of HPV-unrelated HNSCC, experimental and pharmacologic manipulation of cholinergic output, and knockout mouse models, we discovered that activation of cholinergic nerves accelerate tumor growth by directly acting on the adaptive immune system. Cholinergic nerve release of calcitonin gene-related peptide (CGRP) directly inhibited CD8 T cell activity in vitro, and blocking cholinergic nerves pharmacologically or genetically increased CD8 T cell activity in vivo. These data support therapeutic translation of cholinergic nerve targeting of CGRP release to enhance antitumor immune response within the HNSCC TME. Citation Format: Laurel B. Darragh, Tiffany T. Pham, Michael M. Knitz, Alexander Nguyen, Laith A. Mukdad, Yazeed Alhiyari, Maie St. John, Sana D. Karam. Cholinergic nerve release of CGRP increases tumor growth in HNSCC by suppressing TILs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1834.

TYK2 correlates with immune infiltration: A prognostic marker for head and neck squamous cell carcinoma.

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family and is involved in immune and inflammatory signaling. TYK2 is overexpressed in several types of cancers and promotes the invasion and proliferation of cancer cells. Nevertheless, the roles of TYK2 in the prognosis and immune infiltration of head and neck squamous cell carcinoma (HNSCC) remain to be elucidated. In this study, the expression of TYK2 in HNSCC was evaluated based on the data retrieved from multiple databases and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The prognostic potential of TYK2 in patients with HNSCC was analyzed by Kaplan-Meier curves and Cox regression analysis. A TYK2-related risk assessment model was subsequently constructed by Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis and stepwise multivariate Cox regression analysis. The association between the expression of TYK2 and the tumor immune microenvironment, immune checkpoints, and drug sensitivity was explored various packages in R. Cell function assays were finally used for exploring the effects of TYK2 on the growth and metastasis of HNSCC tumors. The expression of TYK2 was significantly upregulated in HNSCC and was found to be closely correlated with HPV status, gender, clinical grade, and TP53 mutation status. Survival analysis suggested that TYK2 is associated with better survival outcomes and acts as an independent prognostic indicator of HNSCC. The model constructed herein also performed well in terms of predicting patient prognosis. The expression of TYK2 was positively associated with the population of tumor-infiltrating immune cells, expression of immune checkpoint genes, and antitumor drug susceptibility. Functionally, TYK2 knockdown significantly promoted the proliferation, migration, and invasion of HNSCC cell lines in vitro. The findings demonstrated that TYK2 could serve as a suppressor of tumor growth and holds significant promise as a novel biomarker for assessing the prognosis of patients with HNSCC and aid in immunotherapy against HNSCC.

LncRNA TM4SF19-AS1 exacerbates cell proliferation, migration, invasion, and EMT in head and neck squamous cell carcinoma via enhancing LAMC1 expression

ABSTRACT Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous and aggressive tumor with high mortality and unfavorable prognosis. Numerous long non-coding RNAs (lncRNAs) have been confirmed to exert pivotal parts in cancers. Nevertheless, the functions of most lncRNAs in HNSCC need deeper exploration. Our present research tried to clarify the biological role of TM4SF19 antisense RNA 1 (TM4SF19-AS1) and investigate its regulatory mechanism in HNSCC. RT-qPCR analysis was done to test TM4SF19-AS1 expression and identify the up-regulation of TM4SF19-AS1 in HNSCC cells. Loss-of-function assays were also involved, and the data implied that TM4SF19-AS1 knockdown hampered the proliferation, migration, invasion, along with epithelial-mesenchymal transition (EMT) of HNSCC cells. In vivo assays revealed TM4SF19-AS1 depletion restrained HNSCC tumor growth. Additionally, mechanism experiments were implemented to uncover the underlying regulatory mechanism of TM4SF19-AS1 in HNSCC cells. It turned out that TM4SF19-AS1 modulated laminin subunit gamma 1 (LAMC1) expression via sequestering microRNA-153-3p (miR-153-3p) and recruiting heterogeneous nuclear ribonucleoprotein C (HNRNPC) protein. Rescue assays confirmed that TM4SF19-AS1 contributed to HNSCC cell malignant behaviors via up-regulating LAMC1. To summarize, TM4SF19-AS1 played an oncogenic role in HNSCC cells, signifying TM4SF19-AS1 may have the potential to be used as a novel molecular target for HNSCC diagnosis.

G-Protein-Coupled Receptor Kinase-Interacting Protein 1 (GIT1) Promotes Head and Neck Squamous Cell Carcinoma Metastases via Activating the PI3K/AKT/mTOR Signal Pathway.

Objective GIT1 is identified as a novel tumor oncogene in breast cancer. In this article, we aimed to explore the role of GIT1 in the progression of head and neck squamous cell carcinoma (HNSCC). Methods GIT1 expression in HNSCC was detected by RT-qPCR, immunohistochemistry assay, and Western blot. HNSCC cell proliferation, migration, and invasion were examined by CCK-8 assay, Wound healing assay, and Transwell assay, respectively. Cell apoptosis was detected by flow cytometric analysis. Results In our study, GIT1 was notably upregulated in HNSCC tissues and cells. Moreover, GIT1 expression level had positive corelation with pathological grade and nodal status of HNSCC. Functional experiments showed that knockdown of GIT1 restrained HNSCC proliferation, invasion, migration, and EMT and facilitated cell apoptosis. Furthermore, GIT1 knockdown was found to restrain HNSCC tumor growth and lung metastasis. Additionally, PI3K/AKT/mTOR signal pathway inhibitors suppressed the effect of GIT1 on HNSCC cell progression. Conclusion GIT1 was upregulated in HNSCC and facilitated HNSCC cell progression by inducing PI3K/AKT/mTOR signal pathway. Therefore, we suggested that GIT1 might be a potential target for HNSCC treatment.

Abstract 13400: STAT3 ASO-Loaded Microbubbles With Pulsed Ultrasound for Enhanced Therapeutic Effect in Head and Neck Tumors

Introduction: Signal transducer and activator of transcription 3 ( STAT3 ) is an oncogenic transcription factor implicated in carcinogenesis, tumor progression, and drug resistance in many cancers, including head and neck squamous cell carcinoma ( HNSCC ). STAT3 antisense oligonucleotide ( ASO ) specifically targeting the nucleotide sequence of STAT3 has therapeutic promise, but effective delivery remains challenging. We developed microbubble ( MB ) delivery vehicles loaded with STAT3 ASO and hypothesized that compared to intravenous ( i.v. ) delivery, ultrasound-targeted microbubble cavitation ( UTMC )-mediated ASO delivery confers superior tumor growth inhibition. Methods: Lipid MBs loaded with STAT3 ASO (S3MB) or mutant ASO (Mut-MB) were prepared. First, murine squamous carcinoma cells (SCC-7) in contact with MBs were exposed to pulsed ultrasound (1 MHz, 0.5 MPa, 100 μs pulse duration, 1 ms pulse interval) for 10 sec to establish efficacy with respect to cytotoxicity, apoptosis, and gene knockdown. Thereafter, the anti-tumor effect of 3 treatments with STAT3 ASO (10 μg) delivered i.v. or via UTMC x 15 min ( f= 1MHz, 0.7MPa, 100 cycles x 5 every 2 sec) was compared in vivo in a murine SCC-7 xenograft model followed up to 17 days. Results: S3MB + UTMC conferred more cytoxocitiy, apoptosis, and downregulation of transcripts for STAT3 and its target genes (Bcl-xL and Cyclin D1) compared to Mut-MB + UTMC in vitro (p<0.05). In tumor bearing mice, compared to i.v. STAT3 ASO (n=8), Mut-MB+UTMC (n=5) or no treatment (n=8), S3MB + UTMC (n=9) significantly attenuated tumor burden, prolonged doubling time, and improved survival (p<0.05). While i.v. STAT3 ASO had no effect on tumor expression of STAT3, Bcl-x, and cyclin D1 at the transcript and protein levels, its delivery via UTMC significantly reduced tumor expression of these genes (p<0.01). Conclusion: At a dose of 10 μg, STAT3 ASO significantly inhibited murine HNSCC tumor growth and downregulated STAT3 and its target genes when delivered via UTMC, compared with intravenous administration alone. Our platform offers a promising imaged-guided, targeted delivery strategy for nucleotide-based therapeutics in cancer applications.

Cancer-associated fibroblasts secrete hypoxia-induced serglycin to promote head and neck squamous cell carcinoma tumor cell growth in vitro and in vivo by activating the Wnt/β-catenin pathway.

The tumor microenvironment (TME) is known to play a prominent role in the pathology of head and neck squamous cell carcinoma (HNSCC). Cancer-associated fibroblasts (CAFs) have been reported to regulate tumor progression, and serglycin (SRGN), one of the paracrine cytokines of CAFs, has been reported to play an important role in various signaling pathways. Hypoxia is a distinct feature of the HNSCC TME. Here, we investigated the mechanism underlying CAF-secreted SRGN leading to HNSCC progression under hypoxia. Immunohistochemical staining was used to detect SRGN expression in clinical HNSCC samples, after which its relation with patient survival was assessed. CAFs were isolated and SRGN expression and secretion by CAFs under normoxia and hypoxia were confirmed using qRT-PCR and ELISA assays, respectively. HNSCC sphere-forming abilities, stemness-related gene expression, and chemoresistance were assessed with or without SRGN treatment. A Wnt/β-catenin pathway inhibitor (PNU-75,654) was used to block its activation, after which nuclear translocation of β-catenin in the presence of SRGN with or without PNU-75,654 was evaluated. shRNAs were used to stably knock down SRGN expression in CAFs. HNSCC tumor cells with or without (SRGN silenced) CAFs were inoculated submucosally in nude mice after which tumor weights and sizes were determined to assess the effects of CAFs and SRGN on tumor growth. We found that SRGN was expressed in both HNSCC tumor and stroma cells, and that high SRGN expression in the stroma cells, but not in the tumor cells, was significantly related to a poor patient survival. After the extraction of CAFs and normal fibroblasts (NFs) from paired tumor samples and adjacent normal tissues, respectively, we found that the expression of CAF-specific genes, including fibroblast activation protein (FAP) and alpha-smooth muscle actin (α-SMA), was clearly upregulated compared to the expression in NFs. The hypoxia marker HIF-1α was found to be expressed in tumor stroma cells. Hypoxyprobe immunofluorescence staining confirmed stromal hypoxia in an orthotopic tongue cancer mouse model. Using qRT-PCR and ELISA we found that a hypoxic TME upregulated SRGN expression and secretion by CAFs. SRGN markedly enhanced the sphere-forming ability, stemness-related gene expression and chemoresistance of HNSCC tumor cells. SRGN activated the Wnt/β-catenin pathway and promoted β-catenin nuclear translocation. An in vivo study confirmed that CAFs can accelerate HNSCC tumor growth, and that this effect can be counteracted by SRGN silencing. Our data indicate that a hypoxic tumor stroma can lead to upregulation of SRGN expression. SRGN secreted by CAFs can promote β-catenin nuclear translocation to activate downstream signaling pathways, leading to enhanced HNSCC cell stemness, chemoresistance and accelerated tumor growth.

A novel diphtheria toxin-based bivalent human EGF fusion toxin for treatment of head and neck squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) is often overexpressed in head and neck squamous cell carcinoma (HNSCC) and represents a top candidate for targeted HNSCC therapy. However, the clinical effectiveness of current Food and Drug Administration (FDA)‐approved drugs targeting EGFR is moderate, and the overall survival rate for HNSCC patients remains low. Therefore, more effective treatments are urgently needed. In this study, we generated a novel diphtheria toxin‐based bivalent human epidermal growth factor fusion toxin (bi‐EGF‐IT) to treat EGFR‐expressing HNSCC. Bi‐EGF‐IT was tested for in vitro binding affinity, cytotoxicity, and specificity using 14 human EGFR‐expressing HNSCC cell lines and three human EGFR‐negative cancer cell lines. Bi‐EGF‐IT had increased binding affinity for EGFR‐expressing HNSCC compared with the monovalent version (mono‐EGF‐IT), and both versions specifically depleted EGFR‐positive HNSCC, but not EGFR‐negative cell lines, in vitro. Bi‐EGF‐IT exhibited a comparable potency to that of the FDA‐approved EGFR inhibitor, erlotinib, for inhibiting HNSCC tumor growth in vivo using both subcutaneous and orthotopic HNSCC xenograft mouse models. When tested in an experimental metastasis model, survival was significantly longer in the bi‐EGF‐IT treatment group than the erlotinib treatment group, with a significantly reduced number of metastases compared with mono‐EGF‐IT. In addition, in vivo off‐target toxicities were significantly reduced in the bi‐EGF‐IT treatment group compared with the mono‐EGF‐IT group. These results demonstrate that bi‐EGF‐IT is more effective and markedly less toxic at inhibiting primary HNSCC tumor growth and metastasis than mono‐EGF‐IT and erlotinib. Thus, the novel bi‐EGF‐IT is a promising drug candidate for further development.

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.

Pharmacological inhibition of CDK7 by THZ1 impairs tumor growth in p53-mutated HNSCC.

Cyclin-dependent kinase 7 (CDK7) has been critically linked to human cancer. However, the roles of CDK7 in head and neck squamous cell carcinoma (HNSCC) remain incompletely known. Here, we sought to dissect the functions of CDK7 underlying HNSCC tumorigenesis and explore whether pharmacological inhibition of CDK7 could induce anti-cancer effects. CDK7 expression was measured in a panel of HNSCC cell lines with p53 mutation and 20 pairs of HNSCC samples and adjacent non-tumor tissues. Genetic targeting and pharmacological inhibition of CDK7 were conducted to dissect the biological roles of CDK7 in p53-mutated HNSCC cells. An HNSCC xenograft model was developed to determine the therapeutic effects of THZ1 in vivo. Potential genes and pathways responsible for therapeutic effects of THZ1 were identified by genome-wide RNA-sequencing and bioinformatics interrogations. CDK7 expression was significantly elevated in cancerous cells and samples as compared with their adjacent non-tumor counterparts. Impaired cell proliferation, migration, and invasion as well increased apoptosis were observed in cells upon CDK7 knockdown or THZ1 exposure. THZ1 administration potently inhibited tumor overgrowth in vivo. Mechanistically, hundreds of genes enriched in cell proliferation, apoptosis, and cancer-related categories were identified to be potentially mediated the therapeutic effects of THZ1 in HNSCC. Our findings reveal that CDK7 might serve as a novel putative pro-oncogenic gene underlying HNSCC tumorigenesis and therapeutic targeting of CDK7 might be a promising strategy for p53-mutated HNSCC.

Elevated RUNX1 is a prognostic biomarker for human head and neck squamous cell carcinoma.

Runt-related transcription factors regulate many developmental processes such as proliferation and differentiation. In this study, the function of the runt-related transcription factor 1 (RUNX1) was investigated in head and neck squamous cell carcinoma (HNSCC). Our results show that RUNX1 expression was elevated in HNSCC patients, which was greatly correlated with the N stage, tumor size, and American Joint Committee on Cancer stage. Cox proportional hazard models showed that RUNX1 could be used as a prognostic indicator for the overall survival of HNSCC patients (hazard ratio, 5.572; 95% confidence interval, 1.860-9.963; P < 0.001). Moreover, suppression of RUNX1 inhibited HNSCC cell proliferation, migration, and invasion. Using the HNSCC xenograft nude mouse model, we found that the shRUNX1-transfected tumor (sh-RUNX1) was significantly smaller both in size and weight than the control vector-transfected tumor (sh-Control). In conclusion, our results show that the elevated RUNX1 expression was correlated with tumor growth and metastasis in HNSCC, indicating that RUNX1 could be used as a biomarker for tumor recurrence and prognosis.

Abstract 6042: TNFa co-activates IKK/NF-kB/RELA prosurvival and WEE1-CDC2 G2/M checkpoint signaling and is targetable by WEE1 antagonist AZD1775 in head and neck cancer

Abstract Cytotoxic and genotoxic therapies are known to induce the prosurvival Inhibitor-kappaB kinase (IKK)-NF-κB/RELA pathway and DNA repair at the G2/M checkpoint, thereby promoting therapeutic resistance, but the coordination of these mechanisms is unclear. Our recent RNAi screen unexpectedly uncovered possible crosstalk between WEE1, a G2/M checkpoint kinase and the TNFα-IKK-NF-κB prosurvival pathway components. siRNAs targeting WEE1 and IKKs both inhibited TNFα-inducible NF-κB activity in a HNSCC reporter line, suggesting a mechanistic linkage. We investigated this potential connection further and studied the effect of WEE1 inhibition using AZD1775 in head and neck squamous cell carcinoma (HNSCC) cells and mouse xenograft models. Increased expression, phosphorylation and cellular localization of IKK-NF-κB and WEE1-CDC2 axis proteins co-occurred in a subset of HNSCC lines, and were inversely co-modulated by TNFα and WEE1 kinase inhibitor AZD1775. WEE1 co-immunoprecipitated with the IKKα/β and RELA protein complex, and AZD1775 or an IKK inhibitor, IKK16, inhibited phosphorylation and kinase activities, further suggesting that these two axes interact with each other. AZD1775 sensitized HNSCC cells to TNFα-induced cytotoxicity, in part by inhibiting IKK-dependent phosphorylation and nuclear localization of NF-κB activation, and expression of target prosurvival protein BCL-2. Combination of AZD1775 with radiation, a known inducer of TNFα- and DNA-mediated cytotoxicity, potently inhibited HPV+/- HNSCC tumor xenografts. Depleting TNFα abolished the anti-tumor activity of combined therapy with AZD1775 and radiation. The findings of our study reveal that TNFα co-modulates a novel interaction between the prosurvival IKK/NF-κB/RELA and WEE1-CDC2 G2/M checkpoint pathway and that components of both the pathways are inhibited by AZD1775. Our mouse xenograft experiments suggest that AZD1775, when combined with radiation, significantly delays the growth of xenograft HNSCC tumors differing in HPV status in a TNFα-dependent manner. The current findings reveal a novel mechanism whereby crosstalk and co-activation of WEE1- and NF-κB-signaling promotes resistance to TNFα-mediated cytotoxicity, which may be targeted using WEE1 inhibitors. Supported by NIDCD projects ZIA-DC-000016, -73 and 74. Citation Format: Ramya Viswanathan, Zhengbo Hu, Jianghong Chen, Xinping Yang, Angel Huynh, Paul Clavijo, Yi An, Yvette Robbins, Christopher Silvin, Clint Allen, Anthony Saleh, Zhong Chen, Carter Van Waes. TNFa co-activates IKK/NF-kB/RELA prosurvival and WEE1-CDC2 G2/M checkpoint signaling and is targetable by WEE1 antagonist AZD1775 in head and neck cancer [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 6042.

Abstract 3579: Pimozide combined with paclitaxel demonstrates a significant antitumor effect in head and neck squamous cell carcinoma

Abstract Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer in the world. Despite the development of various treatments, the five-year survival rate for HNSCC is about 50-60% and has not been improved in recent years. Immune checkpoint inhibitors are promising, but the effect of those is limited. The development of new reagents having different mechanisms against tumor progression is still needed. From the result of drug screening, Pimozide (PMZ), an antipsychotic drug used for the treatment of schizophrenia and chronic psychosis, demonstrated a strong antitumor ability to HNSCC cells expressing high level of CD44v. In this study, we examined the combined effects of PMZ and paclitaxel (PTX) for HNSCC cells. Methods: Using HNSCC cell lines (FaDu, Detroit562, HSC-2, OSC-19), the antitumor effects of PTX, PMZ, and the combination of PTX and PMZ were evaluated by MTS assay. Moreover, the reactive oxygen species (ROS) expression level under each drug administration was measured by flow cytometry. Moreover, PTX, PMZ (0.45mg/kg), PMZ (1mg/kg), PTX+PMZ were administered to the xenograft model by OSC-19. The tumor was resected after those treatments. The expression of CD44v, ALDH3A1, and NRF2 was evaluated by immunohistochemistry (IHC) of FFPE tissues from those extracted tumors. Results: PMZ single-agent inhibited tumor growth in various tumor cell lines, and the antitumor effect was enhanced by concomitant use of PTX. PMZ, which significantly increased ROS of tumor cells, was more effective than PTX in OSC-19, which was a chemoresistant cell line expressing high CD44v. Xenograft model exhibited that tumor growth was suppressed in PTX+PMZ group than in PTX and PMZ single-agent group. IHC staining revealed that PMZ administration restrained CD44v expression, and ALDH3A1 expression markedly decreased in PMZ treated tumors, but not in PTX treated tumors. Conclusions: PMZ dramatically induced ROS production in tumor cells. These data suggest that aldehyde produced by ROS might suppress HNSCC tumor growth. The results from this study indicated that PMZ could be a novel therapeutic reagent for HNSCC in combination with existing anticancer drugs. Citation Format: Shintaro Nakamura, Hiroyuki Ozawa, Juntaro Yamasaki, Shogo Okazaki, Momoko Yoshikawa, Tomoya Soma, Seiji Asoda, Osamu Nagano, Mariko Sekimizu, Shin Saito, Keisuke Yoshihama, Takuya Mikoshiba, Kaoru Ogawa, Hideyuki Saya. Pimozide combined with paclitaxel demonstrates a significant antitumor effect in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3579.

UBE2O Promotes Progression and Epithelial-Mesenchymal Transition in Head and Neck Squamous Cell Carcinoma.

BackgroundUBE2O, as a member of the ubiquitin-conjugating enzyme family, is abnormally expressed and exhibits abnormal functions in human malignancies. However, the function of UBE2O in head and neck squamous cell carcinoma (HNSCC) remains unknown. Therefore, our study aims to investigate the role of UBE2O in HNSCC progression and the underlying mechanisms.MethodsThe expression of UBE2O in HNSCC patients was investigated with data from the Cancer Genome Atlas (TCGA) and from a separate primary tumor cohort. The function of UBE2O in HNSCC cells was studied by cell viability assay, colony formation assay, wound healing assay, and cell migration and invasion chamber assay. The effect of UBE2O on tumor growth in vivo was determined in a subcutaneous xenograft model of HNSCC.ResultsTCGA data showed that UBE2O mRNA expression was dramatically increased in HNSCC tissues and that patients with high expression of UBE2O transcripts had a worse survival prognosis than patients with low expression of UBE2O transcripts. Gain-of-function and loss-of-function analyses revealed that oncogenic UBE2O enhanced the proliferation, migration and invasion of HNSCC cells in vitro. Further, mechanistic analysis revealed that UBE2O induced the epithelial–mesenchymal transition (EMT) phenotype and also potentiated TGF-β1-induced EMT, and thus leading to an enhanced capacity of migration and invasion in HNSCC. Finally, xenograft models showed that UBE2O knockout obviously inhibited the occurrence of EMT, angiogenesis and tumor growth in HNSCC in vivo.ConclusionOur study indicates that UBE2O acts as an oncogene to promote the malignant progression and EMT of HNSCC.

CD244 represents a new therapeutic target in head and neck squamous cell carcinoma

BackgroundDeveloping novel strategies to overcome the immunosuppressive tumor microenvironment is a critically important area of cancer therapy research. Here, we assess the therapeutic potential of CD244 (2B4/signaling lymphocyte activation molecule...

MiR-762 Promotes Malignant Development of Head and Neck Squamous Cell Carcinoma by Targeting PHLPP2 and FOXO4.

BackgroundHead and neck squamous cell carcinoma (HNSCC) is among the most common malignant tumors worldwide. This study, investigated the role of microRNA (miR)-762 in regulating HNSCC progression.Materials and methodsThe expression levels of miR-762 in HNSCC tissues were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Statistical analyses were performed to investigate the association of miR-762 with clinicopathological features in patients with HNSCC. Cell proliferation and migration were examined by cell counting (CCK-8) and IncuCyte assays. Target genes of miR-762 were screened using bioinformatics tools and microarrays, and confirmed using a luciferase activity reporter assay, qRT-PCR and Western blot analysis. Recuse experiments were performed to detect whether target genes mediated the effects of miR-762 on HNSCC cells. The in vivo effects of miR-762 were verified using tumor xenografts.ResultsHNSCC clinical specimens showed high expression levels of miR-762, which positively correlated with tumor-node-metastasis (TNM) stage and poor prognosis of HNSCC. miR-762 overexpression promoted the proliferation and migration of HNSCC cells in vitro. In addition, overexpression of miR-762 upregulated the expression of phosphorylated AKT (p-AKT) and mesenchymal markers (N-cadherin and vimentin), but suppressed epithelial marker (E-cadherin) expression. miR-762 also promoted HNSCC tumor growth in vivo. PH domain and leucine-rich repeat protein phosphatase 2 (PHLPP2) and Forkhead box O4 (FOXO4) were direct target genes of miR-762. HNSCC tissues had low expression levels of PHLPP2 and FOXO4, showing a negative correlation with miR-762 expression. Moreover, silencing of PHLPP2 and FOXO4 mimicked the tumor-promotive effects of miR-762 on HNSCC cells. Notably, overexpression of PHLPP2 and FOXO4 abolished the pro-tumoral function of miR-762 on cell proliferation and migration.ConclusionmiR-762 promotes HNSCC progression by targeting PHLPP2 and FOXO4. Therefore, miR-762 might be a potential diagnostic or therapeutic target for HNSCC.

Targeting of EZH2 inhibits epithelial‑mesenchymal transition in head and neck squamous cell carcinoma via regulating the STAT3/VEGFR2 axis.

Tumor metastasis regulated by epithelial‑mesenchymal transition (EMT) plays a significant role in the development of human cancers, whereas the molecular mechanisms of this process in head and neck squamous cell carcinoma (HNSCC) remain elusive. In this study, we found that inhibition of enhancer of zeste homolog2 (EZH2) resulted in suppressed EMT in HNSCC invitro and invivo. We reported that signal transducer and activator of transcription factor3 (STAT3)/vascular endothelial growth factor receptor2 (VEGFR2) axis served as the downstream signaling of EZH2 and mediated EMT in HNSCC. EZH2 inhibition downregulated the expression of key molecules of the STAT3/VEGFR2 axis and EMT‑related markers, while the expression of E‑cadherin was upregulated in HNSCC cells. Targeting the EZH2/STAT3/VEGFR2 axis significantly reduced motility of HNSCC cells. Furthermore, EZH2 knockdown reduced the growth of xenograft HNSCC tumors via inhibiting the EZH2/STAT3/VEGFR2 axis. In conclusion, we proposed that EZH2 regulates EMT and tumor invasion and metastasis in HNSCC by governing the STAT3/VEGFR2 axis. These findings provide a rationale for developing novel strategies to treat invasive and metastatic HNSCC via targeting the EZH2/STAT3/VEGFR2 pathway.