Head And Neck Cancer Cell Lines Research Articles

PLAU promotes cell proliferation and migration of head and neck cancer via STAT3 signaling pathway

It was reported that within the head and neck cancer (HNC) cell line CAL21 the epithelial-mesenchymal transition (EMT) and cell proliferation were promoted by Urokinase-Type Plasminogen Activator (PLAU) proteinase through TNFRSF12A. Additionally, in this paper HNC cell lines refer to Fadu and Tu686. A novel PLAU-STAT3 axis was found to be involved in HNC cell line proliferation and metastasis. PLAU expression in HNC samples was upregulated, besides, the elevated expression of PLAU was linked to the lower overall survival (OS) and disease-free survival (DFS). Ectopic PLAU expression promoted cell proliferation and migration, while PLAU knockdown exhibited opposite results. RNA-seq data identified the JAK-STAT signaling pathway, confirmed by western blotting. A recovery assay using S3I-201, a selective inhibitor of signal transducer and activator of transcription 3 (STAT3), indicated that PLAU promoted HNC cell line progression via STAT3 signaling in vitro. The oncogenic role of PLAU in HNC tumor growth in vivo was confirmed using xenograft models. In summary, we identified the tumorigenic PLAU function in the HNC progress. PLAU may represent a potential prognostic biomarker of HNC and the PLAU-STAT3 pathway might be considered a therapeutic target of HNC.

IP3R1 dysregulation via mir-200c-3p/SSFA2 axis contributes to taxol resistance in head and neck cancer

Head and neck cancer (HNC) is the sixth most common malignancy worldwide. Although current modalities offer a wide variety of therapy choices, head and neck carcinoma has poor prognosis due to its diagnosis at later stages and development of resistance to current therapeutic tools. In the current study, we aimed at exploring the roles of miR-200c-3p during head and neck carcinogenesis and acquisition of taxol resistance. We analyzed miR-200c-3p levels in HNC clinical samples and cell lines using quantitative real-time polymerase chain reaction and evaluated the effects of differential miR-200c-3p expression on cancer-related cellular phenotypes using in-vitro tools. We identified and characterized a direct target of miR-200c-3p using in-silico tools, luciferase and various in-vitro assays. We investigated potential involvement of miR-200c-3p/SSFA2 axis in taxol resistance in-vitro. We found miR-200c-3p expression as significantly downregulated in both HNC tissues and cells compared to corresponding controls. Ectopic miR-200c-3p expression in HNC cells significantly inhibited cancer-related phenotypes such as viability, clonogenicity, migration, and invasion. We, then, identified SSFA2 as a direct target of miR-200c-3p and demonstrated that overexpression of SSFA2 induced malignant phenotypes in HNC cells. Furthermore, we found reduced miR-200c-3p expression in parallel with overexpression of SSFA2 in taxol resistant HNC cells compared to parental sensitive cells. Both involved in intracellular cytoskeleton remodeling, we found that SSFA2 works collaboratively with IP3R1 to modulate resistance to taxol in HNC cells. When considered collectively, our results showed that miR-200c-3p acts as a tumor suppressor microRNA and targets SSFA2/IP3R1 axis to sensitize HNC cells to taxol.

Abstract 3880: LYTAC targeting galectin-1 to enhance radioimmunotherapy of cancers in the upper aerodigestive tract

Abstract Non-small cell lung cancer (NSCLC) and head and neck cancer (HNC), ranking respectively as a leading cause of cancer-related death and the sixth most common cancer worldwide. Despite treatment advancements, there's a pressing need for more effective strategies to improve survival and quality of life for NSCLC and HNC patients. Carolyn Bertozzi group recently reported in Nature the groundbreaking protein degraders named lysosome-targeted chimeras (LYTACs), which have bispecific binding affinity that drives cell-surface endocytic receptors to drag membrane or extracellular oncogenic proteins to lysosomes for degradation. They have successfully targeted critical oncoproteins such as epidermal growth factor receptor (EGFR) and more. However, current degradation demonstration was limited to in vitro cancer cells, LYTACs' impact on immune cell modulation and their therapeutic potentials in preclinical animal models remain unknown and require extensive evaluation before venturing to clinical trials.Our group and others have identified galectin-1 (Gal-1) as a multifaceted immunosuppressive biomarker overexpressed in NSCLC and HNC. By interacting with glycosylated receptors (e.g. CD45) on immune cells, it results in apoptosis of cytotoxic T cells, expansion of regulatory T cells, and intratumoral recruitment of myeloid-derived suppressor cells. Despite promising as an immune checkpoint target, effective anti-Gal-1 therapies are currently absent in clinical trials.Through histopathological analyses, we have confirmed substantial LYTAC receptor (cation-independent mannose-6-phosphate receptor, CI-M6PR) expression in ~200 HNC and ~400 NSCLC patient samples. Collaborating with Bertozzi group, we have developed the first Gal-1 targeting LYTAC named G-M6Pn which triggers significant target protein degradation in various human HNC and NSCLC cell lines. Such efficient Gal-1 degradation leads to significant immunosuppression reversal by stimulating CD8+ T cell and natural killer (NK) cell activation while dampening regulatory T cell (Treg) activity in both cell experiments and humanized mouse tumor models, resulting in excellent tumor/metastases control and survival improvement on both NSCLC and HNC models. When synergized with stereotactic ablative radiotherapy, the antitumor immune responses are further amplified and G-M6Pn also mediates ‘abscopal effect’ inhibiting non-irradiated tumors. This study thus contributes to expediting clinical translation of LYTACs as novel immune checkpoint degraders to improve personalized radio-immunotherapy of cancer. Citation Format: Yuyan Jiang, Green Ahn, Mobeen Rahman, Hongbin Cao, Carolyn Bertozzi, Qunh Le. LYTAC targeting galectin-1 to enhance radioimmunotherapy of cancers in the upper aerodigestive tract [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3880.

New Propargyloxy Derivatives of Galangin, Kaempferol and Fisetin-Synthesis, Spectroscopic Analysis and In Vitro Anticancer Activity on Head and Neck Cancer Cells.

Head and neck cancer (HNC) therapy is limited; therefore, new solutions are increasingly being sought among flavonoids, which exhibit numerous biological properties, including potential anticancer activity. However, because they are mostly insoluble in water, are unstable and have low bioavailability, they are subjected to chemical modification to obtain new derivatives with better properties. This study aimed to synthesize and analyze new propargyloxy derivatives of galangin, kaempferol and fisetin, and to evaluate their anticancer activity against selected HNC cell lines. The obtained derivatives were assessed by spectroscopic analysis; next, their anticancer activity was evaluated using a flow cytometer and real-time cell analysis. The results showed that only the fisetin derivative was suitable for further analysis, due to the lack of crystal formation of the compound. The fisetin derivative statistically significantly increases the number of cells in the G2/M phase (p < 0.05) and increases cyclin B1 levels. A statistically significant increase in the number of apoptotic cells after being exposed to the tested compound was also observed (p < 0.05). The data indicate that the obtained fisetin derivative exhibits anticancer activity by affecting the cell cycle and increasing apoptosis in selected HNC lines, which suggests its potential use as a new medicinal agent in the future.

Plasma-derived small extracellular vesicles unleash the angiogenic potential in head and neck cancer patients

BackgroundIn Head and neck cancer (HNC) angiogenesis is essential for tumor progression and metastasis. Small extracellular vesicles (sEVs) from HNC cell lines alter endothelial cell (EC) functions towards a pro-angiogenic phenotype. However, the role of plasma sEVs retrieved from HNC patients in this process is not clear so far.MethodsPlasma sEVs were isolated on size exclusion chromatography columns from 32 HNC patients (early-stage UICC I/II: 8, advanced-stage UICC III/IV: 24), 12 patients with no evident disease after therapy (NED) and 16 healthy donors (HD). Briefly, sEVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), BCA protein assays and Western blots. Levels of angiogenesis-associated proteins were determined using antibody arrays. The interaction of fluorescently-labeled sEVs with human umbilical vein ECs was visualized by confocal microscopy. The functional effect of sEVs on tubulogenesis, migration, proliferation and apoptosis of ECs was assessed.ResultsThe internalization of sEVs by ECs was visualized using confocal microscopy. Based on antibody arrays, all plasma sEVs were enriched in anti-angiogenic proteins. HNC sEVs contained more pro-angiogenic MMP-9 and anti-angiogenic proteins (Serpin F1) than HD sEVs. Interestingly, a strong inhibition of EC function was observed for sEVs from early-stage HNC, NED and HD. In contrast, sEVs from advanced-stage HNC showed a significantly increased tubulogenesis, migration and proliferation and induced less apoptosis in ECs than sEVs from HD.ConclusionsIn general, plasma sEVs carry a predominantly anti-angiogenic protein cargo and suppress the angiogenic properties of ECs, while sEVs from (advanced-stage) HNC patients induce angiogenesis compared to HD sEVs. Thus, tumor-derived sEVs within the plasma of HNC patients might shift the angiogenic switch towards angiogenesis.

Abstract 2906: Memory-like differentiation, tumor targeting monoclonal antibodies, and chimeric antigen receptors enhance natural killer cell responses to head and neck cancer

Abstract Background: Head and neck cancer (HNC) is an aggressive tumor with low response rates to frontline PD-1 blockade. Natural killer (NK) cells are a promising cellular therapy for T cell-refractory cancers, but are frequently dysfunctional in patients with HNC. New strategies are needed to enhance NK cell responses against HNC. Activation of NK cells with IL-12, 15 and 18 results in memory-like (ML) differentiation improving NK cell responses in hematological malignancies. We hypothesized that ML NK cell differentiation, tumor targeting with cetuximab, and engineering with α-ephA2 chimeric antigen receptor (CAR) would enhance NK cell responses against HNC. Methods: ML NK and conventional (c) NK cells were generated from healthy donors (HD) and evaluated for their ability to produce IFNγ, TNF, degranulate (CD107a) and kill HNC cell lines and primary HNC cells alone or in combination with cetuximab in vitro and in xenograft models. To identify activating receptors involved in NK cell recognition of HNC, blocking experiments were used. ML and cNK cells were engineered to express α-EphA2 CAR-CD8A-41BB-CD3z and functional responses were assessed in vitro against HNC cell lines and primary HNC targets. Results: ML NK cells generated from HD displayed enhanced IFNγ response compared to cNK cells against HNC cell lines SCC1(11±2% vs. 24±1%, mean ± SEM p&amp;lt;0.001), SCC47 (13±2% vs. 36±2%, p&amp;lt;0.01) and primary HNC targets (17±2% vs. 32±3%, p&amp;lt;0.01) which were further enhanced by cetuximab, SCC1 (cNK + cetuximab vs. ML + cetuximab 16±2% vs. 28±3%, p&amp;lt;0.001), SCC47 (39±3% vs. 56±2%, p&amp;lt;0.001), primary HNC targets (23±2% vs. 40±2%, p&amp;lt;0.0001). The combination of ML NK cells and cetuximab was superior to cNK and cetuximab in 51 Cr release assay and IncuCyte® killing assays for HNC cell lines (p&amp;lt;0.01) and primary cells (p&amp;lt;0.01). Findings were consistent in SCC1 HNC xenograft model, where tumor bearing NSG mice treated with cetuximab and ML NK cells had significantly lower tumor burden at 21 days compared to untreated mice, mice that received cNK cells + cetuximab or cetuximab only (p&amp;lt;0.001). Mechanistically, NK cell IFNγ, CD107a and cytotoxicity against HNC targets is partially dependent on activating receptors NKG2D, CD2 and DNAM-1. ML NK cells expressing α-ephA2 CAR were specific to ephA2+ cell lines and demonstrated increased IFNγ (37±4% vs. 16±3%, p&amp;lt;0.001) and CD107a (36±4% vs. 20±1%, p&amp;lt;0.001) against NK resistant ephA2+ UD-SCC2, compared to CAR negative cells. EphA2 CAR ML NK cells had increased CD107a (26±4% vs. 39±4%, p&amp;lt;0.01) and IFNγ (45±5% vs. 66±3%, p&amp;lt;0.001) against primary ephA2+ HNC targets. Conclusion: These pre-clinical findings show that ML differentiation alone or in concert with cetuximab directed targeting or ephA2 CAR engineering, was effective against HNC and provide the rationale for investigation in early phase clinical trials for HNC patients. Citation Format: Miriam T. Jacobs, Pamela Wong, Alice Zhou, Michelle Becker-Hapak, Nancy Marin, Lynne Marsala, Mark Foster, Jennifer Foltz, Celia Cubitt, Jennifer Tran, Carly Neal, David Russler-Germain, Lily Chang, Timothy Schappe, Ethan McClain, Samantha Kersting-Schadek, Carl DeSelm, Melissa Berrien-Elliott, Sidarth V. Puram, Todd A. Fehniger. Memory-like differentiation, tumor targeting monoclonal antibodies, and chimeric antigen receptors enhance natural killer cell responses to head and neck cancer [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 2906.

Abstract 3501: A genome-wide screen for determinants of radioresistance in head and neck cancer

Abstract Introduction: Head and neck cancer (HNC) is the seventh most common cancer worldwide with over 1 million new cases diagnosed each year. Radioresistance is a major cause of both treatment failure and poor prognosis, with locoregional recurrence after radiotherapy occurring in up to 50% of HNC patients. Despite the discovery of individual genes implicated in the ability of HNC cells to develop radioresistance, a systematic evaluation remains to be undertaken. This project is the first to conduct genome-wide CRISPR screens for regulators of radioresistance in HNC, with the aim to identify phenotype-driving genes and pathways. Experimental Procedures: Genome-wide negative selection screens were conducted on HNC cell lines using the Toronto KnockOut (TKO) CRISPR library. Monoclonal Cas9 cell lines were derived from UT-SCC-42A (42A), FaDu, and PE/CA-PJ41 (clone D2) with Cas9 editing efficiencies &amp;gt;80%. Library-transduced cells were treated with the minimum dose of radiation resulting in cessation of cell growth (10 Gy for 42A and FaDu; 8 Gy for PE/CA-PJ41). Genomic DNA from the resultant radioresistant populations was extracted and sequenced on the Illumina NextSeq 500. MAGeCK analysis of read counts identified gene targets of significantly depleted gRNAs after irradiation. Genes that correlated with overall survival (OS) in the TCGA Pan-Cancer database were selected for further investigation. The top candidate genes in the determination of radioresponse were validated using cell proliferation and clonogenic assays. Effects of loss-of-function on cell migration were assessed using scratch wound and transwell migration assays. Results: 117 putative radioresistance genes were identified in the 42A cell line screen; the top ranked hits were MMP14, CD44, CALR, and HHLA1. RNA expression levels of these genes had a significant correlation with OS of radiation-treated HNC patients in the TCGA Pan-Cancer Atlas. Loss-of-function of the candidate genes was confirmed to increase radiosensitivity through live cell imaging in 42A and PJ41 cells, which were further corroborated with clonogenic assays. Downregulation of at least one gene impaired the migration of 42A and PJ41 cells, suggesting a role in cellular invasion and migration. Conclusion: This study will contribute to a deeper understanding of mechanisms of radioresistance in HNC, which continues to be a leading cause of mortality in HNC patients. Future pathway elucidation through transcriptome analysis and functional characterization may reveal additional therapeutic targets that can improve the outcome for HNC patients treated with radiation. Citation Format: Jacqueline H. Law, Pierre-Antoine Bissey, Isabella Kojundzic, Kenneth W. Yip, Fei-Fei Liu. A genome-wide screen for determinants of radioresistance in head and neck cancer [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 3501.

HPV16 E6 induces chromosomal instability due to polar chromosomes caused by E6AP-dependent degradation of the mitotic kinesin CENP-E

Chromosome segregation during mitosis is highly regulated to ensure production of genetically identical progeny. Recurrent mitotic errors cause chromosomal instability (CIN), a hallmark of tumors. The E6 and E7 oncoproteins of high-risk human papillomavirus (HPV), which causes cervical, anal, and head and neck cancers (HNC), cause mitotic defects consistent with CIN in models of anogenital cancers, but this has not been studied in the context of HNC. Here, we show that HPV16 induces a specific type of CIN in patient HNC tumors, patient-derived xenografts, and cell lines, which is due to defects in chromosome congression. These defects are specifically induced by the HPV16 oncogene E6 rather than E7. We show that HPV16 E6 expression causes degradation of the mitotic kinesin CENP-E, whose depletion produces chromosomes that are chronically misaligned near spindle poles (polar chromosomes) and fail to congress. Though the canonical oncogenic role of E6 is the degradation of the tumor suppressor p53, CENP-E degradation and polar chromosomes occur independently of p53. Instead, E6 directs CENP-E degradation in a proteasome-dependent manner via the E6-associated ubiquitin protein ligase E6AP/UBE3A. This study reveals a mechanism by which HPV induces CIN, which may impact HPV-mediated tumor initiation, progression, and therapeutic response.

Promotion of ferroptosis in head and neck cancer with divalent metal transporter 1 inhibition or salinomycin

Divalent metal transporter 1 (DMT1) inhibitors can selectively kill iron-addicted cancer stem cells by causing lysosomal iron overload, but their role in head and neck cancer (HNC) is unknown. We examined the role of DMT1 inhibition or salinomycin in promoting ferroptosis by lysosomal iron targeting in HNC cells. RNA interference was performed by transfection of siRNA targeting DMT1 or scrambled control siRNA in HNC cell lines. Cell death and viability, lipid peroxidation, iron contents, and molecular expression were compared between the DMT1 silencing or salinomycin group and the control. DMT1 silencing markedly accelerated cell death induced by the ferroptosis inducers. DMT1 silencing marked increases in the labile iron pool, intracellular ferrous and total iron contents, and lipid peroxidation. DMT1 silencing revealed molecular changes in iron starvation response, resulting in increases in TFRC, and decreases in FTH1. Salinomycin treatment also showed similar results to the above DMT1 silencing. DMT1 silencing or salinomycin can promote ferroptosis in HNC cells, suggesting a novel strategy for killing iron-avid cancer cells.

Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer.

Head and neck cancer (HNC) is mainly treated by surgery, radiotherapy, and adjuvant chemotherapy; however, the prognosis of some patients with HNC is poor because of radiotherapy and chemotherapy resistance. In recent years, anti‑PD‑1 monoclonal antibodies have shown certain efficacy, and a change of the tumor immune microenvironment is the main reason for the failure of HNC immunotherapy. The present study aimed to identify and verify that CD38, which is closely related to the prognosis of HNC, is a potential biological marker of radiotherapy and chemotherapy resistance and PD-L1 immunotherapy resistance via a comprehensive bioinformatic analysis in The Cancer Genome Atlas and Gene Expression Omnibus databases. According to the UALCAN database, the transcript level of CD38 in HNC was analyzed using cluster analysis, and the expression of CD38 mRNA in HNC was detected using the Oncomine database. The characteristics of CD38-related oncogenes were identified by gene cluster enrichment analysis in LinkedOmics. The R2 and SEER databases were used to further evaluate the prognostic significance of the CD38 gene in HNC using receiver operating characteristic curve analysis of Kaplan-Meier (KM) survival and the clinical characteristics of the subjects. The protein-protein interaction network of the top 50 genes showing significant positive correlations with CD38 in HNC was analyzed using STRING. Finally, we used a nasopharyngeal carcinoma (NPC) cell line to verify the biological function. The results showed that the levels of CD38 mRNA expression in patients with HNC were significantly higher than those in healthy controls. The levels of CD38 mRNA expression in patients with HNC of different ages, sexes, and races were significantly higher than those in the healthy controls. CD38 is an independent prognostic factor for HNC, and high expression of CD38 indicates poor prognosis. CD38 expression correlated positively with the markers of many kinds of immune cells, and correlated significantly with the expression of PD-L1. We found that the high expression of CD38 suggested a poor prognosis in the subgroup of tumors treated with chemotherapeutic drugs in the G1/S phase. We used HNC cell lines to verify that the high expression of CD38 promoted the proliferation of NPC cells and produced radiotherapy tolerance. Through comprehensive bioinformatics analysis, we suggested that CD38 is a key gene involved in radiotherapy, chemotherapy, and immune drug resistance in HNC. This study provides a reliable biomarker to predict the prognosis of patients with HNC and a reference for clinical comprehensive treatment of HNC. Individualization combined with CD38 monoclonal antibodies might provide a promising treatment strategy for this fatal disease, and this comprehensive treatment might reduce the damage to normal tissue and improve the prognosis and quality of life of patients with HNC.

HPV16 E6 Induces Chromosomal Instability due to Misaligned Chromosomes Caused by E6AP-Dependent Degradation of the Mitotic Kinesin CENP-E

<h3>Purpose/Objective(s)</h3> Chromosome missegregation over multiple cell cycles, termed chromosomal instability (CIN), is common in tumors and often leads to aneuploidy, a hallmark of cancer. High risk human papillomavirus (HPV) causes anogenital and head and neck cancers (HNC). HPV has been shown to cause certain types of CIN, but the mechanisms are unclear and this has not been studied in HNC. Here we describe a specific, novel mechanism of CIN induced by the HPV16 oncoprotein E6, in a physiologically relevant HNC model and in patient tumors. <h3>Materials/Methods</h3> Normal oral keratinocytes (NOKs) were transduced with retrovirus expressing LXSN control vector, HPV16 E6, E7 or E6+E7 and were kept under selection to achieve stable cell lines. CIN was quantified by assessing mitotic defects using immunofluorescence microscopy or H&E-stained slides in the NOKs, HNC cell lines, patient-derived xenografts (PDX), and in patient HNC tumors. Quantitative immunofluorescence and western blotting were used to quantify CENP-E levels. Tetracycline (tet) inducible GFP-CENP-E was introduced into NOKs expressing the Tet repressor using the PiggyBac transposon system. p53 and E6AP null NOKs were created using CRISPR. Significant differences were determined using a two-tailed student's t-test. <h3>Results</h3> HPV+ HNC cell lines have a significant increase specifically in misaligned chromosomes during mitosis, as compared to HPV- cell lines (20% v. 5% of cells, n=4 cell lines each). Using NOKs expressing individual HPV oncogenes, we found that these defects are specifically induced by the HPV oncogene E6 rather than E7. Further studies revealed that HPV16 E6 causes the specific degradation of CENP-E, a mitotic kinesin that plays a critical role in chromosome congression during mitosis. Tetracycline induced over-expression of GFP-CENP-E in LXSN- and E6-expressing NOKs revealed appropriate localization to kinetochores in LXSN cells. Strikingly, E6 mediated degradation of CENP-E was so efficient that CENP-E levels (total and at kinetochores) did not increase even after tet-inducible expression. Degradation of CENP-E occurred post-translationally in a proteasome dependent manner. Proteasome inhibition in E6-expressing NOKs rescued CENP-E protein levels at the kinetochore and rescued the misaligned chromosome phenotype. Furthermore, E6-mediated CENP-E degradation required the ubiquitin ligase E6AP, as evidenced by CENP-E protein restoration and rescue of chromosome alignment in E6AP null NOKs expressing E6. Finally, a significantly increased incidence of misaligned chromosomes was confirmed in HPV+ tumors from HNC patients and HPV+ PDXs. <h3>Conclusion</h3> This study reveals a novel, specific mechanism by which HPV16 E6 induces CIN in HNC, which may play a role in HPV-induced tumor initiation, progression and radiation response. Additionally, there is exciting potential for synergy with a CENP-E inhibitor, currently in clinical trials, and radiation to increase CIN beyond a viable threshold to increase cell death.

A Quadratic Phenotypic Optimization Drug Screening Platform (QPOP) Identified Chromatin Modification as a Potential Strategy to Target Radioresistant (RR) Head and Neck Cancers (HNC)

<h3>Purpose/Objective(s)</h3> Radiotherapy (RT) is a primary treatment modality for most HNC. However, locoregional recurrence post-RT remains a pertinent clinical problem, given that salvage therapeutic options are often limited and ineffective. Resistance to RT is thus an important unmet clinical need in HNC, but therapeutic strategies against radioresistance remain suboptimal, partly owing to the divergent tumor and microenvironmental factors underpinning radioresistance. We had previously developed a high-throughput drug screening method – QPOP that was applied successfully in drug-resistant multiple myeloma (Rashid M. et al., 2018). Here, we investigated the utility of QPOP for the discovery of novel drug combinations in RR-HNC models, and hypothesize that QPOP can accurately identify optimal drug combinations that resensitize RR cells. <h3>Materials/Methods</h3> <i>In vitro</i> isogenic models of RR-HNC were generated by exposing two wild-type (WT) HNC cell lines, FaDu and HK-1, to 90 Gy of 2 Gy irradiation (IR) (45 fr x 2 Gy/fr), and validated using clonogenic assays. QPOP is a complex system analytics platform that rapidly and efficiently ranks and compares all possible system-specific drug combinations from a large drug search set. Pre-experimental selection of drug candidates consisted of standard chemotherapy (cisplatin, carboplatin, and docetaxel), as well as small molecule inhibitors and targeted therapies specific to HNC. Validation of top and lowest QPOP-ranked drug combinations include synergy scoring (Bliss model), combination index theorem of Chou-Talalay, and apoptotic response (Annexin V assay). Drug targeting was validated by western blot analyses, and the Comet assay was used to quantify DNA damage. <h3>Results</h3> A preliminary QPOP-based combinatorial drug screen identified distinct drug sensitivities between WT- and RR-FaDu cells. Validation of the top 5 QPOP-ranked drug combinations revealed that co-treatment of panobinostat (HDAC inhibitor, Pano) and AZD7762 (CHK1 inhibitor) exhibited the most distinct and contrasting response between WT and RR cells. Response surface mapping highlighted Pano and AZD7762 synergy in RR-FaDu, further supported by Bliss synergy scoring that showed synergistic growth inhibition in RR (20.1, synergistic), but not in WT (8.3, additive) cells. These results were corroborated by H3K27 hyperacetylation, and increased downstream caspase-dependent apoptosis and DNA damage in RR-FaDu following combinatorial treatment, compared with single drug treatments. Next, we interrogated if Pano can re-sensitize RR-FaDu and RR-HK-1 to IR. Interestingly, exposure to Pano before 4 Gy IR led to radiosensitization of RR-HNC cells (radiosensitization ratio: FaDu=1.77 [<i>P</i>=0.06]; HK-1=1.81 [<i>P</i>=0.041]), but not WT-HNC cells (FaDu=0.90 [<i>P</i>=0.60]; HK-1=1.26 [<i>P</i>=0.29]). <h3>Conclusion</h3> Herein, QPOP identified the potential of chromatin modification using Pano, in combination with CHK1 blockade or re-IR for targeting RR-HNC.

Mitochondrial dysfunction and epithelial to mesenchymal transition in head neck cancer cell lines

Mitochondrial dysfunction promotes cancer aggressiveness, metastasis, and resistance to therapy. Similar traits are associated with epithelial mesenchymal transition (EMT). We questioned whether mitochondrial dysfunction induces EMT in head and neck cancer (HNC) cell lines. We induced mitochondrial dysfunction in four HNC cell lines with carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial electron transport chain uncoupling agent, and oligomycin, a mitochondrial ATP synthase inhibitor. Extracellular flux analyses and expression of the cystine/glutamate antiporter system xc (xCT) served to confirm mitochondrial dysfunction. Expression of the EMT-related transcription factor SNAI2, the mesenchymal marker vimentin and vimentin/cytokeratin double positivity served to detect EMT. In addition, holotomographic microscopy was used to search for morphological features of EMT. Extracellular flux analysis and xCT expression confirmed that FCCP/oligomycin induced mitochondrial dysfunction in all cell lines. Across the four cell lines, mitochondrial dysfunction resulted in an increase in relative SNAI2 expression from 8.5 ± 0.8 to 12.0 ± 1.1 (mean ± SEM; p = 0.007). This effect was predominantly caused by the CAL 27 cell line (increase from 2.2 ± 0.4 to 5.5 ± 1.0; p < 0.001). Similarly, only in CAL 27 cells vimentin expression increased from 2.2 ± 0.5 × 10–3 to 33.2 ± 10.2 × 10–3 (p = 0.002) and vimentin/cytokeratin double positive cells increased from 34.7 ± 5.1 to 67.5 ± 9.8% (p = 0.003), while the other 3 cell lines did not respond with EMT (all p > 0.1). Across all cell lines, FCCP/oligomycin had no effect on EMT characteristics in holotomographic microscopy. Mitochondrial dysfunction induced EMT in 1 of 4 HNC cell lines. Given the heterogeneity of HNC, mitochondrial dysfunction may be sporadically induced by EMT, but EMT does not explain the tumor promoting effects of mitochondrial dysfunction in general.

Abstract 1403: ROS metabolism and autophagy as potential targets to improve head and neck cancer treatment

Abstract Introduction: Cancer cells exhibit a high degree of plasticity to adapt the rapidly changing tumor microenvironment (TME), in part by altering cellular metabolic pathways at a faster and more efficient pace than normal cells. Cancer cell plasticity, with genetic and epigenetic alterations, promote the diversity of cancer cells contributing to heterogeneity within the tumor. Head and neck cancer (HNC) is a notable example of this diversity; HNC tumors harbor cells with a wide genetic and metabolic heterogeneity, including cancer stem cells (CSC). Despite current treatment approaches, more than 40% of HNC patients have disease recurrence. Thus, we developed a multimodal approach to effectively prevent the development of radio/drug-resistance. Experimental Procedures: Nuclear-localized mkate2 expressing HNC cell numbers detected for every 6 hours by using IncuCyte S3 instrument. Apoptotic cells detected via NucView 488 Caspase-3 substrate and ROS levels determined using CellROX green simultaneously with cell numbers in the IncuCyte S3 instrument. Synergy scores calculated according to viability in SynergyFinder 2.0 web-application. Results: Our preliminary data profiling of metabolic regulators as radiosensitizer indicate that auranofin (AUR, thioredoxin reductase inhibitor) and buthionine sulfoximine (BSO, γ-glutamylcysteine synthetase inhibitor), given in combination with radiation (RT+AUR+BSO): 1) significantly inhibit proliferation in HNC cell lines, 2) increase ROS accumulation, and 3) reduce the surviving fraction of cells in colony forming assays compared to individual/dual agent applications. Furthermore, we added an autophagy inhibitor, SAR405, in our treatment regimen because of the crosstalk between ROS levels and autophagy. Human (FaDu and UM-SCC-47) and murine (MOC1 and MOC2) HNC cell were treated with +/-AUR+/-BSO+/-SAR405 and high-throughput combinatorial screening of cell numbers obtained over time. The combination of AUR+BSO+SAR405 registered a higher synergy score than the double combinations. Furthermore, as compared to single or double drug applications, the AUR+BSO+SAR405 group had a greater apoptotic cell rate and ROS levels. Conclusion: We present here a high-throughput method to screen multi-drug synergy. Multi-modal approach is important to prevent recurrence by providing a broader effect on heterogenous cancer cells in tumor. Moreover, multi-drug combinations allow usage of lower doses of drugs effectively, hence lowers the treatment-related adverse effects. This approach indicated the possibility of HNC treatment by increasing the ROS levels beyond the containable threshold which is consistent with the recent findings about the alterations in redox metabolism, particularly upregulation in thioredoxin and glutathione pathways, in many cancer types. In vivo experiments are ongoing to evaluate the potential of this combination for clinical studies. Citation Format: Zafer Gurel, Qianyun Luo, Michael S. Luy, Randall J. Kimple. ROS metabolism and autophagy as potential targets to improve head and neck cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1403.

Impact of Secretion-Active Osteoblast-Specific Factor 2 in Promoting Progression and Metastasis of Head and Neck Cancer.

Simple SummaryHead and neck cancers (HNC) exhibit poor survival due to metastases. Our study identified osteoblast-specific factor 2 (OSF-2) as overexpressed in primary tumors, lymph node metastases, and the tumor microenvironment. High OSF-2 levels correlate with metastatic disease and reduced survival of HPV-negative HNC patients. OSF-2’s active secretion signal seems to promote metastases by supporting the tumor microenvironment via the ß1 integrin-induced PI3K and Akt/PKB signaling pathway. We suggest OSF-2 as a potential biomarker and drug target to control (HPV-negative) HNC metastasis and disease management.Treatment success of head and neck cancer (HNC) is still hampered by tumor relapse due to metastases. Our study aimed to identify biomarkers by exploiting transcriptomics profiles of patient-matched metastases, primary tumors, and normal tissue mucosa as well as the TCGA HNC cohort data sets. Analyses identified osteoblast-specific factor 2 (OSF-2) as significantly overexpressed in lymph node metastases and primary tumors compared to normal tissue. High OSF-2 levels correlate with metastatic disease and reduced overall survival of predominantly HPV-negative HNC patients. No significant correlation was observed with tumor localization or therapy response. These findings were supported by the fact that OSF-2 expression was not elevated in cisplatin-resistant HNC cell lines. OSF-2 was strongly expressed in tumor-associated fibroblasts, suggesting a tumor microenvironment-promoting function. Molecular cloning and expression studies of OSF-2 variants from patients identified an evolutionary conserved bona fide protein secretion signal (1MIPFLPMFSLLLLLIVNPINA21). OSF-2 enhanced cell migration and cellular survival under stress conditions, which could be mimicked by the extracellular administration of recombinant protein. Here, OSF-2 executes its functions via ß1 integrin, resulting in the phosphorylation of PI3K and activation of the Akt/PKB signaling pathway. Collectively, we suggest OSF-2 as a potential prognostic biomarker and drug target, promoting metastases by supporting the tumor microenvironment and lymph node metastases survival rather than by enhancing primary tumor proliferation or therapy resistance.

MicroRNA-21 induces cisplatin resistance in head and neck squamous cell carcinoma.

Drug resistance, either intrinsic or acquired, can impair treatment effects and result in increased cell motility and death. MicroRNA-21 (miR-21), a proto-oncogene, may facilitate the development or maintenance of drug resistance in cancer cells. Restoring drug sensitivity can improve therapeutic strategies, a possibility that requires functional evaluation and mechanistic exploration. For miR-21 detection, matched tissue samples from 30 head and neck squamous cell carcinoma (HNSCC) patients and 8 head and neck cancer (HNC) cell lines were obtained. Reverse transcription-PCR to detect expression, MTT and clonogenic assays to evaluate cell proliferation, apoptosis assays, resazurin cell viability assays, western blot and luciferase reporter assays to detect protein expression, and flow cytometry to analyse the cell cycle were adopted. Compared to the corresponding normal control (NC) tissues, 25 cancer tissues had miR-21 upregulation among the 30 matched pair tissues (25/30, 83.8%); furthermore, among the 8 HNC cell lines, miR-21 expression that was notably upregulated in three: UPCI-4B, UMSCC-1, and UPCI-15B. In both the UMSCC-1 and UPCI-4B cell lines, the miR-21 mimic enhanced cell proliferation with reduced apoptosis and increased viability, whereas the miR-21 inhibitor resulted in the opposite effects (all P<0.001); additionally, miR-21 directly targeted the tumour suppressor phosphatase and tensin homologue (PTEN) and inhibited PTEN expression. Furthermore, the miR-21 mimic induced cisplatin resistance, while the miR-21 inhibitor restored cisplatin sensitivity. Overexpression of miR-21 can enhance cell proliferation, reduce apoptosis, and induce drug resistance by inhibiting PTEN expression. Targeting miR-21 may facilitate cancer diagnosis, restore drug sensitivity, and improve therapeutic effects.

Poly(rC)-binding protein 1 represses ferritinophagy-mediated ferroptosis in head and neck cancer

A cytosolic iron chaperone poly(rC)-binding protein 1 (PCBP1) is a multifunctional RNA-binding protein involving gene transcription, RNA regulation, and iron loading to ferritins. PCBP1 is also known to repress autophagy, but the role of PCBP1 in ferritinophagy and ferroptosis remains unrevealed. Therefore, we examined the role of PCBP1 in ferritinophagy-mediated ferroptosis in head and neck cancer (HNC) cells. The effects of system xc– cystine/glutamate antiporter (xCT) inhibitors and PCBP1 gene silencing/overexpression were tested on HNC cell lines and mouse tumor xenograft models. These effects were analyzed by assessing cell viability and death, lipid reactive oxygen species and iron production, lipid, malondialdehyde, mRNA/protein expression, and autophagy flux assays. Interaction between PCBP1 and BECN1 mRNA was also examined by luciferase and RNA-protein pull-down assays. PCBP1 gene silencing increased autophagosome generation and autophagic flux. Conversely, PCBP1 upregulation inhibited autophagy activation via direct binding to the CU-rich elements on the 3′-untranslated region (3′-UTR) of BECN1 mRNA. The internal deletion or mutation of the 3′-UTR F2 region recovered BECN1 mRNA stability repressed by PCBP1, resulting in enhanced ferritinophagy-mediated ferroptosis. Besides, PCBP1 knockdown promoted polyunsaturated fatty acid peroxidation by increasing ALOX15 expression. Further, excess iron accumulation caused mitochondrial dysfunction in PCBP1-suppressed cells. A ferroptosis inducer sulfasalazine significantly suppressed tumor growth in mice with the transplantation of PCBP1-silenced HNC. Our data suggest that the dual functions of PCBP1 repressing BECN1 and ALOX15 mRNAs contribute to attenuating cancer susceptibility to ferroptosis inducers.

Radiation dose enhancement using gold nanoparticles with a diamond linear accelerator target: a multiple cell type analysis

Radiotherapy (RT) is an effective cancer treatment modality, but standard RT often causes collateral damage to nearby healthy tissues. To increase therapeutic ratio, radiosensitization via gold nanoparticles (GNPs) has been shown to be effective. One challenge is that megavoltage beams generated by clinical linear accelerators are poor initiators of the photoelectric effect. Previous computer models predicted that a diamond target beam (DTB) will yield 400% more low-energy photons, increasing the probability of interacting with GNPs to enhance the radiation dose by 7.7-fold in the GNP vicinity. After testing DTB radiation coupled with GNPs in multiple cell types, we demonstrate decreased head-and-neck cancer (HNC) cell viability in vitro and enhanced cell-killing in zebrafish xenografts compared to standard RT. HNC cell lines also displayed increased double-stranded DNA breaks with DTB irradiation in the presence of GNPs. This study presents preclinical responses to GNP-enhanced radiotherapy with the novel DTB, providing the first functional data to support the theoretical evidence for radiosensitization via GNPs in this context, and highlighting the potential of this approach to optimize the efficacy of RT in anatomically difficult-to-treat tumors.

DNA Double-strand Break Signaling Is a Therapeutic Target in Head and Neck Cancer.

Head and neck cancer (HNC) is common worldwide. Given poor outcomes for patients with HNC, research into targeted therapies is needed. Ataxia telangiectasia mutated (ATM) is a DNA damage kinase which is activated by double-strand DNA breaks. We tested the effects of a novel ATM inhibitor on HNC cell lines and xenografts. p53-Binding protein 1 and phosphorylated ATM were localized in cultured cells by immunofluorescence microscopy. Protein expression was determined by western blot. Tumor xenografts were established by injecting HNC lines into immunocompromised mice. Tumor sections were characterized by immunohistochemistry. Apoptotic cells were determined by terminal transferase-mediated dUTP nick-end labeling assay. ATM inhibition increased double-strand DNA breaks at replication foci in HNC cell lines. ATM inhibition affected cell-cycle regulatory protein expression, blocked cell-cycle progression at the G2/M phase and resulted in apoptosis. ATM inhibition may be therapeutically useful in treating HNC.

Systematic Analysis and Identification of Dysregulated Panel lncRNAs Contributing to Poor Prognosis in Head-Neck Cancer.

Head and neck cancer (HNC) is one of the most prevalent cancers worldwide, accounting for approximately 5% of all cancers. While the underlying molecules and their pathogenetic mechanisms in HNC have yet to be well elucidated, recent studies have shown that dysregulation of lncRNAs may disrupt the homeostasis of various biological pathways. However, the understanding of lncRNAs in HNC is still limited by the lack of expression profiling. In the present study, we employed a systematic strategy to identify a panel of lncRNA associated with HNC. A cancer-related lncRNA profile PCR array was screened to explore potential molecules specific for HNC. A total of 55 lncRNAs were found to be dysregulated in HNC cells when compared to normal keratinocytes. Further analysis of the prognostic significance using The Cancer Genome Atlas (TCGA) database revealed 15 lncRNAs highly correlated with overall survival in HNC patients. Additionally, clinical sample expression analysis of the TCGA-HNSC cohort revealed 16 highly dysregulated lncRNAs in HNC, resulting in a combined 31-lncRNA signature panel that could predict prognosis. Validation of these molecules confirmed the considerable level of altered expressions in HNC cells, with XIST, HOXA11-AS, TSIX, MALAT1, WT1-AS, and IPW being the most prominently dysregulated. We further selected a molecule from our panel (XIST) to confirm the validity of these lncRNAs in the regulation of cancer aggressiveness. Gene ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses demonstrated that XIST participated in various cancer-related functions, including cell proliferation and metastasis. XIST silencing with the RNAi technique substantially reduced invasion and migration in several HNC cell lines. Thus, our study defined a 31-lncRNA panel as prognostic signatures in HNC. These perspective results provide a knowledge foundation for further application of these molecules in precision medicine.