Head And Neck Squamous Cell Carcinoma Xenografts Research Articles

Radiosensitization approaches for HPV-positive and HPV-negative head and neck squamous carcinomas.

Radiotherapy is one of the most used treatment approaches for head and neck squamous cell carcinoma (HNSCC). Targeted inhibition of DNA repair machinery has the potential to improve treatment response by tailoring treatment to cancer cells lacking specific DNA repair pathways. Human papillomavirus (HPV)‐negative and HPV‐positive HNSCCs respond differently to radiotherapy treatment, suggesting that different approaches of DNA repair inhibition should be employed for these HNSCC groups. Here, we searched for optimal radiosensitization approaches for HPV‐positive and HPV‐negative HNSCCs by performing a targeted CRISPR‐Cas9 screen. We found that inhibition of base excision repair resulted in a better radiotherapy response in HPV‐positive HNSCC, which is correlated with upregulation of genes involved in base excision repair. In contrast, inhibition of nonhomologous end‐joining and mismatch repair showed strong effects in both HNSCC groups. We validated the screen results by combining radiotherapy with targeted inhibition of DNA repair in several preclinical models including primary and recurrent patient‐derived HNSCC xenografts. These findings underline the importance of stratifying HNSCC patients for combination treatments.

Preclinical efficacy of copanlisib in cetuximab sensitive and resistant tumors of HNSCC.

6031 Background: Copanlisib is a highly selective, pan-class I PI3K inhibitor with preferential activity against the p110α and p110δ isoforms that lead to downregulation of PI3K signaling. Copanlisib has been approved for the treatment of follicular lymphoma in the US. Here, we explored the anti-tumor activity of copanlisib in head and neck squamous cell carcinoma (HNSCC), where Pi3K signaling has been defined as alternate signaling in cetuximab resistant tumors. Further, TCGA data show up to 56% of HNSCC display either amplification or mutational changes in the Pi3K pathway making Pi3K an attractive target. Methods: Using a mouse-clinical trial set-up we profiled 20 patient derived HNSCC xenograft models for their sensitivity to cetuximab or copanlisib as single agent as well as in combination. Models were selected from our HNSCC PDX platform based on Pi3K mutational status, with 6 models harboring hot spot mutations, HPV positivity (n=3) and/or cetuximab resistance based on previous drug screenings (n=12). Treatment response was defined as tumor regression, stabilization or progression expressed as relative tumor volume (RTV) after 3 weeks of treatment: RTV<0,7 responder, RTV>1,2 progressor. Results: Copanlisib single agent treatment resulted in moderate activity with 5 responders (25%). In cetuximab resistant tumors (n=12) combined treatment led to an improved tumor response in 75% (n=9) whereas 41% (n=5) resulted in tumor control. Pi3KCA mutation was not predictive for treatment response to either cetuximab or copanlisib. No PTEN mutation was detected in the selected cohort. Increased Pi3K signaling activity evaluated through gene expression profiling and computed with GSEA pathway analyses was positively correlated with response. Conclusions: The anti-tumor responses observed in monotherapy or in combination treatment support further investigation for the potential of PI3K inhibition in HNSCC with high expression of Pi3K pathway signature as a potential predictive biomarker.

The CD98 Heavy Chain Is a Marker and Regulator of Head and Neck Squamous Cell Carcinoma Radiosensitivity.

The heavy chain of the CD98 protein (CD98hc) is encoded by the SLC3A2 gene. Together with the light subunit LAT1, CD98hc constitutes a heterodimeric transmembrane amino acid transporter. High SLC3A2 mRNA expression levels are associated with poor prognosis in patients with head and neck squamous cell carcinoma (HNSCC) treated with radiochemotherapy. Little is known regarding the CD98hc protein-mediated molecular mechanisms of tumor radioresistance. CD98hc protein expression levels were correlated with corresponding tumor control dose 50 (TCD50) in HNSCC xenograft models. Expression levels of CD98hc and LAT1 in HNSCC cells were modulated by siRNA or CRISPR/Cas9 gene editing. HNSCC cell phenotypes were characterized by transcription profiling, plasma membrane proteomics, metabolic analysis, and signaling pathway activation. Expression levels of CD98hc and LAT1 proteins were examined by IHC analysis of tumor tissues from patients with locally advanced HNSCC treated with primary radiochemotherapy (RCTx). Primary endpoint was locoregional tumor control (LRC). High expression levels of CD98hc resulted in an increase in mTOR pathway activation, amino acid metabolism, and DNA repair as well as downregulation of oxidative stress and autophagy. High expression levels of CD98hc and LAT1 proteins were significantly correlated and associated with an increase in radioresistance in HNSCC in vitro and in vivo models. High expression of both proteins identified a poor prognosis subgroup in patients with locally advanced HNSCC after RCTx. We found that CD98hc-associated signaling mechanisms play a central role in the regulation of HNSCC radioresistance and may be a promising target for tumor radiosensitization.

Integrated Genomic and Functional microRNA Analysis Identifies miR-30-5p as a Tumor Suppressor and Potential Therapeutic Nanomedicine in Head and Neck Cancer.

To identify deregulated and inhibitory miRNAs and generate novel mimics for replacement nanomedicine for head and neck squamous cell carcinomas (HNSCC). We integrated miRNA and mRNA expression, copy number variation, and DNA methylation results from The Cancer Genome Atlas (TCGA), with a functional genome-wide screen. We reveal that the miR-30 family is commonly repressed, and all 5 members sharing these seed sequence similarly inhibit HNSCC proliferation in vitro. We uncover a previously unrecognized inverse relationship with overexpression of a network of important predicted target mRNAs deregulated in HNSCC, that includes key molecules involved in proliferation (EGFR, MET, IGF1R, IRS1, E2F7), differentiation (WNT7B, FZD2), adhesion, and invasion (ITGA6, SERPINE1). Reexpression of the most differentially repressed family member, miR-30a-5p, suppressed this mRNA program, selected signaling proteins and pathways, and inhibited cell proliferation, migration, and invasion in vitro. Furthermore, a novel miR-30a-5p mimic formulated into a targeted nanomedicine significantly inhibited HNSCC xenograft tumor growth and target growth receptors EGFR and MET in vivo. Significantly decreased miR-30a/e family expression was related to DNA promoter hypermethylation and/or copy loss in TCGA data, and clinically with decreased disease-specific survival in a validation dataset. Strikingly, decreased miR-30e-5p distinguished oropharyngeal HNSCC with poor prognosis in TCGA (P = 0.002) and validation (P = 0.007) datasets, identifying a novel candidate biomarker and target for this HNSCC subset. We identify the miR-30 family as an important regulator of signal networks and tumor suppressor in a subset of HNSCC patients, which may benefit from miRNA replacement nanomedicine therapy.

Blocking autophagy flux promotes interferon-alpha-mediated apoptosis in head and neck squamous cell carcinoma.

Despite multiple antitumor activities, interferon-alpha (IFNα) therapy alone is less effective in solid tumors. Autophagy has been reported to play a key role in tumor chemoresistance. Therefore, it is meaningful to explore whether autophagy can be activated by IFNα in head and neck squamous cell carcinoma (HNSCC) and serve as a potential target to improve efficacy of IFNα therapy. In this study, we report that IFNα not only exhibits anti-proliferation activity and induces apoptosis, but also activates autophagy in HNSCC cells. Moreover, silencing autophagy-related protein 5 (ATG5) and signal transducer and activator of transcription 1 (STAT1) suppresses autophagy flux. Furthermore, IFNα and autophagy inhibitors (hydroxychloroquine and wortmannin) show clear synergistic effects on inhibiting growth and promoting apoptosis in HNSCC cells and xenograft models. Our findings indicate that IFNα-induced autophagy plays a cytoprotective role and blocking autophagy flux promotes IFNα-mediated apoptosis in HNSCC. These results suggest that the combination of IFNα and autophagy inhibitors represents a novel strategy for HNSCC treatment.

Lymphotoxin-α promotes tumor angiogenesis in HNSCC by modulating glycolysis in a PFKFB3-dependent manner.

Tumor angiogenesis is critical for tumor progression as the new blood vessels supply nutrients and facilitate metastasis. Previous studies indicate tumor associated lymphocytes, including B cells and T cells, contribute to tumor angiogenesis and tumor progression. The present study aims to identify the function of Lymphotoxin-α (LT-α), which is secreted by the activated lymphocytes, in the tumor angiogenesis of head and neck squamous cell carcinoma (HNSCC). The coculture system between HNSCC cell line Cal27 and primary lymphocytes revealed that tumor cells promoted the LT-α secretion in the cocultured lymphocytes. In vitro data further demonstrated that LT-α promoted the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) by enhancing the PFKFB3-mediated glycolytic flux. Genetic and pharmacological inhibition of PFKFB3 suppressed the enhanced proliferation and migration of HUVECs. We further identified that LT-α induced PFKFB3 expression was dependent on the TNFR/NF-κB signaling pathway. In addition, we proved that PFKFB3 blockade decreased the density of CD31 positive blood vessels in HNSCC xenografts. Finally, the results from the human HNSCC tissue array revealed that the expression of LT-α in HNSCC samples positively correlated with microvessel density, lymphocytes infiltration and endothelial PFKFB3 expression. In conclusion, infiltrated lymphocyte secreted LT-α enhances the glycolysis of ECs in a PFKFB3-dependent manner through the classical NF-κB pathway and promotes the proliferation and migration of ECs, which may contribute to the aberrant angiogenesis in HNSCCs. Our study suggests that PFKFB3 blockade is a promising therapeutic approach for HNSCCs by targeting tumor angiogenesis.

Impact of Epigenetic Regulation on Head and Neck Squamous Cell Carcinoma

The most common type of head and neck cancer, head and neck squamous cell carcinoma (HNSCC), can develop therapeutic resistance that complicates its treatment. The 5-y survival rate for HNSCC remains at ~50%, and improving these outcomes requires a better understanding of the pathogenesis of HNSCC. Studies of HNSCC using in vitro, ex vivo, and in vivo approaches provide a novel conceptual framework based on epigenetic mechanisms for developing future clinical applications. Normal oral tissues are influenced by environmental factors that induce pathological changes affecting the network of epigenetic enzymes and signaling pathways to induce HNSCC growth and metastasis. Although various epigenetic regulator families, such as DNA methyltransferases, ten-eleven translocation proteins, histone acetyltransferases, histone deacetylases, BET bromodomain proteins, protein arginine methyltransferases, histone lysine methyltransferases, and histone lysine demethylases, have a role in diverse cancers, specific members have a function in HNSCC. Recently, lysine-specific demethylases have been identified as a potential, attractive, and novel target of HNSCC. Lysine-specific demethylase 1 (LSD1) expression is inappropriately upregulated in HNSCC and an orthotopic HNSCC mouse model. LSD1 can demethylate lysine at specific histone positions to repress gene expression or stimulate transcription, indicating a dual and context-dependent role in transcriptional regulation. Our study showed that LSD1 promotes HNSCC growth and metastasis. Pharmacological attenuation of LSD1 inhibits orthotopic and patient-derived HNSCC xenograft growth-specific target genes and signaling pathways. This review provides recent evidence demonstrating the function of epigenetic regulator enzymes in HNSCC progression, including potential therapeutic applications for such enzymes in combination and immunotherapy.

A controlled trial of HNSCC patient-derived xenografts reveals broad efficacy of PI3Kα inhibition in controlling tumor growth.

Head and neck squamous cell carcinomas (HNSCCs) frequently harbor alterations in the PI3K/AKT/mTOR signaling axis, particularly in the PIK3CA gene. PI3K-targeted agents have therefore gained considerable preclinical and clinical interest as emerging therapies for HNSCC. Identification of predictive biomarkers of response would advance the clinical application of PI3K-targeted drugs for patients, in order to achieve maximal benefit. To date, studies of drug biomarkers have largely focused on screening cell lines, with much more limited in vivo testing, usually only as validation. This approach has rarely enabled accurate predictions of clinical efficacy. Recently, clinical trials of PDX models (PDX clinical trials) have been introduced as a preclinical approach to interrogate interpatient response heterogeneity. Already, PDX clinical trial responses have been demonstrated to correlate closely with patient outcomes. Here, using both an HNSCC specific, 28-cell line panel and a PDX clinical trial of 80 xenografts derived from 20 unique HNSCC tumors, we systematically examine patterns of response to PI3K inhibition in HNSCC. We find EGFR, AKT1 and CSMD1 copy number aberrations, but not PIK3CA mutations, to be associated with responsiveness to PI3K-targeted drugs. Further, we reveal PI3Kα inhibition to be almost globally tumoristatic in HNSCC xenografts regardless of PIK3CA mutational status, emphasizing its potential as a stabilizing neoadjuvant therapy for HNSCC patients.

Intracellular reduction in ATP levels contributes to CYT997-induced suppression of metastasis of head and neck squamous carcinoma.

The incidence rate of head and neck squamous cell carcinoma (HNSCC) has steadily increased over the past decade. However, treatment options for metastatic HNSCC are often limited and the 5‐year survival rate has remained static. Therefore, the development and assessment of more efficient but less toxic therapeutic strategies is an unmet need for treatment of more extensive HNSCC. Here, we report that CYT997, a novel microtubule‐disrupting agent, exerts strong activity in inhibiting HNSCC cell invasion and metastasis. The loss of invasion capacity by CYT997 was accompanied by an associated increase in cell adhesion and the reversal of epithelial‐mesenchymal transition (EMT). Increased expression of E‐cadherin protein and decreased expression of Vimentin protein became evident in HNSCC cells following CYT997 exposure, which were consistently observed in HNSCC xenografts from the mice receiving CYT997. Moreover, the capacity of invasive HNSCC cells to form pulmonary metastases was significantly blocked with CYT997 treatment, indicating that the diminishment of EMT traits contributes to CYT997‐suppressed metastasis. Intriguingly, CYT997 impaired intracellular ATP levels in HNSCC cells, at least in part, through its inhibitory effect on the mitochondrial protein IF1. The addition of ATP attenuated CYT997‐induced suppression of cell invasion, coupled with down‐regulation of E‐Cadherin and up‐regulation of Vimentin. These findings support a critical role of ATP levels in cell invasion and metastasis under the influence of CYT997. Collectively, our data unveil the mechanism involved in mediating CYT997 action, and provide preclinical rationale for possible clinical application of CYT997 as a novel therapeutic strategy against aggressive HNSCC.

Molecular Profiling of Head and Neck Cancer Patient Derived Xenografts Identifies FGFR As a Target for Radiosensitization

The fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3) are frequently altered or overexpressed in head and neck squamous cell carcinomas (HNSCC). Amplification of FGFR1 has been reported to be as high as 17% in this disease. FGFRs activate multiple pathways involved in the radiation response including RAS/RAF/MAPK, PI3K/AKT and STAT. AZD4547 is a potent and selective tyrosine kinase inhibitor of the FGFR family and is currently being investigated in clinical trials. We therefore examined the anti-tumor effects of AZD4547 in combination with radiation in HNSCC model systems. HNSCC patient derived xenografts were screened using FGFR1 and FGFR2 RNA in situ hybridization. A panel of HNSCC cell lines was screened for FGFR1/2 protein and RNA expression and response to AZD4547. Immunoblots were used to examine the effect of AZD4547 on downstream signaling proteins. Radiation clonogenic survival assays and xenograft growth delays experiments were performed to investigate radiosensitization. Out of the 22 HNSCC patient derived xenografts examined, three were found to have FGFR1/2 expression. This included one p16 negative tumor and two p16 positive tumors. Nine cell lines demonstrated varying degree of FGFR1/2 protein and RNA expression. The half-maximal inhibitory concentration (IC50) of AZD4547 in these cell lines ranged from 0.05 to 42.2 uM. Sensitivity to AZD4547 did not directly correlate with protein or RNA expression. In sensitive cell lines, AZD4547 inhibited p-MAPK in a time dependent manner. In vitro clonogenic survival assays showed enhancement in radiosensitivity with AZD4547 in two HNSCC cell lines. AZD4547 induced significant growth inhibition in a FGFR1 expressing patient derived xenograft. Significant tumor growth delay was observed with the combination of radiation and AZD4547 compared to radiation or drug alone in a FGFR2 expressing cell line HNSCC xenograft model. These findings suggest that AZD4547 can augment the response of radiation HNSCC in vivo model systems. FGFR1 and FGFR2 expression may provide a potential target for radiosensitization in HNSCC. Additional studies are underway to understand the mechanism of radiosensitization.

Evaluating Imaging Biomarkers of Acquired Resistance to Targeted EGFR Therapy in Xenograft Models of Human Head and Neck Squamous Cell Carcinoma.

Background: Overexpression of EGFR is a negative prognostic factor in head and neck squamous cell carcinoma (HNSCC). Patients with HNSCC who respond to EGFR-targeted tyrosine kinase inhibitors (TKIs) eventually develop acquired resistance. Strategies to identify HNSCC patients likely to benefit from EGFR-targeted therapies, together with biomarkers of treatment response, would have clinical value.Methods: Functional MRI and 18F-FDG PET were used to visualize and quantify imaging biomarkers associated with drug response within size-matched EGFR TKI-resistant CAL 27 (CALR) and sensitive (CALS) HNSCC xenografts in vivo, and pathological correlates sought.Results: Intrinsic susceptibility, oxygen-enhanced and dynamic contrast-enhanced MRI revealed significantly slower baseline , lower hyperoxia-induced and volume transfer constant Ktrans in the CALR tumors which were associated with significantly lower Hoechst 33342 uptake and greater pimonidazole-adduct formation. There was no difference in oxygen-induced ΔR1 or water diffusivity between the CALR and CALS xenografts. PET revealed significantly higher relative uptake of 18F-FDG in the CALR cohort, which was associated with significantly greater Glut-1 expression.Conclusions: CALR xenografts established from HNSCC cells resistant to EGFR TKIs are more hypoxic, poorly perfused and glycolytic than sensitive CALS tumors. MRI combined with PET can be used to non-invasively assess HNSCC response/resistance to EGFR inhibition.

Targeting CD44v6 for fluorescence-guided surgery in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is an often highly invasive tumor, infiltrating functionally important tissue areas. Achieving complete tumor resection and preserving functionally relevant tissue structures depends on precise identification of tumor-free resection margins during surgery. Fluorescence-guided surgery (FGS), by intraoperative detection of tumor cells using a fluorescent tracer, may guide surgical excision and identify tumor-positive resection margins. Using a literature survey on potential surface molecules followed by immunohistochemical validation, we identified CD44 variant 6 (CD44v6) as a constitutively expressed antigen in the invasion zone of HNSCC lesions. The monoclonal anti-CD44v6 antibody BIWA was labeled with both a near-infrared fluorescent dye (IRDye800CW) and a radioactive label (Indium-111) and dual-modality imaging was applied in a locally invasive tumor mouse model. BIWA accurately detected human HNSCC xenografts in mice with a tumor uptake of 54 ± 11% ID/g and invasion regions with an accuracy of 94%. When dissected under clinical-like conditions, tumor remnants approximately 0.7 mm in diameter consisting of a few thousand cells were identified by fluorescence imaging, resulting in reliable dissection of invasive microregions. These data indicate that CD44v6 is a suitable target for reliable near-infrared detection and FGS of invasive HNSCC lesions in vivo.

Abstract 5884: 4EBP1 is a tumor suppressor gene unleashed by mTOR inhibition in head and neck squamous cell carcinoma

Abstract Persistent aberrant activation of the PI3K/AKT/mTOR signaling pathway occurs in over 80% of head and neck squamous cell carcinomas (HNSCC). In prior studies we have shown that mTOR regulates proliferation, angiogenesis and apoptosis, thus playing an essential role in HNSCC initiation and progression. The frequent over-reliance on this signaling circuit for HNSCC growth may in turn represent a cancer vulnerability that can be exploited therapeutically. Indeed, we have shown that mTOR inhibitors (mTORi) promote tumor regression in multiple genetically defined and chemically induced carcinogenesis animal models, and encouraging results have been recently obtained using mTORi in HNSCC in the clinic. However, the complexity of genomic alterations in HNSCC may facilitate the emergence of drug resistance, especially in cases with patients who may receive multiple rounds of chemotherapy and/or radiation. Understanding the precise molecular processes by which mTOR acts in HNSCC may help predict intrinsic resistance mechanisms and select patients who may benefit the most from the administration of mTORi. Here, we focused on EIF4EBP1 (4EBP1), a direct target of mTOR that serves as key effector for protein synthesis. Remarkably, we found a negative correlation between 4EBP1 gene copy number loss and disease-free survival (p<0.001) and overall survival (p<0.01) in the HNSCC TCGA database (n=181). Furthermore, IHC analysis of 4EBP1 protein expression revealed that 16.3% of HNSCC cases analyzed (n=49) lack 4EBP1, with progressive protein loss in advanced cases. Aligned with a tumor-suppressive role, 4ebp1-knockout mice formed larger and more HNSCC lesions (p<0.001) in 4NQO oral carcinogenesis models. We engineered a mutant 4EBP1 that cannot be phosphorylated by mTOR (4EBP1-mut), resembling de-phospho-4EBP1, and generated HNSCC cells expressing this mutant in a tetracycline-inducible fashion. Protein-protein interactions and assembly of the translational complex were analyzed using 7-methyl GTP pull-down and co-IP assays. Expression of 4EBP1-mut disrupted the function of the translation initiation complex and was sufficient to prevent tumor growth in HNSCC xenograft models. Similarly, mTORi induced the accumulation of dephosphorylated endogenous wild-type 4EBP1 and resulted in HNSCC xenograft regression. Remarkably, CRISPR-CAS9-mediated knockout of 4EBP1 in HNSCC cells caused insensitivity to mTORi in vitro and in vivo. Overall, our findings suggest that 4EBP1 needs to be persistently repressed by mTOR phosphorylation in HNSCC and that mTORi may unleash 4EBP1's tumor-suppressive activity, thereby reducing the translation of growth-promoting and cancer cell stemness-related proteins. In turn, 4EBP1 gene and protein loss may underlie mTORi resistance, supporting that 4EBP1 expression and phosphorylation status may represent a mechanistic biomarker of mTORi sensitivity in HNSCC patients. Citation Format: Zhiyong Wang, Daniel Martin, Lynn Vitale-Cross, Xiaodong Feng, Alfredo A. Molinolo, Michael M. Allevato, Victoria H. Wu, Mara Gilardi, Hao Xu, Qianming Chen, J.Silvio Gutkind. 4EBP1 is a tumor suppressor gene unleashed by mTOR inhibition in head and neck squamous cell carcinoma [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 5884.

Abstract 4401: Integrated genomic and functional microRNA analysis identifies miR-30-5p as a tumor suppressor and novel therapeutic nanomedicine in head and neck cancer

Abstract We have developed an integrative approach to elucidate the functional role of deregulated microRNAs (miRs) and identify candidates for miR replacement therapy in cancer, applied here to head and neck squamous cell carcinomas (HNSCC). Our method involves integration of results from TCGA and other validation databases of genetic and expression data, including: miR expression, mRNA expression, copy number variation, DNA methylation. This data is then intersected with functional in vitro genome-wide miR screening for anti-proliferative miR mimics. We report that the miR-30-5p family is commonly repressed and correlates with overexpression of a broad network of proliferative and metastasis-related oncogenic mRNAs, including the growth receptors EGFR, MET and IGF1R. We show that re-expression of miR-30a-5p repressed this gene program, downstream signaling, proliferation, migration, and invasion in vitro. We have developed a chemically modified mimic of miR-30-5p with 50x improved stability in human serum, and 5X improvement in IC50 in vitro by XTT assay. We have formulated our novel miR-30a-5p mimic into Transferrin Receptor targeted nanoparticles which strongly inhibited HNSCC xenograft tumor growth, and regulated miR-30-5p family targets in vivo. Together with data linking decreased miR-30 family expression with DNA copy loss and promoter hypermethylation, and clinical disease-specific survival, for the first time, we have reported a more global picture of the function of this important tumor suppressor and identified a subset of patients that may benefit from miR replacement therapy with our novel miR-30-5p based nanomedicine. This research was supported by NIDCD intramural projects ZIA-DC-000073, 74, and NCI grant U43CA22156701. Citation Format: Anthony D. Saleh, Robert F. Place, Hui Cheng, Rita Das, Thomas M. Harris, Geoffrey Childs, Gordon A. Robertson, Kathleen F. Pirollo, Esther H. Chang, Zhong Chen, Carter Van Waes. Integrated genomic and functional microRNA analysis identifies miR-30-5p as a tumor suppressor and novel therapeutic nanomedicine in head and neck cancer [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 4401.

Lactoferricin B reverses cisplatin resistance in head and neck squamous cell carcinoma cells through targeting PD-L1.

Head and neck squamous cell carcinoma (HNSCC) ranks among the top most common cancers with a poor prognosis. The mechanism of chemoresistance is still not well known. This study is to investigate the programmed death‐ligand 1 (PD‐L1) expression in HNSCC, and test the effect of lactoferricin B (LfcinB) on chemoresistance and its mechanism. We analyzed 510 HNSCC patients in TCGA database and investigated how CD274 expression was related to patient prognosis. PD‐L1 was verified from HNSCC samples at local hospital with immunohistochemistry. PD‐L1 expression in the acquired cisplatin‐resistant HNSCC cells was examined by PCR and WB in order to test PD‐L1‐induced chemoresistance. LfcinB inoculation in cisplatin‐resistant HNSCC cells and in the nude mice was introduced to test the effect of LfcinB on targeting cisplatin resistance and its mechanism. High CD274 mRNA (>125 FPKM) from TCGA database had a significantly reduced 5‐year survival rate, and a lower 5‐year survival rate in the chemotherapy and radiotherapy‐treated patients (P < .05). PD‐L1 overexpression was further supported from analysis of 40 HNSCC specimens. PD‐L1 and IL‐6 in the established cisplatin‐resistant HNSCC cells were shown significantly higher (P < .05). IL‐6 and PD‐L1 expression were partially inhibited by the anti‐IL‐6/STAT3 antibody. LfcinB displayed a direct cytotoxic effect on cisplatin‐resistant HNSCC cells and HNSCC xenografts of cisplatin‐resistant cells in the nude mice displayed significant reduction in tumor volume after LfcinB injection (P < .05). Besides, the increase of IL‐6 and PD‐L1 in cisplatin‐resistant HNSCC cells was abolished in vitro by LfcinB (P < .05). PD‐L1 expression in HNSCC cells correlates with poor prognosis and chemoresistance, and LfcinB might provide therapeutic potential in HNSCC patients through modulating IL‐6 and PD‐L1.

Impact of Age on Disease Progression and Microenvironment in Oral Cancer

Despite the recognized link between aging and cancer, most preclinical studies in experimental tumor models are conducted with 6- to 8-wk-old rodents. The goal of the present study was to examine the impact of age on tumor incidence, growth, and microenvironmental characteristics in mouse models of head and neck squamous cell carcinoma (HNSCC). Experimental studies were conducted with the 4-nitroquinoline-oxide (4NQO) oral carcinogenesis model and orthotopic FaDu HNSCC xenografts, established in young (7 to 12 wk of age) and old (65 to 70 wk of age) female C57BL/6 mice (n = 44; 4NQO model) and severe combined immunodeficient mice (n = 13; HNSCC xenografts). Noninvasive whole body magnetic resonance imaging revealed increased subcutaneous and visceral fat in aging animals of both strains. On histologic examination, a higher incidence (P < 0.001) of severe dysplasia/invasive squamous cell carcinoma was observed in old mice (92%) as compared with young mice (69%). Old C57BL/6 mice exposed to 4NQO exhibited increased incidence of oral and extraoral (peritoneal masses) neoplasms (42%) versus their young counterparts (P < 0.05). The incidence of extraoral neoplasms was significantly lower (16%) in the younger cohort. Interestingly, no difference in growth rate and oxygen saturation was observed between orthotopic FaDu xenografts established in old and young severe combined immunodeficient mice. Our observations suggest that host age may have an impact on the growth kinetics and progression of HNSCC in the immunocompetent 4NQO model. Further investigation into the impact of aging on tumor response to preventive and therapeutic intervention is warranted.

Visualizing the effects of metformin on tumor growth, vascularity, and metabolism in head and neck cancer.

The antidiabetic drug metformin (Met) is believed to inhibit tumor proliferation by altering the metabolism of cancer cells. In this study, we examined the effects of Met on tumor oxygenation, metabolism, and growth in head and neck squamous cell carcinoma (HNSCC) using non-invasive multimodal imaging. Severe combined immunodeficient (SCID) mice bearing orthotopic FaDu HNSCC xenografts were treated with Met (200 mg/kg, ip) once daily for 5 days. Tumor oxygen saturation (%sO2 ) and hemoglobin concentration (HbT) were measured using photoacoustic imaging (PAI). Fluorescence imaging was employed to measure intratumoral uptake of 2-deoxyglucosone (2-DG) following Met treatment while magnetic resonance imaging (MRI) was utilized to measure tumor volume. Correlative immunostaining of tumor sections for markers of proliferation (Ki67) and vascularity (CD31) was also performed. At 5 days post-Met treatment, PAI revealed a significant increase (P < .05) in %sO2 and HbT levels in treated tumors compared to untreated controls. Fluorescence imaging at this time point revealed a 46% decrease in mean 2-DG uptake compared to controls. No changes in hemodynamic parameters were observed in mouse salivary gland tissue. A significant decrease in Ki-67 staining (P < .001) and MR-based tumor volume was also observed in Met-treated tumors compared to controls with no change in CD31 + vessel count following Met therapy. Our results provide, for the first time, direct in vivo evidence of Met-induced changes in tumor microenvironmental parameters in HNSCC xenografts. Our findings highlight the utility of multimodal functional imaging for non-invasive mapping of the effects of Met in HNSCC.

The Neddylation Inhibitor Pevonedistat (MLN4924) Suppresses and Radiosensitizes Head and Neck Squamous Carcinoma Cells and Tumors.

The cullin RING E3 ubiquitin ligase 4 (CRL4) with its substrate receptor CDT2 (CRL4-CDT2) is emerging as a critical regulator of DNA replication through targeting CDT1, SET8, and p21 for ubiquitin-dependent proteolysis. The aberrant increased stability of these proteins in cells with inactivated CRL4-CDT2 results in DNA rereplication, which is deleterious to cells due to the accumulation of replication intermediates and stalled replication forks. Here, we demonstrate that CDT2 is overexpressed in head and neck squamous cell carcinoma (HNSCC), and its depletion by siRNA inhibits the proliferation of human papilloma virus-negative (HPV-ve) HNSCC cells primarily through the induction of rereplication. Treatment of HNSCC with the NEDD8-activating enzyme inhibitor pevonedistat (MLN4924), which inhibits all cullin-based ligases, induces significant rereplication and inhibits HNSCC cell proliferation in culture and HNSCC xenografts in mice. Pevonedistat additionally sensitizes HNSCC cells to ionizing radiation (IR) and enhances IR-induced suppression of xenografts in mice. Induction of rereplication via CDT2 depletion, or via the stabilization or activation of CDT1, also radiosensitizes HNSCC cells. Collectively, these results demonstrate that induction of rereplication represents a novel approach to treating radioresistant HNSCC tumors and suggest that pevonedistat may be considered as an adjuvant for IR-based treatments. Mol Cancer Ther; 17(2); 368-80. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

Interferon-alpha enhances the antitumour activity of EGFR-targeted therapies by upregulating RIG-I in head and neck squamous cell carcinoma

Background:The epidermal growth factor receptor (EGFR)-targeted therapies have been tested in the clinic as treatments for head and neck squamous cell carcinoma (HNSCC). Owing to intrinsic or acquired resistance, EGFR-targeted therapies often lead to a low response rate and treatment failure. Interferon-alpha (IFNα) is a chemosensitising agent and multi-functional cytokine with a tumour inhibitory effect. However, the synergic effect of IFNα and EGFR-targeted therapies (erlotinib and nimotuzumab) and their mechanisms in HNSCC remain unclear.Methods:The interactions between IFNα, erlotinib, and nimotuzumab were evaluated in vitro in HNSCC cells. The synergistic effect of IFNα (20 000 IU per day, s.c.), erlotinib (50 mg kg−1 per day, i.g.) and nimotuzumab (10 mg kg−1 per day, i.p.) was further confirmed in vivo using HNSCC xenografts in nude mice. The upregulation of retinoic-acid inducible gene I (RIG-I) induced by IFNα and EGFR-targeted therapies and its mechanism were detected in vitro and in vivo.Results:IFNα enhances the antitumour effects of erlotinib and nimotuzumab on HNSCC cells both in vitro and in vivo. Importantly, both IFNα and EGFR-targeted therapies promote the expression of RIG-I by activating signal transducers and activators of transcription 1 (STAT1) in HNSCC cells. RIG-I knockdown reduced the sensitivity of HN4 and HN30 cells to IFNα, erlotinib, and nimotuzumab. Moreover, IFNα transcriptionally induced RIG-I expression in HNSCC cells through STAT1.Conclusions:IFNα enhances the effect of EGFR-targeted therapies by upregulating RIG-I, and its expression may represent a predictor of the effectiveness of a combination treatment including IFNα in HNSCC.

Effect of Adipose-Derived Stem Cells on Head and Neck Squamous Cell Carcinoma.

Objective Patients with head and neck squamous cell carcinoma (HNSCC) have significant wound-healing difficulties. While adipose-derived stem cells (ASCs) facilitate wound healing, ASCs may accelerate recurrence when applied to a cancer field. This study evaluates the impact of ASCs on HNSCC cell lines in vitro and in vivo. Study Design In vitro experiments using HNSCC cell lines and in vivo mouse experiments. Setting Basic science laboratory. Subjects and Methods Impact of ASCs on in vitro proliferation, survival, and migration was assessed using 8 HNSCC cell lines. One cell line was used in a mouse orthotopic xenograft model to evaluate in vivo tumor growth in the presence and absence of ASCs. Results Addition of ASCs did not increase the number of HNSCC cells. In clonogenic assays to assess cell survival, addition of ASCs increased colony formation only in SCC9 cells (maximal effect 2.3-fold, P < .02) but not in other HNSCC cell lines. In scratch assays to assess migration, fluorescently tagged ASCs did not migrate appreciably and did not increase the rate of wound closure in HNSCC cell lines. Addition of ASCs to HNSCC xenografts did not increase tumor growth. Conclusion Using multiple in vitro and in vivo approaches, ASCs did not significantly stimulate HNSCC cell proliferation or migration and increased survival in only a single cell line. These findings preliminarily suggest that the use of ASCs may be safe in the setting of HNSCC but that further investigation on the therapeutic use of ASCs in the setting of HNSCC is needed.