Head And Neck Squamous Cell Carcinoma Cancer Stem Cells Research Articles

Mithramycin targets head and neck cancer stem cells by inhibiting Sp1 and UFMylation

BackgroundThe development of resistance to therapy is characteristic of head and neck squamous cell carcinoma (HNSCC), the 6th most common cancer, and is often attributed to cancer stem cells (CSCs). By proteomic approach, we determined that UFMylation plays an important role in HNSCC CSCs. Because of the necessity for innovative therapeutic strategies, we explore here the therapy targeting CSCs based on mithramycin and its inhibitory effect on Sp1 transcription factor, UFMylation, and CSCs survival and stemness.MethodsHNSCC-derived cancer cell lines Detroit 562, FaDu, and Cal27, and tumor spheres are used as a model for CSCs. Proteomic analysis identified the importance of the UFMylation pathway in CSCs which we further studied by bioinformatics, western blot, immunocytochemistry, and cytotoxicity assay.ResultsProteomic analysis and subsequent confirmation revealed UFSP2 and DDRGK1 were strongly expressed in tumor spheres. Bioinformatic analysis indicated high expression of UFM1 is linked with worse overall and disease-free survival, and it correlated with main EMT proteins (Zeb, Twist, and Fn) in HNSCC. UFM1 was also strongly expressed in tumor spheres compared to the adherent cells. Silencing of UFM1 reduced sphere number, size, and stemness. As Sp1 is the main transcription factor for the genes of the UFMylation system, we explored its inhibitor mithramycin, as a potential drug for CSCs inhibition. We proved mithramycin inhibits CSCs survival, induces apoptosis, and reduces UFMylation and stemness.ConclusionUFMylation is an important process in CSCs, and mithramycin, or its lesser toxic analogs, should be further explored as CSCs targeted therapy in HNSCC.

A bioengineered tumor matrix-based scaffold for the evaluation of melatonin efficacy on head and neck squamous cancer stem cells

Head and neck squamous cell carcinoma (HNSCC) presents a significant challenge worldwide due to its aggressiveness and high recurrence rates post-treatment, often linked to cancer stem cells (CSCs). Melatonin shows promise as a potent tumor suppressor; however, the effects of melatonin on CSCs remain unclear, and the development of models that closely resemble tumor heterogeneity could help to better understand the effects of this molecule. This study developed a tumor scaffold based on patient fibroblast-derived decellularized extracellular matrix that mimics the HNSCC microenvironment. Our study investigates the antitumoral effects of melatonin within this context. We validated its strong antiproliferative effect on HNSCC CSCs and the reduction of tumor invasion and migration markers, even in a strongly chemoprotective environment, as it is required to increase the minimum doses necessary to impact tumor viability compared to the non-scaffolded tumorspheres culture. Moreover, melatonin exhibited no cytotoxic effects on healthy cells co-cultured in the tumor hydrogel. This scaffold-based platform allows an in vitro study closer to HNSCC tumor reality, including CSCs, stromal component, and a biomimetic matrix, providing a new valuable research tool in precision oncology.

Identification of tumor stemness and immunity related prognostic factors and sensitive drugs in head and neck squamous cell carcinoma

The presence of cancer stem cells (CSCs) contributes significantly to treatment resistance in various cancers, including head and neck squamous cell carcinoma (HNSCC). Despite this, the relationship between cancer stemness and immunity remains poorly understood. In this study, we aimed to identify potential immunotherapeutic targets and sensitive drugs for CSCs in HNSCC. Using data from public databases, we analyzed expression patterns and prognostic values in HNSCC. The stemness index was calculated using the single-sample gene set enrichment analysis (ssgsea) algorithm, and weighted gene co-expression network analysis (WGCNA) was employed to screen for key stemness-related modules. Consensus clustering was then used to group samples for further analysis, and prognosis-related key genes were identified through regression analysis. Our results showed that tumor samples from HNSCC exhibited higher stemness indices compared to normal samples. WGCNA identified a module highly correlated with stemness, comprising 187 genes, which were significantly enriched in protein digestion and absorption pathways. Furthermore, we identified sensitive drugs targeting prognostic genes associated with tumor stemness. Notably, two genes, HLF and CCL11, were found to be highly associated with both stemness and immunity. In conclusion, our study identifies a stemness-related gene signature and promising drug candidates for CSCs of HNSCC. Additionally, HLF and CCL11, which are associated with both stemness and immunity, represent potential targets for immunotherapy in HNSCC.

The biomarkers for maintenance Cancer stem cell features can be applicable in precision medicine of head and neck squamous cell carcinoma

Cancer stem cells (CSCs) play a crucial role in tumor relapse, proliferation, invasion, and drug resistance in head and neck squamous cell carcinoma (HNSCC). This narrative review aims to synthesize data from articles published between 2019 and 2023 on biomarkers for detecting CSCs in HNSCC and changes in molecular pathways, genetics, epigenetics, and non-coding RNAs (ncRNAs) in CSCs relevant to precision medicine approaches in HNSCC management. The search encompassed 41 in vitro studies and 22 clinical studies. CSCs exhibit diverse molecular profiles and unique biomarker expression patterns, offering significant potential for HNSCC diagnosis, treatment, and prognosis, thereby enhancing patient survival. Their remarkable self-renewal ability and adaptability are closely linked to tumorigenicity development and maintenance. Assessing biomarkers before and after therapy can aid in identifying various cell types associated with cancer progression and relapse. Screening for CSCs, senescent tumor cells, and cells correlated with the senescence process post-treatment has proven highly beneficial. However, the clinical application of precision medicine in HNSCC management is hindered by the lack of specific and definitive CSC biomarkers. Furthermore, our limited understanding of CSC plasticity, governed by genomic, transcriptomic, and epigenomic alterations during tumorigenesis, as well as the bidirectional interaction of CSCs with the tumor microenvironment, underscores the need for further research. Well-designed studies involving large patient cohorts are, therefore, essential to establish a standardized protocol and address these unresolved queries.

Head and neck cancer stem cell maintenance relies on mTOR signaling, specifically involving the mechanistic target of rapamycin complexes 1 and 2 (mTORC1 and mTORC2)

ObjectiveEmerging evidence suggests that the modest response of head and neck squamous cell carcinoma (HNSCC) to treatment is associated with cancer stem cells (CSC). However, the signaling pathways that play a role in HNSCC CSC maintenance and therapy response are not well-understood. In this study, we investigate the response of CSCs to phosphatase and tensin homolog (PTEN) modulation and its potential dependency on the mammalian target of rapamycin (mTOR) signaling. DesignPTEN deficiency was stably induced using short hairpin RNA (shRNA). Downregulation of RPTOR/mTORC1 and RICTOR/mTORC2 was achieved using small interfering RNA (siRNA). CSCs were evaluated through tumorsphere formation and were classified into various subtypes: parasphere, merosphere, and holosphere. We investigated the effect of rapamycin on CSC properties in both control and PTEN-deficient HNSCC cells. ResultsPTEN deficiency led to an accumulation of CSCs and enhanced a favorable response to rapamycin treatment. The viability of HNSCC CSCs was dependent on mTOR signaling. Deficiencies in both mTORC1 and mTORC2 reduced the number of CSCs. However, CSCs with PTEN deficiency had a greater reliance on mTORC1 signaling. Interestingly, when considering CSC subtypes, a deficiency in mTORC2 led to an increased number of paraspheres in both the control and PTEN-deficient groups. ConclusionsLoss of PTEN signaling increased the HNSCC CSC population, which can be targeted by rapamycin. However, the mTORC2 deficiency can induce a problematic selection of paraspheres CSCs subtype.

Multi-Omics Identification of Genetic Alterations in Head and Neck Squamous Cell Carcinoma and Therapeutic Efficacy of HNC018 as a Novel Multi-Target Agent for c-MET/STAT3/AKT Signaling Axis.

Amongst the most prevalent malignancies worldwide, head and neck squamous cell carcinoma (HNSCC) is characterized by high morbidity and mortality. The failure of standard treatment modalities, such as surgery, radiotherapy, and chemotherapy, demands the need for in-depth understanding of the complex signaling networks involved in the development of treatment resistance. A tumor's invasive growth and high levels of intrinsic or acquired treatment resistance are the primary causes of treatment failure. This may be a result of the presence of HNSCC's cancer stem cells, which are known to have self-renewing capabilities that result in therapeutic resistance. Using bioinformatics methods, we discovered that elevated expressions of MET, STAT3, and AKT were associated with poor overall survival in HNSCC patients. We then evaluated the therapeutic potential of our newly synthesized small molecule HNC018 towards its potential as a novel anticancer drug. Our computer-aided structure characterization and target identification study predicted that HNC018 could target these oncogenic markers implicated in HNSCC. Subsequently, the HNC018 has demonstrated its anti-proliferative and anticancer activities towards the head and neck squamous cell carcinoma cell lines, along with displaying the stronger binding affinities towards the MET, STAT3, and AKT than the standard drug cisplatin. Reduction in the clonogenic and tumor-sphere-forming ability displays HNC018's role in decreasing the tumorigenicity. Importantly, an vivo study has shown a significant delay in tumor growth in HNC018 alone or in combination with cisplatin-treated xenograft mice model. Collectively with our findings, HNC018 highlights the desirable properties of a drug-like candidate and could be considered as a novel small molecule for treating head and neck squamous cell carcinoma.

METTL3-mediated ALDH m6A methylation regulates the malignant behavior of BMI1+ HNSCC stem cells.

To reveal the effect and mechanism of methyltransferase-like 3 (METTL3) on cancer stem cells (CSCs) of head and neck squamous cell carcinoma (HNSCC). First, we analyzed 14-HNSCC-patients' scRNA-seq dataset and TCGA dataset of HNSCC. Then, Mettl3 knockout or overexpression mice models were studied via tracing and staining technologies. In addition, we took flow cytometry sorting and sphere formation assays to observe tumorigenicity and used cell transfection and western blotting to verify target protein expression levels. Furthermore, methylated RNA immunoprecipitation sequencing (MeRIP-seq) and MeRIP-quantitative real-time PCR (MeRIP-qPCR) were taken to identify the mechanism of Mettl3 regulating Bmi1+ CSCs in HNSCC. Due to SOX4 transcriptional regulation, METTL3 regulated the malignant behavior of BMI1+ HNSCC stem cells through cell division pathway. The progression and malignancy of HNSCC were decreased after Mettl3 knocked-out, while increased after Mettl3 knocked-in in Bmi1+ CSCs in vivo. Knockdown of Mettl3 inhibited stemness properties of CSCs in vitro. Mechanically, Mettl3 mediated the m6A modification of ALDH1A3 and ALDH7A1 mRNA in Bmi1+ HNSCC CSCs. Regulated by SOX4, METTL3-mediated ALDH m6A methylation regulates the malignant behavior of BMI1+ HNSCC CSCs through cell division pathway.

YTHDC1 Promotes Stemness Maintenance and Malignant Progression in Head and Neck Squamous Cell Carcinoma.

YTH domain containing 1 (YTHDC1), an N6-methyladenosine (m6A) modification reading protein, plays a key role in regulating RNA translation and degradation. However, the role of YTHDC1 in head and neck squamous cell carcinoma (HNSCC) cancer stem cells remains largely unknown. This study is aimed at investigating the role of YTHDC1 in HNSCC and exploring its role in regulating cancer stem cells. RNA sequencing was used to detect differentially expressed genes (DEGs) between SCC9 spheres and SCC9 cells and to uncover molecular pathways and target molecules associated with CSCs. We detected YTHDC1 expression in The Cancer Genome Atlas (TCGA) database data and clinical samples. Subsequently, YTHDC1 gene suppression assays were performed in HNSCC cell lines to investigate the effect of YTHDC1 on tumor cell stemness maintenance, proliferation, and migration capacity. To further confirm the role of YTHDC1 in regulating cancer stem cells in HNSCC, we analyzed online HNSCC single-cell transcriptomic data to investigate YTHDC1 expression patterns at the single-cell level and the correlation of these levels with the expression of stem cell markers. YTHDC1 expression levels were significantly upregulated in SCC9 spheres, and YTHDC1 was aberrantly expressed in HNSCC tumor tissues. The increased YTHDC1 expression was closely correlated with the clinical characteristics of HNSCC patients. YTHDC1 regulates the malignant phenotype of HNSCC in both in vivo and in vitro studies. Further single-cell transcriptomic data analysis revealed that YTHDC1 positively correlated with malignant epithelial cell stemness capacity at the single-cell level, and that YTHDC1 was involved in regulating stemness maintenance in HNSCC. These findings suggest that YTHDC1 may serve as a biomarker for stem maintenance and malignant progression in HNSCC, providing new insights into the treatment of cancer.

Melatonin and verteporfin synergistically suppress the growth and stemness of head and neck squamous cell carcinoma through the regulation of mitochondrial dynamics.

The prevalence of head and neck squamous cell carcinoma (HNSCC) has continued to rise for decades. However, drug resistance to chemotherapeutics and relapse, mediated by cancer stem cells (CSCs), remains a significant impediment in clinical oncology to achieve successful treatment. Therefore, we focused on analyzing CSCs in HNSCC and demonstrated the effect of melatonin (Mel) and verteporfin (VP) on SCC-25 cells. HNSCC CSCs were enriched in the reactive oxygen species-low state and in sphere-forming cultures. Combination treatment with Mel and VP decreased HNSCC viability and increased apoptosis without causing significant damage to normal cells. Sphere-forming ability and stem cell population were reduced by co-treatment with Mel and VP, while mitochondrial ROS level was increased by the treatment. Furthermore, the expression of mitophagy markers, parkin and PINK1, was significantly decreased in the co-treated cells. Mel and VP induced mitochondrial depolarization and inhibited mitochondrial function. Parkin/TOM20 was localized near the nucleus and formed clusters of mitochondria in the cells after treatment. Moreover, Mel and VP downregulated the expression of markers involved in epithelial-mesenchymal transition and metastasis. The migration capacity of cells was significantly decreased by co-treatment with Mel and VP, accompanied by the down-regulation of MMP-2 and MMP-9 expression. Taken together, these results indicate that co-treatment with Mel and VP induces mitochondrial dysfunction, resulting in the apoptosis of CSCs. Mel and VP could thus be further investigated as potential therapies for HNSCC through their action on CSCs.

Research progress of cancer stem cell biomarkers in head and neck squamous cell carcinoma

Cancer stem cells （CSCs）, as a few amount of tumors, have infinite replication, self-renewal, differentiation and regeneration of cell subsets with tumorigenicity, have close relationship with tumor occurrence and recurrence, which can be found in head and neck squamous cell carcinoma （HNSCC）. One of the important measures to improve the patient prognosis is monitoring cancer stem cells and timely clinical intervention. Biomarker detection of cancer stem cells is an important method for clinical monitoring of cancer stem cells. This article reviews the biomarkers of CSCs in HNSCC, which is consist of membrane surface markers, non-coding RNAs, target genes and proteins.

Precision Medicine Gains Momentum: Novel 3D Models and Stem Cell-Based Approaches in Head and Neck Cancer.

Despite the current progress in the development of new concepts of precision medicine for head and neck squamous cell carcinoma (HNSCC), in particular targeted therapies and immune checkpoint inhibition (CPI), overall survival rates have not improved during the last decades. This is, on the one hand, caused by the fact that a significant number of patients presents with late stage disease at the time of diagnosis, on the other hand HNSCC frequently develop therapeutic resistance. Distinct intratumoral and intertumoral heterogeneity is one of the strongest features in HNSCC and has hindered both the identification of specific biomarkers and the establishment of targeted therapies for this disease so far. To date, there is a paucity of reliable preclinical models, particularly those that can predict responses to immune CPI, as these models require an intact tumor microenvironment (TME). The “ideal” preclinical cancer model is supposed to take both the TME as well as tumor heterogeneity into account. Although HNSCC patients are frequently studied in clinical trials, there is a lack of reliable prognostic biomarkers allowing a better stratification of individuals who might benefit from new concepts of targeted or immunotherapeutic strategies. Emerging evidence indicates that cancer stem cells (CSCs) are highly tumorigenic. Through the process of stemness, epithelial cells acquire an invasive phenotype contributing to metastasis and recurrence. Specific markers for CSC such as CD133 and CD44 expression and ALDH activity help to identify CSC in HNSCC. For the majority of patients, allocation of treatment regimens is simply based on histological diagnosis and on tumor location and disease staging (clinical risk assessments) rather than on specific or individual tumor biology. Hence there is an urgent need for tools to stratify HNSCC patients and pave the way for personalized therapeutic options. This work reviews the current literature on novel approaches in implementing three-dimensional (3D) HNSCC in vitro and in vivo tumor models in the clinical daily routine. Stem-cell based assays will be particularly discussed. Those models are highly anticipated to serve as a preclinical prediction platform for the evaluation of stable biomarkers and for therapeutic efficacy testing.

Abstract PO-006: Single cell transcriptomic analysis of primary head and neck cancer stem cells

Abstract Despite advances in treatment modalities for head and neck squamous cell carcinoma (HNSCC) survival rates remain unchanged. Tumor heterogeneity within HNSCC as well as within the tumor microenvironment (TME) has been shown to be a major driver of treatment failure and poor outcomes. A key component of the TME and a potential key to deciphering tumor heterogeneity are cancer stem cells. While numerous studies have demonstrated the tumorigenicity and malignant potential of these cells, few studies have investigated their expression profiles. The aim of this study is to leverage single cell technology to investigate the expression profile of primary HNSCC cancer stem cells. We performed single cell sequencing of five primary HPV-negative head and neck cancers and two HNSCC cell lines that were presorted into the CSC (CD44high/ALDH+) and non-CSC (CD44low/ALDH-) phenotype. We are able to characterize an expression profile of the CSC TME niche and a novel CSC expression profile (DCN, C110rf96, KLF4, MEG3 and NR4A1) relative to non-CSC. We confirm this CSC profile is conserved across all five primary tumors. Together these data provide some of the first insights into CSC transcriptome, further define the CSC-TME niche, and identify conserved targets within HNSCC CSC as a means to decipher the challenges inherent to tumor heterogeneity. Citation Format: Molly E. Heft Neal, Stephanie The, John Henry J. Owen, Clifford Chang, Mark E.P. Prince, Arvind Rao, Steven B. Chinn. Single cell transcriptomic analysis of primary head and neck cancer stem cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-006.

Cancer Cell CD44 Mediates Macrophage/Monocyte-Driven Regulation of Head and Neck Cancer Stem Cells.

Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K-4EBP1-SOX2 activation and signaling regulate CSC properties, yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME, we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K-4EBP1-SOX2 pathway, possibly by modulating availability of hyaluronic acid (HA), the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships, and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC-derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions, respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K-4EBP1-SOX2 signaling and the CSC fraction, whereas CD44-VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo, targeting CD44 decreased PI3K-4EBP1-SOX2 signaling, tumor growth, and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally, a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes, respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals. SIGNIFICANCE: These findings establish a mechanistic link between tumor cell CD44, TAM, and CSC properties at the tumor-stroma interface that can serve as a vital area of focus for target and drug discovery.

Cancer stem cells enrichment with surface markers CD271 and CD44 in human head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) has a poor 5-year survival rate of 50%. One potential reason for treatment failure is the presence of cancer stem cells (CSCs). Several cell markers, particularly CD44, have been used to isolate CSCs. However, isolating a pure population of CSC in HNSCC still remains a challenging task. Recent findings show that normal oral stem cells were isolated using CD271 as a marker. Thus, we investigated the combined use of CD271 and CD44 to isolate an enriched subpopulation of CSCs, followed by their characterization in vitro, in vivo, and in patients' tissue samples. Fluorescent-activated cell sorting was used to isolate CD44+/CD271+ and CD44+/CD271- from two human HNSCC cell lines. Cell growth and self-renewal were measured with MTT and sphere/colony formation assays. Treatment-resistance was tested against chemotherapy (cisplatin and 5-fluorouracil) and ionizing radiation. Self-renewal, resistance, and stemness-related genes expression were measured with qRT-PCR. In vivo tumorigenicity was tested with an orthotopic immunodeficient mouse model of oral cancer. Finally, we examined the co-localization of CD44+/CD271+ in patients' tissue samples. We found that CD271+ cells were a subpopulation of CD44+ cells in human HNSCC cell lines and tissues. CD44+/CD271+ cells exhibited higher cell proliferation, sphere/colony formation, chemo- and radio-resistance, upregulation of CSCs-related genes, and in vivo tumorigenicity when compared to CD44+/CD271- or the parental cell line. These cell markers showed increased expression in patients with the increase of the tumor stage. In conclusion, using both CD44 and CD271 allowed the isolation of CSCs from HNSCC. These enriched CSCs will be more relevant in future treatment and HNSCC progression studies.

RETRACTED: LINC00355 Promotes Tumor Progression in HNSCC by Hindering MicroRNA-195-Mediated Suppression of HOXA10 Expression

Emerging evidence suggests that long non-coding RNAs (lncRNAs) are involved in the progression of head and neck squamous cell carcinoma (HNSCC). However, the specific role of LINC00355 in HNSCC remains elusive. Here, we identify the relationship between LINC00355 and the development of HNSCC through the interaction of LINC00355 with microRNA-195 (miR-195), which in turn targets homeoboxA10 (HOXA10). First, we identified differentially expressed lncRNAs and genes related to HNSCC. Next, the interaction among LINC00355, miR-195, and HOXA10 was identified. Subsequently, the expression of LINC00355 and miR-195 was altered to evaluate their effects on viability, invasion, migration, epithelial mesenchymal transition (EMT), and apoptosis of cancer stem cells (CSCs) in HNSCC. Finally, we assessed the ability of LINC00355 to alter tumor growth after HNSCC CSCs were injected into nude mice. Our findings indicate that LINC00355 and HOXA10 were highly expressed in HNSCC, while miR-195 was poorly expressed. CSCs with upregulated aldehyde dehydrogenase 1 (ALDH-1) were sorted. Silencing LINC00355 in these cells led to increased miR-195 expression and a reduction in HOXA10 expression, which inhibited viability, invasion, migration, and EMT and promoted apoptosis of CSCs. Silencing LINC00355 in vivo also led to decreased tumor growth. Our study provides evidence that LINC00355 acts as a miR-195 sponge to promote viability, invasion, migration, and EMT and inhibit apoptosis of CSCs by upregulating HOXA10, suggesting that LINC00355 represents a potential therapeutic target in the treatment of HNSCC.

Metformin Results in Diametrically Opposed Effects by Targeting Non-Stem Cancer Cells but Protecting Cancer Stem Cells in Head and Neck Squamous Cell Carcinoma.

Cancer stem cells (CSCs) have been shown as a distinct population of cancer cells strongly implicated with resistance to conventional chemotherapy. Metformin, the most widely prescribed drug for diabetes, was reported to target cancer stem cells in various cancers. In this study, we sought to determine the effects of metformin on head and neck squamous cell carcinoma (HNSCC). CSCs and non-stem HNSCC cells were treated with metformin and cisplatin alone, and in combination, and cell proliferation levels were measured through MTS assays. Next, potential targets of metformin were explored through computational small molecule binding analysis. In contrast to the reported effects of metformin on CSCs in other cancers, our data suggests that metformin protects HNSCC CSCs against cisplatin in vitro. Treatment with metformin resulted in a dose-dependent induction of the stem cell genes CD44, BMI-1, OCT-4, and NANOG. On the other hand, we observed that metformin successfully decreased the proliferation of non-stem HNSCC cells. Computational drug–protein interaction analysis revealed mitochondrial complex III to be a likely target of metformin. Based on our results, we present the novel hypothesis that metformin targets complex III to reduce reactive oxygen species (ROS) levels, leading to the differential effects observed on non-stem cancer cells and CSCs.

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

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) affects 40,000 patients annually and is associated with <50% 5-year survival despite aggressive therapy. Cancer stem cells (CSC) have been reported to contribute to tumor aggressiveness and therapeutic resistance in several tumor types, including HNSCC. Very little is known about the molecular characterization of CSCs in HNSCC. High expression of Doublecortin-like Kinase 1 (DCLK1), a microtubule associated protein that has been previously reported to mark quiescent CSCs of the colon and pancreas, is correlated with poor prognostic outcomes according to TCGA databases. We hypothesized that DCLK1 expression confers stem cell properties and hence is a target for therapy in HNSCC. Methods: HNSCC tumor micro array was stained with DCLK1 to evaluate DCLK1 expression in HNSCC compared to normal mucosal tissue. Additionally, HNSCC cells were screened for expression of DCLK1 to assess expression. HN5 and FaDu cells were treated with DiFiD (0-10µM) and analyzed for cell viability in 2-dimensional and 3-dimensional cultures. Expression for markers of apoptosis, stemness, and cell cycle regulation were determined by western blotting. Furthermore, Caspase 3/7 assay and Annexin V/Pi staining were performed to confirm induction of apoptosis. Thermal shift binding assays (CETSA) were performed to confirm DiFiD:DCLK1 interaction. Lastly, HN5 tumor xenograft toxicity studies were performed in Balb c/ nude FOXN1 mice to determine the antitumor efficacy of DiFiD in vivo. Results: We identified expression of DCLK1 in HNSCC cell lines and patient samples. IHC demonstrated increased expression of DCLK1 in HNSCC compared to normal mucosal tissue. Furthermore, DCLK1 was upregulated in HNSCC cells lines. CETSA assay demonstrated increased thermal stability of DCLK1 following incubation with DiFiD, suggesting DiFiD binds to DCLK1. There was a dose and time dependent reduction in cell viability both in 2-dimensional and 3-dimensional culture models following treatment with DiFiD in both cell lines. DiFiD induced caspase mediated apoptosis following G2/M cell cycle arrest. DiFiD significantly inhibited tumor growth in vivo, as confirmed by reduction in tumor volume and weight. DiFiD also showed little off target toxicity, as mouse weights were not significantly altered following DiFiD treatment. Conclusions: Taken together, these data demonstrate that DiFiD has potent anti-cancer activity and therefore may serve as a potential novel therapy for the treatment of HNSCC. Citation Format: David Standing, Jake New, Prasad Dandawate, Dharmalingam Subramaniam, Vusala Snyder, Jonathan Enders, Afreen Sayed, Shrikant Anant, Sufi M. Thomas. 3,5-bis(2,4-difluorobenzylidene)-4-piperidone, a novel compound potently inhibits HNSCC through a DCLK1 mediated mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5862.

RXRα-enriched cancer stem cell-like properties triggered by CDDP in head and neck squamous cell carcinoma (HNSCC).

Retinoid X receptor-α (RXRα) is a kind of nuclear receptor and is a target of cancer prevention and treatment in various types of cancers. Cancer stem cells (CSCs) are regarded as the main cause of carcinoma metastasis, tumor recurrence and chemotherapy resistance. So far, the mechanism how RXRα regulates CSCs remains unknown. In the present study, we found that RXRα was upregulated in head and neck squamous cell carcinoma (HNSCC) tissues and the enriched HNSCC CSCs. Overexpression of RXRα was able to expand the CSC-like properties in HNSCC cells, whereas knockdown of RXRα could repress the stemness respectively. Meanwhile, low doses of cisplatin (CDDP) increased the CSC-like properties and RXRα expression in HNSCC cells. Also, Wnt signaling pathway played a significant role in CDDP-induced CSCs. Simultaneously, curcumin, a plant polyphenol, which is an effective anticancer compound, exhibited an inhibitory effect in the HNSCC CSCs induced by CDDP in vitro and in vivo. Via inhibition of RXRα, curcumin suppressed CSC-like phenotypes induced by CDDP. These findings may suggest a novel mechanism for HNSCC treatment.

Novel C-Terminal Heat Shock Protein 90 Inhibitors (KU711 and Ku757) Are Effective in Targeting Head and Neck Squamous Cell Carcinoma Cancer Stem cells

Advanced head and neck squamous cell carcinoma (HNSCC) remains a therapeutic challenge due to the development of therapy resistance. Several studies have implicated the development of cancer stem cells as a possible mechanism for therapy resistance in HNSCC. Heat shock protein 90’s (Hsp90’s) molecular chaperone function is implicated in pathways of resistance in HNSCC. Therefore, in the present study, we investigated the efficacy of novel C-terminal Hsp90 inhibitors (KU711 and KU757) in targeting HNSCC cancer stem cells (CSCs). Treatment of HNSCC human cell lines MDA1986, UMSCC 22B, and UMSCC 22B cisplatin-resistant cells with the KU compounds indicated complete blockage of self-renewal for the resistant and parent cell lines starting from 20 μM KU711 and 1 μM KU757. Dose-dependent decrease in the cancer stem cell markers CD44, ALDH, and CD44/ALDH double-positive cells was observed for all cell lines after treatment with KU711 and KU757. When cells were treated with either drug, migration and invasion were downregulated greater than 90% even at the lowest concentrations of 20 μM KU711 and 1 μM KU757. Western blot showed >90% reduction in client protein “stemness” marker BMI-1 and mesenchymal marker vimentin, as well as increase in epithelial marker E-cadherin for both cell lines, indicating epithelial to mesenchymal transition quiescence. Several CSC-mediated miRNAs that play a critical role in HNSCC therapy resistance were also downregulated with KU treatment. In vivo, KU compounds were effective in decreasing tumor growth with no observed toxicity. Taken together, these results indicate that KU compounds are effective therapeutics for targeting HNSCC CSCs.

Inhibitor of DNA binding 2 is a novel therapeutic target for stemness of head and neck squamous cell carcinoma

Background:Head and neck squamous cell carcinomas (HNSCCs) are highly lethal epithelial tumours containing self-renewal cancer stem cells (CSCs). CSCs in HNSCCs are strongly associated with tumour initiation, invasion, and chemoradiation resistance. However, the important factors regulating stemness in HNSCCs remain unclear. Here, we investigated the molecular roles and clinical significance of inhibitor of DNA binding 2 (Id2) protein to determine if it constitutes a novel therapeutic target for ablating HNSCC cells with stemness.Methods:We performed in vitro and in vivo studies of Id2 function and its effects on stemness using HNSCC cells. We also examined whether Id2 expression could be used as a prognostic indicator through immunohistochemical staining of 119 human HNSCC tumours.Results:Expression of Id2 was higher in HNSCC cells with stemness compared with differentiated HNSCC cells. Overexpression of Id2 increased proliferation, self-renewal, and expression of the putative stemness marker CD44 in HNSCC cells in vitro and in vivo. In contrast, silencing of Id2 using short hairpin RNA attenuated the stemness phenotype of HNSCC cells by reducing self-renewal, CD44 expression, cisplatin chemoresistance, and xenograft tumourigenicity. Most importantly, increased expression of Id2 was closely associated with poorer post-treatment survival rates in HNSCC patients.Conclusions:Inhibitor of DNA binding2 represents a novel and promising therapeutic target for treating and improving the clinical outcomes for patients with HNSCC.