Oral Cancer Stem Research Articles

Abstract 6306: Genomic analysis markers of stemness in oral cancer progression

Abstract Introduction: In clinical cancer treatment there is an continuing effort to identify limited gene sets from high dimensional data to predict risk, treatment response, etc. Oral carcinogenesis is a multistep process of mutations, disordered growth, and the development of invasion. In the oral cancer milieu, inflammation and stemness has been demonstrated to be cooperative during carcinogenesis. We hypothesized that differences in inflammation, stemness, and EMT might distinguish lesion and adjacent normal mucosa in our RNA Seq data on preneoplastic lesions. Once candidate genes are identified they might be applied to other oral mucosal specimens to identify risk for malignancy development. Materials and Methods: We collected lesion and adjacent normal mucosal tissues from 16 subjects with high risk leukoplakia and early oral cancer (T1N0 and T2N0). Sequencing libraries were generated using the Illumina Truseq Stranded mRNA library prep kit. Sequencing was performed on an Illumina Hiseq 3000 instrument with paired-end 75bp read and sequenced to an overall depth of 30 million reads per sample. Results: After developing a biospecimen collection procedure that functions in the senior author’s clinic flow, we were able to isolate sequenceable total RNA from 100% of the specimens we collected. We identified several hundred differentially expressed genes between lesion and adjacent normal. With regard to stemness, inflammation, and EMT we multiple candidate genes that could be incorporated into a limited gene set for further studies. Aldehyde dehydrogenase family members were of particular interest in the stemness candidate genes, for example. ALD 1A1 and ALD 3B1, were significantly upregulated in lesion versus normal (P< 0.02). Conclusions: We conclude there are differentially expressed inflammatory, EMT, and stemness in oral preneoplastic specimens. We have identified multiple candidate genes that could be assembled into a limited gene panel (12-24 genes) for further studies of oral cancer risk in mucosal lesions Citation Format: Gretchen Unger, Beverly G. Wuertz, Patrick Gaffney, Frank G. Ondrey. Genomic analysis markers of stemness in oral cancer progression [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 6306.

Application of Random Forest and data integration identifies three dysregulated genes and enrichment of Central Carbon Metabolism pathway in Oral Cancer

BackgroundStudies of epigenomic alterations associated with diseases primarily focus on methylation profiles of promoter regions of genes, but not of other genomic regions. In our past work (Das et al. 2019) on patients suffering from gingivo-buccal oral cancer – the most prevalent form of cancer among males in India – we have also focused on promoter methylation changes and resultant impact on transcription profiles. Here, we have investigated alterations in non-promoter (gene-body) methylation profiles and have carried out an integrative analysis of gene-body methylation and transcriptomic data of oral cancer patients.MethodsTumor and adjacent normal tissue samples were collected from 40 patients. Data on methylation in the non-promoter (gene-body) regions of genes and transcriptome profiles were generated and analyzed. Because of high dimensionality and highly correlated nature of these data, we have used Random Forest (RF) and other data-analytical methods.ResultsIntegrative analysis of non-promoter methylation and transcriptome data revealed significant methylation-driven alterations in some genes that also significantly impact on their transcription levels. These changes result in enrichment of the Central Carbon Metabolism (CCM) pathway, primarily by dysregulation of (a) NTRK3, which plays a dual role as an oncogene and a tumor suppressor; (b) SLC7A5 (LAT1) which is a transporter dedicated to essential amino acids, and is overexpressed in cancer cells to meet the increased demand for nutrients that include glucose and essential amino acids; and, (c) EGFR which has been earlier implicated in progression, recurrence, and stemness of oral cancer, but we provide evidence of epigenetic impact on overexpression of this gene for the first time.ConclusionsIn rapidly dividing cancer cells, metabolic reprogramming from normal cells takes place to enable enhanced proliferation. Here, we have identified that among oral cancer patients, genes in the CCM pathway – that plays a fundamental role in metabolic reprogramming – are significantly dysregulated because of perturbation of methylation in non-promoter regions of the genome. This result compliments our previous result that perturbation of promoter methylation results in significant changes in key genes that regulate the feedback process of DNA methylation for the maintenance of normal cell division.

Abstract 501: Aldh2 mutation promotes oral and esophageal cancer stem cells via autophagy-mediated redox homeostasis

Abstract Introduction: Heavy alcohol (EtOH) drinking and tobacco smoking increase the risk for upper digestive (e.g. oral and esophageal) squamous cell carcinomas (SCC) in individuals with polymorphic mutations in aldehyde dehydrogenase 2 (Aldh2). Mutant Aldh2E487K protein delays mitochondrial clearance of acetaldehyde, the chief metabolite of EtOH and constituents of tobacco smoke, aka a major human carcinogen. The role of Aldh2 in tumor biology remains elusive. Methods: Single cell-derived three-dimensional (3D) organoids were generated from murine oral and esophageal preneoplastic and SCC lesions carrying wild-type Aldh2WT or mutant Aldh2E487K as well as human SCC cell lines with Aldh2E487K, the latter subjected to xenograft transplantation in immunodeficient mice. 3D organoids and xenograft tumors were analyzed by morphology and flow cytometry to determine CD44, autophagy (cyto-ID), proliferation (EdU), and reactive oxygen species (ROS) following EtOH exposure ex vivo (0.1-2% EtOH) or in vivo (10% EtOH in drinking water) along with or without inhibitors of alcohol metabolism (4-methylpyrazole; 4MP, siRNA against Adh1B and Cyp2E1), and autophagy flux (chloroquine; CQ or siRNA against Atg7). Flow cytometry was performed to determine cancer stem-like cells with high CD44 expression (CD44H cells). Results: EtOH stimulated xenograft tumor growth and increased highly proliferative intratumoral CD44H cells with increased autophagy. EtOH-stimulated tumor growth and induction of CD44H cells were sharply suppressed by concurrent 4MP treatment, indicating that EtOH is directly metabolized in SCC cells to promote tumor growth via CD44H cells. These EtOH-induced effects were recapitulated in 3D organoids where Aldh2E487K augmented EtOH-mediated induction of CD44H cells and organoid formation with increased cellular atypia. EtOH induced mitochondrial superoxide and ROS-dependent autophagy activation in CD44H cells. Inhibition of autophagy prevented EtOH from enriching CD44H cells in 3D organoids, suggesting that autophagy may limit alcohol-induced oxidative stress as a cytoprotective mechanism. Conclusions: These studies provide not only mechanistic insights into the role of Aldh2 mutation in oral and esophageal SCC pathogenesis but also establish the utility of the 3D organoid system to test the functional interplay between genetic and environmental factors which may subsequently be targeted for pharmacological intervention in the setting of personalized medicine. Citation Format: Masataka Shimonosono, Takashi Kijima, Hisatsugu Maekawa, Satoshi Takada, Koji Tanaka, Varun Sahu, Manti Guha, Andres J. Klein-Szanto, J. Alan Diehl, Adam J. Bass, Anil K. Rustgi, Elizabeth Philipone, Angela J. Yoon, Hiroshi Nakagawa. Aldh2 mutation promotes oral and esophageal cancer stem cells via autophagy-mediated redox homeostasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 501.

Abstract 2681: Oral micro biome enhances stemness in oral cancer cells by activating Toll like receptor signaling

Abstract Background: The resistance and progression of cancers after chemotherapy to invasive and metastatic stages accounts for the overwhelming majority of cancer deaths. Recent studies suggest, microbiomes can induce a cascade of host events to either support or inhibit tumor growth. Specially, in oral cancer, chemotherapy treatment may alter the oral microbial flora, which may favor or inhibit tumor growth. Hence, it is importantl to develop novel experimental approaches to study the role of oral microbial flora in oral cancer stemness (self-renewal and undifferntiated state of cancer stem cells). Importantly, patients in developing area, including Assam, where KaviKrishna laboratory is located, may have distinct oral microbial flora that could favor oral cancer growth. Hence, it is important to include patients from developing countries for such studies. Our previous research showed that chemotherapy ehances stemenss in many cancer cell types, including oral squamous cell carcinoma cell line SSC-25. The stemness switch is characterized by enhanced expression of stemness associated genes including Nanog, Lin28A/B, Oct-4, MYC, HIF-2alpha and inflammation associated genes including Toll like receptor (TLR) 2/4. Here we investigated the role of oral microbiomes in the TLR mediated stemness switch of oral cancer cells. Methods: SCC-25 oral cancer cell line was treated with bacterial product lipopoly saccharide (LPS), and the stemness switch evaluated by isolation of ABCG2+ cells and expression of stemness associated genes by these cells. Capacity of interaction of tumor stromal cells with mesenchymal stem cells was also evaluated. Additionally, we obtained sputum from oral cancer subjects undergoing chemotherapy. The patients were from the Kamrup district of Assam, where KaviKrishna laboratory is located. The sputum was processed and then added to the culture medium of SCC-25 cells. These post-sputum treated SCC-25 cells were subjected to phenotypic stemness switch analysis. Results: We found, LPS and sputum treatment led to the enhanced stemness of ABCG2+ cells, including the high expression of TLR2/4, MYC, Nanog, Sox-2, and HIF-2alpha. Importantly, sputum derived from oral cancer subjects under remission showed inhibitory activity on ABCG2+ cell self-renewal. In contrast, sputum obtained from oral cancer subjects with relapse showed enhanced stemness of ABCG2+ cells, and also increased tumorigenic potential. The post-sputum treated ABCG2+ cells exhibited high expression of TLR2/4 and associated increase of HIF-2alpha and MYC transcriptional activity. The sputum treated with broad spectrum antibiotic ciprofloxacin did not enhance the stemness and TLR2/4 signaling of SCC-25 cells. Conclusion: These results indicate that oral microbiomes may differentially influence the stemness of oral cancer cells. We also conclude that live bacteria present in the sputum may be required to enhance stemness in a TLR2/4 dependent manner. Note: This abstract was not presented at the meeting. Citation Format: Joyeeta Talukdar, Rashmi Bhuyan, Bidisha Pal, Sorra Sandhya, Hong Li, Seema Bhuyan, Sukanya Garhyan, Debabrat Baishya, Anupam Sarma, Jyotirmoy Phukan, Amal Kataki, Bikul Das. Oral micro biome enhances stemness in oral cancer cells by activating Toll like receptor signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2681. doi:10.1158/1538-7445.AM2017-2681

Abstract 4380: CD133/prominin-1 modulates epithelial-mesenchymal transition, stemness and tumorigenicity of oral squamous cell carcinoma-derived cancer initiating

Abstract Background: Oral squamous carcinoma cell (OSCC) is a lethal cancer with clinical, pathological, phenotypical and biological heterogeneity. Tumor initiating cells (TICs), which are responsible for tumor growth and coupled with epithelial-mesenchymal transition (EMT), have been identified. We have also identified the subpopulation of oral cancer derived tumor initiating cells (OC-TICs), and observed the upregulation of CD133 in OC-TICs. Others demonstrate that CD133 is phosphorylated by Src and Fyn tyrosine kinases. However, the molecular function of CD133 in OC-TICs remains unclear. Herein we determined the critical role of CD133, an important TICs marker, in the maintenance of stemness characteristics and tumorigenic phenotype of OC-TICs. Methods: Stable overexpression and knockdown of CD133 expression in OSCCs and OC-TICs was achieved by lentiviral-mediated system. Consequently, we elucidated the epithelial-mesenchymal transition, the stemness properties and tumorigenicity of OSCCs and OC-TICs with CD133 down-regulation or overexpression, respectively. Consequently, we investigated the role of Src and and Fyn tyrosine kinases in CD133 mediated signaling pathway. Results: Stable over-expression of CD133 induced epithelial-mesenchymal transition, enhanced tumorigenicity and enhanced the TICs properties in OSCCs. In contrast, lentiviral knockdown of CD133 significantly reduced the self-renewal ability, side population cells, and stemness genes in OC-TICs. Additionally, down-regulation of CD133 enhanced the differentiation capability but inversely diminished “stemness” gene expression of OC-TICs. Of note, knockdown of CD133 lessened tumorigenicity of OC-TICs both in vitro and in vivo. Furthermore, over-expression of CD133 increased phosphorylation of Src and Erk1/2. Conclusion: We showed that CD133 contributes to the epithelial-mesenchymal transition and the maintenance of stemness and tumorigenicity of OC-TICs. In addition, Src and Erk1/2 activation was involved in CD133 modulating phenotype. Overall, silencing CD133 might be a potential therapeutic target for OSCC by eliminating TICs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4380. doi:10.1158/1538-7445.AM2011-4380