Ganesan Arunkumar

Oral squamous cell carcinoma: microRNA expression profiling and integrative analyses for elucidation of tumourigenesis mechanism

BackgroundThe advantages and utility of microRNAs (miRNAs) as diagnostic and prognostic cancer markers is at the vanguard in recent years. In this study, we attempted to identify and validate the differential expression of miRNAs in oral squamous cell carcinoma (OSCC), to correlate their expression with the clinico-pathological profile of tumours and to identify the signaling pathways through which the aberrantly expressed miRNAs effect tumourigenesis.MethodsmiRCURY LNA™ array with probes specific to 1168 miRNAs and TaqMan assays specific for 10 miRNAs was employed to evaluate and validate miRNA expression in a discovery cohort (n = 29) and validation cohort (n = 61) of primary OSCC tissue specimens, respectively. A computational pipeline with sequential integration of data from miRTarBase, CytoScape, UniProtKB and DIANA-miRPath was utilized to map the target genes of deregulated miRNAs and associated molecular pathways.ResultsMicroarray profiling identified 46 miRNAs that were differentially expressed in OSCC. Unsupervised clustering demonstrated a high degree of molecular heterogeneity across the tumour samples as the clusters did not represent any of their clinico-pathological characteristics. The differential expression of 10 miRNAs were validated by RT-qPCR (let-7a, let-7d, let-7f and miR-16 were downregulated while miR-29b, miR-142-3p, miR-144, miR-203, and miR-223 were upregulated in OSCC; the expression of miR-1275 was variable in tumours, with high levels associated to regional lymph node invasion; additionally, miR-223 exhibited an association with advanced tumour stage/size). In silico analyses of the experimentally confirmed target genes of miRNAs revamp the relationship of upregulated miRNAs with tumour suppressor genes and of downregulated miRNAs with oncogenes. Further, the differentially expressed miRNAs may play a role by simultaneously activating genes of PI3K/Akt signaling on one hand and by repressing genes of p53 signaling pathway on the other.ConclusionsThe identified differentially expressed miRNAs and signaling pathways deregulated in OSCC have implications for the development of novel therapeutic strategies. To the best of our knowledge, this is the first report to show the association of miR-1275 with nodal invasion and the upregulation of miR-144 in OSCC.

Expression profiling of long non-coding RNA identifies linc-RoR as a prognostic biomarker in oral cancer:

Oral squamous cell carcinoma is the most aggressive cancer that is associated with high recurrence, metastasis, and poor treatment outcome. Dysregulation of long non-coding RNAs has been shown to promote tumor growth and metastasis in several cancers. In this study, we investigated the expression of 11 selected long non-coding RNAs that are associated with cell proliferation, metastasis, and tumor suppression in oral squamous cell carcinomas and normal tissues by quantitative real-time polymerase chain reaction. Out of the 11 long non-coding RNAs profiled, 9 were significantly overexpressed in tumors with tobacco chewing history. Moreover, the long non-coding RNA profile was similar to the head and neck cancer datasets of The Cancer Genome Atlas database. Linc-RoR, a regulator of reprogramming, implicated in tumorigenesis was found to be overexpressed in undifferentiated tumors and showed strong association with tumor recurrence and poor therapeutic response. In oral squamous cell carcinomas, for the first time, we observed linc-RoR overexpression, downregulation of miR-145-5p, and overexpression of c-Myc, Klf4, Oct4, and Sox2, suggesting the existence of linc-RoR-mediated competing endogenous RNA network in undifferentiated tumors. Taken together, this study demonstrated the association of linc-RoR overexpression in undifferentiated oral tumors and its prognostic value to predict the therapeutic response.

LncRNA OIP5-AS1 is overexpressed in undifferentiated oral tumors and integrated analysis identifies as a downstream effector of stemness-associated transcription factors

Long non-coding RNAs (lncRNAs) play an important role in the regulation of key cellular processes in early development and cancer. LncRNA Oip5-as1 facilitates stem cell self-renewal in mouse by sponging mmu-miR-7 and modulating NANOG level, yet its role in cancer is less understood. We analyzed OIP5-AS1 expression in oral tumors and in TCGA datasets. We observed overexpression of OIP5-AS1 in oral tumors (P < 0.001) and in tumors of epithelial origin from TCGA. OIP5-AS1 expression was strongly associated with undifferentiated tumors (P = 0.0038). In silico analysis showed miR-7 binding site is conserved in mouse and human OIP5-AS1. However, human NANOG 3′-UTR lost the binding site for hsa-miR-7a-3. Therefore, we screened for other miRNAs that can be sponged by OIP5-AS1 and identified six potential miRNAs and their downstream target genes. Expression analysis showed downregulation of miRNAs and upregulation of downstream target genes, particularly in undifferentiated tumors with high-level of OIP5-AS1 suggesting OIP5-AS1 could post-transcriptionally modulate the downstream target genes. Further, systematic epigenomic analysis of OIP5-AS1 promoter revealed binding motifs for MYC, NANOG and KLF4 suggesting that OIP5-AS1 could be transactivated by stemness-associated transcription factors in cancer. OIP5-AS1 overexpression in undifferentiated oral tumors may be suggestive of enhanced cancer stemness, and consequently, poor clinical outcome.

Dysregulation of miR-200 family microRNAs and epithelial-mesenchymal transition markers in oral squamous cell carcinoma

MicroRNAs (miRNAs) are reported to function as a major component in the cellular signaling circuit, which regulates epithelial-mesenchymal transition (EMT). Dysregulation of the microRNA-200 (miR-200) family and EMT-associated genes enables tumor metastasis and resistance to therapy. The present study profiled miR-200 family members miR-200a, miR-200b, miR-200c, miR-141 and miR-429, and also several EMT-regulatory genes including zinc finger E-box-binding homeobox (ZEB)1, ZEB2, epithelial cadherin and vimentin in 40 oral primary tumors in order to understand their role(s) in oral squamous cell carcinoma (OSCC). The reverse transcription-quantitative polymerase chain reaction was used to analyze each sample. Results demonstrated a significant downregulation of miR-200 family members in tumors with a history of tobacco chewing/smoking (P<0.0006, P=0.0467, P=0.0014, P=0.0087 and P=0.0230, respectively) and undifferentiated pathology (miR-200a, P=0.0067; miR-200c, P=0.0248). EMT markers ZEB2 (P=0.0451) and vimentin (P=0.0071) were significantly upregulated in the oral tumors. Furthermore, ZEB2 antisense RNA1 was overexpressed in 50% of OSCC samples (P=0.0075). EMT-regulatory genes did not exhibit any association with clinical outcome. The present study also analyzed the expression of EMT-regulatory genes in 523 head and neck squamous cell carcinoma (HNSCC) samples from The Cancer Genome Atlas (TCGA) database, and the association with treatment outcome. Analysis of TCGA datasets also demonstrated no significant association in the expression of EMT markers with disease recurrence and treatment outcome. The results of the present study revealed dysregulation of miR-200 family miRNAs and EMT-regulatory genes in OSCC without any significant effect on treatment outcome.

Absence of the TP53 Poly-A Signal Sequence Variant rs78378222 in Oral, Cervical and Breast Cancers in South India

With recent advancements in genomics, rare genetic variants attract the attention of cancer researchers as they represent one among the many diverse mechanisms that determine cancer risk and outcome. One such rare variant is the Single Nucleotide Polymorphism rs78378222 that alters the polyadenylation signal (AATAAA to AATACA) of TP53. Initially identified in Caucasian population, the variant ‘C’ allele was associated with an increased risk for prostate cancer, brain cancer and colorectal adenoma but not with colorectal cancer, breast cancer and melanoma (Stacey et al., 2011). Further, the risk allele impaired the proper termination of TP53 resulting in ‘cancer promoting’ longer mRNA transcripts that hindered p53 expression and its downstream functions in apoptosis (Stacey et al., 2011; Li et al., 2013). Simultaneously it was reported that rs78378222 increased the risk of esophageal cancer in Han Chinese population (Zhou et al., 2012). A similar study based on Caucasian population, showed that the rare variant was associated with risk for glioma and also significantly improved survival in patients (Egan et al., 2012). However, the association to survival was not observed in glioma patients of Northern European ancestry although it increased glioma risk (Enciso-Mora et al., 2013). A recent study showed that the heterozygous ‘AC’ genotype was not associated with melanoma and lung cancer, but had a protective role against squamous cell carcinoma of head and neck in non-Hispanic whites (Guan et al., 2013). Given the multitude of effects of rs78378222 in cancer patients of different ethnicity and on account of paucity of information on ‘C’ allele frequency in Indian population, we genotyped 439 genomic DNA samples from oral (108), cervical (96) and breast (235) cancer patients of Indian ethnicity. Blood samples were collected from Government Royapettah Hospital & Government Kasturba Gandhi Hospital for Women and Children, Chennai and clinical information from subjects was undertaken with informed consent and relevant ethical review board approval was accorded. We adopted the PCR-RFLP methodology described by Zhou et al (2012), with a single base mismatch in reverse primer creating a Hind III recognition site when the wild type A allele is present (Figure 1). The RFLP results were confirmed by direct sequencing of 10% of case/control samples chosen at random (Figure 2). We found that the ‘C’ allele was totally absent in cancer samples. We also genotyped 504 healthy controls LETTER to the EDITOR

Comprehensive analysis of aberrantly expressed lncRNAs and construction of ceRNA network in gastric cancer

Gastric cancer remains fifth most common cancer often diagnosed at an advanced stage and is the second leading cause of cancer-related death worldwide. Long non-coding RNAs (lncRNAs) involved in various cellular pathways are essential for tumor occurrence and progression and they have high potential to promote or suppress the expression of many genes. In this study, we profiled 19 selected cancer-associated lncRNAs in thirty gastric adenocarcinomas and matching normal tissues by qRT-PCR. Our results showed that most of the lncRNAs were significantly upregulated (12/19). Further, we performed bioinformatic screening of miRNAs that share common miRNA response elements (MREs) with lncRNAs and their downstream mRNA targets. The prediction identified three microRNAs (miR-21, miR-145 and miR-148a) and five gastric cancer-specific target genes (EGFR, KLF4, DNMT1 and AGO4) which also showed strong correlation with lncRNAs in regression analysis. Finally, we constructed an integrated lncRNA-miRNA-mRNA interaction network of the candidate genes to understand the post-transcriptional gene regulation. The ceRNA network analysis revealed that the differentially regulated miR-21 and miR-148a were playing as central candidates coordinating sponging activity of the lncRNAs analyzed (H19, TUG1 and MALAT1) in this study and the overexpression of H19 and miR-21 could be a signature event of gastric tumorigenesis that could serve as prognostic indicators and therapeutic targets.

Absence of the frequently reported PIK3CA, CASP8, and NOTCH1 mutations in South Indian oral cancers

OBJECTIVES Somatic mutations of the PIK3CA, CASP8, and NOTCH1 have been frequently detected in various human cancers. Our study aimed to analyze the mutational status of these genes in South Indian oral cancers. SUBJECTS AND METHODS We performed mutational analysis of the PIK3CA (exons 9 and 20), CASP8 (exon 9), and NOTCH1 (exons 5, 6, 7, 8, and 9) genes in 96, 48, and 44 oral cancer samples, respectively. All the specified exons were PCR (polymerase chain reaction)-amplified and directly sequenced by Sanger sequencing. RESULTS PIK3CA gene mutations were not found; however, a synonymous single nucleotide polymorphism (SNP) [rs17849079] was observed frequently [35/96 (36.4%)] in oral cancer samples. Further, no mutations were detected in the CASP8 gene, but observed a frequent [32/48 (66.6%)] SNP [rs1045487] in the oral cancer samples. We did not detect any mutation in the NOTCH1 gene (exons 5, 6, 7, 8, and 9) in all the [0/44] analyzed oral cancer samples. CONCLUSIONS This is the first study that reports the status of the PIK3CA, CASP8, and NOTCH1 mutations in South Indian oral cancer samples. Our study suggests that either mutations in these genes are uncommon in South Indian oral cancer samples or likely other genes in this pathway might be mutated.

Genotyping of CYP2C9 and VKORC1 polymorphisms predicts south Indian patients with deep vein thrombosis as fast metabolizers of warfarin/acenocoumarin

Deep vein thrombosis (DVT) is a life-threatening disease. Warfarin and acenocoumarol are anticoagulants used to treat DVT and vary among individuals in terms of treatment response/toxicity. Single nucleotide polymorphisms (SNPs) in CYP2C9 and VKORC1 play a role in the pharmacokinetics and dynamics of warfarin and acenocoumarol and they determine the efficacy of treatment by controlling drug clearance in treated individuals. The aim of the current study was to genotype the critical SNPs of CYP2C9 and VKORC1 genes in a south Indian population in order to understand the metabolizer phenotype of patients with DVT. CYP2C9 (rs1799853, rs1057910, rs1057909, rs28371686) and VKORC1 (rs9923231) SNPs were genotyped in 124 cases of DVT. Genomic regions of these SNPs from genomic DNA were amplified with PCR and directly sequenced using Sanger sequencing except for the SNP rs1799853, which was detected using Sau96I restriction endonuclease-based digestion of variant alleles. Among south Indian patients with DVT, 6.5% (8/124) had the rs1799853 SNP of CYP2C9 and 11% (14/124) had the rs1057910 SNP while 16% (20/124) had the rs9923231 SNP of VKORC1 which were associated with the response to warfarin treatment. None of the patients tested positive for poor drug metabolizing genotypes of the CYP2C9 gene and only 1.6% of the south Indian population was sensitive to warfarin treatment. Genotyping results suggest that a relatively greater amount of the therapeutic drug is required to achieve/maintain the international normalized ratio (INR) in south Indian patients with DVT.

Erratum to: TERT promoter hot spot mutations are frequent in Indian cervical and oral squamous cell carcinomas.

The complete Acknowledgement text is shown below. Acknowledgments We thank Dr. Sindhuja, Mrs. Arivazhagi (Arignar Anna Memorial Cancer Hospital & Research Institute, Kanchipuram), and Dr. V. Rajalakshmi (Institute of Obstetrics & Gynaecology and Government General Hospital forWomen and Children, Chennai) for clinical assessment and sample collection. We, VV, KA, MM, AKD, and GA gratefully acknowledge the Government of India’s Council of Scientific and Industrial Research (CSIR) and University Grant Commission (UGC), respectively, for providing research fellowships. We also thank DST-FIST and UGC-SAP infrastructure facilities. SR gratefully acknowledges the research felowship provided by University Grants commission, Government of India.