Roshni Roy

A quest for miRNA bio-marker: a track back approach from gingivo buccal cancer to two different types of precancers.

Deregulation of miRNA expression may contribute to tumorigenesis and other patho-physiology associated with cancer. Using TLDA, expression of 762 miRNAs was checked in 18 pairs of gingivo buccal cancer-adjacent control tissues. Expression of significantly deregulated miRNAs was further validated in cancer and examined in two types of precancer (leukoplakia and lichen planus) tissues by primer-specific TaqMan assays. Biological implications of these miRNAs were assessed bioinformatically. Expression of hsa-miR-1293, hsa-miR-31, hsa-miR-31* and hsa-miR-7 were significantly up-regulated and those of hsa-miR-206, hsa-miR-204 and hsa-miR-133a were significantly down-regulated in all cancer samples. Expression of only hsa-miR-31 was significantly up-regulated in leukoplakia but none in lichen planus samples. Analysis of expression heterogeneity divided 18 cancer samples into clusters of 13 and 5 samples and revealed that expression of 30 miRNAs (including the above-mentioned 7 miRNAs), was significantly deregulated in the cluster of 13 samples. From database mining and pathway analysis it was observed that these miRNAs can significantly target many of the genes present in different cancer related pathways such as “proteoglycans in cancer”, PI3K-AKT etc. which play important roles in expression of different molecular features of cancer. Expression of hsa-miR-31 was significantly up-regulated in both cancer and leukoplakia tissues and, thus, may be one of the molecular markers of leukoplakia which may progress to gingivo-buccal cancer.

Association between risk of oral precancer and genetic variations in microRNA and related processing genes

BackgroundMicroRNAs have been implicated in cancer but studies on their role in precancer, such as leukoplakia, are limited. Sequence variations at eight miRNA and four miRNA processing genes were studied in 452 healthy controls and 299 leukoplakia patients to estimate risk of disease.ResultsGenotyping by TaqMan assay followed by statistical analyses showed that variant genotypes at Gemin3 and mir-34b reduced risk of disease [OR = 0.5(0.3–0.9) and OR = 0.7(0.5–0.9) respectively] in overall patients as well as in smokers [OR = 0.58(0.3–1) and OR = 0.68(0.5–0.9) respectively]. Among chewers, only mir29a significantly increased risk of disease [OR = 1.8(1–3)]. Gene-environment interactions using MDR-pt program revealed that mir29a, mir34b, mir423 and Xpo5 modulated risk of disease (p < 0.002) which may be related to change in expression of these genes as observed by Real-Time PCR assays. But association between polymorphisms and gene expressions was not found in our sample set as well as in larger datasets from open access platforms like Genevar and 1000 Genome database.ConclusionVariations in microRNAs and their processing genes modulated risk of precancer but further in-depth study is needed to understand mechanism of disease process.

Association of DNA sequence variation in mitochondrial DNA polymerase with mitochondrial DNA synthesis and risk of oral cancer.

Enzymes responsible for mitochondrial (mt) DNA synthesis and transcription are encoded by nuclear genome and inherited mutations in these genes may play important roles in enhancing risk of precancer and cancer. Here, genetic variations in 23 functionally relevant tagSNPs in 6 genes responsible for mtDNA synthesis and transcription were studied in 522 cancer and 241 precancer (i.e. leukoplakia) patients and 525 healthy controls using Illumina Golden Gate assay to explore association with risk of oral precancer and cancer. Two SNPs, rs41553913 at POLRMT and rs9905016 at POLG2, significantly increased risk of oral leukoplakia and cancer, respectively, at both genotypic and allelic levels. Gene-environment interaction models also revealed that tobacco habits and SNPs at POLG2 and TFAM may modulate risk of both leukoplakia and cancer. In silico analysis of published data-set also revealed that variant heterozygote (TC) significantly increased transcription of POLG2 compared to wild genotype (p=0.03). Cancer tissues having variant allele genotypes (TC+CC) at POLG2 contained 1.6 times (p<0.01) more mtDNA compared to cancer tissues having wild genotype (TT). In conclusion, polymorphisms at POLG2 and POLRMT increased risk of oral cancer and leukoplakia, respectively, probably modulating synthesis and activity of the enzymes. Enhanced synthesis of mtDNA in cancer tissues may have implication in carcinogenesis, but the mechanism is yet to be explored.

MicroRNA and target gene expression based clustering of oral cancer, precancer and normal tissues

PURPOSE Development of oral cancer is usually preceded by precancerous lesion. Despite histopathological diagnosis, development of disease specific biomarkers continues to be a promising field of study. Expression of miRNAs and their target genes was studied in oral cancer and two types of precancer lesions to look for disease specific gene expression patterns. METHODS Expression of miR-26a, miR-29a, miR-34b and miR-423 and their 11 target genes were determined in 20 oral leukoplakia, 20 lichen planus and 20 cancer tissues with respect to 20 normal tissues using qPCR assay. Expression data were, then, used for cluster analysis of normal as well as disease tissues. RESULTS Expression of miR-26a and miR-29a was significantly down regulated in leukoplakia and cancer tissues but up regulated in lichen planus tissues. Expression of target genes such as, ADAMTS7, ATP1B1, COL4A2, CPEB3, CDK6, DNMT3a and PI3KR1 was significantly down regulated in at least two of three disease types with respect to normal tissues. Negative correlations between expression levels of miRNAs and their targets were observed in normal tissues but not in disease tissues implying altered miRNA-target interaction in disease state. Specific expression profile of miRNAs and target genes formed separate clusters of normal, lichen planus and cancer tissues. CONCLUSION Our results suggest that alterations in expression of selected miRNAs and target genes may play important roles in development of precancer to cancer. Expression profiles of miRNA and target genes may be useful to differentiate cancer and lichen planus from normal tissues, thereby bolstering their role in diagnostics.

Genome-wide mitochondrial DNA sequence variations and lower expression of OXPHOS genes predict mitochondrial dysfunction in oral cancer tissue

Several studies reported that mtDNA mutations may play important roles in carcinogenesis although the mechanism is not clear yet. Most of the studies compared mtDNA sequences in a tumor with those in normal tissues from different individuals ignoring inter-individual variations. In this study, 271 SNPs, 7 novel SNPs (or SNVs), and 15 somatic mutations were detected in mtDNA of 8 oral cancer tissues with respect to reference (rCRS) and adjacent normal tissues, respectively, using Ion PGM next generation sequencing method. Most of the sequence variations (76 SNPs and 1 somatic) are present in D-loop region followed by CyB (36 SNPs), ATP6 (24 SNPs), ND5 (17 SNPs and 5 somatic), ND4 (18 coding and 2 somatic) and other non-coding and coding DNA sequences. A total of 53 and 8 non-synonymous SNPs and somatic mutations, respectively, were detected in tumor tissues and some of these variations may have deleterious effects on the protein function as predicted by bioinformatic analysis. Moreover, significantly low mtDNA contents and expression of several mitochondrial genes in tumor compared to adjacent normal tissues may have also affected mitochondrial functions. Taken together, this study suggests that mtDNA mutations as well as low expression of mtDNA coded genes may play important roles in tumor growth. Although the sample size is low, an important aspect of the study is the use of adjacent control tissues to find out somatic mutations and a change in the expression of mitochondrial genes, to rule out inter-individual and inter-tissue variations which are important issues in the study of mitochondrial genomics.

Expression deregulation of mir31 and CXCL12 in two types of oral precancers and cancer: importance in progression of precancer and cancer.

Oral cancer generally progresses from precancerous lesions such as leukoplakia (LK), lichen planus (LP) and oral submucous fibrosis (OSMF). Since few of these precancers progress to cancers; it is worth to identify biological molecules that may play important roles in progression. Here, expression deregulation of 7 miRNAs (mir204, mir31, mir31*, mir133a, mir7, mir206 and mir1293) and their possible target genes in 23 cancers, 18 LK, 12 LP, 23 OSMF tissues compared to 20 healthy tissues was determined by qPCR method. Expression of mir7, mir31, mir31* and mir1293 was upregulated and that of mir133a, mir204 and mir206 was downregulated in cancer. Expression of most of these miRNAs was also upregulated in LK and LP tissues but not in OSMF. Expression deregulation of some of the target genes was also determined in cancer, LK and LP tissues. Significant upregulation of mir31 and downregulation of its target gene, CXCL12, in cancer, LK and LP tissues suggest their importance in progression of precancer to cancer. Expression upregulation of mir31 was also validated using GEO data sets. Although sample size is low, novelty of this work lies in studying expression deregulation of miRNAs and target genes in oral cancer and three types of precancerous lesions.

Single Nucleotide Polymorphism Network: A Combinatorial Paradigm for Risk Prediction

Risk prediction for a particular disease in a population through SNP genotyping exploits tests whose primary goal is to rank the SNPs on the basis of their disease association. This manuscript reveals a different approach of predicting the risk through network representation by using combined genotypic data (instead of a single allele/haplotype). The aim of this study is to classify diseased group and prediction of disease risk by identifying the responsible genotype. Genotypic combination is chosen from five independent loci present on platelet receptor genes P2RY1 and P2RY12. Genotype-sets constructed from combinations of genotypes served as a network input, the network architecture constituting super-nodes (e.g., case and control) and nodes representing individuals, each individual is described by a set of genotypes containing M markers (M = number of SNP). The analysis becomes further enriched when we consider a set of networks derived from the parent network. By maintaining the super-nodes identical, each network is carrying an independent combination of M-1 markers taken from M markers. For each of the network, the ratio of case specific and control specific connections vary and the ratio of super-node specific connection shows variability. This method of network has also been applied in another case-control study which includes oral cancer, precancer and control individuals to check whether it improves presentation and interpretation of data. The analyses reveal a perfect segregation between super-nodes, only a fraction of mixed state being connected to both the super-nodes (i.e. common genotype set). This kind of approach is favorable for a population to classify whether an individual with a particular genotypic combination can be in a risk group to develop disease. In addition with that we can identify the most important polymorphism whose presence or absence in a population can make a large difference in the number of case and control individuals.

Analysis of the whole transcriptome from gingivo-buccal squamous cell carcinoma reveals deregulated immune landscape and suggests targets for immunotherapy

Background Gingivo-buccal squamous cell carcinoma (GBSCC) is one of the most common oral cavity cancers in India with less than 50% patients surviving past 5 years. Here, we report a whole transcriptome profile on a batch of GBSCC tumours with diverse tobacco usage habits. The study provides an entire landscape of altered expression with an emphasis on searching for targets with therapeutic potential. Methods Whole transcriptomes of 12 GBSCC tumours and adjacent normal tissues were sequenced and analysed to explore differential expression of genes. Expression changes were further compared with those in TCGA head and neck cohort (n = 263) data base and validated in an independent set of 10GBSCC samples. Results Differentially expressed genes (n = 2176) were used to cluster the patients based on their tobacco habits, resulting in 3 subgroups. Immune response was observed to be significantly aberrant, along with cell adhesion and lipid metabolism processes. Different modes of immune evasion were seen across 12 tumours with up-regulation or consistent expression of CD47, unlike other immune evasion genes such as PDL1, FUT4, CTLA4 and BTLA which were downregulated in a few samples. Variation in infiltrating immune cell signatures across tumours also indicates heterogeneity in immune evasion strategies. A few actionable genes such as ITGA4, TGFB1 and PTGS1/COX1 were over expressed in most samples. Conclusion This study found expression deregulation of key immune evasion genes, such as CD47 and PDL1, and reasserts their potential as effective immunotherapeutic targets for GBSCC, which requires further clinical studies. Present findings reiterate the idea of using transcriptome profiling to guide precision therapeutic strategies.

Abstract 1499: Role of mutations and expression change of mitochondrial function related nuclear genes in oral gingivobuccal squamous cell carcinoma

Background: Mitochondria have long been suspected to have contribution in progression and sustenance of cancer. But mitochondria related nuclear genes have not been well-studied in oral cancer. The aim of this study is deciphering the impact of somatic mutations and expression deregulation of mitochondria related nuclear genes in oral gingivobuccal squamous cell carcinoma (GBSCC). Methods: Nuclear-encoded genes which are functionally involved with mitochondria were enlisted from Mitocarta 2.0 and IMPI databases. Whole exome sequencing was performed with 12 paired cancer-normal GBSCC samples and somatic mutations in mitochondria related nuclear genes were extracted from the data. Expression deregulation of mitochondria related nuclear genes were quantified from whole transcriptome data of 12 paired cancer-normal GBSCC samples. Expression was re-validated in another set of 12 cancer-normal paired samples. 5 cancer-normal paired samples with whole transcriptome data were also used for reduced representation bisulfite sequencing (RRBS). Probable impact of non-synonymous somatic mutations were predicted using SIFT and POLYPHEN2 tools. Genes with possible damaging mutations and expression deregulation were taken for literature search and KEGG pathway analysis to understand their contribution in the function of mitochondria in GBSCC. Result: Total 1561 mitochondria related nuclear genes were identified from two databases and included in the study. A total of 977 somatic mutations were identified in 583 such genes in 12 oral GBSCC samples. These mutations were distributed in exonic (346 mutations), intronic (288 mutations), splicing (4 mutations), UTR3’ (270 mutations) and UTR5’ (69 mutations) regions. Ten recurrently mutated genes (AK3, AK4, ATP10D, CASP8, ERBB4, MRPL10, NT5DC3, RYR3, SMURF1and TP53) were identified in at least 25% (3/12) of samples. Significant expression deregulation in 147 mitochondrial related nuclear genes was found from whole transcriptome data. Differential methylation was found in some of these 147 genes in 5 oral GBSCC samples. Thirty-seven genes were selected from 147 genes for expression re-validation by qPCR and expressions of 32 genes were re-validated in another set of 12 GBSCC samples. Somatically mutated and expression deregulated genes were different except two genes, ACADM and NNT, which were mutated and deregulated. Literature survey and KEGG pathway analysis revealed that most of the mutated or deregulated genes were functionally involved with biological pathways like mitochondrial metabolism, oxidative phosphorylation, apoptosis and molecular transport. It indicates possible disruption of these pathways in oral GBSCC. Conclusion: Mitochondrial function related nuclear genes may have significant impact on oral GBSCC in maintenance and progression, but needs to be validated in larger set of samples. Citation Format: Esita Chattopadhyay, Richa Singh, Roshni Roy, Bidyut Roy. Role of mutations and expression change of mitochondrial function related nuclear genes in oral gingivobuccal squamous cell carcinoma [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 1499. doi:10.1158/1538-7445.AM2017-1499

Abstract 5274: Expression of selected miRNA and miRNA processing genes in oral precancer and cancer tissues.

Introduction: Oral leukoplakia is a premalignant lesion that long has been considered to confer increased risk for the development of Oral Cancer which is highly prevalent in India. Oral lichen planus (OLP) on the other hand is a chronic inflammatory disease whose malignant potential is debatable. Since not all oral precancerous lesions progress to oral cancer, molecular markers like miRNA expression could be used to diagnose and predict progression of disease that cannot be detected histopathologically. Objective: Study of intra- and inter-variation in the expression of a few microRNAs (26a1, 423, 34b, 29a, 193a, 137 and 205) and microRNA processing (XPO5, GEMIN3 and RAN) genes in the three disease types, namely, oral cancer, leukoplakia and lichen planus. Methodology: Total RNA was extracted from paired disease and normal tissues from 15 oral cancer, 19 leukoplakia and 19 OLP patients. Expression of microRNAs and microRNA processing genes were studied using TaqMan assay. Subsequent statistical analyses were performed for interpretation of expression data. Results: Expression of mir 26a-1 and mir 137 genes was dysregulated >2 folds in 70% of the cancer samples while expression of remaining microRNAs and mir-processing genes were deregulated in a few samples. In leukoplakia, expression of mir-26a-1 was dysregulated >2 folds in 50% of the samples while dysregulation in expression of the remaining miRNAs was observed in 1/3rd of the patients. In OLP, there was an interesting trend of unidirectional up or down regulation in the expression of mir26a-1, mir29a, mir-34b and mir-137 genes. We also observed that the average expression of miRNA genes differed among the three patient groups. Conclusion: Deregulation in microRNA expression could not be correlated with individual9s tobacco habits. Variation in expression of microRNA as well as miRNA processing genes was observed within individuals of the same patient group (i.e. cancer or leukoplakia or lichen planus) and between any two patients groups. There was correlation in expression of microRNAs which probably explains the unidirectional increase or decrease of miR expression in OLP. Finally, the mean expression level in the three types of disease tissues differed significantly. But expression of more miRNA genes in more samples would be necessary for molecular markers. Citation Format: Bidyut Roy, Roshni Roy, Navonil De Sarkar, Ranjan Rashni Paul. Expression of selected miRNA and miRNA processing genes in oral precancer and cancer tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5274. doi:10.1158/1538-7445.AM2013-5274