Esita Chattopadhyay

Altered Mitochondrial Signalling and Metabolism in Cancer

Mitochondria being the central organelle for metabolism and other cell signalling pathways have remained the topic of interest to tumour biologists. In spite of the wide acceptance of Warburg’s hypothesis, role of mitochondrial metabolism in cancer is still unclear. Uncontrolled growth and proliferation, hallmarks of tumour cells, are maintained when the cells adapt to metabolic reprogramming with the help of altered metabolism of mitochondria. This review has focussed on different aspects of mitochondrial metabolism and inter-related signalling pathways which have been found to be modified in cancer.

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.

Genetic Analysis of Familial Spontaneous Pneumothorax in an Indian Family

Familial spontaneous pneumothorax is one of the phenotypes of Birt–Hogg–Dubé syndrome (BHDS), an autosomal dominant condition associated with folliculin (FLCN). We investigated clinical and genetic data of an Indian family having two patients suffering from spontaneous pneumothorax in the absence of skin lesions or renal tumors. HRCT scan of patient’s lung revealed paracardiac cysts, and DNA sequencing of all 14 exons of FLCN from patients showed the presence of heterozygous “C allele” deletion in the poly-cytosine (poly-C) tract of exon 11 leading to truncated folliculin. This mutation was also observed in four asymptomatic members of the family. Our results confirmed the presence of deletion mutation in poly-C tract of FLCN in members of BHDS family. This is the first report of genetic insight in a BHDS family from India but in-depth studies with a larger sample set are necessary to understand mechanism of familial pneumothorax.

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.

A novel mutation at ANTXR1 in an indian GAPO patient

OBJECTIVE Growth retardation-alopecia-pseudoanodontia-optic atrophy (GAPO) syndrome (Online Mendelian Inheritance in Man [OMIM] ID 230740) is one of the rarest autosomal recessive syndromes. It is characterized by many phenotypes, including wide anterior fontanel, frontal bossing of the face, depressed nasal bridge, along with the 4 classic phenotypes contained in the name of the syndrome. Recent reports identified nonsense, missense, and splicing mutations at different exons of ANTXR1 responsible for GAPO syndrome in patients from different ethnic populations. Here, we are reporting a mutation at ANTXR1 in an Indian patient with GAPO syndrome. STUDY DESIGN We describe an inherited mutation at ANTXR1 in a 6-year-old Indian boy with GAPO syndrome. RESULTS Genomic DNA from the patient with the GAPO syndrome and his family members were screened for previously reported mutations at ANTXR1 by sequencing. Novel homozygous and heterozygous mutations in exon-3 of ANTXR1 (c.265 G > A, p.Gly89 Arg) were identified in the patient and in other members of the family, respectively. However, no mutated allele was identified in genomic DNA from unrelated healthy individuals. Bioinformatic analysis by different tools predicted the deleterious, damaging, or aberrant splicing effect of mutation on the protein. CONCLUSIONS Functional inefficiency of ANTXR1 as a result of mutation might have led to GAPO syndrome.Summary Purpose GAPO (OMIM ID 230740 ) is one of the rarest autosomal recessive syndromes, characterised by eponymic description of G rowth retardation, A lopecia, P seudo anodontia and O ptic atrophy. It is characterized by many phenotypes such as wide anterior fontanel in head, frontal bossing in face, depressed nasal bridge etc. along with above mentioned four classical phenotypes. Recent reports identified nonsense, missense and splicing mutations at different exons of ANTXR1 to be responsible for GAPO syndrome in patients from different ethnic population. Here, we are reporting mutation at ANTXR1 in a GAPO patient from India. Patients, Methods and Results We describe an inherited mutation at ANTXR1 in a GAPO patient (6-year-old boy) from India. Genomic DNA from GAPO patient and other family members were screened for previously reported mutations at ANTXR1 by sequencing. A novel homozygous and heterozygous mutation in exon-3 of ANTXR1 (c.265G>A, p. Gly89Arg ) has been identified in patient and other members of the family, respectively. But no mutated allele was identified in genomic DNA from unrelated healthy individuals. Bioinformatic analysis by different tools predicted deleterious, damaging or aberrant splicing effect of mutation on the protein. Conclusion Functional inefficiency of ANTXR1 due to mutation might have led to GAPO syndrome.

Oral and Maxillofacial PathologyA novel mutation at ANTXR1 in an Indian patient with growth retardation–alopecia–pseudoanodontia–optic atrophy syndrome

OBJECTIVE Growth retardation-alopecia-pseudoanodontia-optic atrophy (GAPO) syndrome (Online Mendelian Inheritance in Man [OMIM] ID 230740) is one of the rarest autosomal recessive syndromes. It is characterized by many phenotypes, including wide anterior fontanel, frontal bossing of the face, depressed nasal bridge, along with the 4 classic phenotypes contained in the name of the syndrome. Recent reports identified nonsense, missense, and splicing mutations at different exons of ANTXR1 responsible for GAPO syndrome in patients from different ethnic populations. Here, we are reporting a mutation at ANTXR1 in an Indian patient with GAPO syndrome. STUDY DESIGN We describe an inherited mutation at ANTXR1 in a 6-year-old Indian boy with GAPO syndrome. RESULTS Genomic DNA from the patient with the GAPO syndrome and his family members were screened for previously reported mutations at ANTXR1 by sequencing. Novel homozygous and heterozygous mutations in exon-3 of ANTXR1 (c.265 G > A, p.Gly89 Arg) were identified in the patient and in other members of the family, respectively. However, no mutated allele was identified in genomic DNA from unrelated healthy individuals. Bioinformatic analysis by different tools predicted the deleterious, damaging, or aberrant splicing effect of mutation on the protein. CONCLUSIONS Functional inefficiency of ANTXR1 as a result of mutation might have led to GAPO syndrome.Summary Purpose GAPO (OMIM ID 230740 ) is one of the rarest autosomal recessive syndromes, characterised by eponymic description of G rowth retardation, A lopecia, P seudo anodontia and O ptic atrophy. It is characterized by many phenotypes such as wide anterior fontanel in head, frontal bossing in face, depressed nasal bridge etc. along with above mentioned four classical phenotypes. Recent reports identified nonsense, missense and splicing mutations at different exons of ANTXR1 to be responsible for GAPO syndrome in patients from different ethnic population. Here, we are reporting mutation at ANTXR1 in a GAPO patient from India. Patients, Methods and Results We describe an inherited mutation at ANTXR1 in a GAPO patient (6-year-old boy) from India. Genomic DNA from GAPO patient and other family members were screened for previously reported mutations at ANTXR1 by sequencing. A novel homozygous and heterozygous mutation in exon-3 of ANTXR1 (c.265G>A, p. Gly89Arg ) has been identified in patient and other members of the family, respectively. But no mutated allele was identified in genomic DNA from unrelated healthy individuals. Bioinformatic analysis by different tools predicted deleterious, damaging or aberrant splicing effect of mutation on the protein. Conclusion Functional inefficiency of ANTXR1 due to mutation might have led to GAPO syndrome.

A novel mutation at ANTXR1 in an Indian patient with growth retardation–alopecia–pseudoanodontia–optic atrophy syndrome

OBJECTIVE Growth retardation-alopecia-pseudoanodontia-optic atrophy (GAPO) syndrome (Online Mendelian Inheritance in Man [OMIM] ID 230740) is one of the rarest autosomal recessive syndromes. It is characterized by many phenotypes, including wide anterior fontanel, frontal bossing of the face, depressed nasal bridge, along with the 4 classic phenotypes contained in the name of the syndrome. Recent reports identified nonsense, missense, and splicing mutations at different exons of ANTXR1 responsible for GAPO syndrome in patients from different ethnic populations. Here, we are reporting a mutation at ANTXR1 in an Indian patient with GAPO syndrome. STUDY DESIGN We describe an inherited mutation at ANTXR1 in a 6-year-old Indian boy with GAPO syndrome. RESULTS Genomic DNA from the patient with the GAPO syndrome and his family members were screened for previously reported mutations at ANTXR1 by sequencing. Novel homozygous and heterozygous mutations in exon-3 of ANTXR1 (c.265 G > A, p.Gly89 Arg) were identified in the patient and in other members of the family, respectively. However, no mutated allele was identified in genomic DNA from unrelated healthy individuals. Bioinformatic analysis by different tools predicted the deleterious, damaging, or aberrant splicing effect of mutation on the protein. CONCLUSIONS Functional inefficiency of ANTXR1 as a result of mutation might have led to GAPO syndrome.Summary Purpose GAPO (OMIM ID 230740 ) is one of the rarest autosomal recessive syndromes, characterised by eponymic description of G rowth retardation, A lopecia, P seudo anodontia and O ptic atrophy. It is characterized by many phenotypes such as wide anterior fontanel in head, frontal bossing in face, depressed nasal bridge etc. along with above mentioned four classical phenotypes. Recent reports identified nonsense, missense and splicing mutations at different exons of ANTXR1 to be responsible for GAPO syndrome in patients from different ethnic population. Here, we are reporting mutation at ANTXR1 in a GAPO patient from India. Patients, Methods and Results We describe an inherited mutation at ANTXR1 in a GAPO patient (6-year-old boy) from India. Genomic DNA from GAPO patient and other family members were screened for previously reported mutations at ANTXR1 by sequencing. A novel homozygous and heterozygous mutation in exon-3 of ANTXR1 (c.265G>A, p. Gly89Arg ) has been identified in patient and other members of the family, respectively. But no mutated allele was identified in genomic DNA from unrelated healthy individuals. Bioinformatic analysis by different tools predicted deleterious, damaging or aberrant splicing effect of mutation on the protein. Conclusion Functional inefficiency of ANTXR1 due to mutation might have led to GAPO syndrome.

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