Mutant p53 reactivation restricts the protumorigenic consequences of wild type p53 loss of heterozygosity in Li-Fraumeni syndrome patient-derived fibroblasts.

The p53 tumor suppressor, encoded by the TP53 gene, serves as a major barrier against malignant transformation. Patients with Li-Fraumeni syndrome (LFS) inherit a mutated TP53 allele from one parent and a wild-type TP53 allele from the other. Subsequently, the wild-type allele is lost and only the mutant TP53 allele remains. This process, which is termed loss of heterozygosity (LOH), results in only mutant p53 protein expression. We used primary dermal fibroblasts from LFS patients carrying the hotspot p53 gain-of-function pathogenic variant, R248Q to study the LOH process and characterize alterations in various pathways before and after LOH. We previously described the derivation of mutant p53 reactivating peptides, designated pCAPs (p53 Conformation Activating Peptides). In this study, we tested the effect of lead peptide pCAP-250 on LOH and on its associated cellular changes. We report that treatment of LFS fibroblasts with pCAP-250 prevents the accumulation of mutant p53 protein, inhibits LOH, and alleviates its cellular consequences. Furthermore, prolonged treatment with pCAP-250 significantly reduces DNA damage and restores long-term genomic stability. pCAPs may thus be contemplated as a potential preventive treatment to prevent or delay early onset cancer in carriers of mutant p53.

Genetic insight into Birt-Hogg-Dubé syndrome in Indian patients reveals novel mutations at FLCN.

BACKGROUND: Birt-Hogg-Dubé syndrome (BHDS) is a rare monogenic condition mostly associated with germline mutations at FLCN. It is characterized by either one or more manifestations of primary spontaneous pneumothorax (PSP), skin fibrofolliculomas and renal carcinoma (chromophobe). Here, we comprehensively studied the mutational background of 31 clinically diagnosed BHDS patients and their 74 asymptomatic related members from 15 Indian families. RESULTS: Targeted amplicon next-generation sequencing (NGS) and Sanger sequencing of FLCN in patients and asymptomatic members revealed a total of 76 variants. Among these variants, six different types of pathogenic FLCN mutations were detected in 26 patients and some asymptomatic family members. Two of the variants were novel mutations: an 11-nucleotide deletion (c.1150_1160delGTCCAGTCAGC) and a splice acceptor mutation (c.1301-1G > A). Two variants were Clinvar reported pathogenic mutations: a stop-gain (c.634C > T) and a 4-nucleotide duplication (c.1329_1332dupAGCC). Two known variants were: hotspot deletion (c.1285delC) and a splice donor mutation (c.1300 + 1G > A). FLCN mutations could not be detected in patients and asymptomatic members from 5 families. All these mutations greatly affected the protein stability and FLCN-FNIP2 interaction as observed by molecular docking method. Family-based association study inferred pathogenic FLCN mutations are significantly associated with BHDS. CONCLUSION: Six pathogenic FLCN mutations were detected in patients from 10 families out of 15 families in the cohort. Therefore, genetic screening is necessary to validate the clinical diagnosis. The pathogenic mutations at FLCN affects the protein-protein interaction, which plays key roles in various metabolic pathways. Since, pathogenic mutations could not be detected in exonic regions of FLCN in 5 families, whole genome sequencing is necessary to detect all mutations at FLCN and/or any undescribed gene/s that may also be implicated in BHDS.

A novel hotspot and rare somatic mutation p.A138V, at TP53 is associated with poor survival of pancreatic ductal and periampullary adenocarcinoma patients.

BACKGROUND: Pancreatic Ductal Adenocarcinoma (PDAC) is a cancer of the exocrine pancreas and 5-year survival rates remain constant at 7%. Along with PDAC, Periampullary Adenocarcinoma (PAC) accounts for 0.5-2% of all gastrointestinal malignancies. Genomic observations were well concluded for PDAC and PACs in western countries but no reports are available from India till now. METHODS: Targeted Next Generation Sequencing were performed in 8 (5 PDAC and 3 PAC) tumour normal pairs, using a panel of 412 cancer related genes. Primary findings were replicated in 85 tumour samples (31 PDAC and 54 PAC) using the Sanger sequencing. Mutations were also validated by ASPCR, RFLP, and Ion Torrent sequencing. IHC along with molecular dynamics and docking studies were performed for the p.A138V mutant of TP53. Key polymorphisms at TP53 and its associated genes were genotyped by PCR-RFLP method and association with somatic mutations were evaluated. All survival analysis was done using the Kaplan-Meier survival method which revealed that the survival rates varied significantly depending on the somatic mutations the patients harboured. RESULTS: Among the total 114 detected somatic mutations, TP53 was the most frequently mutated (41%) gene, followed by KRAS, SMAD4, CTNNB1, and ERBB3. We identified a novel hotspot TP53 mutation (p.A138V, in 17% of all patients). Low frequency of KRAS mutation (33%) was detected in these samples compared to patients from Western counties. Molecular Dynamics (MD) simulation and DNA-protein docking analysis predicted p.A138V to have oncogenic characteristics. Patients with p.A138V mutation showed poorer overall survival (p = 0.01). So, our finding highlights elevated prevalence of the p53p.A138V somatic mutation in PDAC and pancreatobiliary PAC patients. CONCLUSION: Detection of p.A138V somatic variant in TP53 might serve as a prognostic marker to classify patients. It might also have a role in determining treatment regimes. In addition, low frequency of KRAS hotspot mutation mostly in Indian PDAC patient cohort indicates presence of other early drivers in malignant transformation.

Genome-wide miRNA methylome analysis in oral cancer: possible biomarkers associated with patient survival.

AIM: The methylome associated with miRNA loci was investigated in oral cancer to explore tobacco specific methylation and potential biomarkers for patient survival. METHODS: Methylome data was generated from 16 pairs of cancer-normal tissues by reduced representation bisulfite sequencing method. Differentially methylated regions were identified using the DMAP pipeline. In silico validation and Kaplan-Meier survival analyses were performed on The Cancer Genome Atlas data based on our miRNA methylome data. RESULTS: A total of 4310 unique differentially methylated regions, mapping to 144 miRNA loci, were identified. Three distinct groups of miRNAs were differentially methylated in cancer tissues from smokers, chewers and mixed habitués. Hypermethylation of miR-503, miR-200a/b, miR-320b and miR-489 was associated with worse 5-year survival. CONCLUSION: Differential methylation patterns in miRNA loci are associated with poor survival underscoring their potential as predictive and prognostic biomarkers in oral cancer.

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.

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.

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.

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.

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.

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.

D-loop somatic mutations and ∼5 kb "common" deletion in mitochondrial DNA: important molecular markers to distinguish oral precancer and cancer.

Apart from genomic DNA, mutations at mitochondrial DNA (mtDNA) have been hypothesized to play vital roles in cancer development. In this study, ∼5 kb deletion and D-loop mutations in mtDNA and alteration in mtDNA content were investigated in buccal smears from 104 healthy controls and 74 leukoplakia and 117 cancer tissue samples using Taqman-based quantitative assay and re-sequencing. The ∼5 kb deletion in mtDNA was significantly less (9.8 and 10.5 folds, P < 0.0001) in cancer tissues compared to control and leukoplakia tissues, respectively. On the other hand, somatic mutations in D-loop, investigated in 54 controls, 50 leukoplakias and 56 cancer patients, were found to be significantly more in cancer tissues, but not in leukoplakia tissues, compared to control (Z-score = 5.4). MtDNA contents were observed to be significantly more in leukoplakia (2.1 folds, P = 0.004) and cancer (1.6 folds, P = 0.03) tissues compared to control tissues. So, D-loop somatic mutations and ∼5 kb deletion patterns could be used as distinguishing markers between precancer and cancer tissues. This observation further suggests that somatic mutations in D-loop may facilitate carcinogenesis and cancer cells with less ∼5 kb deletion, i.e., intact mtDNA, may become resistant to apoptosis.