Murali D. Bashyam

Convergent structural alterations define SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeler as a central tumor suppressive complex in pancreatic cancer

Defining the molecular genetic alterations underlying pancreatic cancer may provide unique therapeutic insight for this deadly disease. Toward this goal, we report here an integrative DNA microarray and sequencing-based analysis of pancreatic cancer genomes. Notable among the alterations newly identified, genomic deletions, mutations, and rearrangements recurrently targeted genes encoding components of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex, including all three putative DNA binding subunits (ARID1A, ARID1B, and PBRM1) and both enzymatic subunits (SMARCA2 and SMARCA4). Whereas alterations of each individual SWI/SNF subunit occurred at modest-frequency, as mutational “hills” in the genomic landscape, together they affected at least one-third of all pancreatic cancers, defining SWI/SNF as a major mutational “mountain.” Consistent with a tumor-suppressive role, re-expression of SMARCA4 in SMARCA4-deficient pancreatic cancer cell lines reduced cell growth and promoted senescence, whereas its overexpression in a SWI/SNF-intact line had no such effect. In addition, expression profiling analyses revealed that SWI/SNF likely antagonizes Polycomb repressive complex 2, implicating this as one possible mechanism of tumor suppression. Our findings reveal SWI/SNF to be a central tumor suppressive complex in pancreatic cancer.

Hormonal regulation of gluconeogenic gene transcription in the liver

Glucose homeostasis in mammals is achieved by the actions of counterregulatory hormones, namely insulin, glucagon and glucocorticoids. Glucose levels in the circulation are regulated by the liver, the metabolic centre which produces glucose when it is scarce in the blood. This process is catalysed by two rate-limiting enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) whose gene expression is regulated by hormones. Hormone response units (HRUs) present in the two genes integrate signals from various signalling pathways triggered by hormones. How such domains are arranged in the regulatory region of these two genes, how this complex regulation is accomplished and the latest advancements in the field are discussed in this review.

Genomic Profiling Identifies GATA6 as a Candidate Oncogene Amplified in Pancreatobiliary Cancer

Pancreatobiliary cancers have among the highest mortality rates of any cancer type. Discovering the full spectrum of molecular genetic alterations may suggest new avenues for therapy. To catalogue genomic alterations, we carried out array-based genomic profiling of 31 exocrine pancreatic cancers and 6 distal bile duct cancers, expanded as xenografts to enrich the tumor cell fraction. We identified numerous focal DNA amplifications and deletions, including in 19% of pancreatobiliary cases gain at cytoband 18q11.2, a locus uncommonly amplified in other tumor types. The smallest shared amplification at 18q11.2 included GATA6, a transcriptional regulator previously linked to normal pancreas development. When amplified, GATA6 was overexpressed at both the mRNA and protein levels, and strong immunostaining was observed in 25 of 54 (46%) primary pancreatic cancers compared to 0 of 33 normal pancreas specimens surveyed. GATA6 expression in xenografts was associated with specific microarray gene-expression patterns, enriched for GATA binding sites and mitochondrial oxidative phosphorylation activity. siRNA mediated knockdown of GATA6 in pancreatic cancer cell lines with amplification led to reduced cell proliferation, cell cycle progression, and colony formation. Our findings indicate that GATA6 amplification and overexpression contribute to the oncogenic phenotypes of pancreatic cancer cells, and identify GATA6 as a candidate lineage-specific oncogene in pancreatobiliary cancer, with implications for novel treatment strategies.

Understanding cancer metastasis: An urgent need for using differential gene expression analysis

Cancer is a multistep process and occurs as a result of the loss of control of cell division, leading to the initial tumor formation, which is followed by metastatic spread. Recent years have witnessed a vast improvement in the understanding of the molecular mechanisms regulating cell division and their links to tumorigenesis. The process of metastasis involves an intricate interplay between cell adhesion, proteolysis, migration, and angiogenesis. However, there is little knowledge of how these events are coordinately regulated in the tumor cell. Given that the uncontrolled spread of the tumor to distant organs is usually lethal, a study of the molecular mechanisms regulating metastasis assumes great significance. Recently, several technologies have been developed for analyzing differential gene expression. The current review discusses the importance of these technologies in the molecular analyses of metastasis. Cancer 2002;94:1821–9.

Molecular genetics of familial hypertrophic cardiomyopathy (FHC).

AbstractFamilial hypertrophic cardiomyopathy is an autosomal dominant disease with a wide range of clinical features from benign to severe, and is the most common cause of sudden death in otherwise healthy individuals. The two prominent clinical features are left ventricular hypertrophy and myocyte/myofibrillar disarray. The former is responsible for clinical symptoms such as breathlessness and angina, whereas the latter may lead to sudden cardiac death. The last decade has seen an enormous improvement in our understanding of the molecular genetics of this disorder. The clinical heterogeneity has been linked to genetic heterogeneity; mutations in nine genes encoding sarcomere proteins have been shown to be the molecular basis for the disorder. However, attempts to establish a genotype-phenotype correlation for each of the more than 100 mutations that have been identified have not been highly successful. Additional genetic loci, as well as nongenetic factors such as lifestyle, sex, and age, have also been shown to play a role in modulating the clinical presentation of the disease. How each mutation results in hypertrophy and/or myofibrillar disarray is unclear. The present review discusses the current status of the molecular genetic characterization of this important disorder.

Molecular genetic analyses of β-thalassemia in South India reveals rare mutations in the β-globin gene

Abstractβ-thalassemia is the most prevalent single-gene disorder. Since no viable forms of treatment are available, the best course is prevention through prenatal diagnosis. In the present study, the prevalence of β-thalassemia was extensively investigated in the South Indian population, especially from the state of Andhra Pradesh. Screening for causal mutations was carried out on genomic DNA isolated from patient blood samples by using the routine reverse dot blot (RDB) and amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) techniques. DNA sequencing was performed wherever necessary. Among the nine mutations identified, four, including IVS-1-5 (G-C) (IVS1+5G>T), codon 41/42 (-TTCT) (c.124_127delTTCT), codon 15 (G-A) (c.47G>A), and HbS (sickle mutation) (c.20A>T) mutations, accounted for about 98% of the total positive cases. Two mutations viz. codon 8/9 (+G) (c.27_28insG) and HbE (codon 26 G-A) (c.79G>A) exhibited a very low frequency of occurrence, whereas the IVS-1-1 (G-T) (IVS1+1G>T) and the 619 bp deletion (c.366_494del) mutations were absent. We also identified certain rare mutations during the diagnostic evaluation. Gene sequencing confirmed the codon 30 (G-C) (c.92G>C) mutation and the rare codon 5 (-CT) (c.17_18delCT) and IVS-II-837 (T-G) (IVSII-14T>G) mutations. This is the first report of the IVS II 837 mutation in the Indian population. We also report a novel diagnostic application during RDB-based screening for the detection of the (c.92G>C) mutations. Such a comprehensive mutation screening is essential for prenatal diagnosis of β-thalassemia and control of this highly prevalent monogenic disorder in the Indian population.

The Homologous Region Sequence (hr1) of Autographa californica Multinucleocapsid Polyhedrosis Virus Can Enhance Transcription from Non-baculoviral Promoters in Mammalian Cells

The Autographa californica multinucleocapsid polyhedrosis virus homologous region sequence hr1 enhances transcription from the viral polyhedrin promoter in Spodoptera frugiperda insect cells and independently functions as an origin of replication (ori) sequence. The binding of the host nuclear protein, hr1-binding protein (hr1-BP), is crucial for the enhancer activity (Habib, S., Pandey, S., Chatterji, U., Burma, S., Ahmad, R., Jain, A., and Hasnain, S. E. (1996) DNA Cell Biol. 15, 737–747 and Habib, S., and Hasnain, S. E. (1996) J. Biol. Chem. 271, 28250–28258). We demonstrate that hr1 can also enhance transcription from non-baculoviral promoters like cytomegalovirus and hsp70 in mammalian cells but does not support ori activity in these cells. Unlike insect cells, hr1 can also function in mammalian cells as an enhancer when present in trans. hr1 DNA sequence binds with high affinity and specificity to nuclear factors in the mammalian cells. The insect hr1-BP- and the hr1-BP-like proteins from mammalian cells (mhr1-BP) have different properties with respect to ion requirements, DNA groove binding, and molecular size. When mammalian cells are infected with a recombinant baculovirus containing two promoters, the baculovirus polyhedrin and Drosophila hsp70 gene promoter, the hsp70 gene promoter alone is active in these cells, and this activity is further enhanced by the presence of an additional hr1 in the recombinant virus. hr1 may thus also have a role in baculovirus-mediated gene delivery in mammalian cells.

SMURF1 amplification promotes invasiveness in pancreatic cancer.

Pancreatic cancer is a deadly disease, and new therapeutic targets are urgently needed. We previously identified DNA amplification at 7q21-q22 in pancreatic cancer cell lines. Now, by high-resolution genomic profiling of human pancreatic cancer cell lines and human tumors (engrafted in immunodeficient mice to enrich the cancer epithelial fraction), we define a 325 Kb minimal amplicon spanning SMURF1, an E3 ubiquitin ligase and known negative regulator of transforming growth factor β (TGFβ) growth inhibitory signaling. SMURF1 amplification was confirmed in primary human pancreatic cancers by fluorescence in situ hybridization (FISH), where 4 of 95 cases (4.2%) exhibited amplification. By RNA interference (RNAi), knockdown of SMURF1 in a human pancreatic cancer line with focal amplification (AsPC-1) did not alter cell growth, but led to reduced cell invasion and anchorage-independent growth. Interestingly, this effect was not mediated through altered TGFβ signaling, assayed by transcriptional reporter. Finally, overexpression of SMURF1 (but not a catalytic mutant) led to loss of contact inhibition in NIH-3T3 mouse embryo fibroblast cells. Together, these findings identify SMURF1 as an amplified oncogene driving multiple tumorigenic phenotypes in pancreatic cancer, and provide a new druggable target for molecularly directed therapy.

ARID1B, a member of the human SWI/SNF chromatin remodeling complex, exhibits tumour-suppressor activities in pancreatic cancer cell lines

Background:The human ATP-dependent SWItch/sucrose nonfermentable (SWI/SNF) complex functions as a primary chromatin remodeler during ontogeny, as well as in adult life. Several components of the complex have been suggested to function as important regulators of tumorigenesis in various cancers. In the current study, we have characterised a possible tumour suppressor role for the largest subunit of the complex, namely the AT-rich interaction domain 1B (ARID1B).Methods:We performed Azacytidine and Trichostatin A treatments, followed by bisulphite sequencing to determine the possible DNA methylation-induced transcription repression of the gene in pancreatic cancer (PaCa) cell lines. Functional characterisation of effect of ARID1B ectopic expression in MiaPaCa2 PaCa cell line, which harboured ARID1B homozygous deletion, was carried out. Finally, we evaluated ARID1B protein expression in pancreatic tumour samples using immunohistochemistry on a tissue microarray.Results:ARID1B was transcriptionally repressed due to promoter hypermethylation, and ectopic expression severely compromised the ability of MiaPaCa2 cells to form colonies in liquid culture and soft agar. In addition, ARID1B exhibited significantly reduced/loss of expression in PaCa tissue, especially in samples from advanced-stage tumours, when compared with normal pancreas.Conclusion:The results therefore suggest a possible tumour-suppressor function for ARID1B in PaCa, thus adding to the growing list of SWI/SNF components with a similar function. Given the urgent need to design efficient targeted therapies for PaCa, our study assumes significance.

Farber lipogranulomatosis : clinical and molecular genetic analysis reveals a novel mutation in an Indian family

AbstractFarber disease is a rare lysosomal storage disorder caused by a deficiency of the acid ceramidase enzyme, leading to the accumulation of ceramide in various tissues. It usually manifests within a few months after birth with a unique triad of symptoms, including painful and progressive deformed joints, progressive hoarseness and subcutaneous nodules. The disease is inherited as an autosomal recessive trait, and mutations in the N-acylsphingosine amidohydrolase (ASAH1) gene, which codes for the acid ceramidase enzyme, have been shown to cause the disease. In the current study, we report the identification of a novel disease-causing mutation in the ASAH1 gene that results in Farber disease in an Indian family. The mutation was identified in the eighth exon and is a missense mutation resulting in replacement of Valine by Leucine at codon 182. Two affected siblings harboured the identical mutation. The possible mechanism(s) of disease caused by this mutation are discussed.