Hitendra S. Solanki

Identification of differentially expressed serum proteins in gastric adenocarcinoma.

UNLABELLED Gastric adenocarcinoma is an aggressive cancer with poor prognosis. Blood based biomarkers of gastric cancer have the potential to improve diagnosis and monitoring of these tumors. Proteins that show altered levels in the circulation of gastric cancer patients could prove useful as putative biomarkers. We used an iTRAQ-based quantitative proteomic approach to identify proteins that show altered levels in the sera of patients with gastric cancer. Our study resulted in identification of 643 proteins, of which 48 proteins showed increased levels and 11 proteins showed decreased levels in serum from gastric cancer patients compared to age and sex matched healthy controls. Proteins that showed increased expression in gastric cancer included inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Mannose-binding protein C (MBL2), sex hormone-binding globulin (SHBG), insulin-like growth factor-binding protein 2 (IGFBP2), serum amyloid A protein (SAA1), Orosomucoid 1 (ORM1) and extracellular superoxide dismutase [Cu-Zn] (SOD3). We used multiple reaction monitoring assays and validated elevated levels of ITIH4 and SAA1 proteins in serum from gastric cancer patients. BIOLOGICAL SIGNIFICANCE Gastric cancer is a highly aggressive cancer associated with high mortality. Serum-based biomarkers are of considerable interest in diagnosis and monitoring of various diseases including cancers. Gastric cancer is often diagnosed at advanced stages resulting in poor prognosis and high mortality. Pathological diagnosis using biopsy specimens remains the gold standard for diagnosis of gastric cancer. Serum-based biomarkers are of considerable importance as they are minimally invasive. In this study, we carried out quantitative proteomic profiling of serum from gastric cancer patients to identify proteins that show altered levels in gastric cancer patients. We identified more than 50 proteins that showed altered levels in gastric cancer patient sera. Validation in a large cohort of well classified patient samples would prove useful in identifying novel blood based biomarkers for gastric cancers. This article is part of a Special Issue entitled: Proteomics in India.

Dysregulation of splicing proteins in head and neck squamous cell carcinoma

ABSRTRACT Signaling plays an important role in regulating all cellular pathways. Altered signaling is one of the hallmarks of cancers. Phosphoproteomics enables interrogation of kinase mediated signaling pathways in biological systems. In cancers, this approach can be utilized to identify aberrantly activated pathways that potentially drive proliferation and tumorigenesis. To identify signaling alterations in head and neck squamous cell carcinoma (HNSCC), we carried out proteomic and phosphoproteomic analysis of HNSCC cell lines using a combination of tandem mass tag (TMT) labeling approach and titanium dioxide-based enrichment. We identified 4,920 phosphosites corresponding to 2,212 proteins in six HNSCC cell lines compared to a normal oral cell line. Our data indicated significant enrichment of proteins associated with splicing. We observed hyperphosphorylation of SRSF protein kinase 2 (SRPK2) and its downstream substrates in HNSCC cell lines. SRPK2 is a splicing kinase, known to phosphorylate serine/arginine (SR) rich domain proteins and regulate splicing process in eukaryotes. Although genome-wide studies have reported the contribution of alternative splicing events of several genes in the progression of cancer, the involvement of splicing kinases in HNSCC is not known. In this study, we studied the role of SRPK2 in HNSCC. Inhibition of SRPK2 resulted in significant decrease in colony forming and invasive ability in a panel of HNSCC cell lines. Our results indicate that phosphorylation of SRPK2 plays a crucial role in the regulation of splicing process in HNSCC and that splicing kinases can be developed as a new class of therapeutic target in HNSCC.

Chronic exposure to cigarette smoke leads to activation of p21 (RAC1)-activated kinase 6 (PAK6) in non-small cell lung cancer cells

Epidemiological data clearly establishes cigarette smoking as one of the major cause for lung cancer worldwide. Recently, targeted therapy has become one of the most preferred modes of treatment for cancer. Though certain targeted therapies such as anti-EGFR are in clinical practice, they have shown limited success in lung cancer patients who are smokers. This demands discovery of alternative drug targets through systematic investigation of cigarette smoke-induced signaling mechanisms. To study the signaling events activated in response to cigarette smoke, we carried out SILAC-based phosphoproteomic analysis of H358 lung cancer cells chronically exposed to cigarette smoke. We identified 1,812 phosphosites, of which 278 phosphosites were hyperphosphorylated (≥ 3-fold) in H358 cells chronically exposed to cigarette smoke. Our data revealed hyperphosphorylation of S560 within the conserved kinase domain of PAK6. Activation of PAK6 is associated with various processes in cancer including metastasis. Mechanistic studies revealed that inhibition of PAK6 led to reduction in cell proliferation, migration and invasion of the cigarette smoke treated cells. Further, siRNA mediated silencing of PAK6 resulted in decreased invasive abilities in a panel of non-small cell lung cancer (NSCLC) cells. Consistently, mice bearing tumor xenograft showed reduced tumor growth upon treatment with PF-3758309 (group II PAK inhibitor). Immunohistochemical analysis revealed overexpression of PAK6 in 66.6% (52/78) of NSCLC cases in tissue microarrays. Taken together, our study indicates that PAK6 is a promising novel therapeutic target for NSCLC, especially in smokers.

Phosphotyrosine profiling of curcumin-induced signaling

BackgroundCurcumin, derived from the rhizome Curcuma longa, is a natural anti-cancer agent and has been shown to inhibit proliferation and survival of tumor cells. Although the anti-cancer effects of curcumin are well established, detailed understanding of the signaling pathways altered by curcumin is still lacking. In this study, we carried out SILAC-based quantitative proteomic analysis of a HNSCC cell line (CAL 27) to investigate tyrosine signaling in response to curcumin.ResultsUsing high resolution Orbitrap Fusion Tribrid Fourier transform mass spectrometer, we identified 627 phosphotyrosine sites mapping to 359 proteins. We observed alterations in the level of phosphorylation of 304 sites corresponding to 197 proteins upon curcumin treatment. We report here for the first time, curcumin-induced alterations in the phosphorylation of several kinases including TNK2, FRK, AXL, MAPK12 and phosphatases such as PTPN6, PTPRK, and INPPL1 among others. Pathway analysis revealed that the proteins differentially phosphorylated in response to curcumin are known to be involved in focal adhesion kinase signaling and actin cytoskeleton reorganization.ConclusionsThe study indicates that curcumin may regulate cellular processes such as proliferation and migration through perturbation of the focal adhesion kinase pathway. This is the first quantitative phosphoproteomics-based study demonstrating the signaling events that are altered in response to curcumin. Considering the importance of curcumin as an anti-cancer agent, this study will significantly improve the current knowledge of curcumin-mediated signaling in cancer.

Whole Genome Sequencing of Mycobacterium tuberculosis Isolates From Extrapulmonary Sites.

Tuberculosis (TB) remains one of the leading causes of morbidity and mortality worldwide. Extrapulmonary tuberculosis (EPTB) constitutes around 15-20% of TB cases in immunocompetent individuals. Extrapulmonary sites that are affected by TB include bones, lymph nodes, meningitis, pleura, and genitourinary tract. Whole genome sequencing has emerged as a powerful tool to map genetic diversity among Mycobacterium tuberculosis (MTB) isolates and identify the genomic signatures associated with drug resistance, pathogenesis, and disease transmission. Several pulmonary isolates of MTB have been sequenced over the years. However, availability of whole genome sequences of MTB isolates from extrapulmonary sites is limited. Some studies suggest that genetic variations in MTB might contribute to disease presentation in extrapulmonary sites. This can be addressed if whole genome sequence data from large number of extrapulmonary isolates becomes available. In this study, we have performed whole genome sequencing of five MTB clinical isolates derived from EPTB sites using next-generation sequencing platform. We identified 1434 nonsynonymous single nucleotide variations (SNVs), 143 insertions and 105 deletions. This includes 279 SNVs that were not reported before in publicly available datasets. We found several mutations that are known to confer resistance to drugs. All the five isolates belonged to East-African-Indian lineage (lineage 3). We identified 9 putative prophage DNA integrations and 14 predicted clustered regularly interspaced short palindromic repeats (CRISPR) in MTB genome. Our analysis indicates that more work is needed to map the genetic diversity of MTB. Whole genome sequencing in conjunction with comprehensive drug susceptibility testing can reveal clinically relevant mutations associated with drug resistance.

Long-Term Cigarette Smoke Exposure and Changes in MiRNA Expression and Proteome in Non-Small-Cell Lung Cancer

Chronic exposure to cigarette smoke markedly increases the risk for lung cancer. Regulation of gene expression at the post-transcriptional level by miRNAs influences a variety of cancer-related interactomes. Yet, relatively little is known on the effects of long-term cigarette smoke exposure on miRNA expression and gene regulation. NCI-H292 (H292) is a cell line sensitive to cigarette smoke with mucoepidermoid characteristics in culture. We report, in this study, original observations on long-term (12 months) cigarette smoke effects in the H292 cell line, using microarray-based miRNA expression profiling, and stable isotopic labeling with amino acids in cell culture-based quantitative proteomic analysis. We identified 112 upregulated and 147 downregulated miRNAs (by twofold) in cigarette smoke-treated H292 cells. The liquid chromatography-tandem mass spectrometry analysis identified 3,959 proteins, of which, 303 proteins were overexpressed and 112 proteins downregulated (by twofold). We observed 39 miRNA target pairs (proven targets) that were differentially expressed in response to chronic cigarette smoke exposure. Gene ontology analysis of the target proteins revealed enrichment of proteins in biological processes driving metabolism, cell communication, and nucleic acid metabolism. Pathway analysis revealed the enrichment of phagosome maturation, antigen presentation pathway, nuclear factor erythroid 2-related factor 2-mediated oxidative stress response, and cholesterol biosynthesis pathways in cigarette smoke-exposed cells. In conclusion, this report makes an important contribution to knowledge on molecular changes in a lung cell line in response to long term cigarette smoke exposure. The findings might inform future strategies for drug target, biomarker and diagnostics innovation in lung cancer, and clinical oncology. These observations also call for further research on the extent to which continuing or stopping cigarette smoking in patients diagnosed with lung cancer translates into molecular and clinical outcomes.

Signaling network map of the aryl hydrocarbon receptor

The aryl hydrocarbon receptor (AHR) is a multi-domain cytosolic protein that belongs to the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors. Ligand binding induces a conformational change in AHR and promotes nuclear translocation of the receptor (Kewley et al. 2004). AHR can either bind to exogenous (polycyclic aromatic hydrocarbons, dioxins, cigarette smoke) or endogenous ligands (arachidonic acid and leukotrienes, heme metabolites, UV photoproducts of tryptophan) within the cytoplasm. Exogenous ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene are known to activate AHR and mediate cellular toxic response. AHR is also activated by dietary compounds including indole-3-carbinol and flavonoids that mediate various physiological activities in the body (Nguyen and Bradfield 2008; Marconett et al. 2010). AHR ligands are classified as agonists and antagonists depending on the ability of ligands to activate or inhibit AHR induced activity. Previous reports indicate that ligands such as kaempferol, resveratrol, galangin, chrysin and quercetin act either as agonists or antagonists based on the ligand concentration and type of cells induced (Zhang et al. 2003). Thus, diversity of ligands makes AHR signaling a very dynamic and complex. AHR in its inactive state is located in the cytoplasm and forms a complex with molecular chaperones, such as heat shock protein 90 (HSP90) and co-chaperons such as p23 and AHR-interacting protein (AIP) (Trivellin and Korbonits 2011). In presence of ligand, AHR undergoes nuclear translocation where it interacts with AHR nuclear transporter (ARNT) or AHR repressor (AHRR) through the PAS domain. It has been reported that nucleocytoplasmic transport mechanism of AHR varies between humans and mice. In humans, AHR both in stimulated or unstimulated state can undergo nuclear translocation complexed with AIP. In contrast, association of AIP prevents nucleocytoplasmic shuttling of AHR in mice in both stimulated and unstimulated states (Ramadoss et al. 2004). Once AHR is translocated to the nucleus it forms a heterodimer complex with ARNT and binds to xenobiotic response elements located in the promoter region of the target genes. This complex induces coordinated transcription of detoxifying enzymes for efficient absorption, distribution and elimination of xenobiotics from the body (Abel and Haarmann-Stemmann 2010). Apart from this, AHR is known to exhibit endogenous functions such as cell proliferation, cell differentiation and apoptosis. It also acts as an endogenous regulator in several developmental and physiological processes including neurogenesis, hematopoietic stem cell regulation, cellular stress response, immunoregulation and reproductive health (Lindsey and Papoutsakis 2012; Kadow et al. 2011; Hansen et al. 2014). AHR is associated with various pathological and physiological disorders in the body including autoimmune diseases (Veldhoen et al. 2008), inflammation (Podechard et al. 2008; Ovrevik et al. 2014), cardiovascular diseases (Kerley-Hamilton et al. 2012; Savouret et al. 2003) and cancer. Activation of AHR in presence of cigarette smoke has been well documented in lung cancer (Martey et al. 2005; Tsay et al. 2013). Cigarette smoke induced AHR is also known to mediate immune signaling mechanism in chronic obstructive pulmonary disease (COPD) (Chen et al. 2011). Apart from playing an essential role in COPD and lung cancer, AHR expression has been reported in other cancers and adenomas. AHR is reported to be downregulated in growth hormone secreting pituitary adenomas (Jaffrain-Rea et al. 2009), while, increased expression levels of AHR are associated with tumorigenesis in medulloblastoma (Dever and Opanashuk 2012). The enhancement of AHR levels under ligand stimulation induced cell cycle arrest has been reported in pancreatic and gastric cancer (Koliopanos et al. 2002; Peng et al. 2009). Depending upon the type of ligand stimulation, AHR is either known to promote or inhibit tumor progression. Stimulation of AHR with TCDD, 2,3,7,8-tetrachlorodibenzofuran and 3,3′-diindolylmethane inhibits invasiveness and cell growth in breast cancer (Hall et al. 2010). In contrast, stimulation of AHR with n-butyl benzyl phthalate and dibutyl phthalate ligands enhances tumorigenic properties in breast cancer cells (Hsieh et al. 2012). Therefore, AHR could serve as a potential therapeutic target in several cancers (Murray et al. 2014) and hence it is important to develop AHR signaling pathway to understand the mechanism of AHR mediated tumor progression and regression. Although, the diverse role of AHR is documented in literature however a detailed network of AHR signaling is lacking. In this study, we have curated literature information pertaining to AHR induced signaling and developed a pathway map to facilitate better understanding of this receptor.

miRNA and Proteomic Dysregulation in Non-Small Cell Lung Cancer in Response to Cigarette Smoke

BACKGROUND Dysregulation of miRNAs is associated with the development of non-small cell lung cancer (NSCLC). It is imperative to study the dysregulation of miRNAs by cigarette smoke which will affect their targets, either leading to the overexpression of oncoproteins or downregulation of tumor suppressor proteins. OBJECTIVE AND METHODS In this study, we carried out miRNA sequencing and SILAC-based proteomic analysis of H358 cells chronically exposed to cigarette smoke condensate. Using bioinformatics analysis, we mapped the dysregulated miRNAs to differentially expressed target proteins identified in our data. Gene ontology-based enrichment and pathway analysis was performed using the deregulated targets to study the role of cigarette smoke-mediated miRNA dysregulation in NSCLC cell line. RESULTS miRNA sequencing resulted in the identification of 208 miRNAs, of which 6 miRNAs were found to be significantly dysregulated (2 fold, Log Base 2; p-value ≤ 0.05) in H358-Smoke cells. Proteomic analysis of the smoke exposed cells compared to the untreated parental cells resulted in the quantification of 2,610 proteins, of which 690 proteins were found to be differentially expressed (fold change ≥ 2). Gene ontology based analysis of target proteins revealed enrichment of proteins driving metabolism and a decrease in expression of proteins associated with immune response in the cells exposed to cigarette smoke. Pathway study using Ingenuity Pathway Analysis (IPA) revealed activation of NRF2-mediated oxidative stress response and actin-cytoskeleton signaling, and repression of protein kinase A signaling in H358-Smoke cells. We also identified 5 novel miRNAs in H358-Smoke cells using unassigned reads of small RNA-Seq dataset. CONCLUSION In summary, this study indicates that chronic exposure to cigarette smoke leads to widespread dysregulation of miRNAs and their targets, resulting in signaling aberrations in NSCLC cell line. The miRNAs and their targets identified in the study need to be further investigated to explore their role as potential therapeutic targets and/or molecular markers in NSCLC especially in smokers.

Cigarette smoke induces mitochondrial metabolic reprogramming in lung cells

Cellular transformation owing to cigarette smoking is due to chronic exposure and not acute. However, systematic studies to understand the molecular alterations in lung cells due to cigarette smoke are lacking. To understand these molecular alterations induced by chronic cigarette smoke exposure, we carried out tandem mass tag (TMT) based temporal proteomic profiling of lung cells exposed to cigarette smoke for upto 12months. We identified 2620 proteins in total, of which 671 proteins were differentially expressed (1.5-fold) after 12months of exposure. Prolonged exposure of lung cells to smoke for 12months revealed dysregulation of oxidative phosphorylation and overexpression of enzymes involved in TCA cycle. In addition, we also observed overexpression of enzymes involved in glutamine metabolism, fatty acid degradation and lactate synthesis. This could possibly explain the availability of alternative source of carbon to TCA cycle apart from glycolytic pyruvate. Our data indicates that chronic exposure to cigarette smoke induces mitochondrial metabolic reprogramming in cells to support growth and survival.

Chronic exposure of cigarette smoke and chewing tobacco alters expression of microRNAs in esophageal epithelial cells.

BACKGROUND Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers with high mortality rate. Cigarette smoke and chewing tobacco are well known risk factors associated with ESCC. However, molecular mechanisms associated with development of ESCC among smokers and chewers are poorly understood. MicroRNAs play an important role in regulating physiological and disease processes including esophageal cancer. OBJECTIVE AND METHODS In this study, we developed an in vitro model by treating non-neoplastic Het- 1A esophageal cell line with cigarette smoke and chewing tobacco. We carried out miRNA sequencing on Illumina HiSeq 2500 platform and compared miRNA expression pattern across cigarette smoke and chewing tobacco treated Het-1A cells with untreated cells. RESULTS We identified and quantified 433 miRNAs in both smoke exposed and chewing tobacco treated cells, of which 13 miRNAs showed significantly altered expression in cigarette smoke exposed cells while 25 miRNAs showed significantly altered expression in chewing tobacco treated cells. In addition, we predicted novel miRNAs from these data-sets. We evaluated miRNAs that showed selective or context dependent expression pattern in cigarette smoke exposed or chewing tobacco treated cells. CONCLUSION In this study, we have comprehensively mapped miRNA expression pattern in response to cigarette smoke and chewing tobacco in Het-1A cells. We identified miRNAs that show altered expression in these cell models.