Search

In cancer management, the cyclooxygenase (COX)–targeted approach has shown great promise in anticancer therapeutics. However, the use of COX-2 inhibitors has side effects and health hazards; thus, targeting its major metabolite prostaglandin E2 (PGE2)–mediated signaling pathway might be a rational approach for the next generation of cancer management. Recent studies on several in vitro and in vivo models have revealed that elevated expression of COX-2 correlates with prostate tumor growth and angiogenesis. In this study, we have shown the in-depth molecular mechanism and the PGE2 activation of the epidermal growth factor receptor and β3 integrin through E prostanoid 2 (EP2)–mediated and EP4-mediated pathways, which lead to activator protein-1 (AP-1) activation. Moreover, PGE2 also induces activating transcription factor-4 (ATF-4) activation and stimulates cross-talk between ATF-4 and AP-1, which is unidirectional toward AP-1, which leads to the increased expressions of urokinase-type plasminogen activator and vascular endothelial growth factor and, eventually, regulates prostate tumor cell motility. In vivo Matrigel angiogenesis assay data revealed that PGE2 induces angiogenesis through EP2 and EP4. Human prostate cancer specimen analysis also supported our in vitro and in vivo studies. Our data suggest that targeting PGE2 signaling pathway (i.e., blocking EP2 and EP4 receptors) might be a rational therapeutic approach for overcoming the side effects of COX-2 inhibitors and that this might be a novel strategy for the next generation of prostate cancer management. [Cancer Res 2008;68(19):7750–9]

Supplementary Figures 1-6 from Prostaglandin E2 Regulates Tumor Angiogenesis in Prostate Cancer

Resistance to cancer chemotherapy is a major global health burden. Epidermal growth factor receptor (EGFR) is a proven therapeutic target for multiple cancers of epithelial origin. Despite its overexpression in >90% of head and neck squamous cell carcinoma (HNSCC) patients, tyrosine kinase inhibitors such as erlotinib have shown a modest response in clinical trials. Cellular heterogeneity is thought to play an important role in HNSCC therapeutic resistance. Genomic alterations alone cannot explain all resistance mechanisms at play in a heterogeneous system. It is thus important to understand the biochemical mechanisms associated with drug resistance to determine potential strategies to achieve clinical response. We investigated tyrosine kinase signaling networks in erlotinib-resistant cells using quantitative tyrosine phosphoproteomics approach. We observed altered phosphorylation of proteins involved in cell adhesion and motility in erlotinib-resistant cells. Bioinformatics analysis revealed enrichment of pathways related to regulation of the actin cytoskeleton, extracellular matrix (ECM)–receptor interaction, and endothelial migration. Of importance, enrichment of the focal adhesion kinase (PTK2) signaling pathway downstream of EGFR was also observed in erlotinib-resistant cells. To the best of our knowledge, we present the first report of tyrosine phosphoproteome profiling in erlotinib-resistant HNSCC, with an eye to inform new ways to achieve clinical response. Our findings suggest that common signaling networks are at play in driving resistance to EGFR-targeted therapies in HNSCC and other cancers. Most notably, our data suggest that the PTK2 pathway genes may potentially play a significant role in determining clinical response to erlotinib in HNSCC tumors.

BackgroundIncreased infiltration of T cells into ovarian tumors has been repeatedly shown to be predictive of enhanced patient survival. However, despite the evidence of an active immune response in ovarian cancer (OC), the frequency of responses to immune checkpoint blockade (ICB) therapy in OC is much lower than other cancer types. Recent studies have highlighted that deficiencies in the DNA damage response (DDR) can drive increased genomic instability and tumor immunogenicity, which leads to enhanced responses to ICB. Protein phosphatase 4 (PP4) is a critical regulator of the DDR; however, its potential role in antitumor immunity is currently unknown.ResultsOur results show that the PP4 inhibitor, fostriecin, combined with carboplatin leads to increased carboplatin sensitivity, DNA damage, and micronuclei formation. Using multiple OC cell lines, we show that PP4 inhibition orPPP4Cknockdown combined with carboplatin triggers inflammatory signaling via Nuclear factor kappa B (NF-κB) and signal transducer and activator of transcription 1 (STAT1) activation. This resulted in increased expression of the pro-inflammatory cytokines and chemokines:CCL5,CXCL10, andIL-6. In addition,IFNB1expression was increased suggesting activation of the type I interferon response. Conditioned media from OC cells treated with the combination of PP4 inhibitor and carboplatin significantly increased migration of both CD8 T cell and natural killer (NK) cells over carboplatin treatment alone. Knockdown of stimulator of interferon genes (STING) in OC cells significantly abrogated the increase in CD8 T-cell migration induced by PP4 inhibition. Co-culture of NK-92 cells and OC cells withPPP4CorPPP4R3Bknockdown resulted in strong induction of NK cell interferon-γ, increased degranulation, and increased NK cell-mediated cytotoxicity against OC cells. Stable knockdown of PP4C in a syngeneic, immunocompetent mouse model of OC resulted in significantly reduced tumor growthin vivo. Tumors with PP4C knockdown had increased infiltration of NK cells, NK T cells, and CD4+T cells. Addition of low dose carboplatin treatment led to increased CD8+T-cell infiltration in PP4C knockdown tumors as compared with the untreated PP4C knockdown tumors.ConclusionsOur work has identified a role for PP4 inhibition in promoting inflammatory signaling and enhanced immune cell effector function. These findings support the further investigation of PP4 inhibitors to enhance chemo-immunotherapy for OC treatment.

Background: Increased infiltration of T cells into ovarian tumors has been repeatedly shown to be predictive of enhanced patient survival. However, despite the evidence of an active immune response in OC, immune checkpoint blockade therapy has been ineffective. Recently studies have shown that deficiencies in the DNA damage response (DDR) can drive increased genomic instability and tumor immunogenicity. One target involved in the DDR that has the potential to be of therapeutic value is protein phosphatase 4 (PP4); however, the effect of PP4 deficiency on anti-tumor immunity remain unknown. Results: Our results show that PP4 inhibition combined with carboplatin leads to increased carboplatin sensitivity, DNA damage, and micronuclei formation. Using a panel of ovarian cancer cells, we show that PP4 inhibition triggers inflammatory signaling via NF-κB and STAT1 activation resulting in increased expression of pro-inflammatory cytokines including IFNβ1, CCL5, CXCL10, and IL-6. Moreover, conditioned media from OC cells treated with the combination of PP4 inhibitor and carboplatin significantly increased migration of both CD8 T cell and NK cells over carboplatin treatment alone. Knockdown of STING in OC cells significantly abrogated the increase in CD8 T cell migration induced by PP4 inhibition. PPP4C or PPP4R3B knockdown resulted in strong induction of NK cell IFN-γ, increased degranulation, and increased NK cell-mediated cytotoxicity against OC cells. Stable knockdown of PP4C in a syngeneic, immunocompetent mouse model of ovarian cancer resulted in significant reduction of tumor growth in vivo. PP4C low tumors had increased infiltration of CD161+ NK cells and CD4+ T cells. Addition of low dose carboplatin treatment in vivo led to increased CD8+ T cell infiltration in PP4C low tumors when compared to the untreated groups. Conclusions: Our work has identified a role for PP4 inhibition in promoting anti-tumor immune activation. These findings provide the rationale for combining PP4 inhibitors with immunotherapy as a new approach in ovarian cancer treatment. Citation Format: Remya Raja, Christopher Wu, Esen Y. Bassoy, Thomas Rubino, Emma Utagawa, Paul Magtibay, Kristina Butler, Marion Curtis. Protein phosphatase 4 inhibition stimulates anti-tumor immunity in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4455.

Simple Summary Metabolism is the process by which living organisms and cells generate energy to sustain life. At the organismal level, metabolic homeostasis is a tightly controlled balance between energy consumption and energy expenditure. Many studies have shown that disruption of this homeostasis leads to an inflammatory phenotype within adipose tissue. The aim of this review is to provide an overview of the dynamic metabolic interplay within adipose tissue and its implications for cancer progression and metastasis. Abstract Disruption of metabolic homeostasis at the organismal level can cause metabolic syndrome associated with obesity. The role of adipose tissue in cancer has been investigated over the last several decades with many studies implicating obesity as a risk factor for the development of cancer. Adipose tissue contains a diverse array of immune cell populations that promote metabolic homeostasis through a tightly controlled balance of pro- and anti-inflammatory signals. During obesity, pro-inflammatory cell types infiltrate and expand within the adipose tissue, exacerbating metabolic dysfunction. Some studies have now shown that the intracellular metabolism of immune cells is also deregulated by the lipid-rich environment in obesity. What is not fully understood, is how this may influence cancer progression, metastasis, and anti-tumor immunity. This review seeks to highlight our current understanding of the effect of adipose tissue on immune cell function and discuss how recent results offer new insight into the role that adipose tissue plays in cancer progression and anti-tumor immunity.

Gallbladder cancer (GBC) is a rare malignancy, associated with poor disease prognosis with a 5-year survival of only 20%. This has been attributed to late presentation of the disease, lack of early diagnostic markers and limited efficacy of therapeutic interventions. Elucidation of molecular events in GBC can contribute to better management of the disease by aiding in the identification of therapeutic targets. To identify aberrantly activated signaling events in GBC, tandem mass tag-based quantitative phosphoproteomic analysis of five GBC cell lines was carried out. Proline-rich Akt substrate 40 kDa (PRAS40) was one of the proteins found to be hyperphosphorylated in all the invasive GBC cell lines. Tissue microarray-based immunohistochemical labeling of phospho-PRAS40 (T246) revealed moderate to strong staining in 77% of the primary gallbladder adenocarcinoma cases. Regulation of PRAS40 activity by inhibiting its upstream kinase PIM1 resulted in a significant decrease in cell proliferation, colony forming and invasive ability of GBC cells. Our results support the role of PRAS40 phosphorylation in GBC cell survival and aggressiveness. This study also elucidates phospho-PRAS40 as a clinical marker in GBC and the role of PIM1 as a therapeutic target in GBC. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12079-018-00503-5) contains supplementary material, which is available to authorized users.

Background Increased infiltration of T cells into ovarian tumors has been repeatedly shown to be predictive of enhanced patient survival. However, despite the evidence of an active immune response in OC, immune checkpoint blockade therapy has been ineffective. Recently studies have shown that deficiencies in the DNA damage response (DDR) can drive increased genomic instability and tumor immunogenicity. One target involved in the DDR that has the potential to be of therapeutic value is protein phosphatase 4 (PP4); however, the effect of PP4 deficiency on anti-tumor immunity remain unknown. Results Our results show that PP4 inhibition combined with carboplatin leads to increased carboplatin sensitivity, DNA damage, and micronuclei formation. Using a panel of ovarian cancer cells, we show that PP4 inhibition triggers inflammatory signaling via NFκB and STAT1 activation resulting in increased expression of pro-inflammatory cytokines including IFNβ1, CCL5, CXCL10, and IL-6. We further show that PP4 inhibition and carboplatin-induced STAT1 activation is significantly blunted upon STING knockdown in ovarian cancer cells. Moreover, using mouse ID8 ovarian cancer cell line and OT-I cells, we demonstrate that the combination of PP4 inhibition and carboplatin significantly increased CD8 T cell migration over carboplatin treatment alone. In addition, the same treatment resulted in enhanced NK cell-mediated tumor cell killing and T cell killing in an autologous human organoid model. Conclusions Our work has identified a role for PP4 inhibition in promoting anti-tumor immune activation. These findings provide the rationale for combining PP4 inhibitors with immunotherapy as a new approach in ovarian cancer treatment. Supported by DOD W81XWH2110489

Epidermal growth factor receptor (EGFR) targeted therapies have shown limited efficacy in head and neck squamous cell carcinoma (HNSCC) patients despite its overexpression. Identifying molecular mechanisms associated with acquired resistance to EGFR-TKIs such as erlotinib remains an unmet need and a therapeutic challenge. In this study, we employed an integrated multi-omics approach to delineate mechanisms associated with acquired resistance to erlotinib by carrying out whole exome sequencing, quantitative proteomic and phosphoproteomic profiling. We observed amplification of several genes including AXL kinase and transcription factor YAP1 resulting in protein overexpression. We also observed expression of constitutively active mutant MAP2K1 (p.K57E) in erlotinib resistant SCC-R cells. An integrated analysis of genomic, proteomic and phosphoproteomic data revealed alterations in MAPK pathway and its downstream targets in SCC-R cells. We demonstrate that erlotinib-resistant cells are sensitive to MAPK pathway inhibition. This study revealed multiple genetic, proteomic and phosphoproteomic alterations associated with erlotinib resistant SCC-R cells. Our data indicates that therapeutic targeting of MAPK pathway is an effective strategy for treating erlotinib-resistant HNSCC tumors.

BackgroundGene expression profiling is emerging as a tool for tuberculosis diagnosis and treatment response monitoring, but limited data specific to Indian children and incident tuberculosis infection (TBI) exist.MethodsSixteen pediatric Indian tuberculosis cases were age- and sex-matched to 32 tuberculosis-exposed controls (13 developed incident TBI without subsequent active tuberculosis). Longitudinal samples were collected for ribonucleic acid sequencing. Differential expression analysis generated gene lists that identify tuberculosis diagnosis and tuberculosis treatment response. Data were compared with published gene lists. Population-specific risk score thresholds were calculated.ResultsSeventy-one genes identified tuberculosis diagnosis and 25 treatment response. Within-group expression was partially explained by age, sex, and incident TBI. Transient changes in gene expression were identified after both infection and treatment. Application of 27 published gene lists to our data found variable performance for tuberculosis diagnosis (sensitivity 0.38–1.00, specificity 0.48–0.93) and treatment response (sensitivity 0.70–0.80, specificity 0.40–0.80). Our gene lists found similarly variable performance when applied to published datasets for diagnosis (sensitivity 0.56–0.85, specificity 0.50–0.85) and treatment response (sensitivity 0.49– 0.86, specificity 0.50–0.84).ConclusionsGene expression profiles among Indian children with confirmed tuberculosis were distinct from adult-derived gene lists, highlighting the importance of including distinct populations in differential gene expression models.

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.

Tobacco in its smoke and smokeless form are major risk factors for esophageal squamous cell carcinoma (ESCC). However, molecular alterations associated with smokeless tobacco exposure are poorly understood. In the Indian subcontinent, tobacco is predominantly consumed in chewing form. An understanding of molecular alterations associated with chewing tobacco exposure is vital for identifying molecular markers and potential targets. We developed an in vitro cellular model by exposing non-transformed esophageal epithelial cells to chewing tobacco over an eight-month period. Chronic exposure to chewing tobacco led to increase in cell proliferation, invasive ability and anchorage independent growth, indicating cell transformation. Molecular alterations associated with chewing tobacco exposure were characterized by carrying out exome sequencing and quantitative proteomic profiling of parental cells and chewing tobacco exposed cells. Quantitative proteomic analysis revealed increased expression of cancer stem cell markers in tobacco treated cells. In addition, tobacco exposed cells showed the Oxidative Phosphorylation (OXPHOS) phenotype with decreased expression of enzymes associated with glycolytic pathway and increased expression of a large number of mitochondrial proteins involved in electron transport chain as well as enzymes of the tricarboxylic acid (TCA) cycle. Electron micrographs revealed increase in number and size of mitochondria. Based on these observations, we propose that chronic exposure of esophageal epithelial cells to tobacco leads to cancer stem cell-like phenotype. These cells show the characteristic OXPHOS phenotype, which can be potentially targeted as a therapeutic strategy.

Context Inactivating germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene are linked to pituitary adenoma predisposition. Here, we present the youngest known patient with AIP-related pituitary adenoma. Case Description The patient presented at the age of 4 years with pituitary apoplexy and left ptosis with severe visual loss following a 1-year history of abdominal pain, headaches, and rapid growth. His IGF-1 level was 5× the upper limit of normal, and his random GH level was 1200 ng/mL. MRI showed a 43 × 24 × 35‒mm adenoma with suprasellar extension invading the left cavernous sinus (Knosp grade 4). After transsphenoidal surgery, histology showed a grade 2A sparsely granulated somatotropinoma with negative O6-methylguanine-DNA methyltransferase and positive vascular endothelial growth factor staining. Genetic testing identified a heterozygous germline nonsense AIP mutation (p.Arg81Ter). Exome sequencing of the tumor revealed that it had lost the entire maternal chromosome-11, rendering it hemizygous for chromosome-11 and therefore lacking functional copies of AIP in the tumor. He was started on octreotide, but because the tumor rapidly regrew and IGF-1 levels were unchanged, temozolomide was initiated, and intensity-modulated radiotherapy was administered 5 months after surgery. Two months later, bevacizumab was added, resulting in excellent tumor response. Although these treatments stabilized tumor growth over 4 years, IGF-1 was normalized only after pegvisomant treatment, although access to this medication was intermittent. At 3.5 years of follow-up, gamma knife treatment was administered, and pegvisomant dose increase was indicated. Conclusion Multimodal treatment with surgery, long-acting octreotide, radiotherapy, temozolomide, bevacizumab, and pegvisomant can control genetically driven, aggressive, childhood-onset somatotropinomas., AIP mutation-driven gigantism with pituitary apoplexy and aggressive tumor growth was controlled by multimodal treatment: surgery, octreotide-LAR, radiotherapy, temozolomide, bevacizumab, and pegvisomant.

Cigarette smoking has been associated with multiple negative effects on human skin. Long-term physiological effects of cigarette smoke are through chronic and not acute exposure. Molecular alterations due to chronic exposure to cigarette smoke remain unclear. Primary human skin keratinocytes chronically exposed to cigarette smoke condensate (CSC) showed a decreased wound-healing capacity with an increased expression of NRF2 and MMP9. Using quantitative proteomics, we identified 4728 proteins, of which 105 proteins were overexpressed (≥2-fold) and 41 proteins were downregulated (≤2-fold) in primary skin keratinocytes chronically exposed to CSC. We observed an alteration in the expression of several proteins involved in maintenance of epithelial barrier integrity, including keratin 80 (5.3 fold, p value 2.5 × 10-7), cystatin A (3.6-fold, p value 3.2 × 10-3), and periplakin (2.4-fold, p value 1.2 × 10-8). Increased expression of proteins associated with skin hydration, including caspase 14 (2.2-fold, p value 4.7 × 10-2) and filaggrin (3.6-fold, p value 5.4 × 10-7), was also observed. In addition, we report differential expression of several proteins, including adipogenesis regulatory factor (2.5-fold, p value 1.3 × 10-3) and histone H1.0 (2.5-fold, p value 6.3 × 10-3) that have not been reported earlier. Bioinformatics analyses demonstrated that proteins differentially expressed in response to CSC are largely related to oxidative stress, maintenance of skin integrity, and anti-inflammatory responses. Importantly, treatment with vitamin E, a widely used antioxidant, could partially rescue adverse effects of CSC exposure in primary skin keratinocytes. The utility of antioxidant-based new dermatological formulations in delaying or preventing skin aging and oxidative damages caused by chronic cigarette smoke exposure warrants further clinical investigations and multi-omics research.

// Remya Raja 1, * , Nandini A. Sahasrabuddhe 1, * , Aneesha Radhakrishnan 1, 2, * , Nazia Syed 1, 2 , Hitendra S. Solanki 1, 3 , Vinuth N. Puttamallesh 1, 4 , Sai A. Balaji 5 , Vishalakshi Nanjappa 1, 4 , Keshava K. Datta 1, 3 , Niraj Babu 1 , Santosh Renuse 1, 4 , Arun H. Patil 1, 3 , Evgeny Izumchenko 6 , T.S. Keshava Prasad 1, 4, 11, 12 , Xiaofei Chang 6 , Annapoorni Rangarajan 5 , David Sidransky 6 , Akhilesh Pandey 7, 8, 9, 10 , Harsha Gowda 1, 11 , Aditi Chatterjee 1, 11 1 Institute of Bioinformatics, International Tech Park, Bangalore, 560 066, India 2 Department of Biochemistry and Molecular Biology, Pondicherry University, Puducherry, 605014, India 3 School of Biotechnology, KIIT University, Bhubaneswar, Odisha, 751024, India 4 Amrita School of Biotechnology, Amrita University, Kollam, 690 525, India 5 Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560012, India 6 Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21231, USA 7 McKusick-Nathans Institute of Genetic Medicine, Baltimore, Maryland, 21205, USA 8 Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205, USA 9 Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205, USA 10 Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205, USA 11 YU-IOB Center for Systems Biology and Molecular Medicine, Yenepoya University, Mangalore, 575018, India 12 NIMHANS-IOB Proteomics and Bioinformatics Laboratory, Neurobiology Research Centre, National Institute of Mental Health and Neurosciences, Bangalore, 560029, India * These authors contributed equally to this work Correspondence to: Aditi Chatterjee, email: aditi@ibioinformatics.org Keywords: mass spectrometry, NSCLC, p21 (RAC1)-activated kinase 6, smoking Received: January 27, 2016 Accepted: August 08, 2016 Published: August 16, 2016 ABSTRACT 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.

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.

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.

Chewing tobacco is a common practice in certain socio-economic sections of southern Asia, particularly in the Indian subcontinent and has been well associated with head and neck squamous cell carcinoma. The molecular mechanisms of chewing tobacco which leads to malignancy remains unclear. In large majority of studies, short-term exposure to tobacco has been evaluated. From a biological perspective, however, long-term (chronic) exposure to tobacco mimics the pathogenesis of oral cancer more closely. We developed a cell line model to investigate the chronic effects of chewing tobacco. Chronic exposure to tobacco resulted in higher cellular proliferation and invasive ability of the normal oral keratinocytes (OKF6/TERT1). We carried out quantitative proteomic analysis of OKF6/TERT1 cells chronically treated with chewing tobacco compared to the untreated cells. We identified a total of 3,636 proteins among which expression of 408 proteins were found to be significantly altered. Among the overexpressed proteins, stearoyl-CoA desaturase (SCD) was found to be 2.6-fold overexpressed in the tobacco treated cells. Silencing/inhibition of SCD using its specific siRNA or inhibitor led to a decrease in cellular proliferation, invasion and colony forming ability of not only the tobacco treated cells but also in a panel of head and neck cancer cell lines. These findings suggest that chronic exposure to chewing tobacco induced carcinogenesis in non-malignant oral epithelial cells and SCD plays an essential role in this process. The current study provides evidence that SCD can act as a potential therapeutic target in head and neck squamous cell carcinoma, especially in patients who are users of tobacco.

Dysregulation of protein expression is associated with most diseases including cancer. MS-based proteomic analysis is widely employed as a tool to study protein dysregulation in cancers. Proteins that are differentially expressed in head and neck squamous cell carcinoma (HNSCC) cell lines compared to the normal oral cell line could serve as biomarkers for patient stratification. To understand the proteomic complexity in HNSCC, we carried out iTRAQ-based MS analysis on a panel of HNSCC cell lines in addition to a normal oral keratinocyte cell line. LC-MS/MS analysis of total proteome of the HNSCC cell lines led to the identification of 3263 proteins, of which 185 proteins were overexpressed and 190 proteins were downregulated more than twofold in at least two of the three HNSCC cell lines studied. Among the overexpressed proteins, 23 proteins were related to DNA replication and repair. These included high-mobility group box 2 (HMGB2) protein, which was overexpressed in all three HNSCC lines studied. Overexpression of HMGB2 has been reported in various cancers, yet its role in HNSCC remains unclear. Immunohistochemical labeling of HMGB2 in a panel of HNSCC tumors using tissue microarrays revealed overexpression in 77% (54 of 70) of tumors. The HMGB proteins are known to bind to DNA structure resulting from cisplatin-DNA adducts and affect the chemosensitivity of cells. We observed that siRNA-mediated silencing of HMGB2 increased the sensitivity of the HNSCC cell lines to cisplatin and 5-FU. We hypothesize that targeting HMGB2 could enhance the efficacy of existing chemotherapeutic regimens for treatment of HNSCC. All MS data have been deposited in the ProteomeXchange with identifier PXD000737 (http://proteomecentral.proteomexchange.org/dataset/PXD000737).

Objectives: Curcumin has been shown to exhibit anti-neoplastic effects. However, due to its poor bioavailability, the use of curcumin as an anti-cancer drug is limited. Thus, it is necessary to identify molecules as an alternative to curcumin that could serve as anti-cancer targets. In this study, we attempted to understand the underlying curcumin-mediated signalling pathways contributing to anti-neoplastic effects of curcumin. Methods: We carried out mass spectrometry-based phosphoproteomic analysis of head and neck cancer cell line, CAL 27, treated with and without curcumin to identify curcumin-mediated signalling pathways. Serine/threonine kinases were enriched using titanium dioxide. Results: This resulted in the identification of 5921 phosphopeptides corresponding to 1878 proteins. Of these, 275 and 183 phosphopeptides corresponding to 335 and 242 proteins (≥2.0-fold) were found to be hyper- and hypo-phosphorylated, respectively, in response to curcumin treatment. Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), a serine/threonine kinase, and its downstream target protein kinase AMP-activated non-catalytic subunit beta 1 (PRKAB1) were found to be hypo-phosphorylated when treated with curcumin. Further, silencing or inhibiting CaMKK2 resulted in decreased invasion and colony forming ability of not only CAL 27 cells but also other head and neck squamous cell carcinoma (HNSCC) cell lines. Further, Western blot analysis showed that curcumin-mediated signalling is corroborated by CaMKK2. Conclusions: Taken together, our results suggest that CaMKK2 could be a novel therapeutic target in HNSCC and can serve as an alternative to curcumin.

EGFR-based targeted therapies have shown limited success in smokers. Identification of alternate signaling mechanism(s) leading to TKI resistance in smokers is critically important. We observed increased resistance to erlotinib in H358 NSCLC (non-small cell lung carcinoma) cells chronically exposed to cigarette smoke (H358-S) compared to parental cells. SILAC-based mass-spectrometry approach was used to study altered signaling in H358-S cell line. Importantly, among the top phosphosites in H358-S cells we observed hyperphosphorylation of EGFR (Y1197) and non-receptor tyrosine kinase FAK (Y576/577). Supporting these observations, a transcriptomic-based pathway activation analysis of TCGA NSCLC datasets revealed that FAK and EGFR internalization pathways were significantly upregulated in smoking patients, compared to the never-smokers and were associated with elevated PI3K signaling and lower level of caspase cascade and E-cadherin pathways activation. We show that inhibition of FAK led to decreased cellular proliferation and invasive ability of the smoke-exposed cells, and restored their dependency on EGFR signaling. Our data suggests that activation of focal adhesion pathway significantly contributes to erlotinib resistance, and that FAK is a potential therapeutic target for management of erlotinib resistance in smoke-induced NSCLC.

Despite advances in clinical management, 5-year survival rate in patients with late-stage head and neck squamous cell carcinoma (HNSCC) has not improved significantly over the past decade. Targeted therapies have emerged as one of the most promising approaches to treat several malignancies. Though tyrosine phosphorylation accounts for a minority of total phosphorylation, it is critical for activation of signaling pathways and plays a significant role in driving cancers. To identify activated tyrosine kinase signaling pathways in HNSCC, we compared the phosphotyrosine profiles of a panel of HNSCC cell lines to a normal oral keratinocyte cell line. Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) was one of the kinases hyperphosphorylated at Tyr-321 in all HNSCC cell lines. Inhibition of DYRK1A resulted in an increased apoptosis and decrease in invasion and colony formation ability of HNSCC cell lines. Further, administration of the small molecular inhibitor against DYRK1A in mice bearing HNSCC xenograft tumors induced regression of tumor growth. Immunohistochemical labeling of DYRK1A in primary tumor tissues using tissue microarrays revealed strong to moderate staining of DYRK1A in 97.5% (39/40) of HNSCC tissues analyzed. Taken together our results suggest that DYRK1A could be a novel therapeutic target in HNSCC.

Robust diagnostics for many human genetic disorders are much needed in the pursuit of global personalized medicine. Next-generation sequencing now offers new promise for biomarker and diagnostic discovery, in developed as well as resource-limited countries. In this broader global health context, X-linked intellectual disability (XLID) is an inherited genetic disorder that is associated with a range of phenotypes impacting societies in both developed and developing countries. Although intellectual disability arises due to diverse causes, a substantial proportion is caused by genomic alterations. Studies have identified causal XLID genomic alterations in more than 100 protein-coding genes located on the X-chromosome. However, the causes for a substantial number of intellectual disability and associated phenotypes still remain unknown. Identification of causative genes and novel mutations will help in early diagnosis as well as genetic counseling of families. Advent of next-generation sequencing methods has accelerated the discovery of new genes involved in mental health disorders. In this study, we analyzed the exomes of three families from India with nonsyndromic XLID comprising seven affected individuals. The affected individuals had varying degrees of intellectual disability, microcephaly, and delayed motor and language milestones. We identified potential causal variants in three XLID genes, including PAK3 (V294M), CASK (complex structural variant), and MECP2 (P354T). Our findings reported in this study extend the spectrum of mutations and phenotypes associated with XLID, and calls for further studies of intellectual disability and mental health disorders with use of next-generation sequencing technologies.

Complementing genome sequence with deep transcriptome and proteome data could enable more accurate assembly and annotation of newly sequenced genomes. Here, we provide a proof-of-concept of an integrated approach for analysis of the genome and proteome of Anopheles stephensi, which is one of the most important vectors of the malaria parasite. To achieve broad coverage of genes, we carried out transcriptome sequencing and deep proteome profiling of multiple anatomically distinct sites. Based on transcriptomic data alone, we identified and corrected 535 events of incomplete genome assembly involving 1196 scaffolds and 868 protein-coding gene models. This proteogenomic approach enabled us to add 365 genes that were missed during genome annotation and identify 917 gene correction events through discovery of 151 novel exons, 297 protein extensions, 231 exon extensions,192 novel protein start sites,19 novel translational frames, 28 events of joining of exons, and 76 events of joining of adjacent genes as a single gene. Incorporation of proteomic evidence allowed us to change the designation of more than 87 predicted "noncoding RNAs" to conventional mRNAs coded by protein-coding genes. Importantly, extension of the newly corrected genome assemblies and gene models to 15 other newly assembled Anopheline genomes led to the discovery of a large number of apparent discrepancies in assembly and annotation of these genomes. Our data provide a framework for how future genome sequencing efforts should incorporate transcriptomic and proteomic analysis in combination with simultaneous manual curation to achieve near complete assembly and accurate annotation of genomes. Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India [EMR/2014/000444]; DBT Program Support grant on "Development of infrastructure and a computational framework for analysis of proteomic data" [BT/01/COE/08/05]; Infosys Foundation; pilot grant from the Johns Hopkins Malaria Research Institute; Council of Scientific and Industrial Research, Department of Biotechnology, University Grants Commission; Department of Science and Technology, Government of India; Indian Council of Medical Research This paper is funded by the joint research project to NIMR and IOB entitled "Characterization of Malaria vector Anopheles stephensi Proteome and Transcriptome" (EMR/2014/000444) from the Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India. T.S.K.P. is also supported by the DBT Program Support grant on "Development of infrastructure and a computational framework for analysis of proteomic data" (BT/01/COE/08/05). We also thank Infosys Foundation for financial support to IOB. A.P. and P.S. were funded by a pilot grant from the Johns Hopkins Malaria Research Institute. This paper bears the NIMR publication screening committee approval No. 009/2015. H.G. is a Wellcome Trust-DBT India Alliance Early Career Fellow. We thank the Council of Scientific and Industrial Research, Department of Biotechnology, University Grants Commission, Indian Council of Medical Research and Department of Science and Technology, Government of India for research fellowships to M.K., S.K.S., G.D., R.S.N., S.M.P., A.K. M. (IOB), S.S.M., M.K.G., S.B.D., D.S.K., P.R., N.S., S.D.Y., K.K.D., R.R., A.A.K., A.R., G.J.S., S.C., and R.V. M.D. is funded by the Faculty Improvement Program of Siddaganga Institute of Technology, Tumkur.

Hypoxia is a salient feature of most solid tumors, and hypoxic adaptation of cancer cells has crucial implications in propagation of malignant clonal cell population. Osteopontin (OPN) has been identified as a hypoxia-responsive gene, but the mechanistic and regulatory role of OPN under hypoxia is less characterized. The present study identifies the existence of a positive inter-regulatory loop between hypoxia and OPN. We have shown that hypoxia induces OPN expression in breast cancer cells; however, the expression was found to be HIF1α independent. OPN enabled transcriptional upregulation of HIF1α expression both under normoxia and hypoxia, whereas stability of HIF1α protein in breast cancer cells remained unaffected. Moreover, we have shown that OPN induces integrin-linked kinase (ILK)/Akt-mediated nuclear factor (NF)-κB p65 activation leading to HIF1α-dependent vascular endothelial growth factor (VEGF) expression and angiogenesis in response to hypoxia. These in vitro data are biologically important as OPN expressing cells induce greater tumor growth and angiogenesis through enhanced expressions of proangiogenic molecules as compared with control. Immunohistochemical analysis of human breast cancer specimens revealed significant correlation between OPN and HIF1α but not HIF2α. Elevated expression of HIF1α and OPN was observed in pre-neoplastic and early stage infiltrating ductal carcinoma implicating the role of these proteins in neoplastic progression of breast cancer. Together, our results substantiate the prime role of OPN in cellular adaptation through ILK and NF-κB-mediated HIF1α-dependent VEGF expression in response to hypoxia that ultimately controls breast cancer progression and angiogenesis. Our study reinforces the fact that targeting OPN and its regulated signaling network hold important therapeutic implications.

Despite advances in clinical management, 5-year survival rate in patients with late-stage head and neck squamous cell carcinoma (HNSCC) has not improved significantly over the past decade. Targeted therapies have emerged as one of the most promising approaches to treat several malignancies. Though tyrosine phosphorylation accounts for a minority of total phosphorylation, it is critical for activation of signaling pathways and plays a significant role in driving cancers. To identify activated tyrosine kinase signaling pathways in HNSCC, we compared the phosphotyrosine profiles of a panel of HNSCC cell lines to a normal oral keratinocyte cell line. Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) was one of the kinases hyperphosphorylated at Tyr-321 in all HNSCC cell lines. Inhibition of DYRK1A resulted in an increased apoptosis and decrease in invasion and colony formation ability of HNSCC cell lines. Further, administration of the small molecular inhibitor against DYRK1A in mice bearing HNSCC xenograft tumors induced regression of tumor growth. Immunohistochemical labeling of DYRK1A in primary tumor tissues using tissue microarrays revealed strong to moderate staining of DYRK1A in 97.5% (39/40) of HNSCC tissues analyzed. Taken together our results suggest that DYRK1A could be a novel therapeutic target in HNSCC.

Objectives: To identify potential candidate proteins which are secretory in nature and present at a higher abundance in oral cancer patients with tobacco habits. Methods: Conditioned media of tobacco-treated and -untreated non-neoplastic oral keratinocytes were analyzed using iTRAQ-based mass spectrometry. Hypersecreted proteins; SPARC (osteonectin), cwcv and kazal like domains proteoglycan 1 (SPOCK1); prosaposin (PSAP); and protein tyrosine phosphatase, receptor type S (PTPRS) were validated by enzyme-linked immunosorbent assay (ELISA) using saliva samples from oral cancer patients who are tobacco users. Results: Proteomic analysis of tobacco-treated and -untreated cells led to the identification of 2873 proteins. Among these, 378 proteins showed high abundance and 253 proteins showed low abundance (2-fold cutoff) in conditioned-media of tobacco-treated cells. ELISA-based validation showed significantly higher levels of SPOCK1, PSAP, and PTPRS in oral cancer patients with tobacco chewing habits compared to healthy controls. However, PSAP showed low specificity compared to SPOCK1 and PTPRS. Conclusions: This study indicates significantly increased levels of SPOCK1, PSAP, and PTPRS in saliva of oral cancer patients with tobacco habits. These protein biomarkers might be useful to identify tobacco users with high risk of developing oral cancers.

Cigarette smoking remains the leading cause of non-small cell lung carcinoma. Studies involving acute exposure of smoke on lung cells revealed induction of pre- cancerous state in lung cells. Recently few studies have reported the chronic effect of cigarette smoke in inducing cellular transformation. Yet no systemic study has been performed to understand the molecular alterations in lung cells due to cigarette smoke. Hence it is both important and necessary to study the chronic effect of cigarette smoke in a temporal setting to understand the molecular alterations. In this study, we carried out TMT based proteomic profiling of lung cells which were exposed to cigarette smoke condensate (CSC) for upto 12 months. We identified 2621 proteins in total, of which 145, 114, 87, 169 and 671 proteins were differentially expressed (p

In cancer management, the cyclooxygenase (COX)–targeted approach has shown great promise in anticancer therapeutics. However, the use of COX-2 inhibitors has side effects and health hazards; thus, targeting its major metabolite prostaglandin E2 (PGE2)–mediated signaling pathway might be a rational approach for the next generation of cancer management. Recent studies on several in vitro and in vivo models have revealed that elevated expression of COX-2 correlates with prostate tumor growth and angiogenesis. In this study, we have shown the in-depth molecular mechanism and the PGE2 activation of the epidermal growth factor receptor and β3 integrin through E prostanoid 2 (EP2)–mediated and EP4-mediated pathways, which lead to activator protein-1 (AP-1) activation. Moreover, PGE2 also induces activating transcription factor-4 (ATF-4) activation and stimulates cross-talk between ATF-4 and AP-1, which is unidirectional toward AP-1, which leads to the increased expressions of urokinase-type plasminogen activator and vascular endothelial growth factor and, eventually, regulates prostate tumor cell motility. In vivo Matrigel angiogenesis assay data revealed that PGE2 induces angiogenesis through EP2 and EP4. Human prostate cancer specimen analysis also supported our in vitro and in vivo studies. Our data suggest that targeting PGE2 signaling pathway (i.e., blocking EP2 and EP4 receptors) might be a rational therapeutic approach for overcoming the side effects of COX-2 inhibitors and that this might be a novel strategy for the next generation of prostate cancer management. [Cancer Res 2008;68(19):7750–9]

Background Poor prognosis in gallbladder cancer is due to late presentation of the disease, lack of reliable biomarkers for early diagnosis and limited targeted therapies. Early diagnostic markers and novel therapeutic targets can significantly improve clinical management of gallbladder cancer. Methods Proteomic analysis of four gallbladder cancer cell lines based on the invasive property (non-invasive to highly invasive) was carried out using the isobaric tags for relative and absolute quantitation labeling-based quantitative proteomic approach. The expression of macrophage migration inhibitory factor was analysed in gallbladder adenocarcinoma tissues using immunohistochemistry. In vitro cellular assays were carried out in a panel of gallbladder cancer cell lines using MIF inhibitors, ISO-1 and 4-IPP or its specific siRNA. Results The quantitative proteomic experiment led to the identification of 3,653 proteins, among which 654 were found to be overexpressed and 387 were downregulated in the invasive cell lines (OCUG-1, NOZ and GB-d1) compared to the non-invasive cell line, TGBC24TKB. Among these, macrophage migration inhibitory factor (MIF) was observed to be highly overexpressed in two of the invasive cell lines. MIF is a pleiotropic proinflammatory cytokine that plays a causative role in multiple diseases, including cancer. MIF has been reported to play a central role in tumor cell proliferation and invasion in several cancers. Immunohistochemical labeling of tumor tissue microarrays for MIF expression revealed that it was overexpressed in 21 of 29 gallbladder adenocarcinoma cases. Silencing/inhibition of MIF using siRNA and/or MIF antagonists resulted in a significant decrease in cell viability, colony forming ability and invasive property of the gallbladder cancer cells. Conclusions Our findings support the role of MIF in tumor aggressiveness and suggest its potential application as a therapeutic target for gallbladder cancer. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1855-z) contains supplementary material, which is available to authorized users.

Gastric cancer is one of the most common gastrointestinal malignancies and is associated with poor prognosis. Exploring alterations in the proteomic landscape of gastric cancer is likely to provide potential biomarkers for early detection and molecules for targeted therapeutic intervention. Using iTRAQ-based quantitative proteomic analysis, we identified 22 proteins that were overexpressed and 17 proteins that were downregulated in gastric tumor tissues as compared to the adjacent normal tissue. Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) was found to be 7-fold overexpressed in gastric tumor tissues. Immunohistochemical labeling of tumor tissue microarrays for validation of CAMKK2 overexpression revealed that it was indeed overexpressed in 94% (92 of 98) of gastric cancer cases. Silencing of CAMKK2 using siRNA significantly reduced cell proliferation, colony formation and invasion of gastric cancer cells. Our results demonstrate that CAMKK2 signals in gastric cancer through AMPK activation and suggest that CAMKK2 could be a novel therapeutic target in gastric cancer.

(a) Knockdown ofMIFusing its specific siRNA in a panel of GBC cell lines. Western blot analysis was performed using anti-MIF antibody. β-Actin was used as a loading control. (b) Colony forming ability and invasive property of GBC cell lines. (c) Scatter plot representing invasive ability versus the colony forming ability of GBC cell lines. (PDF 1399 kb)

Gene ontology-based classification of proteins identified in the study. (a) Subcellular localization (b) Molecular function (c) Biological process. (d) Graphical representation of the top 12 canonical pathways of the differentially expressed proteins identified in this study generated using Ingenuity Pathway Analysis. The columns represent the â log of the p-value for all of the genes in each particular canonical pathway family calculated based on Fisherâ s exact test. The line graph represents the ratio plot indicating the number of genes differentially expressed in this study relative to the total number of genes in that particular canonical pathway. (PDF 311 kb)

A graphical representation of the proteins related to the MIF nexus identified in our study. (PDF 764 kb)