The role of proteomics in the multiplexed analysis of gene alterations in human cancer.

INTRODUCTION: Proteomics has played a pivotal role in identifying proteins perturbed in disease conditions when compared with healthy samples. Study of dysregulated proteins aids in identifying diagnostic markers and potential therapeutic targets. Cancer is an outcome of interplay of several such disarrayed proteins and molecular pathways which perturb cellular homeostasis, resulting in transformation. In this review, we discuss various facets of proteomic approaches, including tools and technological advancements, aiding in understanding differentially expressed molecules and signaling mechanisms. AREAS COVERED: In this review, we have taken the approach of documenting the different methods of proteomic studies, ranging from labeling techniques, data analysis methods, and the nature of molecule detected. We summarize each technique and provide a glimpse of cancer research carried out using them, highlighting the advantages and drawbacks in comparison with others. Literature search using online resources, such as PubMed and Google Scholar were carried out for this approach. EXPERT OPINION: Technological advancements in proteomics studies have come a long way from the study of two-dimensional mapping of proteins separated on gels in the early 1970s. Higher precision in molecular identification and quantification (high throughput), and greater number of samples analyzed have been the focus of researchers.

Profiling Kinase Activities for Precision Oncology in Diffuse Gastric Cancer.

Gastric cancer (GC) remains a leading cause of cancer-related mortality globally. This is due to the fact that majority of the cases of GC are diagnosed at an advanced stage when the treatment options are limited and prognosis is poor. The diffuse subtype of gastric cancer (DGC) under Lauren's classification is more aggressive and usually occurs in younger patients than the intestinal subtype. The concept of personalized medicine is leading to the identification of multiple biomarkers in a large variety of cancers using different combinations of omics technologies. Proteomic changes including post-translational modifications are crucial in oncogenesis. We analyzed the phosphoproteome of DGC by using paired fresh frozen tumor and adjacent normal tissue from five patients diagnosed with DGC. We found proteins involved in the epithelial-to-mesenchymal transition (EMT), c-MYC pathway, and semaphorin pathways to be differentially phosphorylated in DGC tissues. We identified three kinases, namely, bromodomain adjacent to the zinc finger domain 1B (BAZ1B), WNK lysine-deficient protein kinase 1 (WNK1), and myosin light-chain kinase (MLCK) to be hyperphosphorylated, and one kinase, AP2-associated protein kinase 1 (AAK1), to be hypophosphorylated. LMNA hyperphosphorylation at serine 392 (S392) was demonstrated in DGC using immunohistochemistry. Importantly, we have detected heparin-binding growth factor (HDGF), heat shock protein 90 (HSP90), and FTH1 as potential therapeutic targets in DGC, as drugs targeting these proteins are currently under investigation in clinical trials. Although these new findings need to be replicated in larger study samples, they advance our understanding of signaling alterations in DGC, which could lead to potentially novel actionable targets in GC.

Proteomic Signatures of Diffuse and Intestinal Subtypes of Gastric Cancer.

Gastric cancer is a leading cause of death from cancer globally. Gastric cancer is classified into intestinal, diffuse and indeterminate subtypes based on histology according to the Laurén classification. The intestinal and diffuse subtypes, although different in histology, demographics and outcomes, are still treated in the same fashion. This study was designed to discover proteomic signatures of diffuse and intestinal subtypes. Mass spectrometry-based proteomics using tandem mass tags (TMT)-based multiplexed analysis was used to identify proteins in tumor tissues from patients with diffuse or intestinal gastric cancer with adjacent normal tissue control. A total of 7448 or 4846 proteins were identified from intestinal or diffuse subtype, respectively. This quantitative mass spectrometric analysis defined a proteomic signature of differential expression across the two subtypes, which included gremlin1 (GREM1), bcl-2-associated athanogene 2 (BAG2), olfactomedin 4 (OLFM4), thyroid hormone receptor interacting protein 6 (TRIP6) and melanoma-associated antigen 9 (MAGE-A9) proteins. Although GREM1, BAG2, OLFM4, TRIP6 and MAGE-A9 have all been previously implicated in tumor progression and metastasis, they have not been linked to intestinal or diffuse subtypes of gastric cancer. Using immunohistochemical labelling of a tissue microarray comprising of 124 cases of gastric cancer, we validated the proteomic signature obtained by mass spectrometry in the discovery cohort. Our findings should help investigate the pathogenesis of these gastric cancer subtypes and potentially lead to strategies for early diagnosis and treatment.

Proteomic and phosphoproteomic profiling of shammah induced signaling in oral keratinocytes.

Shammah is a smokeless tobacco product often mixed with lime, ash, black pepper and flavorings. Exposure to shammah has been linked with dental diseases and oral squamous cell carcinoma. There is limited literature on the prevalence of shammah and its role in pathobiology of oral cancer. In this study, we developed a cellular model to understand the effect of chronic shammah exposure on oral keratinocytes. Chronic exposure to shammah resulted in increased proliferation and invasiveness of non-transformed oral keratinocytes. Quantitative proteomics of shammah treated cells compared to untreated cells led to quantification of 4712 proteins of which 402 were found to be significantly altered. In addition, phosphoproteomics analysis of shammah treated cells compared to untreated revealed hyperphosphorylation of 36 proteins and hypophosphorylation of 83 proteins (twofold, p-value ≤ 0.05). Bioinformatics analysis of significantly altered proteins showed enrichment of proteins involved in extracellular matrix interactions, necroptosis and peroxisome mediated fatty acid oxidation. Kinase-Substrate Enrichment Analysis showed significant increase in activity of kinases such as ROCK1, RAF1, PRKCE and HIPK2 in shammah treated cells. These results provide better understanding of how shammah transforms non-neoplastic cells and warrants additional studies that may assist in improved early diagnosis and treatment of shammah induced oral cancer.

Proteomic Alterations Associated with Oral Cancer Patients with Tobacco Using Habits.

Tobacco abuse is a major risk factor associated with the development of oral squamous cell carcinoma. Differences in molecular aberrations induced by tobacco exposure by chewing or smoking form are not well studied in case of oral cancer. We used tandem mass tag-based quantitative proteomic approach to delineate proteomic alterations in oral cancer patients based on their history of tobacco using habits (patients who chewed tobacco, patients who smoked tobacco, and those with no history of tobacco consumption). Our data identified distinct dysregulation of biological processes and pathways in each patient cohort. Bioinformatics analysis of dysregulated proteins identified in our proteomic study revealed dysregulation of collagen formation and antigen processing/presentation pathway in oral cancer patients who smoked tobacco, whereas proteins associated with the process of keratinization showed enrichment in patients who chewed tobacco. In addition, we identified overexpression of proteins involved in immune pathways and downregulation of muscle contraction-mediated signaling events in all three cohorts, irrespective of tobacco using habits. This study lays the groundwork for identification of protein markers that may aid in identification of high-risk patients for cancer development based on the history of tobacco exposure habits.

Comprehensive network map of interferon gamma signaling.

Interferon gamma (IFN-γ), is a cytokine, which is an important regulator of host defense system by mediating both innate and adaptive immune responses. IFN-γ signaling is primarily associated with inflammation and cell-mediated immune responses. IFN-γ is also represented as antitumor cytokine which facilitates immunosurveillance in tumor cells. In addition, IFN-γ mediated signaling also elicits pro-tumorigenic transformations and promotes tumor progression. Impact of IFN-γ signaling in mammalian cells has been widely studied which indicate that IFN-γ orchestrates distinct cellular functions including immunomodulation, leukocyte trafficking, apoptosis, anti-microbial, and both anti- and pro-tumorigenic role. However, a detailed network of IFN-γ signaling pathway is currently lacking. Therefore, we systematically curated the literature information pertaining to IFN-γ signaling and develop a comprehensive signaling network to facilitate better understanding of IFN-γ mediated signaling. A total of 124 proteins were catalogued that were experimentally proven to be involved in IFN-γ signaling cascade. These 124 proteins were found to participate in 81 protein-protein interactions, 94 post-translational modifications, 20 translocation events, 54 activation/inhibiton reactions. Further, 236 differential expressed genes were also documented in IFN-γ mediated signaling. IFN-γ signaling pathway is made freely available to scientific audience through NetPath at ( http://www.netpath.org/pathways?path_id=NetPath_32 ). We believe that documentation of reactions pertaining to IFN-γ signaling and development of pathway map will facilitate further research in IFN-γ associated human diseases including cancer.

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.

Cigarette smoke and chewing tobacco alter expression of different sets of miRNAs in oral keratinocytes.

Carcinogenic effect of tobacco in oral cancer is through chewing and/or smoking. Significant differences exist in development of oral cancer between tobacco users and non-users. However, molecular alterations induced by different forms of tobacco are yet to be fully elucidated. We developed cellular models of chronic exposure to chewing tobacco and cigarette smoke using immortalized oral keratinocytes. Chronic exposure to tobacco resulted in increased cell scattering and invasiveness in immortalized oral keratinocytes. miRNA sequencing using Illumina HiSeq 2500 resulted in the identification of 10 significantly dysregulated miRNAs (4 fold; p ≤ 0.05) in chewing tobacco treated cells and 6 in cigarette smoke exposed cells. We integrated this data with global proteomic data and identified 36 protein targets that showed inverse expression pattern in chewing tobacco treated cells and 16 protein targets that showed inverse expression in smoke exposed cells. In addition, we identified 6 novel miRNAs in chewing tobacco treated cells and 18 novel miRNAs in smoke exposed cells. Integrative analysis of dysregulated miRNAs and their targets indicates that signaling mechanisms leading to oncogenic transformation are distinct between both forms of tobacco. Our study demonstrates alterations in miRNA expression in oral cells in response to two frequently used forms of tobacco.