Pan-cancer quantitation of epithelial-mesenchymal transition dynamics using parallel reaction monitoring-based targeted proteomics approach.

Epithelial-mesenchymal transition (EMT) is a dynamic and complex cellular process that is known to be hijacked by cancer cells to facilitate invasion, metastasis and therapeutic resistance. Several quantitative measures to assess the interplay between EMT and cancer progression are available, based on large scale genome and transcriptome data. However, these large scale multi-omics studies have repeatedly illustrated a lack of correlation in mRNA and protein abundances that may be influenced by diverse post-translational regulation. Hence, it is imperative to understand how changes in the EMT proteome are associated with the process of oncogenic transformation. To this effect, we developed a parallel reaction monitoring-based targeted proteomics method for quantifying abundances of EMT-associated proteins across cancer cell lines. Our study revealed that quantitative measurement of EMT proteome which enabled a more accurate assessment than transcriptomics data and revealed specific discrepancies against a backdrop of generally strong concordance between proteomic and transcriptomic data. We further demonstrated that changes in our EMT proteome panel might play a role in tumor transformation across cancer types. In future, this EMT panel assay has the potential to be used for clinical samples to guide treatment choices and to congregate functional information for the development and advancing novel therapeutics.

Integrated proteomics and phosphoproteomics revealed druggable kinases in neoadjuvant chemotherapy resistant tongue cancer.

Tongue squamous cell carcinoma is an aggressive oral cancer with a high incidence of metastasis and poor prognosis. Most of the oral cavity cancer patients present in clinics with locally advanced unresectable tumors. Neoadjuvant treatment is beneficial for these individuals as it reduces the tumor size aiding complete resection. However, patients develop therapy resistance to the drug regimen. In this study, we explored the differential expression of proteins and altered phosphorylation in the neoadjuvant chemotherapy resistant tongue cancer patients. We integrated the proteomic and phosphoproteomic profiles of resistant (n = 4) and sensitive cohorts (n = 4) and demonstrated the differential expression and phosphorylation of proteins in the primary tissue of the respective subject groups. We observed differential and extensive phosphorylation of keratins such as KRT10 and KRT1 between the two cohorts. Furthermore, our study revealed a kinase signature associated with neoadjuvant chemotherapy resistance. Kinases such as MAPK1, AKT1, and MAPK3 are predicted to regulate the resistance in non-responders. Pathway analysis showed enrichment of Rho GTPase signaling and hyperphosphosphorylation of proteins involved in cell motility, invasion, and drug resistance. Targeting the kinases could help with the clinical management of neoadjuvant chemotherapy-resistant tongue cancer.

Circulating tumor cells as an emerging tool in cancer therapy.

Majority of the cancer-related deaths are related to metastasis during which cancer cells invade the surrounding tissues, enter (intravasation) and exit (extravasation) the peripheral circulation and seed distant organs. The Circulating Tumor Cells (CTCs) exist in peripheral blood as single cells or as oligoclonal clusters of tumor cells along with platelets and lymphocytes. Detection of CTCs allows characterizing the tumors by their genotype and in predicting the prognosis and response to therapy and explants derived from these cells can be used in drug screening. In this review, we highlight the methods used for isolation and culture of CTCs and their clinical use.

Phosphoproteomics Profiling to Identify Altered Signaling Pathways and Kinase-Targeted Cancer Therapies.

Phosphorylation is one of the most extensively studied posttranslational modifications (PTM), which regulates cellular functions like cell growth, differentiation, apoptosis, and cell signaling. Kinase families cover a wide number of oncoproteins and are strongly associated with cancer. Identification of driver kinases is an intense area of cancer research. Thus, kinases serve as the potential target to improve the efficacy of targeted therapies. Mass spectrometry-based phosphoproteomic approach has paved the way to the identification of a large number of altered phosphorylation events in proteins and signaling cascades that may lead to oncogenic processes in a cell. Alterations in signaling pathways result in the activation of oncogenic processes predominantly regulated by kinases and phosphatases. Therefore, drugs such as kinase inhibitors, which target dysregulated pathways, represent a promising area for cancer therapy.

Targeted Proteomics as a Tool for Quantifying Urine-Based Biomarkers.

Mass spectrometry based proteomics approaches are routinely used to discover candidate biomarkers. These studies often use small number of samples to discover candidate proteins that are later validated on a large cohort of samples. Targeted proteomics has emerged as a powerful method for quantification of multiple proteins in complex biological matrix. Parallel reaction monitoring (PRM) and selected reaction monitoring (SRM) are two main methods of choice for quantifying and validating proteins across hundreds to thousands of samples. Over the years, many software tools have become available that enable the users to carry out the analysis. In this chapter, we describe selection of proteotypic peptides, sample preparation, generating a response curve, data acquisition and analysis of PRM data using Skyline software for targeted proteomics to quantify candidate markers in urine.

Urinary glycoproteomic profiling of non-muscle invasive and muscle invasive bladder carcinoma patients reveals distinct N-glycosylation pattern of CD44, MGAM, and GINM1.

Clinical management of bladder carcinomas (BC) remains a major challenge and demands comprehensive multi-omics analysis for better stratification of the disease. Identification of patients on risk requires identification of signatures predicting prognosis risk of the patients. Understanding the molecular alterations associated with the disease onset and progression could improve the routinely used diagnostic and therapy procedures. In this study, we investigated the aberrant changes in N-glycosylation pattern of proteins associated with tumorigenesis as well as disease progression in bladder cancer. We integrated and compared global N-glycoproteomic and proteomic profile of urine samples from bladder cancer patients at different clinicopathological stages (non-muscle invasive and muscle-invasive patients [n = 5 and 4 in each cohort]) with healthy subjects (n = 5) using SPEG method. We identified 635 N-glycopeptides corresponding to 381 proteins and 543 N-glycopeptides corresponding to 326 proteins in NMIBC and MIBC patients respectively. Moreover, we identified altered glycosylation in 41 NMIBC and 21 MIBC proteins without any significant change in protein abundance levels. In concordance with the previously published bladder cancer cell line N-glycoproteomic data, we also observed dysregulated glycosylation in ECM related proteins. Further, we identified distinct N-glycosylation pattern of CD44, MGAM, and GINM1 between NMIBC and MIBC patients, which may be associated with disease progression in bladder cancer. These aberrant protein glycosylation events would provide a novel approach for bladder carcinoma diagnosis and further define novel mechanisms of tumor initiation and progression.

Somatic Mutations Profile of a Young Patient With Metastatic Urothelial Carcinoma Reveals Mutations in Genes Involved in Ion Channels.

Background: Urothelial carcinoma is the most common malignancy of the bladder and is primarily considered as a disease of the elderly. Studies that address bladder tumor occurrence in young age groups are rare. Case Presentation: A 19-year-old male presented with a gross total painless hematuria. A histology after biopsy revealed a high-grade transitional cell carcinoma with lymph node metastasis. The patient succumbed to the disease on day 72 of the treatment. Here, we used whole-exome sequencing of a paired tumor-normal sample to identify the somatic mutations and the possible targets of treatment. Result: We predicted eight potential driver mutations (TP53 p.V157L, RB1 c.1498+1G>T, MED23 p.L1127P, CTNND1 p.S713C, NSD1 p.P2212A, MED17 p.G556V, DPYD p.Q814K, and SPEN p.S1078*). In addition, we predicted deleterious mutations in genes involved in the ion channels (CACNA1S p.E1581K, CACNG1 p.P71T, CACNG8 p.G404W, GRIN2B p.A1096T, KCNC1 p.G16V, KCNH4 p.E874K, KCNK9 p.R131S, P2RX7 p.A296D, and SCN8A p.R558H). Conclusions: Most likely, mutations in genes involved in ion channels may be responsible for the aggressive behavior of a tumor. Ion channels are the second largest class of drug targets, and may thus serve as a putative potential therapeutic target in advanced stage urothelial carcinoma.