Background Phosphorylation is an important regulatory mechanism of protein activity in cells. Studies in various cancers have reported perturbations in kinases resulting in aberrant phosphorylation of oncoproteins and tumor suppressor proteins. Methods In this study, we carried out quantitative phosphoproteomic analysis of gastric cancer tissues and corresponding xenograft samples. Using these data, we employed bioinformatics analysis to identify aberrant signaling pathways. We further performed molecular inhibition and silencing of the upstream regulatory kinase in gastric cancer cell lines and validated its effect on cellular phenotype. Through an ex vivo technology utilizing patient tumor and blood sample, we sought to understand the therapeutic potential of the kinase by recreating the tumor microenvironment. Results Using mass spectrometry-based high-throughput analysis, we identified 1,344 phosphosites and 848 phosphoproteins, including differential phosphorylation of 177 proteins (fold change cut-off ≥ 1.5). Our data showed that a subset of differentially phosphorylated proteins belonged to splicing machinery. Pathway analysis highlighted Cdc2-like kinase (CLK1) as upstream kinase. Inhibition of CLK1 using TG003 and CLK1 siRNA resulted in a decreased cell viability, proliferation, invasion and migration as well as modulation in the phosphorylation of SRSF2. Ex vivo experiments which utilizes patient's own tumor and blood to recreate the tumor microenvironment validated the use of CLK1 as a potential target for gastric cancer treatment. Conclusions Our data indicates that CLK1 plays a crucial role in the regulation of splicing process in gastric cancer and that CLK1 can act as a novel therapeutic target in gastric cancer. Keywords Phosphoserine/threonine • Spliceosome complex • Targeted therapy • Biomarker • PDX in vivo models List of abbreviations CLK Cdc2-like kinase PDX Patient-derived xenografts IHC Immunohistochemistry TMT Tandem Mass Tag bRPLC Basic pH reverse phase chromatography HCD Higher energy collision dissociation IPA Ingenuity pathway analysis FBS Fetal bovine serum Electronic supplementary material The online version of this article (
KRAS mutation status can distinguish between metastatic colorectal carcinoma (mCRC) patients who may benefit from therapies that target the epidermal growth factor receptor (EGFR), such as cetuximab. However, patients whose tumors harbor mutant KRAS (codons 12/13, 61 and 146) are often excluded from EGFR-targeted regimens, while other patients with wild type KRAS will sometimes respond favorably to these same drugs. These conflicting observations suggest that a more robust approach to individualize therapy may enable greater frequency of positive clinical outcome for mCRC patients. Here, we utilized alive tumor tissues in ex-vivo platform termed CANscript, which preserves the native tumor heterogeneity, in order to interrogate the antitumor effects of EGFRtargeted drugs in mCRC (n = 40). We demonstrated that, irrespective of KRAS status, cetuximab did not induce an antitumor response in a majority of patient tumors. In the subset of non-responsive tumors, data showed that expression levels of EGFR ligands contributed to a mechanism of resistance. Transcriptomic and phosphoproteomic profiling revealed deregulation of multiple pathways, significantly the Notch and Erbb2. Targeting these nodes concurrently resulted in antitumor efficacy in a majority of cetuximab-resistant tumors. These findings highlight the importance of integrating molecular profile and functional testing tools for optimization of alternate strategies in resistant population.Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide with a 5-year survival rate of less than 10% 1 . An important molecular target implicated in disease progression is Epidermal Growth Factor Receptor (EGFR) signaling, which after ligand binding triggers two main pathways: the RAS-RAF-MAPK cascade leading to cell proliferation, survival, invasion and metastasis; and the PI3K-PTEN-AKT pathway which controls cell survival, motility and neo-angiogenesis 2 . Notably, EGFR is overexpressed in 60-80% of colorectal tumors 3 . Current chemotherapeutic options include 5FU + leucovorin, XELOX, XELIRI, FOLFOX and FOLFIRI which are combinations of capecitabine, 5-fluorouracil, leucovorin and oxaliplatin or irinotecan. Two classes of anti-EGFR monoclonal antibodies (mAbs) are at present prescribed in combination with conventional chemotherapy for the treatment of CRC. However the underlying problem of using cetuximab (a chimeric-IgG1mAb) is that it has only 8.8% efficacy when used in monotherapy, and 22.9% when used in combination therapy for
Paragangliomas are rare neuroendocrine neoplasms arising in extra-adrenal chromaffin cells of autonomic nervous system and histologically akin to chemodectomas. They are rare, affecting about 1 in 2,000,000 population. It is a generic term applied to tumors of paraganglia regardless of the location. In rare instances, paragangliomas present around and involve the pancreas, thereby mimicking any one of the more common primary pancreatic lesions. Pancreatic paraganglioma is an extremely rare tumor. It grows slowly, so radical resection is recommended to achieve curability with good prognosis. These neoplasms present considerable diagnostic difficulty not only for the clinician and radiologist but also for the pathologist. Here, we report a case of a 55-year-old woman who presented with a left-sided abdominal swelling for 3 months duration, initially having clinical suspicion of an ovarian tumor. The radiological imaging revealed a lesion in the tail of pancreas with a differential diagnosis of pancreatic carcinoma and metastatic tumor. Only after exploratory laparotomy, the diagnosis was made as a rare case of pancreatic paraganglioma on the basis of histological examination and immunohistochemistry.
Sinonasal malignant melanoma is of unusual occurrence. Common sites for melanomas are head, neck region, and the lower extremities as they are exposed to sunlight, which is one of the predisposing factors. We report a case of primary mucosal malignant melanoma of the nasal cavity in a 68-year-old male for its rare occurrence. The primary knowledge of its existence and evaluation of its cytological features are important for a correct preoperative cytological diagnosis and thereby clinical implications for appropriate therapeutic intervention. The cytological features when evaluated along with clinical, histopathological and immunohistochemical features are sufficiently diagnostic. The rarity of its occurrence warrants its mention.
Colorectal cancer is the third major cause of cancer-related mortality worldwide. The upward trend in incidence and mortality rates, poor sensitivity to conventional therapies and a dearth of early diagnostic parameters pose a huge challenge in the management of colorectal cancer in India. Due to the high level of genetic diversity present in the Indian population, unraveling the genetic contributions toward pathogenesis is key for understanding the etiology of colorectal cancer and in reversing this trend. We have established a novel cell line, MBC02, from an Indian colorectal cancer patient and have carried out extensive molecular characterization to unravel the pathological alterations in this cell line. In-depth molecular analysis of MBC02 revealed suppression of E-cadherin expression, concomitant with overexpression of EMT related molecules, which manifested in the form of highly migratory and invasive cells. Loss of membrane-tethered E-cadherin released β-catenin from the adherens junction resulting in its cytoplasmic and nuclear accumulation and consequently, upregulation of c-Myc . MBC02 also showed dramatic transcriptional upregulation of β-catenin. Remarkably, we observed significantly elevated proteasome activity that perhaps co-evolved to compensate for the unnaturally high mRNA level of β-catenin to regulate the increased protein load. In addition, there was substantial misregulation of other clinically relevant signaling pathways that have clinical relevance in the pathogenesis of colorectal cancer. Our findings pave the way toward understanding the molecular differences that could define pathogenesis in cancers originating in the Indian population.
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