Differential Expression of SRY-Related HMG-Box Transcription Factor 2, Oligodendrocyte Lineage Transcription Factor 2, and Zinc Finger E-Box Binding Homeobox 1 in Serum-Derived Extracellular Vesicles: Implications for Mithramycin Sensitivity and Targeted Therapy in High-Grade Glioma.

Glioblastoma multiforme (GBM) is the most aggressive type of glioma and is often resistant to traditional therapies. Evidence suggests that glioma stem cells (GSCs) contribute to this resistance. Mithramycin (Mit-A) targets GSCs and exhibits antitumor activity in GBM by affecting transcriptional targets such as SRY-related HMG-box transcription factor 2 (SOX2), oligodendrocyte lineage transcription factor 2 (OLIG2), and zinc finger E-box binding homeobox 1 (ZEB1). However, its clinical use has been limited by toxicity. This study explored the diagnostic potential of serum extracellular vesicles (EVs) to identify Mit-A responders. Serum EVs were isolated from 70 glioma patients, and targeted gene expression was analyzed using qRT-PCR. Using chemosensitivity assay, we identified 8 Mit-A responders and 17 nonresponders among 25 glioma patients. The M-score showed a significant correlation (p = 0.045) with isocitrate dehydrogenase 1 mutation but not other clinical variables. The genes SOX2 (p = 0.005), OLIG2 (p = 0.003), and ZEB1 (p = 0.0281) were found to be upregulated in the responder EVs. SOX2 had the highest diagnostic potential (AUC = 0.875), followed by OLIG2 (AUC = 0.772) and ZEB1 (AUC = 0.632).The combined gene panel showed significant diagnostic efficacy (AUC = 0.956) through logistic regression analysis. The gene panel was further validated in the serum EVs of 45 glioma patients. These findings highlight the potential of Mit-A as a targeted therapy for high-grade glioma based on differential gene expression in serum EVs. The gene panel could serve as a diagnostic tool to predict Mit-A sensitivity, offering a promising approach for personalized treatment strategies and emphasizing the role of GSCs in therapeutic resistance.

JAK-STAT inhibitor as a potential therapeutic opportunity in AML patients resistant to cytarabine and epigenetic therapy.

The prognosis of AML is generally poor, with 5-year survival rate of 25%. There has been substantial progress in identification of new therapeutic targets, along with approval of at least three targeted therapies for AML in recent years. Nevertheless, treatment has largely remained unchanged over couple of decades, with ~40% patients not achieving remission. AML is a highly heterogenous disease and there is a need for a preclinical platform to understand the heterogeneity and tumor microenvironment that can guide therapy selection. In this study, we employed an ex vivo tumor explant model to study tumor microenvironment and to select a treatment course for AML patients. Our data reveal dysregulation of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) in a subset of AML patients. Based on this observation, epigenetic modulators azacitidine and panobinostat alone and in combination, were evaluated as treatment regimens in cytarabine refractory tumors. More than 50% of the treated samples showed response to the combination therapy. In order to explore alternate treatment modalities for tumors refractory to these epigenetic modulators, TCGA data analysis was done which revealed increased expression and hypomethylation of IFNGR1/2, suggesting activation of JAK/STAT pathway in AML. This was further interrogated ex vivo, with p-STAT3 expression in patients' samples. Fedratinib, a JAK/STAT inhibitor was evaluated and 78% tumor efficacy response was achieved. Taken together, our data indicate that ex vivo platform derived from patient samples is capable in guiding optimal therapy selection for various classes of drugs including identification of novel targeted therapies.

Multi-Omics Analysis to Characterize Cigarette Smoke Induced Molecular Alterations in Esophageal Cells.

Though smoking remains one of the established risk factors of esophageal squamous cell carcinoma, there is limited data on molecular alterations associated with cigarette smoke exposure in esophageal cells. To investigate molecular alterations associated with chronic exposure to cigarette smoke, non-neoplastic human esophageal epithelial cells were treated with cigarette smoke condensate (CSC) for up to 8 months. Chronic treatment with CSC increased cell proliferation and invasive ability of non-neoplastic esophageal cells. Whole exome sequence analysis of CSC treated cells revealed several mutations and copy number variations. This included loss of high mobility group nucleosomal binding domain 2 (HMGN2) and a missense variant in mediator complex subunit 1 (MED1). Both these genes play an important role in DNA repair. Global proteomic and phosphoproteomic profiling of CSC treated cells lead to the identification of 38 differentially expressed and 171 differentially phosphorylated proteins. Bioinformatics analysis of differentially expressed proteins and phosphoproteins revealed that most of these proteins are associated with DNA damage response pathway. Proteomics data revealed decreased expression of HMGN2 and hypophosphorylation of MED1. Exogenous expression of HMGN2 and MED1 lead to decreased proliferative and invasive ability of smoke exposed cells. Immunohistochemical labeling of HMGN2 in primary ESCC tumor tissue sections (from smokers) showed no detectable expression while strong to moderate staining of HMGN2 was observed in normal esophageal tissues. Our data suggests that cigarette smoke perturbs expression of proteins associated with DNA damage response pathways which might play a vital role in development of ESCC.

Phosphoproteomic analysis identifies CLK1 as a novel therapeutic target in gastric cancer.

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.

Activation of Epithelial-Mesenchymal Transition and Altered ß-Catenin Signaling in a Novel Indian Colorectal Carcinoma Cell Line.

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 ß-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 ß-catenin. Remarkably, we observed significantly elevated proteasome activity that perhaps co-evolved to compensate for the unnaturally high mRNA level of ß-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.

Identification of potential biomarkers of head and neck squamous cell carcinoma using iTRAQ based quantitative proteomic approach.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers in India. Despite improvements in treatment strategy, the survival rates of HNSCC patients remain poor. Thus, it is necessary to identify biomarkers that can be used for early detection of disease. In this study, we employed iTRAQ-based quantitative mass spectrometry analysis to identify dysregulated proteins from a panel of head and neck squamous cell carcinoma (HNSCC) cell lines. We identified 2468 proteins, of which 496 proteins were found to be dysregulated in at least two out of three HNSCC cell lines compared to immortalized normal oral keratinocytes. We detected increased expression of replication protein A1 (RPA1) and heat shock protein family H (Hsp110) member 1 (HSPH1), in HNSCC cell lines compared to control. The differentially expressed proteins were further validated using parallel reaction monitoring (PRM) and western blot analysis in HNSCC cell lines. Immunohistochemistry-based validation using HNSCC tissue microarrays revealed overexpression of RPA1 and HSPH1 in 15.7% and 32.2% of the tested cases, respectively. Our study illustrates quantitative proteomics as a robust approach for identification of potential HNSCC biomarkers. The proteomic data has been submitted to ProteomeXchange Consortium (http://www.proteomecentral.proteomexchange.org) via the PRIDE public data repository accessible using the data identifier - PXD009241.

Rationally co-targeting divergent pathways in KRAS wild-type colorectal cancers by CANscript technology reveals tumor dependence on Notch and Erbb2.

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 EGFR-targeted 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.

Pancreatic paraganglioma: An extremely rare entity and crucial role of immunohistochemistry for diagnosis.

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.

Cytodiagnosis of mucosal malignant melanoma of nasal cavity: A case report with review of literature.

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.