Debasish Paul

Mass Spectrometry-Based Proteomics in Molecular Diagnostics: Discovery of Cancer Biomarkers Using Tissue Culture

Accurate diagnosis and proper monitoring of cancer patients remain a key obstacle for successful cancer treatment and prevention. Therein comes the need for biomarker discovery, which is crucial to the current oncological and other clinical practices having the potential to impact the diagnosis and prognosis. In fact, most of the biomarkers have been discovered utilizing the proteomics-based approaches. Although high-throughput mass spectrometry-based proteomic approaches like SILAC, 2D-DIGE, and iTRAQ are filling up the pitfalls of the conventional techniques, still serum proteomics importunately poses hurdle in overcoming a wide range of protein concentrations, and also the availability of patient tissue samples is a limitation for the biomarker discovery. Thus, researchers have looked for alternatives, and profiling of candidate biomarkers through tissue culture of tumor cell lines comes up as a promising option. It is a rich source of tumor cell-derived proteins, thereby, representing a wide array of potential biomarkers. Interestingly, most of the clinical biomarkers in use today (CA 125, CA 15.3, CA 19.9, and PSA) were discovered through tissue culture-based system and tissue extracts. This paper tries to emphasize the tissue culture-based discovery of candidate biomarkers through various mass spectrometry-based proteomic approaches.

Correlation of hydrogen-bonding propensity and anticancer profile of tetrazole-tethered combretastatin analogues.

A series of 1,5-disubstituted tetrazole-tethered combretastatin analogues with extended hydrogen-bond donors at the ortho-positions of the aryl A and B rings were developed and evaluated for their antitubulin and antiproliferative activity. We wanted to test whether intramolecular hydrogen-bonding used as a conformational locking element in these analogues would improve their activity. The correlation of crystal structures with the antitubulin and antiproliferative profiles of the modified analogues suggested that hydrogen-bond-mediated conformational control of the A ring is deleterious to the bioactivity. In contrast, although there was no clear evidence that intramolecular hydrogen bonding to the B ring enhanced activity, we found that increased substitution on the B ring had a positive effect on antitubulin and antiproliferative activity. Among the various analogues synthesized, compounds 5d and 5e, having hydrogen-bonding donor groups at the ortho and meta-positions on the 4-methoxy phenyl B ring, are strong inhibitors of tubulin polymerization and antiproliferative agents having IC50 value in micromolar concentrations.

Global proteomic profiling identifies etoposide chemoresistance markers in non-small cell lung carcinoma.

UNLABELLED Chemoresistance is one of the leading health concerns in cancer treatment. Understanding the mechanism of chemoresistance is the best way to improve the survival of the patient. Etoposide and its analogues are widely used as antitumor drugs in lung cancer but many etoposide resistant lung cancer cases has been identified in recent years. The present study aims to explore the cellular response of lung cancer cell lines to etoposide and finding the potential chemoresistant marker proteins. Multiple proteomic platforms like 2-DE, DIGE and iTRAQ have been used to study the global proteome profile of NCI-H460 and etoposide resistant NCI-H460R cell lines. Our study revealed that etoposide treatment leads to alteration of 83 proteins in NCI-H460R cell lines. The functional analysis highlighted the role of the differential expressed proteins in cellular signaling, apoptosis, and cytoskeleton reorganization. Our study has identified several new proteins like RHOC, DLG5, UGDH, TMOD3 in addition to known chemoresistance associated proteins. In silico prediction of the important selected candidates are further validated at protein and mRNA level. Further, functional studies of newly identified candidate genes RHOC and DLG5 revealed that chemotherapeutic resistance is associated with their elevated level and may serve as novel targets for therapeutic intervention. BIOLOGICAL SIGNIFICANCE Etoposide and its analogues have been used for lung cancer treatment for a while and it was reported that many non small cell lung carcinoma patients are resistant to etoposide. Although etoposide show drug resistance, the exact mechanism was not well understood. The present study focused on the global proteome analysis of NCI-H460 and NCI-H460R cell lines using multiple proteomic platforms to understand the potential chemoresistant markers for etoposide. Our multi-proteomic analysis has showed differential expression of 83 proteins involved in oxidative phosphorylation, metabolic, protein folding, cytoskeleton associated protein along with apoptotic pathway has been identified. In addition, quite a few interesting proteins such as RHOC, DLG5, HSP90, citrate synthase, UDP-glucose-6-dehydrogenase, Tropomodulin-3 are involved in chemoresistance has been observed. Overall, this is the first comprehensive proteomic study on etoposide resistant cell line NCI-H460 to explore the mechanism of chemoresistance in lung cancer.

Cdc20 directs proteasome-mediated degradation of the tumor suppressor SMAR1 in higher grades of cancer through the anaphase promoting complex

The Tumor suppressor SMAR1 (scaffold matrix attachment region binding protein 1) has a crucial role in maintaining genomic stability, cell cycle progression and apoptosis.Our previous finding showed that it is highly suppressed in higher grade of cancer. However, the underlying mechanism of this suppression was not well understood. In this study, we show that SMAR1 expression levels are controlled at the proteasomal level by five RING finger E3 ubiquitin ligases including, Cdc20, a substrate receptor of ubiquitin ligase APC/C complex. We found that Cdc20 binds and promotes proteasomal degradation of SMAR1 in a D-box motif dependent manner. Further, our results demonstrated that Cdc20 promotes proteasomal degradation of SMAR1 through K48-linked specific polyubiquitylation, and that short hairpin RNA mediated inactivation of Cdc20 leads to significant stabilization of SMAR1. These findings suggest that Cdc20 is responsible for maintaining the cellular levels of SMAR1. However, since Cdc20 fails to target SMAR1 upon exposure to genotoxic stresses, SMAR1 helps to maintain genomic stability under these conditions through its DNA damage repair activity. Interestingly, Cdc20-mediated degradation of SMAR1 promotes cell migration and invasion.The reciprocal relationship of the duo is evident in breast cancer cell lines as well as in patient samples, suggesting that Cdc20 functions as an important negative regulator of SMAR1 in higher grades of cancer. Our study reveals for the first time, the molecular mechanism associated with lower levels of expression of the important tumor suppressor SMAR1 in higher grades of breast cancer.

Insights into the inhibitory mechanism of triazole-based small molecules on phosphatidylinositol-4,5-bisphosphate binding pleckstrin homology domain

Background Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important regulator of several cellular processes and a precursor for other second messengers which are involved in cell signaling pathways. Signaling proteins preferably interact with PI(4,5)P2 through its pleckstrin homology (PH) domain. Efforts are underway to design small molecule-based antagonist, which can specifically inhibit the PI(4,5)P2/PH-domain interaction to establish an alternate strategy for the development of drug(s) for phosphoinositide signaling pathways. Methods Surface plasmon resonance, molecular docking, circular dichroism, competitive Förster resonance energy transfer, isothermal titration calorimetric analyses and liposome pull down assay were used. Results In this study, we employed 1,2,3-triazol-4-yl methanol containing small molecule (CIPs) as antagonists for PI(4,5)P2/PH-domain interaction and determined their inhibitory effect by using competitive-surface plasmon resonance analysis (IC50 ranges from 53 to 159 nM for PI(4,5)P2/PLCδ1-PH domain binding assay). We also used phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], PI(4,5)P2 specific PH-domains to determine binding selectivity of the compounds. Various physicochemical analyses showed that the compounds have weak affect on fluidity of the model membrane but, strongly interact with the phospholipase C δ1 (PLCδ1)-PH domains. The 1,2,3-triazol-4-yl methanol moiety and nitro group of the compounds are essential for their exothermic interaction with the PH-domains. Potent compound can efficiently displace PLCδ1-PH domain from plasma membrane to cytosol in A549 cells. Conclusions Overall, our studies demonstrate that these compounds interact with the PIP-binding PH-domains and inhibit their membrane recruitment. General significance These results suggest specific but differential binding of these compounds to the PLCδ1-PH domain and emphasize the role of their structural differences in binding parameters. These triazole-based compounds could be directly used/further developed as potential inhibitor for PH domain-dependent enzyme activity.

A MicroRNA/Ubiquitin Ligase Feedback Loop Regulates Slug-Mediated Invasion in Breast Cancer

The transformation of a normal cell to cancer requires the derail of multiple pathways. Normal signaling in a cell is regulated at multiple stages by the presence of feedback loops, calibration of levels of proteins by their regulated turnover, and posttranscriptional regulation, to name a few. The tumor suppressor protein FBXO31 is a component of the SCF E3 ubiquitin ligase and is required to arrest cells at G1 following genotoxic stresses. Due to its growth-suppression activity, it is underexpressed in many cancers. However, the molecular mechanism underlying the translational regulation of FBXO31 remains unclear. Here we show that the oncogenic microRNAs miR-93 and miR-106a repress FBXO31, resulting in the upregulation of Slug, which is involved in epithelial-mesenchymal transition and cell invasion. FBXO31 targets and ubiquitylates Slug for proteasomal degradation. However, this mechanism is repressed in breast tumors where miR-93 and miR-106a are overexpressed. Our study further unravels an interesting mechanism whereby Slug drives the expression of miR-93 and miR-106a, thus establishing a positive feedback loop to maintain an invasive phenotype. Together, these results establish the presence of interplay between microRNAs and the ubiquitination machinery, which together regulate cancer cell invasion.

FBXO32 activates NF-κB through IκBα degradation in inflammatory and genotoxic stress

In response to diverse stresses, the canonical NF-κB pathway gets activated primarily to protect the cells and maintain their genomic integrity. It activates the cell cycle checkpoints allowing the cells with limited damage to restore a normal life cycle. One of the key events in activation of the canonical NF-κB pathway is the selective proteasomal degradation of IκBα. It has been previously shown that F-box protein βTRCP1 has limited role in directing the proteasomal degradation of IκBα during stress conditions. Here, we report another member of F-box family proteins, FBXO32, as a potential activator of NF-κB signaling during genotoxic stress and inflammatory response. Following genotoxic or inflammatory stress, FBXO32 is stabilized, which leads to polyubiquitination and proteasome mediated degradation of IκBα. We also found that FBXO32 is required for physiological regulation of IκBα levels in unstressed cells. Thus, we decipher the new role of FBXO32 in regulation of NF-κB signaling pathway.