Muralidharan Jayaraman

Molecular and serum markers in hepatocellular carcinoma: Predictive tools for prognosis and recurrence

With increased understanding of cancer biology, a multitude of pathological, genetic, and molecular events that drive hepatocarcinogenesis, including angiogenesis, invasion, and metastasis, has been identified. Lately, they are being aggressively evaluated due to challenges involved in establishing early diagnosis, optimizing therapy for cancer inducing hepatotrophic viruses, minimizing the emergence of new tumors, and preventing recurrence after surgical resection or liver transplantation. This comprehensive review examines and critiques the evidence from published manuscripts reporting various tissue and serum biomarkers involved in hepatocellular carcinoma. These markers not only help in prediction of prognosis or recurrence, but may also assist in deciding appropriate modality of therapy and represent novel targets for potential therapeutic agents.

Mitogenic signaling by lysophosphatidic acid (LPA) involves Gα12

Lysophosphatidic acid (LPA), a major G protein coupled receptor (GPCR)-activating ligand present in serum, elicits growth factor like responses by stimulating specific GPCRs coupled to heterotrimeric G proteins such as Gi, Gq, and G12/13. Previous studies have shown that the overexpression of wild-type Gα12 (Gα12WT) results in the oncogenic transformation of NIH3T3 cells (Gα12WT-NIH3T3) in a serum-dependent manner. Based on the potent growth-stimulating activity of LPA and the presence of LPA and LPA-like molecules in the serum, we hypothesized that the serum-dependent neoplastic transformation of Gα12WT-NIH3T3 cells was mediated by the stimulation of LPA-receptors (LPARs) by LPA in the serum. In the present study, using guanine nucleotide exchange assay and GST-TPR binding assay, we show that the treatment of Gα12WT-NIH3T3 with 2 μM LPA leads to the activation of Gα12. Stimulation of these cells with LPA promotes JNK-activation, a critical component of Gα12-response and cell proliferation. We also show that LPA can substitute for serum in stimulating JNK-activity, DNA synthesis, and proliferation of Gα12WT-NIH3T3 cells. LPA-mediated proliferative response in NIH3T3 cells involves Gα12, but not the closely related Gα13. Pretreatment of Gα12WT-NIH3T3 cells with suramin (100 μM), a receptor-uncoupling agent, inhibited LPA-stimulated proliferation of these cells by 55% demonstrating the signal coupling between cell surface LPAR and Gα12 in the neoplastic proliferation of NIH3T3 cells. As LPA and LPAR mediated mitogenic pathways have been shown to play a major role in tumor genesis and progression, a mechanistic understanding of the signal coupling between LPAR, Gα12, and the downstream effectors is likely to unravel additional targets for novel cancer chemotherapies.

Lysophosphatidic Acid Stimulates the Proliferation of Ovarian Cancer Cells via the gep Proto-Oncogene Gα 12

Lysophosphatidic acid (LPA), an agonist that activates specific G protein-coupled receptors, is present at an elevated concentration in the serum and ascitic fluid of ovarian cancer patients. Although the increased levels of LPA have been linked to the genesis and progression of different cancers including ovarian carcinomas, the specific signaling conduit utilized by LPA in promoting different aspects of oncogenic growth has not been identified. Here, we show that LPA stimulates both migration and proliferation of ovarian cancer cells. Using multiple approaches, we demonstrate that the stimulation of ovarian cancer cells with LPA results in a robust and statistically significant proliferative response. Our results also indicate that Gα(12), the gep proto-oncogene, which can be stimulated by LPA via specific LPA receptors, is overtly activated in a large array of ovarian cancer cells. We further establish that LPA stimulates the rapid activation of Gα(12) in SKOV-3 cells and the expression of CT12, an inhibitory minigene of Gα(12) that disrupts LPAR-Gα(12) interaction and potently inhibits such activation. Using this inhibitory molecule as well as the shRNA approach, we show that the inhibition of Gα(12) or silencing of its expression drastically and significantly attenuates LPA-mediated proliferation of ovarian cancer cell lines such as SKOV3, Hey, and OVCAR-3. Together with our findings that the silencing of Gα(12) does not have any significant effect on LPA-mediated migratory response of SKOV3 cells, our results point to a critical role for LPA-LPAR-Gα(12) signaling in ovarian cancer cell proliferation and not in migration. Thus, results presented here for the first time demonstrate that the gep proto-oncogene forms a specific node in LPA-LPAR-mediated mitogenic signaling in ovarian cancer cells.

LPA-mediated migration of ovarian cancer cells involves translocalization of Gαi2 to invadopodia and association with Src and β-pix

Lysophosphatidic acid (LPA) plays a critical role in the migration and invasion of ovarian cancer cells. However, the downstream spatiotemporal signaling events involving specific G protein(s) underlying this process are largely unknown. In this report, we demonstrate that LPA signaling causes the translocation of Gαi2 into the invadopodia leading to its interaction with the tyrosine kinase Src and the Rac/CDC42-specific guanine nucleotide exchange factor, β-pix. Our results establish that Gαi2 activates Rac1 through a p130Cas-dependent pathway in ovarian cancer cells. Moreover, our report reveals that knockdown of Gαi2 leads to loss of β-pix and active-Rac association in the invadopodia. We also show that knockdown of Gαi2 leads to the complete loss of translocation to p130Cas to focal adhesions. Finally, when Gαi2 is knocked down, this led to the total distribution of Src being shifted primarily from invadopodia and the leading edge of the cells to the perinuclear region, suggesting that Src is inactive in the absence of Gαi2. Overall, our report provides tantalizing evidence that Gαi2 is a critical signaling component of a large signaling complex in the invadopodia that if disrupted could serve as an excellent target for therapy in ovarian and potentially other cancers.

The gep proto-oncogene Gα13 Mediates Lysophosphatidic acid-mediated Migration of Pancreatic Cancer Cells

Objectives Tumor microenvironment, defined by a variety of growth factors including lysophosphatidic acid (LPA), whose levels are increased in pancreatic cancer patients, plays a major role in the genesis and progression of pancreatic cancer. Because the gep proto-oncogenes, G&agr;12 and G&agr;13, are implicated in LPA-stimulated oncogenic signaling, this study is focused on evaluating the role of these proto-oncogenes in LPA-stimulated invasive migration of pancreatic cancer cells. Methods Effect of LPA on the migration and proliferation of pancreatic cancer cells was assessed using BxPC3, Dan-G, MDAPanc-28, Panc-1, and PaCa-2 cell lines. The role of G&agr;13 in the migration of pancreatic cancer cells was interrogated by disrupting lysophosphatidic acid receptor-G&agr;13 interaction using CT13, a dominant negative mutant of G&agr;13, and by silencing the expression of G&agr;13. Results Results indicate that LPA stimulates the migration of pancreatic cancer cells and such LPA-stimulated migratory response is mediated by G&agr;13. Furthermore, the results establish that the silencing of G&agr;13, but not G&agr;12, abrogates LPA-stimulated invasive migration of pancreatic cancer cells. Conclusions These results report for the first time a critical role for G&agr;13 in LPA-stimulated invasive migration of pancreatic cancer cells. These findings identify LPA-lysophosphatidic acid receptor-G&agr;13 signaling node as a novel therapeutic target for pancreatic cancer treatment and control.

Lysophosphatidic acid stimulates epithelial to mesenchymal transition marker Slug/Snail2 in ovarian cancer cells via Gαi2, Src, and HIF1α signaling nexus

Recent studies have identified a critical role for lysophosphatidic acid (LPA) in the progression of ovarian cancer. Using a transcription factor activation reporter array, which analyzes 45 distinct transcription factors, it has been observed that LPA observed robustly activates the transcription factor hypoxia-induced factor-1α (HIF1α) in SKOV3.ip ovarian cancer cells. HIF1α showed 150-fold increase in its activation profile compared to the untreated control. Validation of the array analysis indicated that LPA stimulates a rapid increase in the levels of HIF1α in ovarian cancer cells, with an observed maximum level of HIF1α-induction by 4 hours. Our report demonstrates that LPA stimulates the increase in HIF1α levels via Gαi2. Consistent with the role of HIF1α in epithelial to mesenchymal transition (EMT) of cancer cells, LPA stimulates EMT and associated invasive cell migration along with an increase in the expression levels N-cadherin and Slug/Snail2. Using the expression of Slug/Snail2 as a marker for EMT, we demonstrate that the inhibition of Gαi2, HIF1α or Src attenuates this response. In line with the established role of EMT in promoting invasive cell migration, our data demonstrates that the inhibition of HIF1α with the clinically used HIF1α inhibitor, PX-478, drastically attenuates LPA-stimulates invasive migration of SKOV3.ip cells. Thus, our present study demonstrates that LPA utilizes a Gαi2-mediated signaling pathway via Src kinase to stimulate an increase in HIF1α levels and downstream EMT-specific factors such as Slug, leading to invasive migration of ovarian cancer cells.

Heterologous Expression of Olfactory Receptors for Targeted Chemosensing

With the broad objective of developing a heterologous expression system for the mammalian olfactory signaling pathway, we have engineered yeast cells in which the mammalian olfactory signaling pathway is genetically integrated. Our results demonstrate that the prototypic “olfactory yeast” strain WIF‐1α can sense and report the presence of defined chemical agents through the engineered mammalian olfactory system. In this heterologous Saccharomyces cerevisiae‐based expression system, the primary components of the mammalian olfactory signaling pathway have been engineered, and signaling by the rat olfactory receptor is coupled to the expression of green fluorescent protein. By shuttling a library of olfactory receptor ligand‐binding pockets into the pre‐engineered signaling units of WIF‐1 yeast cells, we further demonstrate the ability of these olfactory yeast cells to detect 2,4‐dinitrotoluene. Using this approach, our results have identified the novel rat olfactory receptor Olfr226 as a 2,4‐dinitrotoluene‐responsive receptor. Genetic integration of a highly discriminatory olfactory system into biologically stable and biochip‐adaptable yeast cells, as presented here, can provide an ideal targeted chemosensing platform for detecting diverse chemical molecules. In addition to their potential use in deorphanizing the superfamily of olfactory receptors, the engineered olfactory yeast cells should be amenable for high‐throughput screening to identify receptor‐specific molecular targets.

Identification of novel diagnostic and prognostic miRNA signatures in endometrial cancer

With the goal of identifying diagnostic and prognostic biomarkers in endometrial cancer, miRNA-profiling was carried out with formalin-fixed paraffin embedded (FFPE) tissue samples from 49 endometrial cancer patients. Results using an 84-cancer specific miRNA panel identified the upregulation of miR-141-3p and miR-96-5p along with a downregulation of miR-26, miR-126-3p, miR-23b, miR-195-5p, miR-374a and let-7 family of miRNAs in endometrial cancer. We validated the dysregulated expression of the identified miRNAs in a panel of endometrial cancer cell-lines. Immunohistochemical analysis of the tissue micro array derived from these patients established the functional correlation between the decreased expression of tumor suppressive miRNAs and their target oncogenes: ERBB2, EGFR, EPHA2, BAX, GNA12, GNA13, and JUN. Comparative analysis of the samples from the patients with extended progression-free survival (PFS) ( > 21 months) versus the patients with the PFS of < 21 months indicated increased expression of tumor suppressive miR-142-3p, miR-142-5p, and miR-15a-5p in samples from extended PFS patients. In addition to defining a specific set of miRNAs and their target genes as potential diagnostic biomarkers, our studies have identified tumor suppressive miR-142 cluster and miR-15a as predictors of favorable prognosis for therapy response in endometrial cancer.

Engineered Saccharomyces cerevisiae strain BioS-OS1/2, for the detection of oxidative stress.

One of the major stress factors during space and high‐altitude flight is the oxidative damage caused by the release of reactive oxygen intermediates (ROIs) in human tissues. ROIs are released in response to several stress factors including radiation in space. Since ROIs contribute to several pathological conditions, there has been a great interest in developing a biosensor that can monitor the impact of ROIs on biological systems. Toward this goal, we sought to engineer a yeast stain that can monitor oxidative stress and be easily integrated into a biosensor platform. Saccharomyces cerevisiae respond to hyperoxidative stress by activating the expression of many proteins including the transcription factor, Yap1. Activated Yap1 primarily binds to the Yap‐1 response elements in the promoters of genes that combat oxidative stress. Based on these observations, we genetically altered the Yap‐1 pathway in the YCR094W BY4742 strain of S. cerevisiae by fusing the YREs in the promoter region of TRX2 gene to a cDNA‐insert encoding green fluorescent protein (GFP). Exposure of this engineered yeast strain BioS‐OS1 to varying levels of oxidative stress, as generated by different concentrations of H2O2 or diamide, elicits robust expression of GFP that can be monitored by the fluorescence of GFP by as early as 1 h. BioS‐OS1 can detect a H2O2 concentration from 300 μM onward. We also show that the signaling strength of the strain can be increased by engineering multiple YREs in the upstream of the cDNA‐insert encoding GFP. Thus, the results presented here demonstrate that the engineered BioS‐OS yeast strain can detect ROI‐generating oxidative stress and validate the use of this prototypic strain for the development of a biosensor to detect and monitor oxidative stress factors during space and high altitude flights.

MICU1 drives glycolysis and chemoresistance in ovarian cancer.

Cancer cells actively promote aerobic glycolysis to sustain their metabolic requirements through mechanisms not always clear. Here, we demonstrate that the gatekeeper of mitochondrial Ca2+ uptake, Mitochondrial Calcium Uptake 1 (MICU1/CBARA1) drives aerobic glycolysis in ovarian cancer. We show that MICU1 is overexpressed in a panel of ovarian cancer cell lines and that MICU1 overexpression correlates with poor overall survival (OS). Silencing MICU1 in vitro increases oxygen consumption, decreases lactate production, inhibits clonal growth, migration and invasion of ovarian cancer cells, whereas silencing in vivo inhibits tumour growth, increases cisplatin efficacy and OS. Mechanistically, silencing MICU1 activates pyruvate dehydrogenase (PDH) by stimulating the PDPhosphatase-phosphoPDH-PDH axis. Forced-expression of MICU1 in normal cells phenocopies the metabolic aberrations of malignant cells. Consistent with the in vitro and in vivo findings we observe a significant correlation between MICU1 and pPDH (inactive form of PDH) expression with poor prognosis. Thus, MICU1 could serve as an important therapeutic target to normalize metabolic aberrations responsible for poor prognosis in ovarian cancer.