Browsing by Subject "MIEN1"
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Item EFFECT OF 4-HYDROXYNONENAL ON MIGRATION AND INVASION ENHANCER PROTEIN 1 (MIEN1) IN COLORECTAL CANCER(2014-03) Raychaudhuri, Urmimala; Vishwanatha, Jamboor K.MIEN1 could be a potential target for therapy in colorectal cancer. Purpose (a): Colorectal cancer (CRC) is the second leading cause of death in the United States. It is believed that the intestinal mucosa is constantly challenged with diet- and bacterial-derived oxidants and carcinogens. Chronic exposure of such challenging conditions may lead to the generation of reactive oxygen species (ROS). ROS initiate an autocatalytic chain of lipid peroxidation (LPO) of polyunsaturated fatty acids, resulting in the formation of large amounts of toxic electrophilic species and free radicals that may play important roles in various human diseases, including carcinogenesis. Consequently, even a minimal transient exposure of cells to ROS causes substantial lipid peroxidation, leading to a significant rise in the level of LPO end product, 4-hydroxynonenal (4-HNE), which is considered to be one of the most abundant cytotoxic aldehydes. HNE reacts not only with DNA but also with proteins and other molecules containing thiol and other nucleophilic groups and can alter the protein structure and functions. We have identified a novel protein called Migration and Invasion Enhancer protein 1 (MIEN1), is highly overexpressed in cancer cells and modulates the AKT activity as a membrane bound adaptor protein. Ectopic expression of MIEN1 activates Akt mediated downstream signaling through NF-kB pathway and induces the expression of several migratory and invasive proteins. However, 4-HNE has also been reported to induce the expression of various proteins involved in cell proliferation and migration. We hypothesize that 4-HNE mediated oxidative stress plays an important role in the etiology of colorectal cancer by modulating the expression and function of MIEN1. In the present studies, we have addressed this question by investigating the effect of 4-HNE on MIEN1 expression in colorectal cancer cell lines SW480 and HT29. Methods (b): Colorectal cancer cell lines, SW480 and HT29 were grown in RPMI-1640 medium containing 10% fetal bovine serum, in a humidified incubator at 37°C with 5% CO2. The toxicity of 4HNE in SW480 cells was determined by MTT assay. The effect of 4HNE on MIEN1 expression was determined by Western blotting in HT29. The effect of 4HNE on SW480 cell migration was examined by scratch wound assay. The LigandFit docking program available in the Accelrys molecular modeling software – Discovery studio, was used to carry out a docking study for the protein MIEN1 and substrate 4HNE. The 3D structure of the protein was obtained from PDB. Results (c): Our results demonstrated that exposure of 4HNE to SW480 cells is toxic. 4HNE concentrations ranging from 0 to 250 μM gradually decreased cell viability in SW480 cells corresponding to an IC50 value of 160 μM. Furthermore, our Western blot analysis demonstrated that treatment of 4HNE increased the expression of MIEN1 at the protein level in HT-29 cells. The scratch wound healing assay showed an increase in migration after treatment with low doses of 4HNE. The docking study produced 10 top scoring(dock score) poses. The poses indicate a possible interaction between the protein binding sites and the substrate (4HNE) including formation of hydrogen bonds between them. Conclusions (d): Together, these results suggest that 4HNE induced cell migration could be mediated via MIEN1.Item Effect of CRISPR MIEN1 Knockout in Metastatic Breast Cancer Cells(2018-12-01) Van Treuren, Timothy R.; Vishwanatha, Jamboor K.; Basu, Alakananda; Basha, RiyazMigration and Invasion Enhancer 1 (MIEN1) is an oncogene which is involved in facilitating the migration and invasion of cancer cells through actin dynamics and gene expression. Increased MIEN1 expression in many types of tumors correlates with disease progression and metastatic propensity. The precise mechanism by which MIEN1 functions is yet to be understood. The goal of these studies is to progress toward determination of the mechanisms and genetic context in which MIEN1 functions contribute to cancer progression. It was hypothesized that Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) mediated knockout of MIEN1 in metastatic breast cancer cells would result in reduced migration and invasion. CRISPR genome editing effectively produced specific genomic deletions in the MIEN1 gene which led to the elimination of its expression in these breast cancer cells. Migration in MDA-MB-231 (231) MIEN1 knockout (MIEN1-KO) cells exhibited no difference when compared to parental 231, which was in contrast with previous siRNA studies. Signaling in several MIEN1-KO pools was inconsistent. Knocking out MIEN1 in 231 derivative cell lines showed few significant alterations in the growth, migration, invasion, signaling, despite significant changes in metabolism. However, re-expression of the MIEN1 protein containing a mutant immunoreceptor tyrosine-based activation motif (ITAM) domain resulted in significantly decreased invasion. This revealed that MIEN1-KO 231 derivative cells were susceptible to interference of compensatory mechanisms and demonstrates the importance of the migration and invasion pathways in which MIEN1 participates in breast cancer metastasis. These findings also suggest MIEN1 may still be a promising therapeutic target to inhibit metastasis if inhibitors can be developed which block ITAM function without affecting localization or expression.Item Effect of CRISPR MIEN1 knockout in metastatic breast cancer cells(2018-12) Van Treuren, Timothy; Vishwanatha, Jamboor K.; Basu, Alakananda; Basha, RiyazMigration and Invasion Enhancer 1 (MIEN1) is an oncogene which is involved in facilitating the migration and invasion of cancer cells through actin dynamics and gene expression. Increased MIEN1 expression in many types of tumors correlates with disease progression and metastatic propensity. The precise mechanism by which MIEN1 functions is yet to be understood. The goal of these studies is to progress toward determination of the mechanisms and genetic context in which MIEN1 functions contribute to cancer progression. It was hypothesized that Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) mediated knockout of MIEN1 in metastatic breast cancer cells would result in reduced migration and invasion. CRISPR genome editing effectively produced specific genomic deletions in the MIEN1 gene which led to the elimination of its expression in these breast cancer cells. Migration in MDA-MB-231 (231) MIEN1 knockout (MIEN1-KO) cells exhibited no difference when compared to parental 231, which was in contrast with previous siRNA studies. Signaling in several MIEN1-KO pools was inconsistent. Knocking out MIEN1 in 231 derivative cell lines showed few significant alterations in the growth, migration, invasion, signaling, despite significant changes in metabolism. However, re-expression of the MIEN1 protein containing a mutant immunoreceptor tyrosine-based activation motif (ITAM) domain resulted in significantly decreased invasion. This revealed that MIEN1-KO 231 derivative cells were susceptible to interference of compensatory mechanisms and demonstrates the importance of the migration and invasion pathways in which MIEN1 participates in breast cancer metastasis. These findings also suggest MIEN1 may still be a promising therapeutic target to inhibit metastasis if inhibitors can be developed which block ITAM function without affecting localization or expression.Item ITAM PHOSPHORYLATION OF THE ADAPTER PROTEIN MIEN1 CONTRIBUTES TO ITS MIGRATION AND INVASION POTENTIAL(2013-04-12) Kpetemey, MarilynePurpose: Metastasis is one of the major causes of treatment failure in cancer patients. This process involves cell migration, stromal invasion, intravasation into the circulatory system leading to cancer proliferation. Identifying metastasis promoting and suppressing genes and their mechanisms of action will provide new insights into the pathogenesis and management of cancer. Previously known as C17orf37 or C35, Migration and Invasion enhancer 1 (MIEN1) is a novel gene that is over-expressed in a wide range of tumors. MIEN1 has been reported to be a critical regulator of cell migration and invasion. MIEN1 has a prenylation motif and an immunoreceptor tyrosine-based activation motif (ITAM), commonly found in signaling chains and receptors. The presence of a prenylation and ITAM domains in MIEN1 suggests that prenylation and/or tyrosine phosphorylation of the ITAM motif are important for its functions. The objective of the present study is to elucidate the molecular mechanisms through which MIEN1 promotes migration in breast cancer and whether or not tyrosine phosphorylation is important. Methods: We knockdown the expression of MIEN1 in MDA-MB231 and MCF10CA1 by siRNA and compared the migration of these cells with their respective controls. To study the importance of tyrosine phosphorylation of MIEN1- ITAM in migration and invasion, NIH3T3 were stably transfected with wild type MIEN1 or its phospho-mutants and used for migration and invasion assays. Results: Silencing of MIEN1 expression in MDA-MB231 and MCF10CA1 led to a significant decrease in breast cancer cell migration, conforming to our previous results in prostate cancer cells. Analyses of in-vitro migration and invasion assays revealed that NIH3T3 cells over-expressing MIEN1 phospho mutants failed to induce significant migration and invasion when compared with NIH3T3 over-expressing MIEN1 wild type. Conclusions: Our results show that MIEN1 plays an important role in the migration and invasion of breast cancer cells and tyrosine phosphorylation of MIEN1-ITAM significantly contributes to its functionsItem MICRO RNA MEDIATED REGULATION OF MIEN1 IN PROSTATE CANCER(2013-04-12) Rajendiran, SmrithiPurpose: MIEN1 is a membrane bound signaling molecule that triggers downstream signaling through the AKT/NF-𝛋B pathway by up-regulating key proteases and has a role in migration and invasion of cancer cells. The overall objective of this study is to identify the mechanisms that lead to the differential regulation of MIEN1 in normal and cancer cells. Though there are multiple mechanisms that are commonly studied, here, we propose to focus on the post-transcriptional regulation of MIEN1. Our data leads to the hypothesis that MIEN1 is post-transcriptionally regulated by a specific microRNA (miR) that is down-regulated in cancer, thus explaining aberrant increased expression of MIEN1 in cancer. Methods: We have performed miR in silico analysis, expression profiling, northern blotting, qPCRs, western blotting, luciferase reporter assays, migration and invasion assays, colony formation assays and stability assays to determine the effects of the miR on different aspects of MIEN1 regulation. Results: Our data indicate that MIEN1 is post-transcriptionally regulated by a specific miR which is lost in cancer cells. This miR is highly expressed in normal cells compared to a decrease in various cancer cells and this expression is inversely correlated to the expression of MIEN1. Introduction of the mimic (precursor) or inhibitor (antagomiR) led to a decrease or increase in MIEN1 expression respectively at both the RNA and protein levels. The downstream effectors were also similarly affected. The luciferase activity significantly reduced when the 3'UTR of MIEN1 was transfected with the target miR compared to the control miR, validating the direct binding of the miR. The miR decreases the migration and invasion of cells as well as the stability of MIEN1 RNA. There was a significant reduction in the colony formation capabilities and the morphology of the colonies differed in cells that were transfected with the miR along with a decrease in E-cadherin, suggesting potential involvement of the miRNA in reducing epithelial to mesenchymal transition and hence inhibiting metastasis. Conclusions: Our results demonstrate that aberrant expression of MIEN1 in cancer is attributed to a specific miR. We are currently exploring the potential of using this miR as a biomarker in prostate cancer. Since the importance of MIEN1 as a key signaling molecule in cancer is well established, understanding the mechanisms involved in its regulation will aid in designing novel and effective therapeutic strategies to treat cancer patients.Item MIEN1 Drives Breast Cancer Invasion by Regulating Cytoskeletal-Focal Adhesions Dynamics(2015-05-01) Kpetemey, Marilyne F.; Vishwanatha, Jamboor K.; Clark, Abbot F.; Basu, AlakanandaIn the recent years, Migration and Invasion Enhancer 1(MIEN1) has emerged as a potential biomarker and a plausible target in breast cancer. Located in the 17q12-21 region of the human chromosome, next to the Her-2/neu loci, MIEN1 presents a robust expression in breast carcinomas; however is completely absent or low in the normal tissues. MIEN1 is post-translationally modified by geranyl-geranyl transferase-I (GgtaseI), which adds isoprenyl group to the carboxyl-terminal of the protein. Prenylated MIEN1 then associates with the inner leaflet of the plasma membrane and acts as an adaptor protein triggering downstream signaling through the Akt/NF-kB axis to regulate the expression of key proteases and angiogenic factors like MMP-9, uPA and VEGF. In migrating cells, MIEN1 enhances filopodium formation at the leading edge. Aside from its prenylation and redox-active motifs, MIEN1 also contains a canonical ITAM, reported to be associated with epithelial-to-mesenchymal transition. Although the role MIEN1 in cell migration and invasion is well known, the underlying molecular mechanisms remain elusive. Here, we show that MIEN1 interacts with Annexin A2, a cytoskeletal protein and a regulator of the plasminogen/plasmin system in breast cancer cells to increase migration and invasion. We confirmed that MIEN1 regulates actin dynamics by associating with cytoskeletal effectors in the lamellum. We also show that MIEN1 expression redirects breast tumor cell migration toward a collective migration. Our studies validate MIEN1-ITAM and CAAX as key motifs to MIEN1-induced functions. In conclusion, our findings confirm the role of MIEN1 in the remodeling of the actin cytoskeleton during motility. Furthermore it attests to previous findings suggesting that motility patterns depend on various environmental factors along with regulatory genes involved. Our study demonstrates an interesting example from cell biology where adaptor proteins regulate various signaling pathways and control cellular processes through protein-protein interactions.Item MIEN1 PROMOTES CANCER CELL MIGRATION AND INVASION THROUGH ENHANCED ACTIN DYNAMICS(2014-03) Kpetemey, Marilyne; Dasgupta, Subhamoy; Vishwanatha, JamboorSurgical resection and adjuvant therapies have so far, only incrementally improved patient survival; as metastatic disease remains incurable. Knowledge has been in part limited because metastasis is a ‘hidden’ process that occurs inside the body and is inherently difficult to observe. Migration and invasion are critical parameters in the dissemination of cancer cells and the formation of distant metastases. Hence identifying migratory and invasive genes and their action mechanisms may provide new insights into the pathogenesis and management of tumor metastasis. In the present investigation, we report functional studies of one of the prime regulators of cancer cell migration and invasion, which is also prenylated and contains an ITAM like the atypical Rho GTPase, RhoH. Migration and invasion enhancer1 (MIEN1) also known as C35, C17orf37, RDX12, and MGC14832, is frequently amplified and overexpressed in breast tumors. Purpose (a): Migration and Invasion Enhancer 1 (MIEN1), previously known as C35, C17orf37, RDX12 and MGC14832, is a novel gene located in the chromosomal region 17q12-21. While absent or low in normal tissues, MIEN1 is abundantly expressed in multiple cancers; including breast, prostate, oral and gastrointestinal carcinomas. A membrane-bound signaling adaptor, MIEN1 localizes to the leading edge of migrating cells and promotes migration and invasion by increasing filopodium formation. MIEN1 contains several functional motifs including a prenylation motif and an immunoreceptor tyrosine-based activation motif (ITAM). While prenylation of MIEN1 is shown to be important for its functions, little is known about the importance of its ITAM. The overall goal of the present study is to dissect the mechanisms by which MIEN1 regulates breast cell motility and whether the ITAM is important. Methods (b): Using site-directed mutagenesis, we introduced point mutations in amino acid sequences in MIEN1-ITAM domains. We established NIH3T3 stable cell lines over-expressing the wild type or mutant proteins. We performed immunofluorescence, migration and invasion assays using the established stable cell lines and breast cancer cells to investigate the mechanisms by which different domains of MIEN1 potentiate cell motility. Results (c): Analyses of in vitro migration and invasion assays showed that stable cells over-expressing MIEN1 phosphorylation mutants failed to induce significant migration and invasion compared to cells over-expressing MIEN1 wild type protein. Immunofluorescence staining with rhodamine conjugated-phalladoin confirmed that MIEN1 induced- migration is associated with actin filaments; and post-translational modifications at the ITAM domains is critical for eliciting MIEN1 functions. Conclusions (d): Our results confirm that MIEN1 regulates cancer cell migration and invasion through filopodia formation. Furthermore we showed that MIEN1 is involved in cell-cell adhesion, a process required for cell motility. MIEN1 is a prime regulator of cancer cell motility; hence understanding the molecular mechanisms by which it is aiding the invasion-metastasis cascade will enable the design of novel and effective treatments for metastatic tumors.Item Post-transcriptional and Epigenetic Regulation of MIEN1 in Prostate Cancer(2014-08-01) Rajendiran, Smrithi; Vishwanatha, Jamboor K.; Basu, Alakananda; Berg, Rance E.Migration and invasion enhancer 1 (MIEN1), a gene located in the 17q12 region of the human chromosome, enhances migratory and invasive potential of cancer cells via two mechanisms; activating the Akt dependent NF-κB downstream signaling and facilitating filopodia formation; thereby playing an important role in cancer progression. MIEN1 is highly expressed in many cancers including prostate and breast, but its expression is very basal to null in a variety of normal tissues making it a plausible target for cancer therapy. Though the functions of MIEN1 are known, the reasons for its increased expression in cancer is unknown. Determining the molecular gene regulatory mechanisms by which expression of MIEN1 is curtailed in normal cells will help in developing better targeting strategies. Among the different gene regulatory mechanisms including transcriptional regulation, post-transcriptional modifications and histone and DNA alterations, here we focus on post transcriptional and DNA methylation based regulation of MIEN1. We show that MIEN1 is downregulated post-transcriptionally by miRNA-940 which itself is present in low amounts in cancer cells and tissues compared to the normal counterparts. The miR-940 also contributes to inhibition of cancer progression by attenuating the migration, invasion, anchorage-independent growth and epithelial-to-mesenchymal transition, when ectopically re-introduced into cancer cells. The miR-940 can be detected in circulation and its elevated levels in serum from cancer patients than normal subjects, suggest its potential as a biomarker for prostate cancer diagnosis. MIEN1, like urokinase plasminogen activator, is also suppressed by DNA methylation in normal cells. On the contrary, hypomethylation in cancer, results in its overexpression. The current approach of using global demethylating agents to activate the expression of hypermethylated tumor suppressor genes may in the long run activate tumor promoting genes like MIEN1. Thus, our study supports the notion that gene-centric hypomethylating agents may be a better epigenetic targeting approach to treat cancer. In conclusion, our data confirm the role of post-transcriptional and DNA methylation mediated mechanisms in the regulation of MIEN1.Item REGULATION OF MIEN1 IN PROSTATE CANCER(2014-03) Rajendiran, Smrithi; Parwani, Anil; Hare, Richard; Treuren, Timothy Van; Vishwanatha, JamboorMigration and Invasion ENhancer 1 (MIEN1) is a novel gene located in the 17q12 region of the human chromosome. While there is minimal expression of MIEN1 in multiple normal tissues and cells, it is abundantly elevated in many human cancers including the breast, prostate, gastrointestinal and oral. MIEN1 is a membrane bound signaling molecule that triggers downstream signaling through the AKT/NF-κB pathway (common oncogenic pathways) by up-regulating key proteases (thus aiding cancer progression). MIEN1 has also been shown to have a role in migration and invasion (key processes in cancer spread) of cancer cells by enabling filopodia formation (extensions that enable a cell to move). Thus, by various known and unknown mechanisms, MIEN1 promotes prostate cancer progression. While the cellular functions of MIEN1 have been deciphered, the reasons for its aberrant increased expression in cancer cells are still unclear. Understanding the mechanism(s) involved in the regulation of MIEN1 will aid in developing diagnostic marker(s) or in designing effective therapeutic approach(es) to treat prostate cancer patients. Purpose (a): The overall objective of this study is to identify the deregulated mechanisms leading to the differential regulation of MIEN1 between normal and cancer cells. Commonly deregulated mechanisms encompass alterations at DNA (chromosome) to destabilization at protein (translational) levels. Our study focuses on regulation by microRNA (miR) and methylation. Our hypothesis is that MIEN1 is post-transcriptionally regulated by a specific miR and its proximal putative promoter region is hypermethylated in normal cells. Deregulation of these mechanisms together explain the aberrant increased expression of MIEN1 in cancer. Methods (b): To validate the role of miR in MIEN1 regulation, we have performed various in vitro studies. To determine the global role of the miR, we ectopically expressed it in cancer cells. Additionally, we have used human tissue and serum samples to predict the use of miR-MIEN1 as biomarkers. To demonstrate the importance of methylation in the regulation of MIEN1, we performed global methylation inhibition and specific methyltransferase knockdown. Results (c): Our data indicate that MIEN1 is post-transcriptionally regulated by a specific miR which is highly expressed in normal cells compared to various cancer cells, inversely correlating with MIEN1. Ectopic expression of the miR led to decrease in MIEN1 expression, migratory and invasive potential and anchorage dependent growth of cells and impeded mesenchymal transition. Additionally, the miR expression was higher in the normal glands of prostate tissue compared to the tumor; while the secreted/circulating miR was higher in serum from cancer patients, much like PSA expression patterns; but with more significance than PSA. Inhibition of methylation by pharmacological inhibitors or by individually knocking down the methyltransferases increased MIEN1 in normal cells, indicating the role of methylation in the regulation of this gene. Conclusions (d): After proving our results in a larger cohort of patient specimen, this miR could be a useful non-invasive diagnostic biomarker. Additionally, understanding the regulation of MIEN1 by methylation will provide reasons to revisit the current strategies of methylation inhibition for cancer treatment. Overall, since the importance of MIEN1 as a key signaling molecule in cancer is well established, understanding the mechanisms involved in the regulation will aid in designing more effective therapeutic strategies to treat cancer patients.