Browsing by Author "Trivedi, Rucha"
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Item 3D TUMOR MODELING TO IMPROVE BIOMARKER IDENTIFICATION AND CANCER OUTCOMES.(2022) Mylabathula, Preteesh Leo; Ranade, Payal; Trivedi, Rucha; Vishwanatha, JamboorPurpose: Cancer-associated proteins annexina2 (ANXA2) and MIEN1 are overexpressed in various cancers including triple negative breast cancer, prostate, and colorectal cancers. Therefore, these proteins are being investigated as potential biomarkers and therapeutic targets. We will use a 3D tumor model to mimic in vivo tumor growth and evaluate changes in tumor characteristics after silencing ANXA2 and MIEN1 to understand their correlative/causal relation to tumor behavior. Methods: Ultra-low attachment 3D culture plates were used to culture tumor spheroids in breast cancer cells(MDA-MD-231) with ANXA2 knock-down using sh-RNA and colorectal cancer cells(HT29) with MIEN1 knock-out (KO) using CRISPR-Cas9. Changes in spheroid growth, migration and invasion, drug sensitivity, and immune cell killing will be evaluated. Boyden chambers with Matrigel inserts were used to evaluate migration and invasion. Spheroids were imaged using confocal microscopy for size estimation. Results: Optimal initial seeding density was 5000 cells and spheroids were grown for 7-days. Average tumor spheroid size was 1360±420µm with no differences in spheroid size with MIEN1 KO. 50% reduction in migration and invasion potential were observed after MIEN1 KO in colorectal cancer cells in a 96-hour time-course experiment. Experiments with ANXA2 knock-down are ongoing with spheroid sizes averaging 1700±260µm. Future experiments include evaluating changes in protein expression, drug sensitivity, immune cell killing as a result of knockout of MIEN1. Conclusion: We expect the results to identify functional role of cancer-associated proteins ANXA2 and MIEN1 in tumor spheroid growth, metastasis, drug sensitivity, and susceptibility to immune cell killing. Funding: Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number R01CA220273 (JKV).Item Annexin A2 in tumor-derived extracellular vesicles: Molecular contributions in metastatic triple negative breast cancer(2023) Trivedi, Rucha; Vishwanatha, JamboorPurpose Tumor-derived extracellular vesicles (TEV) are highly implicated in tissue-specific metastasis. Additionally, TEV interacts with the distant microenvironment to shape a pre-metastatic niche (PMN) for homing the tumor cells. Annexin A2 (AnxA2) is a plasma and endosomal membrane-associated protein. Its high levels have been correlated with poor distant metastasis-free survival and poor overall survival in triple negative breast cancer (TNBC) patients. It is also abundantly present in TEV and recruits TEV-associated cargo such as proteins and microRNAs. Our lab reported that in vivo education with AnxA2 depleted EV led to reduced TNBC metastasis to lungs and brain suggesting a key role in the formation of a PMN. While the presence of AnxA2 in EV has been reported, its contribution in the formation & development of PMN via EV is still unexplored. We aim to evaluate the implications of AnxA2 in EV & elucidate the mechanisms promoting TNBC metastasis. Methods We used shRNA- mediated gene silencing to stably downregulate AnxA2 in organotropic TNBC cell lines derived from the parent MDA MB 231 cells. Differential ultracentrifugation was used to isolate EV from cell culture supernatant & size analysis was done using NTA. Biological characterization was done in concordance with MISEV 2018 guidelines using immunoblotting. Additionally, the EVs will be subjected to quantitative proteomic and transcriptomic analysis to identify differentially expressed proteins and genes upon loss of AnxA2. Results Upon depletion of AnxA2 protein, we observed a significant effect of AnxA2 depletion on its physiological role in plasmin generation. We observed a size distribution of the isolated EV between 30-300 nm. Using immunoblotting we confirmed reduced levels of AnxA2 in EVs derived from AnxA2 depleted TNBC cells. We verified their purity using EV enriched markers - ESCRT, Heat shock proteins & tetraspanins such as CD81, CD9, CD63 & confirmed the absence of negative markers - GM130, calnexin & cytochrome c. Interestingly, we observed a reduced yield of EV with AnxA2 depletion indicating a potential effect on EV biogenesis & release. Conclusion The role of AnxA2 in TEV biogenesis, release and selective cargo loading will lead to potential identification and understanding of the novel secretory and EV protein that may act as a functional regulator in promoting advanced metastasis in TNBC.Item Annexin A2 mediated TNBC metastasis via small extracellular vesicles: A multi-faceted omics-based characterization(2024-03-21) Trivedi, Rucha; Ranade, Payal; Vishwanatha, Jamboor K.Purpose – Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer poses a high risk of metastasis due to a lack of targeted therapy and reliance on conventional treatment approaches. Among the organs susceptible to metastasis, the lungs are most frequently affected sites, accounting for approximately 60% of cases. Extracellular vesicles (EV) play a crucial role in establishing a pre-metastatic niche (PMN) for tumor cell homing to secondary sites. Identifying the tumor-derived EV-specific cargo that may potentially alter the lung microenvironment could establish an understanding of the molecular targets contributing to lung PMN development and consequently improving the poor TNBC prognosis and diagnosis. TNBC exhibits elevated expression of Annexin A2 (AnxA2), a plasma and endosome membrane-associated protein. The abundant expression of AnxA2 in tumor tissues and EVs derived from TNBC patients is correlated with poor overall survival and poor distant metastasis-free survival. In previous studies, depletion of AnxA2 protein levels in TNBC-derived EVs resulted in an altered PMN establishment, leading to significantly lower metastatic lesions in the lungs and brain in in vivo system. We aim to evaluate the molecular effects mediated by AnxA2 in TNBC-derived EVs and their cargo that aid the PMN formation and identify underlying molecular targets creating a favorable microenvironment by interaction with lung stroma. Methods and Results – To assess the role of AnxA2 in TNBC lung metastasis, we utilized a lung selective metastatic TNBC cell line, MDA MB 4175 (LM2). We used the shRNA-mediated stable knockdown technique to downregulate AnxA2 protein levels in LM2 cells. We then performed expressional analysis as well as its cell surface functionality using a plasmin generation assay. Additionally, we conducted tumor cell functional assays such as proliferation and invasion assays. Decreased AnxA2 protein levels in LM2 cells significantly reduced its plasmin generation, proliferative ability, and invasive potential. Subsequently, we isolated cell derived small EVs using differential ultracentrifugation and characterized them following the MISEV 2018 guidelines. We confirmed the purity, yield, size, presence of EV-enriched proteins (ESCRT, Hsp, CD9, CD81), and absence of negative proteins (Calnexin, GM130). We then subjected the EVs to quantitative proteomic analysis and identified the differentially expressed proteins upon AnxA2 depletion. We further exposed the healthy lung stromal fibroblasts and the local less-aggressive TNBC cells with EVs to assess the differences in local and distant site uptake and cargo transfer mediated by AnxA2. Conclusion – This comprehensive approach will determine the novel EV-associated proteins that act as functional regulators in promoting TNBC metastasis to the lungs. [This work is supported by the NCI R01CA220273 awarded to Dr. Jamboor K. Vishwanatha]Item Elucidating the role of MIEN1 in colorectal cancer metastasis(2024-03-21) Ranade, Payal; Trivedi, RuchaPurpose: Colorectal cancer (CRC) ranks among the foremost contributors to global cancer-related fatalities, with its progression marked by hyperproliferation of epithelial cells culminating in polyp formation. Stages 3 and 4 of CRC are notably associated with metastasis. Despite existing chemotherapy focusing on symptom attenuation, a definitive cure remains elusive. This study addresses the imperative need to comprehend the underlying mechanisms of CRC progression for timely diagnosis and effective treatment. The process of metastasis involves a complex interplay of signaling pathways, where various proteins play crucial roles. Migration and Invasion ENhancer 1 (MIEN1) is a significant protein facilitating migration, and its expression is notably upregulated in CRC tissue compared to normal colorectal tissue, correlating with invasive behavior. The exact mechanism of MIEN1 involvement in metastasis remains unexplored. MIEN1 overexpression results from 17q12 chromosomal amplification, and dysregulated expression is associated with trans-regulation of its minimal promoter region. This study aims to investigate the impact of MIEN1 promoter ablation on CRC migration properties. Methods: Utilizing CRISPR-Cas9 gene editing, we deleted the MIEN1 promoter region in the CRC cell line HT29. RNA sequencing and bioinformatics tools revealed differentially expressed genes (DEGs) associated with essential biological processes and molecular pathways, including cell adhesion, migration, invasion, and angiogenesis. Evaluation of critical genes in CRC biogenesis at RNA and protein levels demonstrated that MIEN1 knock-out significantly reduced the migration potential of HT29 cells, as evidenced by functional assays such as wound healing and Matrigel invasion. Furthermore, MIEN1 deletion disrupted cytoskeletal rearrangement, affecting F-actin reorganization, confirmed through phalloidin staining. Confocal staining of proteins involved in actin cytoskeleton rearrangement provided insights into MIEN1's role in mediating phosphorylation of FAK at different sites. Immunoblotting analysis further substantiated MIEN1's involvement in actin cytoskeleton dynamics. Results: Our findings indicate a significant reduction in the migration potential of HT29 cells in the absence of MIEN1 protein. Additionally, we demonstrated the disruption of cytoskeletal rearrangement, specifically affecting F-actin reorganization, upon MIEN1 deletion using phalloidin staining. Confocal staining of various proteins involved in actin cytoskeleton rearrangement, such as FAK and cofilin, further strengthened the evidence of MIEN1 in actin reorganization. Immunoblotting analysis of a diverse set of proteins further confirmed the involvement of MIEN1 in the dynamics of actin cytoskeleton. Conclusion: MIEN1, an influential metastatic protein overexpressed in cancerous cells, plays a crucial role in various signaling pathways governing CRC migration. Deletion of MIEN1 perturbs biological processes, particularly actin cytoskeleton rearrangement crucial for metastasis. Thus, targeting MIEN1 emerges as a promising therapeutic strategy for CRC patients.Item First-in-class Peptide Molecules Targeting the MIEN1 Cancer signaling Pathway for which no inhibitors are currently identified(2024-03-21) Tripathi, Amit; Ranade, Payal; Trivedi, Rucha; Vishwanatha, Jamboor K.Migration and invasion enhancer 1 (MIEN1) overexpression characterizes several cancers and facilitates cancer cell migration and invasion. Leveraging conserved ITAM and prenylation motifs within MIEN1, we identified potent anti-cancer peptides. Among them, bioactive peptides LA3IK and RP-7 induced pronounced transcriptomic and protein expression changes at sub-IC50 concentrations. The peptides effectively inhibited genes and proteins driving cancer cell migration, invasion, and EMT pathways, concurrently suppressing EGF-induced NF-kB nuclear translocation in metastatic breast cancer cells. Specifically, peptides targeted the same signal transduction pathway initiated by MIEN1. Molecular docking and circular dichroism spectroscopy indicated the formation of MIEN1-peptide complexes. The third-positioned isoleucine in LA3IK and CVIL motif in RP-7 were crucial for inhibiting breast cancer cell migration. This is evident from the limited migration inhibition observed when MDA-MB-231 cells were treated with scrambled peptides LA3IK SCR and RP-7 SCR. Additionally, LA3IK and RP-7 effectively suppressed tumor growth in an orthotopic breast cancer model. Notably, mice tolerated high peptide doses of up to 90 mg/Kg well, surpassing significantly lower doses of 5 mg/Kg intravenously (iv) and 30 mg/Kg intraperitoneally (ip) used in both in vivo pharmacokinetic studies and orthotopic mouse model assays. D-isomers of LA3IK and RP-7 showed enhanced anti-cancer activity compared to their L-isomers. D-LA3IK remained stable in mouse plasma for 24 h with 75% remaining, exhibiting superior pharmacokinetic properties over D/L-RP-7. In summary, our findings mark the first report of short peptides based on MIEN1 protein sequence capable of inhibiting cancer signaling pathways, effectively impeding cancer progression both in vitro and in vivo.Item Higher Expression of Annexin A2 in Metastatic Bladder Urothelial Carcinoma Promotes Migration and Invasion(MDPI, 2022-11-27) Guo, Christina; Trivedi, Rucha; Tripathi, Amit K.; Nandy, Rajesh; Wagner, Diana C.; Narra, Kalyani; Chaudhary, PankajIn this study, we aim to evaluate the significance of AnxA2 in BLCA and establish its metastatic role in bladder cancer cells. Analysis of TCGA data showed that AnxA2 mRNA expression was significantly higher in BLCA tumors than in normal bladder tissues. High mRNA expression of AnxA2 in BLCA was significantly associated with high pathological grades and stages, non-papillary tumor histology, and poor overall survival (OS), progression-free survival (PFS), and diseases specific survival (DSS). Similarly, we found that AnxA2 expression was higher in bladder cancer cells derived from high-grade metastatic carcinoma than in cells derived from low-grade urothelial carcinoma. AnxA2 expression significantly mobilized to the surface of highly metastatic bladder cancer cells compared to cells derived from low-grade tumors and associated with high plasmin generation and AnxA2 secretion. In addition, the downregulation of AnxA2 cells significantly inhibited the proliferation, migration, and invasion in bladder cancer along with the reduction in proangiogenic factors and cytokines such as PDGF-BB, ANGPT1, ANGPT2, Tie-2, bFGF, GRO, IL-6, IL-8, and MMP-9. These findings suggest that AnxA2 could be a promising biomarker and therapeutic target for high-grade BLCA.Item Identifying the role of Annexin A2 in acquired chemoresistance in triple negative breast cancer (TNBC)(2021) Trivedi, Rucha; Vishwanatha, JamboorPurpose Triple negative breast cancer accounts for 20% of all breast cancers. Chemotherapy remains the standard of care due to lack of targeted therapy, but patients frequently develop resistance which renders the tumors untreatable. AnnexinA2, a Ca2+-dependent phospholipid binding protein is abundant in TNBC cells. In chemoresistant breast cancer cells, AnnexinA2 is highly overexpressed and shown to reduce sensitivity to chemotherapy drugs by inhibiting apoptosis. Here, we demonstrate the role AnnexinA2 in imparting chemoresistance in TNBC cells. Methods MDA-MB-231 and MCF-7 breast cancer cells were treated with frontline chemotherapy agents to determine the half-maximal inhibitory (IC50) concentration using cell cytotoxicity assay. The cells will be treated with predetermined concentration of drugs and AnnexinA2 protein levels will be analyzed using western blot. AnnexinA2 will be knocked-down or overexpressed to detect the change in sensitivity to the drugs. Results MDA-MB-231 cells were treated with Doxorubicin at concentration of 50 to 10,000 nM for 48 hours and inhibited cell viability in a dose-dependent manner yielding an IC50 of 2,351 nM. AnnexinA2 expression analysis upon Doxorubicin treatment is currently ongoing. AnnexinA2 expression as a function of Doxorubicin, Paclitaxel and Carboplatin exposure to MDA-MB-231 and MCF-7 cells will be presented. Conclusion Preliminary data indicate that Doxorubicin has significant anti-cancer activity on MDA-MB-231 cells with concentrations above 2,000 nM. Future experiments will determine the effect of other chemotherapeutic drugs on TNBC cells and the role of AnnexinA2 in chemoresistance in TNBC.Item Phosphorylation of Tyrosine 23 in Annexin A2 is Essential for its Association with Exosomes and for Imparting Invasive and Proliferative Capacity to other Cells(2020) Chaudhary, Pankaj; Vishwanatha, Jamboor; Sun, Xiangle; Trivedi, Rucha; Prakash Desai, PriyankaPurpose: Triple negative breast cancer (TNBC) accounts for 15%-20% of all breast cancer cases. The lack of targeted-based therapies highlights the importance of studying TNBC. Elevated levels of Annexin A2 (AnxA2), a Ca+2-dependent phospholipid binding protein, has been correlated with worse overall survival in TNBC patients. Our previous data implicate that exosomal AnxA2 is involved in creating a pre-metastatic niche and facilitating metastasis in TNBC. Here, we demonstrated that phosphorylation of AnxA2 at Tyrosine23 (Tyr23) is important for its association with exosomes which imparts invasive and proliferative phenotype to other cells. Methods: pN1-EGFP plasmids expressing the phosphomimetic (AnxA2-Y23E) and non-phosphomimetic mutant (AnxA2-Y23F) gene at Tyr23 were overexpressed in MDA-MB-231 TNBC cells. Exosomes were isolated from the mutant cells by ultracentrifugation. Surface exosomal AnxA2 was detected by flowcytometry. CAL-148 cells were treated with exosomes to analyze invasion and proliferation by transwell invasion and proliferation assay, respectively. Transfer of exosomal AnxA2 (Exo-AnxA2) and signaling mechanism in CAL-148 were studied by immunofluorescence and Western blot, respectively. Results: Exo-AnxA2-Y23E-GFP had elevated surface AnxA2 expression compared to exo-AnxA2-Y23F-GFP. CAL-148 cells treated with Exo-AnxA2-Y23E-GFP showed high invasive and proliferative characteristics, with a higher expression of p-AnxA2, p-Src and p-Paxillin. Conclusion: Phosphorylation of Tyr23 in AnxA2 in TNBC cells is essential for its association with exosomes and for conferring increased invasive and proliferative capacity to other breast cancer cells.