In 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.

The validated assay was applied for clinical pharmacokinetics of ZYAN1 in healthy volunteers.

Colorectal cancer (CRC) is one of the leading causes of cancer associated mortalities worldwide. It starts with hyperproliferation of epithelial cells forming a polyp & is characterized by four stages, where stages 3 & 4 are associated with acquisition of metastasis. Chemotherapy majorly focuses on attenuating symptoms with no promising cure. Hence, studying the underlying mechanism of CRC progression & identifying novel therapeutic targets is critical for early diagnosis & treatment. The process of metastasis involves a complex interplay of signaling pathways, where various proteins play crucial roles. One such important protein that aids the process of migration is Migration & Invasion ENhancer 1 (MIEN1). In CRC, MIEN1 expression is predominantly upregulated in cancerous tissue in comparison to normal colorectal tissue, which is closely associated with invasive behavior. But the exact mechanism involved in the process of metastasis is yet unexplored. It is established that MIEN1 overexpression is a result of 17q12 chromosomal amplification, & such dysregulated expression is linked to the trans-regulation of its minimal promoter region. Therefore, we aim to investigate the effect of MIEN1 promoter ablation on CRC migration properties. CRISPR-Cas9 gene editing technology was used for deleting MIEN1 promoter region in the CRC cell line HT29. RNA seq & bioinformatics tools were employed to assess the transcriptomic consequences of genome-editing. Several DEGs discerned by RNA seq analysis were involved in different biological processes & molecular pathways such as cell adhesion, migration, invasion, & angiogenesis. Out of these the genes vital to CRC biogenesis were evaluated at both RNA & protein levels. Additionally, we analyzed the effect of MIEN1 knock-out on CRC metastatic potential using functional assays such as wound healing, Matrigel invasion, hanging drop cell - cell adhesion, & found that migration potential of HT29 cells was significantly reduced in absence of MIEN1 protein. We also successfully demonstrated that MIEN1 deletion disrupts the cytoskeletal rearrangement by affecting F-actin reorganization using phalloidin staining. Confocal staining of different proteins participating in actin cytoskeleton rearrangement such as paxillin, FAK, MIEN1 gave us an insight about the role of MIEN1 in mediating phosphorylation of FAK at different phosphorylation sites such as Tyr 397 & 925. Immunoblotting analysis of an array of proteins further confirmed the role of MIEN1 in actin cytoskeleton dynamics. Taken together, our results prove that MIEN1 is involved in different signaling pathways responsible for CRC migration & its deletion leads to perturbation of several biological processes especially the actin cytoskeleton rearrangement, involved in metastasis. Hence, targeting MIEN1 would be a potentially effective therapeutic strategy for CRC patients. Citation Format: Payal Ranade, Rucha Trivedi, Jamboor K. Vishwanatha. MIEN1 promoter ablation provides novel evidence for colorectal cancer genome editing-based therapeutics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3623.

Triple negative breast cancer (TNBC) is the most lethal subtype of all breast cancers leading to high mortality rates. Tumor-derived extracellular vesicles (TEV) along with their cell-type specific cargo are greatly implicated in tissue-specific metastasis. In addition to metastasis, TEV play a crucial role in interacting with the distant microenvironment & shaping a pre-metastatic niche (PMN) for tumor cells to home. Understanding the mechanisms by which TEV promote PMN development has great potential to improve the diagnosis, prognosis, & treatment of TNBC. Annexin A2 (AnxA2) is a plasma & endosomal membrane-associated protein. High levels of AnxA2 in TNBC patients have been correlated with poor distant metastasis-free survival and poor overall survival. It is abundantly present in the TEV & recruits cargo such as proteins and microRNAs. Our lab reported that in vivo education with AnxA2 depleted TNBC-derived extracellular vesicles (EVs) led to reduced metastasis in lungs & brain suggesting a key role in the formation of a PMN. While the presence of AnxA2 in TNBC-derived EVs has been reported, its molecular role in the formation & development of PMN via EVs is still unexplored. We aim to evaluate the implications of AnxA2 in EVs at distant sites to mediate tumor cell homing & elucidate mechanisms promoting TNBC metastasis. We have employed RNA interference-mediated gene silencing to stably downregulate AnxA2 in organotropic TNBC cell lines derived from the parent MDA MB 231 cells. We observed a significant effect of AnxA2 depletion on its physiological role in plasmin generation. Differential ultracentrifugation was used to isolate EVs from cell culture supernatant and the physical characterization was done using Nanoparticle Tracking Analyzer. Biological characterization was done in concordance with MISEV 2018 guidelines. 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 & cytochrome c. Interestingly, a reduced yield of EV was observed with AnxA2 depletion indicating a potential effect on EV biogenesis and release. Additionally, the EVs will be subjected to quantitative proteomic analysis to identify differentially expressed proteins upon loss of AnxA2. We will further carry out transcriptome profiling of the AnxA2 depleted TNBC cells and -derived EVs to identify the differentially expressed genes. 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. [This research is supported by the NCI under Award Number R01CA220273 (JKV), awarded to Dr. Jamboor K. Vishwanatha.] Citation Format: Rucha Trivedi, Jamboor K. Vishwanatha. Molecular contribution of Annexin A2 in triple negative breast cancer metastasis via tumor-derived extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1314.