Abstract B017: MICAL2 promotes pancreatic cancer growth and metastasis independent of MRTF-A signaling
Authors: Khan Sohini , Sharma Shweta , Sood Divya , Garg Bharti , Jaquish Dawn , Mose Evangeline , Esparza Edgar , Tiriac Herve , Lowy Andrew M. 
Published: November 2022

Abstract
Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in large part due to its propensity for early metastasis. We used ChIP-seq to identify differentially tagged super-enhancer associated genes in PDAC and identified MICAL2 as a gene of interest. MICAL (molecule interacting with CasL) proteins are flavin monooyxgenases that induce actin depolymerization. MICAL2 canonically modulates nuclear actin to regulate SRF/MRTF-A (myocardin related transcription factor) mediated transcription to drive EMT (epithelial-mesenchymal transition) and fibrosis. In this study, we sought to determine the impact of MICAL2 on PDAC progression and its dependence on SRF/MRTF-A signaling. Methods: Studies were performed in human and murine PDAC models. MICAL2 and MRTF-A were knocked down with shRNA and gene expression quantitated with qPCR. In vitro cell viability was assessed by MTT colorimetric assay. Cell migration was tested with in vitro wound scratch assays. Cell cycle analysis was done with flow cytometry-based DNA content measurement. Subcutaneous, orthotopic, and intrasplenic injections of PDAC cells were performed to assess in vivo growth and metastasis. Protein expression was measured with Western blotting. Student’s t-test was used to confirm statistical significance of experimental results. Results: In human PDAC cell lines, MICAL2 knockdown decreased cell viability, reduced cell migration and wound healing, and induced cell cycle arrest at G0/G1. MICAL2 inhibition was associated with decreased p-AKT, c-Myc, and increased p27 by Western blot. Orthotopic and subcutaneous injections of KPC46 shMICAL2 cells were performed in syngeneic mice; orthotopic injections of shMICAL2 cells led to decreased tumor growth compared to control (0.26 gm vs. 1 gm, p = 0.008), not observed with shMRTF-A (1.032 gm vs. 1 gm, p = 0.95). Subcutaneous injections of MICAL2 knockdown cells led to no tumor growth; injections of MRTF-A knockdown cells again led to comparable tumor growth as control (3.02 gm vs. 1.72 gm, p = 0.24). GSEA (Gene Set Enrichment Analysis) of RNAseq data demonstrated a correlation between MICAL2 knockdown and downregulation of Hallmark EMT pathways. Intrasplenic injection studies in syngeneic mice revealed grossly decreased liver metastases after intrasplenic injection of shMRTF-A cells, with total abrogation of metastases after injection of shMICAL2 cells. Conclusions: MICAL2 promotes pancreatic cancer cell growth and metastasis. While MICAL2’s effect on metastasis appears largely dependent on SRF/MRTF-A, tumor growth appears to be MRTF-A independent, suggesting putative novel functions of MICAL2. Given its potentially targetable enzymatic domain and important role in promoting cell growth in vitro and tumor progression and metastasis in vivo, MICAL2 holds promise as a novel tractable therapeutic target in pancreatic cancer.
Citation Format: Sohini Khan, Shweta Sharma, Divya Sood, Bharti Garg, Dawn Jaquish, Evangeline Mose, Edgar Esparza, Herve Tiriac, Andrew M. Lowy. MICAL2 promotes pancreatic cancer growth and metastasis independent of MRTF-A signaling [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B017.