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Cellular and Molecular Bioengineering

MRTF-SRF interaction promotes breast cancer cell migration in a DIAPH3-dependent manner

(I-343) MRTF-SRF interaction promotes breast cancer cell migration in a DIAPH3-dependent manner

Saturday, October 14, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row I - Poster # 343

Presenting Author(s)

VY

Virginia Yu (they/them/theirs)

Undergraduate student
University of Pittsburgh
Pittsburgh, Pennsylvania, United States

Co-Author(s)

IE

Ian Eder

Graduate student
University of Pittsburgh, United States
DG

David Gau

Lecturer
University of Pittsburgh, United States
DB

David Boone

Professor
University of Pittsburgh, United States

Primary Investigator(s)

PR

Partha Roy

Professor
University of Pittsburgh, United States

Introduction:: Breast cancer accounts for the second most cancer-related deaths in women in the United States. Mortality associated with breast cancer (BC) is due to the metastatic spread of tumor cells whose aberrant motility arises from dysregulated actin cytoskeleton. Myocardin-related transcription factor (MRTF) belongs to a family of actin-regulated transcriptional co-factors of serum-response factor (SRF) that control gene expression in both SRF-dependent and –independent (through its SAP domain interaction) manners and is critical to invasion and metastatic outgrowth of breast cancer cells. In the present study, we sought to investigate the mechanistic underpinnings of how MRTF-SRF interaction regulates motility of breast cancer cells.

Materials and Methods:: Sub-lines of MDA-MB-231 (a metastatic triple-negative breast cancer cell line) that are genetically engineered to overexpress either wild-type (WT) or mutants of MRTF-A (one of the two major isoforms of MRTF) were used to investigate the role of specific functional interactions of MRTF-A on various modes of cell migration. Specifically, we performed live cell imaging of cells over a duration of 3 hours and analyzed time-lapse images by Ilastik in an automated fashion to study the impact of specific functional perturbations of MRTF-A on 2D random migration of MDA-MB-231 cells. In the same setting, shorter duration time-lapse movies were utilized to perform kymography analyses of membrane dynamics. In addition, transwell assays were performed to assess the impact of MRTF-perturbation on serum-induced chemotactic migration. To gain insights into potential molecular mechanisms of MRTF’s regulation of cell migration, RNA sequencing followed by Ingenuity Pathway Analysis (IPA) of 2D and 3D cultures of various MRTF-engineered sublines of MDA-MB-231 cells were performed. Differentially expressed genes of interest were confirmed by qRT-PCR and immunoblot analyses. Gene silencing experiments were performed to establish a mechanistic connection between target gene regulation and MRTF-dependent modulation of cell migration.

Results, Conclusions, and Discussions:: We found that overexpression of WT MRTF-A significantly enhanced both 2D random migration and chemotactic migration of MDA-MB-231 cells. MRTF-A-induced increase in migration is reversed upon disruption of either SAP-domain or SRF’s interaction of MRTF-A but more prominently by the latter, suggesting that the SRF-mediated transcriptional arm plays the predominant role in MRTF-dependent control of cell migration. Kymography analyses showed that a distinguishing phenotypic feature of cells with disrupted MRTF-SRF interaction is dramatic suppression of the dynamic behavior of the leading edge. Consistent with this phenotype, IPA analyses of differentially expressed genes revealed “Actin Cytoskeleton Signaling” and the “Actin Nucleation” pathways to be significantly impacted by MRTF. In particular, we identified dramatic transcriptional repression of DIAPH3, a member of the formin family of actin-nucleating and -elongating proteins, in breast cancer cells, specifically induced by disruption of the MRTF-SRF interaction, a finding that we further corroborated by qRT-PCR and immunoblot analyses. Finally, we showed that transient knockdown of DIAPH3 alone was sufficient to prevent MRTF-A induced increase in cell migration, further suggesting that MRTF promotes breast cancer cell migration through transcriptional regulation of DIAPH3 in a SRF-dependent manner. In conclusion, this study for the first time links regulation of specific actin-nucleating/elongating proteins to MRTF-dependent control of cell migration. These findings also pave the way to our ongoing effort to discover highly specific inhibitor of MRTF-SRF interaction as a novel pharmacological tool to curb migration and metastatic ability of breast cancer cells.

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