Transgelin Depletion is Critical for the TGFβ1‐mediated Initiation of PLCγ1‐Cofilin‐driven Morphological and Migratory Changes in MDA‐MB‐231 Cells

Abstract

The role of transgelin (TAGLN) in cancer has been discussed; however, the mechanisms underlying its regulation and correlation with MDA‐MB‐231 cell plasticity and migratory patterns remain unclear. We generated stable TAGLN‐knockdown MDA‐MB‐231 cells and treated them with phorbol 12‐myristate 13‐acetate or transforming growth factor (TGF)‐β. Chemotaxis, morphology, and invasion were assayed using three‐dimensional matrices to evaluate cytoskeletal remodeling and migratory changes. Wound healing assays were conducted using cell inserts. TAGLN knockdown cells exhibited altered morphology due to cytoskeletal remodeling, yet only untreated and TGFβ1‐treated cells exhibited enhanced migration. Untreated and TGFβ1‐treated TAGLN knockdown cells showed increased N‐WASP, ROCK1, and ROCK2 protein levels, which induce cytoskeletal remodeling. Evaluating phospholipase Cγ1 (PLCγ1)‐cofilin signaling‐related proteins revealed that only TGFβ1‐treated TAGLN knockdown cells were influenced by PLCγ1‐cofilin signaling. Taken together, TAGLN knockdown is necessary for the TGFβ1‐mediated activation of PLCγ1‐cofilin pathway‐driven amoeboid morphology and enhanced migratory properties in MDA‐MB‐231 cells.

Transgelin (TAGLN) Regulates IQGAP1 and Alters the Functions of Breast Cancer Cells

“…We recently reported that a loss of TAGLN results in increased PIP2 levels and enhanced migratory patterns. 19 The binding of IQGAP1 to the phosphoinositide PIP2 through its RasGAP C-terminal (RGCT) influences cytoskeletal dynamics and migration. 21 We have postulated possible molecular interplay between TAGLN, PIP2, and IQGAP1, with subsequent effects on critical functions such as proliferation, chemotaxis, and invasion of MDA-MB-231 cells.…”

“…The human breast cancer cell lines MDA-MB-231 and MCF-7 were obtained from the Korean Cell Line Bank (Seoul, Korea) and cultured as previously described. 19 TAGLN Cloning and Transfection. The 611-bp BamHI-HindIII fragment of transgelin full-length cDNA was cloned into the plasmid pEGFP N1.…”

“…Chemotaxis assays were performed as previously described. 19 Separation filters were obtained from Neuro-Probe (Neuro Probe Inc. Biotechnology, Gaithersburg, MD, USA); fibronectin was procured from Sigma-Aldrich. Transwell Invasion Assays.…”

“…Invasion assays were performed as previously described. 19 Transwell cell-culture inserts and Matrigel Matrix were purchased from BD Biosciences (BD Biosciences, NJ, USA). Proliferation Assays.…”

“…Western blotting was carried out as previously described. 19 The following antibodies were purchased: anti-PIP2, Echelon Biosciences (Salt Lake City, Utah, USA); anti-TAGLN, Abcam (Cambridge, UK); anti-Rac1/anti-Cdc42, Cell Signaling Technology (Danvers, MA, USA); and anti-HSP90, anti-Raf-1, anti-IQGAP1, Santa Cruz Biotechnology (Dallas, TX, USA). The corresponding secondary antibodies were purchased from Santa Cruz Biotechnology.…”