Abstract
Here we report the identification and characterization of a novel high molecular weight isoform of tropomyosin, Tpm4.1, expressed from the human TPM4 gene. Tpm4.1 expression is down-regulated in a subset of breast cancer cells compared with untransformed MCF10A breast epithelial cells and in highly metastatic breast cancer cell lines derived from poorly metastatic MDA-MD-231 cells. In addition, patients with invasive ductal breast carcinoma show decreased TPM4 expression compared with patients with ductal breast carcinoma in situ, and low TPM4 expression is associated with poor prognosis. Loss of Tpm4.1 using siRNA in MCF10A cells increases cell migration in wound-healing and Boyden chamber assays and invasion out of spheroids as well as disruption of cell-cell adhesions. Down-regulation of Tpm4.1 in MDA-MB-231 cells leads to disruption of actin organization and increased cell invasion and dissemination from spheroids into collagen gels. The down-regulation of Tpm4.1 induces Rac1-mediated alteration of myosin IIB localization, and pharmacologic inhibition of Rac1 or down-regulation of myosin IIB using siRNA inhibits the invasive phenotypes in MCF10A cells. Thus Tpm4.1 plays an important role in blocking invasive behaviors through Rac1-myosin IIB signaling and our findings suggest that decreased expression of Tpm4.1 might play a crucial role during tumor progression.
Highlights
Tropomyosins are a family of actin filament binding proteins
In experiments comparing the expression of tropomyosins in various human breast cancer cell lines with untransformed MCF10A breast epithelial cells we observed that the LC24 antibody that was raised against sequences in the carboxy-terminal domain of the TPM4 gene detected the well-characterized LMW Tpm4 isoform, Tpm4.2, and a protein with the same mobility as a HMW tropomyosin (Figure 1A)
Previous immunoblot studies have suggested that the LC24 antibody cross-reacts with Tpm2.1, a HMW tropomyosin isoform encoded by the TPM2 gene, and a HMW tropomyosin band detected by LC24 is Tpm2.1 [15, 16]
Summary
Tropomyosins are a family of actin filament binding proteins. Humans contain four tropomyosin genes, TPM1, TPM2, TPM3, and TPM4. The coiled-coil structure is based on a repeated pattern of seven amino acids with hydrophobic residues at the first and fourth positions and is highly conserved in all tropomyosin isoforms from yeast to human. They appear to exhibit a relatively simple protein structure, molecular and genetic studies revealed a level of functional complexity among metazoan tropomyosins. Different tropomyosin isoforms have distinct properties and cellular functions including regulation of contractility, actin filament dynamics, cell motility, intracellular transport and regulation of cell signaling [2,3,4,5]
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