The actin filament cross‐linker L‐plastin confers resistance to TNF‐α in MCF‐7 breast cancer cells in a phosphorylation‐dependent manner

Abstract

We used a tumour necrosis factor (TNF)-α resistant breast adenocarcinoma MCF-7 cell line to investigate the involvement of the actin cytoskeleton in the mechanism of cell resistance to this cytokine. We found that TNF resistance correlates with the loss of cell epithelial properties and the gain of a mesenchymal phenotype, reminiscent of an epithelial-to-mesenchymal transition (EMT). Morphological changes were associated with a profound reorganization of the actin cytoskeleton and with a change in the repertoire of expressed actin cytoskeleton genes and EMT markers, as revealed by DNA microarray-based expression profiling. L-plastin, an F-actin cross-linking and stabilizing protein, was identified as one of the most significantly up-regulated genes in TNF-resistant cells. Knockdown of L-plastin in these cells revealed its crucial role in conferring TNF resistance. Importantly, overexpression of wild-type L-plastin in TNF-sensitive MCF-7 cells was sufficient to protect them against TNF-mediated cell death. Furthermore, we found that this effect is dependent on serine-5 phosphorylation of L-plastin and that non-conventional protein kinase C isoforms and the ceramide pathway may regulate its phosphorylation state. The protective role of L-plastin was not restricted to TNF-α resistant MCF-7 cells because a correlation between the expression of L-plastin and the resistance to TNF-α was observed in other breast cancer cell lines. Together, our study discloses a novel unexpected role of the actin bundling protein L-plastin as a cell protective protein against TNF-cytotoxicity.

L‐plastin Ser5 phosphorylation in breast cancer cells andin vitrois mediated by RSK downstream of the ERK/MAPK pathway

“…Because a correlation between the invasive capacity of melanoma cells and the phosphorylation state of L‐plastin on Ser5 has been suggested before (10), we continued by investigating L‐plastin Ser5 phosphorylation in the 4 breast cancer cell lines. To this end, we used an antibody specifically recognizing Ser5‐P‐LPL (anti‐Ser5‐ P antibody) raised and characterized by our group (3, 7, 14). Although the L‐plastin expression level is higher in the invasive compared with the noninvasive cell lines, our results clearly show high‐baseline L‐plastin Ser5 phosphorylation in the invasive cell lines BT‐20 and MDA‐MB‐435S as compared to absent or extremely weak phosphorylation in the noninvasive cell lines MCF7 and SK‐BR‐3 (Fig.…”

“…PI3K has also been reported to play a role in L‐plastin phosphorylation in human neutrophils (13, 16), but not in T lymphocytes (18). Although an siRNA knockdown of PKCδ (3, 14) and PKCβ II (17) reduced L‐plastin Ser5 phosphorylation, it has to be taken into account that most reports are based on activation and/or inhibition studies. Strikingly, all inhibitors used to demonstrate the involvement of PKA and PKC (H89, GF109203X, Gö6976, and Ro‐31–8220) in L‐plastin phosphorylation also strongly inhibit RSK2 (47, 48).…”

“…In cells, by contrast, distinct protein kinases have been reported to play a role in triggering L-plastin phosphorylation depending on the cell type and environment. Most frequent are reports of the involvement of PKA (7,12) and PKC (3,(13)(14)(15)(16)(17)(18). PI3K has also been reported to play a role in L-plastin phosphorylation in human neutrophils (13,16), but not in T lymphocytes (18).…”

The acquisition of resistance to TNFα in breast cancer cells is associated with constitutive activation of autophagy as revealed by a transcriptome analysis using a custom microarray

“…These categories are highly enriched in genes linked to cell adhesion, actin cytoskeleton dynamics and TGFβ-dependent induction of EMT. 22 While the autophagy gene function category appeared at rank #189 (p-value 3.24E-02) in the operon platform, it was the most regulated pathway in the dataset obtained with the autophagy-dedicated array (p-value 2.74E-14) (Sup. Table 5).…”

Identifying Differentially Expressed Genes and Screening Small Molecule Drugs for Lapatinib-resistance of Breast Cancer by a Bioinformatics Strategy

“…Then, 29 DEGs enriched in the category of actin cytoskeleton, while abnormity of structure or function of actin cytoskeleton is related to drug resistance of many cancers including breast cancer. Previous reports indicated that the molecular motor recruitment in the microtubule environment was associated with Taxol resistance of breast cancer (Froidevaux-Klipfel et al, 2011), and the actin filament cross-linker L-plastin was related to TNFalpha resistance in breast cancer (Janji et al, 2010). We speculated that actin cytoskeleton disorder might also play important roles in Lapatinib-resistance, but concrete mechanism is still worth further studying.…”