Phosphorylation and Stabilization of Topoisomerase IIα Protein by p38γ Mitogen-activated Protein Kinase Sensitize Breast Cancer Cells to Its Poisons

Abstract

Cancer drugs suppress tumor cell growth by inhibiting specific cellular targets. However, most drugs also activate several cellular nonspecific stress pathways, and the implications of these off-target effects are mostly unknown. Here, we report that p38␥, but not p38␣, MAPK is specifically activated by treatment of breast cancer cells with topoisomerase II (Topo II) drugs, whereas paclitaxel (Taxol) does not have this effect. The activated p38␥ in turn phosphorylates and stabilizes Topo II␣ protein, and this enhances the growth inhibition by Topo II drugs. Moreover, p38␥ activity was shown to be necessary and sufficient for Topo II␣ expression, the drug-p38␥-Topo II␣ axis is only detected in intrinsically sensitive but not resistant cells, and p38␥ is co-overexpressed with Topo II␣ protein in primary breast cancers. These results reveal a new paradigm in which p38␥ actively regulates the drug-Topo II␣ signal transduction, and this may be exploited to increase the therapeutic activity of Topo II drugs.

p38γ Mitogen-activated Protein Kinase Signals through Phosphorylating Its Phosphatase PTPH1 in Regulating Ras Protein Oncogenesis and Stress Response

“…S3). These results are similar to our recent observation that p38␥ is specifically activated by DNA topoisomerase II␣ inhibitors in breast cancer, and resultant p38␥ phosphorylates topoisomerase II␣ at Ser-1524 leading to the stabilization of topoisomerase II␣ protein (33). Thus, p38␥ may be a noncanonical MAPK that functions to mediate stress signaling to cellular targets via phosphorylation and stabilization by a stimulus-specific mechanism.…”

“…To express p38␥ in HCT116 cells, the retroviral vector pLHCX and pLHCX-p38␥ were used (18). Tet-inducible expression of MKK6-p38␥ fusion protein in MCF-7 cells was described recently (33), whereas Tet-On shRNA against the mutated K-Ras was performed as reported previously (34).…”

p38γ Mitogen-activated Protein Kinase (MAPK) Confers Breast Cancer Hormone Sensitivity by Switching Estrogen Receptor (ER) Signaling from Classical to Nonclassical Pathway via Stimulating ER Phosphorylation and c-Jun Transcription

“…To measure in vivo ER phosphorylation, V5-tagged ER constructs were co-expressed with the indicated CA kinases in 293T cells, and phosphorylated ER/Ser-118 was assessed by direct Western blotting. Moreover, endogenous p-ER in MCF-7 cells was assessed by Western blotting of cells in which CA p38␥ was expressed by the Tet-on system (29). Additional methods are described under supplemental Experimental Procedures.…”

“…The vectors expressing the fusion protein constructs including the constitutively active (CA) p38␥ (MKK6-p38␥), its nonphosphorable dominant negative mutant (MKK6-p38␥/AGF), and their p38␣ counterparts were described previously (24). The tetracycline-inducible system (Tet-on; Invitrogen) was used to express the MKK6-p38 fusion protein in MCF-7 cells and to express ER and ER/S118A in 231 cells, as described previously (23,29,35). To deplete p38␥ expression, lentiviral vectors expressing shp38␥ or the control shLuc were transfected into packaging cells, and supernatants were collected for infecting target cells, followed by an antibiotic selection (25).…”

“…Whereas both p38␥ and p38␣ can be similarly activated in response to stress, p38␥ plays an anti-apoptotic role (24,26,27). In addition, recent studies from others (28) and us (29) have shown that p38␥ can be selectively activated by certain stress stimuli, indicating its specific role in stress response. Because all MAPKs can phosphorylate the S/TP motif (30) such as Ser-118 in ER (10), p38␥ can stimulate breast cancer invasion and/or metastasis through binding and antagonizing ER (23), and/or additional pathways (31,32), p38␥ may therefore be a key MAPK to phosphorylate ER/Ser-118 in breast cancer (22).…”