ABSTRACT
IRS-1 (Insulin Receptor Substrate-1) is an adaptor protein important for insulin and IGF-I receptor (Insulin-like Growth Factor-IR) transduction to downstream targets. One mechanism recently identified to downregulate IGF-I or insulin receptor signaling in diabetic models is IRS-1 Ser(312) phosphorylation. To date, the importance of this residue in cancer is unknown. This paper identifies mechanisms leading to Ser(312) regulation in MCF-7 breast cancer cells. Whereas IGF-I phosphorylation of IRS(312) is PI (phosphatidylinositol) 3-kinase dependent, anisomycin stress treatment requires JNK activation to induce phosphorylation of IRS(312). We show that both IGF-I and anisomycin stress treatment converge downstream onto mTOR (Mammalian Target of Rapamycin) and PKCdelta (Protein Kinase C-delta) to induce IRS-1 Ser(312) phosphorylation. mTOR associates with IRS-1 and is primarily required for Ser(312) phosphorylation in response to stress or IGF-I treatment. PKCdelta binds to mTOR and its activity is also important for stress or IGF-I mediated Ser(312) phosphorylation. Thus, mTOR and PKCdelta convey diverse signals to regulate IRS-1 function.
Subject(s)
Breast Neoplasms/metabolism , Insulin-Like Growth Factor I/pharmacology , Phosphoproteins/metabolism , Protein Kinase C/metabolism , Protein Kinases/metabolism , Anisomycin/pharmacology , Breast Neoplasms/pathology , Enzyme Activation , Female , Humans , Insulin Receptor Substrate Proteins , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Kinase 4 , Mitogen-Activated Protein Kinase Kinases/metabolism , Oligonucleotides, Antisense/pharmacology , Phosphorylation , Protein Kinase C-delta , Protein Kinases/chemistry , Protein Kinases/genetics , Protein Synthesis Inhibitors/pharmacology , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Serine/chemistry , Signal Transduction , TOR Serine-Threonine Kinases , Tumor Cells, Cultured , Tyrosine/metabolismABSTRACT
c-Jun N-terminal kinase (JNK) is activated by diverse cell stimuli, including stress, growth factors, and cytokines. Traditionally, activation of JNK by stress treatment is thought to induce cell death. However, our recent data indicate that JNK's ability to sensitize cells to apoptosis may be, in part, cell cycle dependent. Here, we show that the majority of both paclitaxel- and UV-induced apoptosis can be inhibited by the pharmacological JNK inhibitor, SP600125, in MCF-7 cells. However, inhibition of JNK does little to reverse doxorubicin-induced apoptosis in MCF-7 cells or doxorubicin- and UV-mediated death in MDA MB-231 cells. SP treatment causes G2/M arrest of three breast cancer cell lines and results in the endoreduplication (cellular DNA content >4N) of MCF-7 and MDA MB-231 cells. These effects on cell cycle and apoptosis are not significantly altered by the inhibition of p53, indicating that JNK is functioning independently of p53. Lastly, inhibition of JNK using both SP and antisense oligonucleotides targeted to JNK1 and JNK2 reduced proliferation of all three breast cancer cell lines. Taken together, these results suggest that the activation of JNK is important for the induction of apoptosis following stresses that function at different cell cycle phases, and that basal JNK activity is necessary to promote proliferation and maintain diploidy in breast cancer cells.
Subject(s)
Apoptosis , Breast Neoplasms/pathology , DNA Replication , G2 Phase , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitosis , Tumor Suppressor Protein p53/metabolism , Anthracenes/pharmacology , Apoptosis/drug effects , Apoptosis/radiation effects , Breast Neoplasms/genetics , Cell Division/drug effects , Cell Division/radiation effects , Cell Line, Tumor , DNA Replication/drug effects , DNA Replication/radiation effects , Doxorubicin/pharmacology , G2 Phase/drug effects , G2 Phase/radiation effects , Humans , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Mitosis/drug effects , Mitosis/radiation effects , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/metabolism , Paclitaxel/pharmacology , Tumor Suppressor Protein p53/antagonists & inhibitors , Ultraviolet RaysABSTRACT
Insulin-like growth factor-I receptor (IGF-IR) is frequently overexpressed in a variety of cancer types. Since many breast tumors and cancer cell lines overexpress IGF-IR, we tested IGF-I effects on chemotherapy-treated breast cancer cells. IGF-I protects from chemotherapy-induced apoptosis, suggesting that overlapping signaling pathways modulate IGF-I and chemotherapy treatment outcomes. Taxol and other chemotherapy drugs induce c-Jun N-terminal kinase (JNK), a kinase that conveys cellular stress and death signals. Notably, in this paper we show that IGF-I alone induces a potent JNK response and this activity is reversed by inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) with LY294002 in MCF-7 but not T47D cells. Cotreatment of cells with chemotherapy and IGF-I leads to additive JNK responses. Using cells overexpressing Akt, we confirm that IGF-I-mediated survival is Akt dependent. In contrast, overexpression of JNK significantly enhances Taxol-induced apoptosis and inhibits IGF-I survival effects. Further, JNK attenuates anchorage-independent growth of MCF-7 cells. The inhibitory effect of JNK appears to be mediated by serine phosphorylation of IRS-1 (insulin receptor substrate) since both Taxol and IGF-I treatment enhanced Ser(312) IRS-1 phosphorylation, while LY294002 blocked IGF-I-mediated phosphorylation. Taken together, these data provide a mechanism whereby stress or growth factors activate JNK to reduce proliferation and/or survival in breast cancer cells.
