ABSTRACT
Mitogen-activated kinase activating death domain containing protein (MADD) is abundantly expressed in cancer cells and necessary for maintaining cancer cell survival. However, this survival function of MADD is dependent upon its phosphorylation by protein kinase B (Akt). The tumour suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a lipid phosphatase that negatively regulates the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. The downstream targets of PTEN in triggering apoptosis have not yet been completely identified. Here, we report that MADD can act as a pro-apoptotic factor to initiate TRAIL-induced apoptosis when its phosphorylation is attenuated by PTEN. Our data show that tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) induced a reduction in MADD phosphorylation with a concomitant up-regulation of PTEN. Knock down of PTEN using a specific siRNA prevented TRAIL-induced reduction in pMADD levels. Surprisingly, Akt non-phosphorylated MADD translocated from the plasma membrane to cytoplasm where it bound to 14-3-3 and displaced 14-3-3 associated Bax, which translocated to mitochondria resulting in cytochrome c release. Taken together, our data reveal that PTEN can convey the death signal by preventing MADD phosphorylation by Akt.
Subject(s)
Apoptosis/genetics , Death Domain Receptor Signaling Adaptor Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , PTEN Phosphohydrolase/metabolism , Tumor Necrosis Factor-alpha/metabolism , 14-3-3 Proteins/metabolism , Cell Line, Tumor , Death Domain Receptor Signaling Adaptor Proteins/genetics , Guanine Nucleotide Exchange Factors/genetics , Humans , Mitochondria/metabolism , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering , Tumor Necrosis Factor-alpha/geneticsABSTRACT
The Map kinase Activating Death Domain containing protein (MADD) isoform of the IG20 gene is over-expressed in different types of cancer tissues and cell lines and it functions as a negative regulator of apoptosis. Therefore, we speculated that MADD might be over-expressed in human breast cancer tissues and that MADD knock-down might synergize with chemotherapeutic or TRAIL-induced apoptosis of breast cancer cells. Analyses of breast tissue microarrays revealed over-expression of MADD in ductal and invasive carcinomas relative to benign tissues. MADD knockdown resulted in enhanced spontaneous apoptosis in human breast cancer cell lines. Moreover, MADD knockdown followed by treatment with TRAIL or doxorubicin resulted in increased cell death compared to either treatment alone. Enhanced cell death was found to be secondary to increased caspase-8 activation. These data indicate that strategies to decrease MADD expression or function in breast cancer may be utilized to increase tumor cell sensitivity to TRAIL and doxorubicin induced apoptosis.
Subject(s)
Apoptosis/drug effects , Breast Neoplasms/pathology , Death Domain Receptor Signaling Adaptor Proteins/deficiency , Death Domain Receptor Signaling Adaptor Proteins/genetics , Doxorubicin/pharmacology , Gene Knockdown Techniques , Guanine Nucleotide Exchange Factors/deficiency , Guanine Nucleotide Exchange Factors/genetics , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Amino Acid Sequence , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Death Domain Receptor Signaling Adaptor Proteins/chemistry , Gene Expression Regulation, Neoplastic/drug effects , Guanine Nucleotide Exchange Factors/chemistry , Humans , Molecular Sequence Data , RNA, Small Interfering/genetics , Receptors, Death Domain/metabolismABSTRACT
OBJECTIVE: The clinical utility of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the treatment of established human malignancies is limited by the development of resistance to TRAIL. We hypothesized that knockdown of map-kinase activating death domain containing protein (MADD), a TRAIL-resistance factor, may overcome TRAIL resistance in ovarian cancer cells. STUDY DESIGN: MADD expression in resected ovarian cancer specimens and cell lines was quantified with the use of polymerase chain reaction. Sensitivity of ovarian cancer cell lines to TRAIL, with or without MADD knockdown, was assessed. RESULTS: MADD is expressed at relatively higher levels in human malignant ovarian cancer tissues and cell lines, compared with normal ovarian tissues. The cell lines OVCA429 and OVCAR3 were susceptible, and cell lines CAOV-3 and SKOV-3 were resistant to TRAIL. MADD knockdown in CAOV-3 cells, but not in SKOV-3 cells, conferred TRAIL sensitivity. Knockdown of cellular Fas-associated death domain-like interleukin-1 beta-converting enzyme-inhibitory protein (c-FLIP) in SKOV-3 cells increased spontaneous and TRAIL-induced apoptosis, which was further increased on MADD knockdown. CONCLUSION: MADD/c-FLIP(L) knockdown can render TRAIL-resistant ovarian cancer cells susceptible to TRAIL.
Subject(s)
Apoptosis/physiology , CASP8 and FADD-Like Apoptosis Regulating Protein/physiology , Death Domain Receptor Signaling Adaptor Proteins/physiology , Guanine Nucleotide Exchange Factors/physiology , Ovarian Neoplasms/pathology , TNF-Related Apoptosis-Inducing Ligand/physiology , Female , Humans , Tumor Cells, CulturedABSTRACT
MADD plays an essential role in cancer cell survival. Abrogation of endogenous MADD expression results in significant spontaneous apoptosis and enhanced susceptibility to tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, the regulation of MADD function is largely unknown. Here, we demonstrate that endogenous MADD is phosphorylated at three highly conserved sites by Akt, and only the phosphorylated MADD can directly interact with the TRAIL receptor DR4 thereby preventing Fas-associated death domain recruitment. However, in cells susceptible to TRAIL treatment, TRAIL induces a reduction in MADD phosphorylation levels resulting in MADD dissociation from, and Fas-associated death domain association with DR4, which allows death-inducing signaling complex (DISC) formation leading to apoptosis. Thus, the pro-survival function of MADD is dependent upon its phosphorylation by Akt. Because Akt is active in most cancer cells and phosphorylated MADD confers resistance to TRAIL-induced apoptosis, co-targeting Akt-MADD axis is likely to increase efficacy of TRAIL-based therapies.
