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 ␣-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.A fine balance between opposing signaling events that regulate survival and death appears to determine the outcome of cell fate. A complex array of genes regulates the above process, and one such gene we previously identified is IG20 (insulinomaglucagonoma 20) (1). The IG20 can profoundly affect cancer cell survival and death through alternative splicing (2, 3). The IG20 gene encodes six different splice variants (SVs).3 The KIAA and IG20-SV4 isoforms are selectively expressed only in neuronal tissues (4). The KIAA is analogous to rat Rab3a GEP (also referred to as MADD/DENN) and plays an important role in neuronal vesicular trafficking and is required for animal survival (5-7). The IG20pa, MADD, IG20-SV2, and DENN-SV are more ubiquitously expressed. Of these, MADD is physiologically the most important isoform. MADD is expressed at very low levels in a variety of healthy tissues; however, its expression levels are much higher in many types of human tumors and tumor cell lines (1, 8). Knockdown of endogenous MADD or all IG20 SVs results in enhanced spontaneous as well as tumor necrosis factor ␣-related apoptosis-inducing ligand (TRAIL)-induced apoptosis (9 -11). Interestingly, expression of exogenous MADD, and not other SVs, in the absence of endogenous IG20 SVs can rescue the cells from undergoing apoptosis and indicates that only the MADD isoform is required and sufficient to promote cancer cell survival (10, 11).The extrinsic apoptotic pathway is initiated by death ligands such as Fas ligand, TRAIL, or tumor necrosis factor ␣ (12-15). Unlike Fas ligand, TRAIL can induce cancer cell death with little or no effect on most normal cells (16). However, a number of different factors can confer resistance to TRAIL-induced apoptosis in different cells (17). TRAIL binding to death receptors 4 and 5 (DR4/DR5) induces receptor trimerization and recruitment of FADD (16, 18 -20). This facilitates recruitment of procasp...

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.

OBJECTIVE The clinical utility of TRAIL in the treatment of established human malignancies is limited by the development of resistance to TRAIL. We hypothesized that knockdown of 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 using q-RT-PCR. 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 to normal ovarian tissues. The cell lines OVCA429 and OVCAR3 were susceptible, and 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 c-FLIP in SKOV-3 cells increased spontaneous and TRAIL-induced apoptosis, which was further increased upon MADD knockdown. CONCLUSION MADD/c-FLIPL knockdown can render TRAIL-resistant ovarian cancer cells susceptible to TRAIL.

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 signalling 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 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-phopshorylated 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.