The IG20 gene is overexpressed in human tumors and cancer cell lines, and encodes at least four splice variants (SVs) namely, IG20pa, MADD, IG20-SV2 and DENN-SV. Earlier, gain-of-function studies showed that IG20-SVs can exhibit diverse functions and play a critical role in cell proliferation and apoptosis. Expression of exogenous IG20pa or DENN-SV rendered cells either susceptible or resistant to induced apoptosis, respectively, whereas MADD and IG20-SV2 had no apparent effect. In order to understand the contrasting effects of the IG20-SVs in a physiologically more relevant system, we expressed exonspecific small hairpin RNAs (shRNAs) to selectively knockdown specific IG20-SVs. Consistent with an earlier study, knockdown of all IG20-SVs resulted in spontaneous apoptosis of HeLa and PA-1 cells. In addition, we unambiguously demonstrated that knockdown of MADD can render cells susceptible to spontaneous apoptosis but had no discernible effect on cell proliferation, colony size or cell cycle progression. Moreover, expression of MADD alone, and not DENN-SV, in the absence of endogenous IG20-SVs was sufficient to prevent spontaneous apoptosis. Our results show the utility of shRNAs for selective knockdown of particular IG20-SVs and their potential therapeutic value in cancer. Further, they demonstrate that MADD alone is sufficient and necessary for cancer cell survival.

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...

In a mouse model of autoimmune Graves' disease, stimulatory anti-TSH receptor (TSHR) antibodies (TSAbs) slowly evolve upon repeated immunization with TSHR and lead to hyperthyroidism. Although all immunized mice developed high levels of TSH-binding inhibitory Ig (TBII), only a subset of these mice become hyperthyroid, suggesting that the generation of pathogenic antibodies (Abs) may require affinity maturation. We analyzed the complementarity-determining region 3 (CDR3) of IGHV1 and IGHV5 heavy chains from mice at different stages of disease development. Subcloned CDR3 PCR products were amplified from RNA isolated from enriched splenic B/plasma cells of a control mouse, and mice with low TBII and normal T(4) levels (LTNT(4)), high TBII and normal T(4) levels (HTNT(4)), and high TBII and high T(4) levels (HTHT(4)). Using statistical analyses, we correlated usage of D and J genes and the amino acid composition and length of and mutations within the CDR3 with different outcomes after TSHR immunization. CDR3 sequences from TSHR-immunized mice contained a higher frequency of D gene SP2.9 relative to control, whereas sequences from HTHT(4) contained a higher frequency of D gene Q52 compared with sequences from LTNT(4). Furthermore, HTHT(4) sequences also contained higher CDR3 replacement mutations, relative to LTNT(4) and HTNT(4) mice, that are indicative of somatic hypermutation. Collectively, our results suggest that higher somatic mutations within the CDR3 may correlate with pathogenic antibodies against the TSHR.