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Several MADD/MADD related cDNAs have been cloned(1-9) that represent the human MADD/IG20/DENN gene, which can undergo alternate splicing (7-14). The IG20 gene is expressed in all tissues at low levels however its expression levels are relatively high in most tumors and cancer cell lines (13-16). The human IG20 gene can encode 6 different splice variants. Of these the KIAA and the IG20-SV4 isoforms are expressed only in certain neuronal tissues such as cerebral cortex, hippocampus and spinal cord (19) and play important roles in regulating neurotransmission neuronal cell survival (17-27). The other 4 SVs of the IG20 gene are expressed at barely detectable levels in normal tissues. The IG20pa and IG20-SV2 isoforms may or may not be expressed depending upon the cell type, indicating that their expression is not essential for cancer cell survival or proliferation. In contrast, the MADD and DENN-SV isoforms are constitutively expressed in all cells and tissues, and are expressed at much higher levels in human tumors and cancer cell lines relative to normal tissues and other splice variants (8,9).

The 4 IG20 isoforms are distinguished by the differential splicing of exons 13L and 16. The MADD, IG20-SV2 and DENN-SV isoforms, in contrast to the IG20pa, lack exon 16 or 13L, or both, respectively (9). Consistent with a regulated functional role, the IG20 splicing pattern is conserved between humans and mouse. It is apparent that different IG20 SVs can contribute to multiple cellular functions (survival/proliferation/apoptosis) mediated through the death receptors (19-27). Gain of function studies reveal contrasting effects of IG20pa and DENN-SV:In order to delineate the role of individual IG20 SVs, gain of function studies through cDNA encoded protein expression was carried out in HeLa cells. These cells normally express all four IG20 SVs, and expression of exogenous IG20pa rendered these cells highly susceptible to TNFα (9) and TRAIL–induced apoptosis (18). IG20pa-enhanced TRAIL-induced apoptosis was accompanied by increased formation of DISC and caspase activation (18). Co-localization and co-precipitation studies indicated that IG20pa can interact directly with the DR4/5 (28). A critical step in TRAIL-induced signaling, like in CD95L-induced signaling (28-31), is DISC formation characterized by the recruitment of FADD, recruitment and activation of caspase-8, and caspase-10 (32-34). Caspase-10 does not functionally substitute caspase-8 (35); complete inhibition of death in the presence of CrmA further indicated that caspase-8 was sufficient for TRAIL-induced apoptosis.

Additionally, IG20pa expression led to increased susceptibility to various intrinsic apoptotic stimuli (gamma-irradiation, etoposide and vinblastine) and also suppressed cell proliferation. In contrast, DENN-SV not only conferred resistance to both extrinsic and intrinsic apoptotic stimuli, it also enhanced cell proliferation (9, 13,14). Thus, upon over-expression, the IG20pa acts as a “tumor suppressor” while the DENN-SV acts as a “tumor promoter”; these findings suggested that the IG20 gene could differentially regulate cell proliferation and death through alternative splicing (13, 14). Interestingly, expression of exogenous MADD and IG20-SV2 showed little or no effect on cell proliferation or induced apoptosis (19, 23). The unique functions of different SVs could result from the expected differences in their conformations and consequent interactions with different receptors and/or intracellular signaling molecules. Although studies involving over-expression of exogenous proteins may be helpful in gaining initial insights, the above noted functional consequences may or may not be physiologically relevant. In contrast, loss of function studies using dominant negatives or knockdown approach may provide physiologically more relevant insights. Therefore, MADD knockdown approach was used to investigate the role of this over expressed protein in breast cancers.

The physiological role of the IG20 gene in cell survival is underscored by studies using antisense oligodeoxynucleotides (ODNs) that abrogate the expression of all isoforms (15, 16, 24, 25). When cancer cells were treated with anti-sense ODNs, it increased cell death and reduced cell proliferation. A limitation of these studies (15, 16, 24, 25) is that the ODNs used for silencing could knockdown all IG20 SVs, and thus could not identify which specific isoform was required for cell survival. Nevertheless, these studies showed that the IG20 gene plays a critical role in cell survival and death.

To more precisely characterize the physiological functions of IG20 SVs, a novel approach to selectively down-modulate either all or a select combination of IG20 SVs using small hairpin RNA (shRNA) (36-40)was employed.The Mid-shRNA was used to abrogate the expression of all IG20 SVs, the 13L shRNA to down-modulate IG20pa and MADD, and the 16E shRNA to target IG20pa and IG20-SV2. As noted earlier(15, 16, 24, 25), knockdown of all IG20 SVs led to significant increase in spontaneous as well as TRAIL and TNFα-induced apoptosis (41). Interestingly, knockdown of MADD/IG20pa, and not IG20pa/IG20-SV2, was sufficient to render cells more susceptible to apoptosis. In contrast to HeLa cells that express all 4 isoforms of IG20, PA-1 ovarian carcinoma cells and WRO and FRO thyroid cancer cells express MADD and DENN-SV. In these cells, we specifically suppressed only MADD expression using 13L shRNA (Fig-1C) and conclusively demonstrated that MADD, and not the DENN-SV, is necessary for cell survival (42, 43). The above finding is intriguing, given the gain of function studies in which over-expression of MADD had no discernable effect on induced apoptosis and cell proliferation (9, 13, 14). Therefore, we determined whether expression of MADD alone would be sufficient to prevent spontaneous apoptosis. This was achieved by constructing a mutant MADD cDNA (with 3rd base substitution in the targeted sequence) that could not be down-modulated by the Mid-shRNA. Our studies clearly demonstrated that in the absence of endogenous IG20 SVs, only expression of MADD, and not other isoforms, is sufficient to prevent spontaneous apoptosis (42, 43).

Apoptosis induced upon MADD knockdown can be inhibited by CrmA or a dominant-negative FADD, thereby suggesting that endogenous MADD may be preventing caspase-8 activation (42), which was confirmed by caspase-8 activation upon MADD knockdown. Additionally, we found that MADD can directly interact with DRs, and not with either caspase-8 or FADD, and inhibit caspase-8 activation. More recently, we have shown that MADD knockdown can also render cells susceptible to TNFα-induced apoptosis (41). These results clearly demonstrated the importance of MADD in the control of cancer cell survival/death and in conferring significant resistance to TRAIL/TNFα-induced apoptosis. These results also indicated the therapeutic potential of MADD abrogation in enhancing TRAIL/TNFα-induced cancer cell apoptosis (41-43).

Earlier studies had clearly shown that MADD mediates TRAIL resistance by preventing DISC formation and capse-8 activation (42,43) but its mechanism of action in conferring resistance against TNFα-induced apoptosis was unknown. Previous studies had shown that MADD can interact with the cytoplasmic tail of the TNFR1 (9) therefore, the physiological role of endogenous MADD in TNFα-induced activation of NF-kB and the mitogen activated protein kinases (MAPK) ERK1/2, JNK and p38 was investigated. Abrogation of MADD expression rendered cells highly susceptible to TNFα–induced apoptosis, in the absence of cycloheximide. This also resulted in a dramatic loss in TNFα-induced activation of MAPK without any apparent effect on NF-kB activation. This observation was substantiated by loss of activation of p90RSK, a key downstream target of MAPK, in MADD knockdown cells; whereas the NF-kB regulated IL6 levels remained unaffected. Although EGF is a potent activator of MAPK, knock down of endogenous MADD did not affect EGF-induced MAPK activation. Re-expression of SiR-MADD in the absence of endogenous IG20 expression rescued the cells from TNFα-induced apoptosis. The requirement for MADD was highly specific for TNFα-induced activation of MAPK but not the related JNK and p38 kinases. Loss of MADD expression resulted in reduced GRB2 and SOS1/2 recruitment to TNFR1 complex, and decreased Ras and Erk1/2 activation. These results demonstrated the essential role of MADD in protecting cancer cells from TNFα-induced apoptosis by specifically activating MAPKs through GRB2 and SOS1/2 recruitment (44).

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