Mice engineered for an obligatory Mdm4 exon skipping express higher levels of the Mdm4-S isoform but exhibit increased p53 activity

Abstract

Mdm4, a protein related to the ubiquitin-ligase Mdm2, is an essential inhibitor of tumor suppressor protein p53. In both human and mouse cells, the Mdm4 gene encodes two major transcripts: one encodes the full-length oncoprotein (designated below as Mdm4-FL), whereas the other, resulting from a variant splicing that skips exon 6, encodes the shorter isoform Mdm4-S. Importantly, increased Mdm4-S mRNA levels were observed in several human cancers, and correlated with poor survival. However, the role of Mdm4-S in cancer progression remains controversial, because the Mdm4-S protein appeared to be a potent p53 inhibitor when overexpressed, but the splice variant also leads to a decrease in Mdm4-FL expression. To unambiguously determine the physiological impact of the Mdm4-S splice variant, we generated a mouse model with a targeted deletion of the Mdm4 exon 6, thereby creating an obligatory exon skipping. The mutant allele (Mdm4(ΔE6)) prevented the expression of Mdm4-FL, but also led to increased Mdm4-S mRNA levels. Mice homozygous for this allele died during embryonic development, but were rescued by a concomitant p53 deficiency. Furthermore in a hypomorphic p53(ΔP/ΔP) context, the Mdm4(ΔE6) allele led to p53 activation and delayed the growth of oncogene-induced tumors. We next determined the effect of Mdm4(+/ΔE6) heterozygosity in a hypermorphic p53(+/Δ31) genetic background, recently shown to be extremely sensitive to Mdm4 activity. Mdm4(+/ΔE6) p53(+/Δ31) pups were born, but suffered from aplastic anemia and died before weaning, again indicating an increased p53 activity. Our results demonstrate that the main effect of a skipping of Mdm4 exon 6 is not the synthesis of the Mdm4-S protein, but rather a decrease in Mdm4-FL expression. These and other data suggest that increased Mdm4-S mRNA levels might correlate with more aggressive cancers without encoding significant amounts of a potential oncoprotein. Hypotheses that may account for this apparent paradox are discussed.

Tumorigenesis promotes Mdm4-S overexpression

“…Moreover, Mdm4-S protein was not detected in mouse tumors that exhibited high levels of Mdm4-S mRNA (data not shown). This is in agreement with the recent reports that suggest that the endogenous Mdm4-S transcript is susceptible to nonsense mediated decay [18, 24, 29]. …”

“…Surprisingly, decreasing the levels of full-length Mdm4 by 50% and thereby increasing the Mdm4-S/Mdm4 ratio also failed to promote B-cell linked tumorigenesis. These results are similar to the previous report that showed decrease in Mdm4-S/Mdm4 ratio had little effect on adult mouse tissues [24]. Furthermore, genetic crosses with a prototypical B-CLL mouse model also showed that Mdm4-S expression does not cooperate with other oncogenic insults such as TCL1 transgene expression to alter B-cell tumor latency in mice.…”

“…In the absence of prospective animal studies, it is not clear whether Mdm4-S overexpression is a cause or a consequence of tumorigenesis. To address this issue, a knock-in mouse was recently generated [24]. Unfortunately, the mouse was embryonic lethal due to excessive p53 activity.…”

Antisense oligonucleotide–mediated MDM4 exon 6 skipping impairs tumor growth

“…Consistently, we found that MDM4-S was readily detectable in melanoma, and inhibition of NMD by cyclohexamide did not lead to a consistent increase in MDM4-S levels (data not shown). Importantly, previous evidence indicates that MDM4-S is inefficiently translated (26) and that the MDM4-S peptide is highly unstable (26,27,45). Together, these observations provide a rational explanation for our inability to detect the MDM4-S protein in melanoma or other cancers (data not shown and ref.…”

“…In addition, we previously demonstrated that defects in constitutive splicing efficiency decrease Mdm4 exon 6 inclusion, leading to the production of an unstable transcript known as Mdm4-S (lacking exon 6 in humans or exon 7 in mice), which contains a premature termination codon (25) and is targeted for nonsense-mediated decay (NMD) (26). Finally, homozygous mouse embryos engineered to skip Mdm4 exon 7 die in utero, just like Mdm4-null embryos, as a result of ectopic p53 activation (27). These data raise the possibility that human MDM4 exon 6/mouse exon 7 functions as an "NMD switch" exon (24) and that regulation of this splicing event may directly impact MDM4 protein expression levels.…”