Widespread Overexpression of Epitope-Tagged Mdm4 Does Not Accelerate Tumor Formation In Vivo

Abstract

Mdm2 and Mdm4 are critical negative regulators of p53. A large body of evidence indicates that elevated expression of either Mdm2 or Mdm4 may favor tumor formation by inhibiting p53 tumor suppression function. To explore this possibility in vivo, we generated conditional Mdm2 and Mdm4 transgenic mice. We show that although both transgenes are designed to be expressed ubiquitously and at comparable levels, only the Mdm4 transgenic protein is produced at high levels in vivo. In contrast, exogenous Mdm2 is constitutively degraded in a proteasome-dependent manner, indicating that cells are equipped with efficient mechanisms that prevent Mdm2 accumulation in vivo. Mice that are homozygous for the Mdm4 transgene die during embryogenesis owing to severe vascular maturation defects. Importantly, this lethality is not rescued on a p53-null background, indicating that high levels of Mdm4 impact on a pathway(s) other than p53 that controls vascular and embryonic development. Mice expressing a single copy of the Mdm4 transgene are viable and, surprisingly, are not prone to spontaneous, radiation-induced or E-myc-induced tumor formation. The findings have clear implications for cancer etiology as well as for cancer therapy.The p53 tumor suppressor pathway is altered in virtually all human cancers (26). While the p53 gene itself is mutated or deleted in roughly 50% of cases, other mechanisms that disable p53 function are selected for in tumors with wild-type p53. Overexpression of MDM2, a critical negative regulator of p53, represents one way by which tumor cells escape p53-mediated tumor suppression (49). MDM2 inhibits p53 mainly, if not exclusively, by promoting its proteasome-dependent degradation through its E3 ubiquitin ligase activity (17,19,24,29).MDM2 is overexpressed in one third of all human sarcomas (37), 10% of glioblastomas and astrocytomas (41), 40% of oral squamous cell carcinomas (30), and 53% of myeloid (4) and 28% of B-cell (56) lymphomas. In addition, high levels of MDM2 are associated with poor prognosis in patients with non-Hodgkin's lymphoma (33). It is believed that overexpression of MDM2 is, at least in part, a direct consequence of gene amplification (34). However, high MDM2 levels have also been seen in many tumors that do not harbor gene amplification, indicating that there are additional mechanisms leading to MDM2 overexpression in human tumors.Ubiquitous overexpression of an Mdm2 transgene, under the control of its own promoter and regulatory regions, predisposes mice to spontaneous tumor formation (21). Tissuespecific Mdm2 overexpression in the basal layer of the epidermis induces hyperplasia and predisposes the epidermis to the formation of premalignant lesions and squamous cell carcinomas (11). These data establish a clear causal link between Mdm2 overexpression and increased tumor susceptibility in vivo. However, this effect is not clear-cut in all tissues: overexpression of Mdm2 in the granular layer of the epidermis does not promote tumorigenesis but rather affects its differentiation in a p...

Antisense oligonucleotide–mediated MDM4 exon 6 skipping impairs tumor growth

“…In order to accurately do this, we developed quantitative PCR-based (qPCR-based) methods, which are illustrated in Supplemental Figure 1, A and B; supplemental material available online with this article; doi:10.1172/JCI82534DS1. As expected, whereas MDM4 protein was detectable in embryonic tissues, it was undetectable in most adult tissues, except brain, thymus, and colon (2). Remarkably, this decrease in MDM4 protein expression was accompanied by a reduced percentage spliced in (PSI), defined as the percentage of full-length Mdm4 mRNA (Mdm4-FL) over the total of all isoforms [Mdm4-FL/(Mdm4-FL + Mdm4-S)] ( Figure 1A and Supplemental Figure 1C).…”

“…An increasing body of evidence has shown that MDM4 also possesses p53-independent oncogenic functions (2,(18)(19)(20)(21). Since ASO-mediated exon 6 skipping directly affects MDM4 abundance, and not its ability to interact with p53, this approach is expected to also have an impact on the growth of MDM4-expressing, TP53-mutant melanoma cells.…”

“…In contrast to MDM2, however, MDM4 is only expressed at low or undetectable levels in most adult tissues (2) and is largely dispensable for adult tissue homeostasis (3)(4)(5)(6). Therefore, whereas MDM2 functions in both proliferating and terminally differentiated cells, MDM4 assists MDM2 in suppressing p53 only in highly proliferating cells such as those found during embryonic development or in the proliferative compartment of the intestinal epithelium (7).…”

“…Low, 3 Cheryl M. Koh, 3 Florian Rambow, 1,2 Mark Fiers, 2 Aljosja Rogiers, 1,2 Enrico Radaelli, 5 Muthafar Al-Haddawi, 6 Soo Yong Tan, 6,7 Els Hermans, 8 Frederic Amant, 8,9 Hualong Yan, 10 Manikandan Lakshmanan, 11 Ratnacaram Chandrahas Koumar, 11 Soon Thye Lim, 12 Frederick A. Derheimer, 13 Robert M. Campbell, 13 Zahid Bonday, 13 Vinay Tergaonkar, 11 Mark Shackleton, resents a unique therapeutic opportunity to reactivate suppressed p53 function in the context of antimelanoma combination therapy. Unfortunately, small molecules that selectively and efficiently disrupt the MDM4-p53 complexes have so far not been identified or introduced into the clinic.…”

“…Unfortunately, small molecules that selectively and efficiently disrupt the MDM4-p53 complexes have so far not been identified or introduced into the clinic. Moreover, an increasing body of evidence has shown that MDM4 possesses p53-independent oncogenic functions (2,(18)(19)(20)(21). Consistently, in addition to inducing p53-dependent apoptosis, MDM4 silencing in melanoma cells also caused cell-cycle arrest that could not be rescued upon concomitant inactivation of p53 (13).…”