Significance of HDMX‐S (or MDM4) mRNA splice variant overexpression and HDMX gene amplification on primary soft tissue sarcoma prognosis

Abstract

The product of the HDMX (or MDM4) gene is structurally related to the MDM2 oncoprotein and is also capable of interacting with the tumor suppressor protein p53. The aim of our study was to determine the amplification status of the HDMX gene and the expression of the HDMX mRNA (particularly that of the HDMX-S splice variant) in soft-tissue sarcomas (STS). Patients with STS were evaluated for the status of HDMX gene amplification (n 5 66) and HDMX-S mRNA expression (n 5 57) within their tumors. DNA, total RNA and protein were isolated from frozen tumor tissue. We determined that the HDMX-S splice variant transcript was predominant in a subset (14%) of tumor samples and that its expression was correlated with decreased patient survival (15 vs. 53 months, p < 0.0001, log-rank test) and with a 17-fold increased risk of a tumor-related death (p < 0.0001, multivariate Cox's regression model). The tumors from these patients also expressed elevated levels of HDMX-S protein. The HDMX gene was amplified in 17% of STSs, and the gene amplification was associated with poor prognosis (RR 5 6.5, p < 0.0001). There was no correlation between the HDMX gene amplification and overexpression of the HDMX-S splice variant. In summary, our data indicate that both the overexpression of the HDMX-S transcript as well as HDMX gene amplification are important prognostic markers for STS. ' 2005 Wiley-Liss, Inc.Key words: HDMX; amplification; overexpression; oncogene; prognosis; alternative splicing; cancer Recently, a protein related to the product of the murine double minute gene 2 (MDM2), designated as HDMX, has been identified, and the HDMX gene was mapped to chromosome region 1q32. 1,2 It has been shown that HDMX (also referred to as MDM4 or MDMX in the mouse) is able to bind to p53, thereby inhibiting its activity. However, it fails to induce the nuclear export of p53 or its proteosomal degradation. 3 Although HDMX bears resemblance to MDM2, it has some distinct features. For example, whereas expression of MDM2 can be induced by DNA damaging agents, HDMX levels are not. 1 This suggests that the HDMX promoter, unlike the P2-promoter of the MDM2 gene, is not transcriptionally transactivated by p53. Data from gene knockout experiments indicate that embryonic lethality of both MDMX 2/2 and MDM2 2/2 mice can be rescued by the loss of p53. 4,5 Therefore, it seems likely that MDM2 and MDMX act in nonoverlapping pathways to regulate p53. 5 However, data also show that HDMX actively regulates p53 activity through its interaction with MDM2, 6 suggesting that HDMX/MDMX is another important regulator of the p53-MDM2 network (reviewed in Michael and Oren 7 ). These data raise the possibility that increased levels of HDMX result in the inactivation of p53 that may contribute to tumor development. In fact, several studies have revealed that the HDMX gene is a target of amplification at the 1q32 gene locus in malignant gliomas that have no p53 mutations or MDM2 amplification. 8,9 In addition, the HDMX gene has been shown to be amplified in breast canc...

In vitro and in silico studies of MDM2/MDMX isoforms predict Nutlin-3A sensitivity in well/de-differentiated liposarcomas

“…38 and that is expected to correlate with a more aggressive behavior. In line with recent sarcoma genetic data, 22 all of the analyzed WD/DD LPS were disomic for MDMX.…”

“…12 Finally, there are no published data regarding the presence of MDMX-S in WD/DD LPS, although it has been identified in 70% of soft tissue sarcomas. 22 We have now found that MDM2 and full-length MDMX, together with their MDM2-B and MDMX-S isoforms, are expressed and translated in WD/DD LPS surgical specimens. Furthermore, the findings of our in vitro and in silico experiments show that they contribute differently to Nutlin-3A sensitivity and resistance.…”

“…Previously described specific primers 22 were used to coamplify full-length mdmx and mdmx-S transcripts, and the PCR products were cloned and sequenced (Supplementary Figure 3). As shown in Figures 3a and b, both full-length mdmx and mdmx-s transcripts were present in all of the WD/ DD samples.…”

“…It has been reported that MDMX protein expression is very low in WD/DD LPS cell lines, 21 and MDMX gene amplification has been described in association with MDM2 co-amplification in one WD/DD LPS surgical specimen. 22 However, in a recently published series of 28 WD/DD cases, only one carried a low level of MDMX gene amplification (between three-and six-fold). 12 Finally, there are no published data regarding the presence of MDMX-S in WD/DD LPS, although it has been identified in 70% of soft tissue sarcomas.…”

Prognostic factors in soft-tissue sarcomas: what have we learnt?

“…Aneuploid DNA content has been correlated with a poorer prognosis [70]; proliferative activity, as reflected by S-phase fraction, silver staining for nucleolar organizer regions, Ki-67 and PCNA, has also been correlated with survival [71][72][73][74]. The expression of oncogenic and tumor-suppressor proteins, such as Bcl-2, MDM2, retinoblastoma gene product and p53, are some of the more well-published molecules that have been associated with prognosis in soft-tissue sarcoma [24,[75][76][77][78].…”