Amplification of Mdmx and overexpression of MDM2 contribute to mammary carcinogenesis by substituting for p53 mutations

The p53 tumor suppressor gene has a central role in maintaining the integrity of the genome and the defense against cancer. The tumor protein 53 (TP53) becomes stabilized and regulates numerous downstream targets to induce cell cycle arrest, senescence, apoptosis, and DNA repair in response to diverse stresses. Mutation and LOH at the p53 locus occur as tumors progress under conditions of increasing genomic instability[1‐3]. p53 is mutated in nearly half of all human cancers, and it is functionally abrogated in much of the remaining 50% of cancers through signaling pathways[4]. In breast cancer, approximately about 15% to 50% of the cases carry a mutant p53 gene and/or loss of heterozygosity (LOH) at chromosome location 17p13, where the p53 gene is located[5‐7].

TP53 is negatively regulated by numerous factors. The murine double minute 2 (Mdm2) and Mdmx represent two important cellular regulators of p53. The Mdm2 gene was identified as one of three unknown genes (Mdm1-3) coamplified in the spontaneously transformed 3T3-DM mouse cell line[8]. Acting as an ubiquitin (Ub) protein ligase (E3), MDM2 (also called HDM2) can bind and ubiquitinate TP53 and promote rapid degradation of TP53 through the ubiquitin proteolysis pathway, which keeps TP53 at low levels in the absence of stress signals. MDM2 overexpression has been observed in about a third of human sarcomas that retained wild-type TP53[3, 9‐11].

MDMx (also known as MDM4), a TP53 binding protein structurally homologous to MDM2, was more recently identified[12]. MDMx was found amplified or overexpressed in 10–20% of breast cancers, glioblastomas, retinoblastomas, and soft tissue sarcomas[3, 13‐15] in the presence of wild-type TP53, which confirmed that aberrant expression of MDMx may contribute to tumor formation by inhibiting TP53 activity. But the molecular details of the role of MDMx in the control of P53 and tumorigenesis are not well understood.

Although MDM2 and MDMx are overexpressed in many malignancies, data were mainly from cell-based studies and in vivo studies on mouse models. Limited studies using human archived tissue of breast cancers revealed the roles of the above molecular markers in carcinogenesis and the relationship among them as well as the relationship with the clinicopathologic characteristics. And to our knowledge, there was no study on these molecular markers using invasive carcinoma and carcinoma in situ in the same tumors. In present study our aims were to assess the genetic instability at p53 (located on 17p13.1), Mdmx (located on 1q32.1) and Mdm2 (located on 12q15) and address the roles of these proteins in breast cancer progression. Using multi-color fluorescence in situ hybridization (FISH) protocols in 115 primary breast cancers, we examined the genetic changes at p53, Mdmx and Mdm2 loci in archived breast cancers. The expression of these proteins was evaluated by immunohistochemistry.

Breast cancer is characterized by a number of genetic aberrations[23‐25]. Although improvements have been achieved in recent years, few genetic biomarkers are available to easily identify individuals at risk for breast cancer or breast cancer progression[26‐28]. A better understanding of the molecular mechanisms involved in breast cancer initiation and progression will likely contribute to providing useful prognostic biomarker and therapeutic target for breast cancer therapy.

FISH is considered to be the most precise method for amplification and deletion detection. In this study, we successfully detected the gene copy number changes of p53, Mdmx and Mdm2 by multi-color FISH, which enables investigators obtaining far more information from one specific cell at one time, rather than carrying out separate experiments on multiple specimens prepared from the same sample, then extrapolating results.

Dysfunction of p53 often occurs as a result of mis-sense mutation, but can also result from nonsense or deletion mutation[3]. TP53 is a product of the mutated gene. Therefore here TP53 overexpression using immunohistochemical method was adopted to investigate p53 mutation. For scattered weak to moderate staining can be observed in normal breast tissue, hyperplasia breast tissue and tumor tissue without p53 mutation, only tumor tissues with diffuse strong nuclear staining for TP53 was considered to show mutant p53 as in serious papillary carcinoma in ovary. In this study, FISH was adopted to detect p53 allelic loss as a co-criterion for identifying the dysfunctional p53 phenotype. Allelic loss of p53 was detected in 23% of all analyzed samples in present study while 17% of cases showed TP53 overexpression indicating p53 mutation. The presence of p53 allelic loss and/or TP53 overexpression was observed in 38% out of all patients, within the range of frequencies reported by others[29, 30]. Because of the involvement of DCIS, p53 alteration must be an early event in breast carcinogenesis. According to previous reports, mutation of p53 is associated with increased tumor size and tumor grade, axillary lymph node metastases and ki67 expression[30‐32]. But data presented here indicate there was no significant relationship between p53 allelic loss or TP53 overexpression and tumor node status, whereas p53 dysfunction was detected significantly more often in larger (d > 20 mm), high grade (grade 3), ER negative and high ki67 index tumors.

LOH and mutation of p53 only present in part of breast cancers. It is believed that tumors retaining wild type p53 contain abnormalities in other genes that interact with p53 or are downstream of p53 and result in an identical physiological defect within the cells. One of the best examples of the latter class is amplification or overexpression of Mdm2, which was observed in a subset of human tumors, some of which retain wild-type p53[33] leading to the conclusion that increased levels of MDM2 directly contribute to human tumor formation by substituting for mutations in p53 gene, which represents an alternative mechanism by which tumor cells escape from the tumor suppressive activities of p53. Overexpression of MDM2 can impair wild-type TP53 function in two ways by binding to the transactivation site of TP53 in the nucleus or by targeting TP53 for ubiquitination and degradation in the cytoplasm, and linked with low levels of TP53 immunostaining in human breast cancers[34]. Frequent overexpression of MDM2 in advanced breast tumors was observed[34]. It was reported MDM2 protein overexpression often due to gene amplifications[35, 36]. But Mdm2 amplification was found only in two samples while its overexpression was observed in 38% of all tumors in this study, which supported Mdm2 amplification occurs at a lower frequency than increased transcription or enhanced translation in breast cancers. So in this study MDM2 overexpression was used to reflect Mdm2 abnormality. Importantly, we found most tumors contained either a TP53 alteration or a MDM2 alteration, but not both. This distribution was significant (P< 0.05), and strongly suggests alterations of these two genes are mutually exclusive. Our data support the hypothesis that the major effect of MDM2 overexpression is identical to that resulting from p53 mutation. One would expect that either TP53 or MDM2 would be altered and alterations of both genes should be examined in a given breast cancer. There were few tumors observed without TP53 dysfunction and MDM2 overexpression in this study. So there might be other alterations of TP53 or MDM2, or some of these tumors might progress through genetic events that involve a totally different pathway. More detailed analyses of such tumors are needed to reveal.

Several studies together now also implicate aberrant expression of MDMx could thus contribute to tumor formation[37]. Unlike MDM2, MDMx does not have intrinsic E3 ligase activity and does not promote TP53 degradation. However, MDMx binds TP53 in its transactivation domain and is thereby able to inhibit its transcriptional activity. Amplification of Mdmx was found in several tumor types. Migliorini found amplification of Mdmx correlated with a wild-type p53 status and lack of Mdm2 amplification[38]. In this study Mdmx was amplified in 57% of all cases, while it was overexpressed in 65% of all tumors, indicating MDMx overexpression was mainly due to its amplification. Mdmx amplification was adopted in data analyses. Although not all the tumors with Mdmx amplification show wild type p53, significant inverse correlation between Mdmx amplification and TP53 overexpression was still observed. We did not find correlation between Mdmx amplification and MDM2 overexpression, but tumors with Mdmx amplification were more likely lack of MDM2 overexpression. Up to now, it was still controversial whether each plays its own distinct role or MDM2 and MDMx function together as one heterocomplex in p53 regulation. Our data seems to support MDMx may contribute to the regulation of TP53 independently of MDM2. A recent evidence demonstrated that endogenous level of MDMx could regulate transformation and chromosomal stability in TP53-deficient cells and these MDMx functions were not shared by MDM2, and were distinct from the well-established ability of MDMx to complex with and inhibit TP53 activity[39]. Together these data strongly indicate that Mdmx amplification is a common event in breast carcinogenesis, even in most tumorigenesis and MDMx probably functions as an oncogene through a quite different way compared to MDM2. The molecular mechanism that prevents TP53 activation and carcinogenesis in the presence of high level of MDMx is largely unknown, which need to be elucidated in the future. Furthermore we found Mdmx amplification was seen in more invasive breast cancers. So we speculate amplified Mdmx more likely to be associated with tumor progression. Larger sample sizes to provide more definitive data on the potential role of genetic changes of Mdmx in breast cancer progression should be performed in the future.

In conclusion, we detected genetic alterations of p53, Mdmx and Mdm2 by multi-color FISH in one specific cell simultaneously and compared these genetic alterations in DCIS and invasive lesions in individual cases for the first time. Our data indicate that disruption of the TP53 pathway by high levels of MDM2 and MDMx are common events in the breast tumorigenesis and Mdmx amplifies quite frequently with low-level increase of gene copy number while few tumors with Mdm2 amplification are seen in primary breast cancers. Mdmx amplification is seen in invasive breast cancer but not in DCIS in some cases and likely to be associated with tumor progression. Moreover, high levels of both genes in breast cancer that retain wild-type p53 suggest that these inhibitors may substitute for mutations in p53, therefore, contribute to the severity and progression of the disease. But as an oncogene, high level of MDMx may act through a quite different way compared to MDM2. Together, our data strongly suggest that overexpression of MDM2 or MDMx and p53 mutations in primary breast cancer are mutually exclusive events. The presence of high levels of MDM2 and MDMx in many breast cancers suggests that these data should be considered in the treatment of breast cancer.