Stjepan Uldrijan Group: Cancer Biology


About our research

Regulation of the p53 tumour suppressor by oncoproteins MDM2 and MDMX

Tumour suppressor p53 is a transcription factor that has a key role in the regulation of cellular responses to various types of stress. Many of these stress stimuli contribute to tumour development and progression and it is therefore not surprising that p53 function is usually lost in cancer cells. The most common single cause of p53 inactivation in tumours is mutation in the DNA binding domain of p53 protein that leads to the loss of the ability of p53 to transactivate its target genes. Another very common cause of p53 inactivation in cancer cells is overexpression of inhibitory proteins Mdm2 or MdmX (Mdm4). These two proteins are critical regulators of p53 in normal untransformed cells and their function is required for normal embryonic development and for the survival of normal somatic cells.

Despite significant homology between Mdm2 and MdmX, notably in their N-terminal p53 binding domain and C-terminal RING finger domain, each of the two proteins adopts a different strategy to suppress p53 function. While Mdm2 serves as an RING finger E3 ubiquitin ligase for p53 and is responsible for keeping p53 protein levels low by targeting it for degradation in 26S proteasome, the main function of MdmX was believed to be a direct inhibition of p53 activity as a transcription factor. However, we have shown recently that MdmX, whose RING domain lacks E3 activity, is able to directly cooperate with Mdm2 in p53 ubiquitylation and degradation. We have also shown that a previously unidentified C-terminal region in both Mdm2 and MdmX is required for their physical and functional interaction. In our current projects, we are studying the molecular mechanisms of the Mdm2/MdmX complex function in p53 ubiquitylation and degradation, and possible role of MdmX-binding proteins in the regulation of the Mdm2/MdmX complex activity.

Central acidic domain is another key region of Mdm2 that is required for both p53 ubiquitylation and degradation p53. The central part of Mdm2 also mediates binding to numerous positive and negative regulators of Mdm2 function. For example, the positive regulators include transcription factor YY1 and transcription co-activator p300, both of which enhance p53 ubiquitylation by Mdm2, the negative regulators include tumour suppressor p14Arf whose expression is induced by oncogene activation, the promyelocytic leukaemia (PML) tumour suppressor and ribosomal proteins L5, L11 and L23 that inhibit Mdm2 function in response to ribosomal stress. Recently, using proteomic approaches, we have identified novel binding partners for the central region and we are currently studying their role in the regulation of Mdm2-mediated p53 degradation. Interestingly, the exact role of the acidic domain of Mdm2 in p53 ubiquitylation and degradation remains unclear. In order to clarify the function of Mdm2 central domain in p53 regulation, we are also performing mutational and functional analyses of the Mdm2 acidic domain itself.

Significance of the project

The overexpression of oncoproteins Mdm2 and MdmX is a common cause of p53 inactivation in human cancers. It is evident that there is a complex and dynamic relationship between p53, Mdm2, MdmX, and their regulators which is a key to the regulation of p53 stability and activity under normal as well as stress conditions. Understanding the functions of these proteins, and how they might interact with each other, will be critical to exploiting them as potential therapeutic targets for the reactivation of p53 function in tumours.

Targeted therapy for malignant melanoma

The incidence of melanoma has been increasing rapidly in Caucasian populations worldwide over the last 25 years at an annual rate of 3-7%. In the Czech Republic the incidence of melanoma has increased five fold between years 1970 and 2002. Despite such sharp increase in melanoma incidence, it still accounts for less than 5% of all dermatologic cancers. However, it is responsible for about 80 % of all deaths from skin cancers. Moreover, unlike most other cancers, melanoma is relatively frequent among young and middle-aged adults. In the United States, melanoma is the second leading cause of lost productive years of all cancers and it is the most common cancer among women 20 to 29 years of age.

Although the large majority of people diagnosed with early melanoma are cured after surgical excision of the primary tumour, advanced metastatic disease is usually refractory to all current forms of systemic therapy and has a very poor prognosis. The 5-year survival rate is currently estimated at only 6%, with a median survival time of 6 months. Among cytotoxic agents, dacarbazine (DTIC) and its analogue temozolomide remain the chemotherapy of choice but they produce objective response in only 15 to 20% of patients and the median duration of the response is only 4 months. New immunotherapeutic approaches have been also tested. High doses of interleukin-2 or interferon alpha as single agents have induced responses in 10 to 16% of patients but they are associated with significant toxicity and serious side effects. A potential benefit of combining chemotherapy with immunotherapy has been tested in several phase III trials. However, these trials failed to consistently demonstrate an improvement in either response rates or overall survival.

Currently the most intensely tested targeted therapeutic strategy for malignant melanoma is the inhibition of the RAS/RAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway (ERK pathway). This signal transduction pathway, which normally regulates cell proliferation and survival mainly in response to extracellular signals, has been found constitutively activated in the majority of human melanomas, most commonly by activating mutations of N-Ras and B-RAF proto-oncogenes that are found in approx. 80% of malignant melanomas. Sorafenib (BAY 43-9006), an oral inhibitor of RAF kinase has demonstrated activity against melanoma in preclinical tests and it is currently the most promising drug in clinical trials for the targeted treatment of malignant melanoma. Even though early clinical studies failed to show efficacy of sorafenib as a single agent, preliminary results suggest that combinations of sorafenib with various chemotherapeutics are more effective in the treatment of advanced melanoma than chemotherapeutics alone. Interestingly, results of a recent research comparing the sensitivity to ERK pathway inhibitors of melanoma cell lines derived from different stages of melanoma progression suggest that multiple cellular signalling pathways may need to be targeted in order to achieve maximal therapeutic efficacy against the most aggressive metastatic melanomas.

While the activation of the ERK pathway is believed to be a critical early event in the development of melanoma, the role of other MAPK pathways (and other cellular signaling pathways generally) in the development of this cancer and its response to therapy remains largely unclear. That is why we are studying the role of selected cellular signaling pathways in melanoma cell survival and response to DNA-damaging chemotherapy, and in the regulation of p53 tumour suppressor activity in malignant melanoma cells. Our aim is to identify suitable cellular targets for the development of novel, more efficient targeted therapies for malignant melanoma.

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