Analysis of human MDM4 variants in papillary thyroid carcinomas reveals new potential markers of cancer properties

A wild-type (wt) p53 gene characterizes thyroid tumors, except for the rare anaplastic histotype. Because p53 inactivation is a prerequisite for tumor development, alterations of p53 regulators represent an alternative way to impair p53 function. Indeed, murine double minute 2 (MDM2), the main p53 negative regulator, is overexpressed in many tumor histotypes including those of the thyroid. A new p53 regulator, MDM4 (a.k.a. MDMX or HDMX) an analog of MDM2, represents a new oncogene although its impact on tumor properties remains largely unexplored. We estimated levels of MDM2, MDM4, and its variants, MDM4-S (originally HDMX-S) and MDM4-211 (originally HDMX211), in a group of 57 papillary thyroid carcinomas (PTC), characterized by wt tumor protein 53, in comparison to matched contra-lateral lobe normal tissue. Further, we evaluated the association between expression levels of these genes and the histopathological features of tumors. Quantitative real-time polymerase chain reaction revealed a highly significant downregulation of MDM4 mRNA in tumor tissue compared to control tissue (P < 0.0001), a finding confirmed by western blot on a subset of 20 tissue pairs. Moreover, the tumor-to-normal ratio of MDM4 levels for each individual was significantly lower in late tumor stages, suggesting a specific downregulation of MDM4 expression with tumor progression. In comparison, MDM2 messenger RNA (mRNA) and protein levels were frequently upregulated with no correlation with MDM4 levels. Lastly, we frequently detected overexpression of MDM4-S mRNA and presence of the aberrant form, MDM4-211 in this tumor group. These findings indicate that MDM4 alterations are a frequent event in PTC. It is worthy to note that the significant downregulation of full-length MDM4 in PTC reveals a novel status of this factor in human cancer that counsels careful evaluation of its role in human tumorigenesis and of its potential as therapeutic target

Abstract P3-06-01: ATM Heterozygosity as a Breast Cancer-Susceptibility Factor in the General Population

Abstract Background: Do ATM heterozygous (ATM-htz) carriers have an increased risk of developing breast cancer? Do they have an increased sensitivity to ionizing radiation (IR)? Definitive information does not yet exist to answer these questions. Case-control studies have suggested a link between single copy ATM gene mutation and increased breast cancer risk. Lymphoblasts and skin fibroblasts from ATM-htz carriers are less sensitive to IR than cells from Ataxia Telangiectasia (AT) patients, but more sensitive than cells from the general population. Because of the clinical and environmental implication of these aspects, large studies are in progress to better understand the interaction between ATM-htz and increased breast cancer risk or radio-sensitivity. However, to be performed on the general population rather than on the obligate ATM-htz carriers, i.e., the rare parents of children affected by AT, these studies must relay on the expensive sequencing of the entire ATM gene (approximately 160Kb of genomic DNA, a 13Kb transcript of 66 exons, and more than 400 unique mutations that extend across the full length of the gene). Materials and Methods: Based on a serendipitous discovery, we have development of a new, non-invasive, easy, quick, and inexpensive test to identify AT patients and, more interestingly, the ATM-htz carriers. In particular, we found that, in mitosis, p53 localizes at centrosomes, the cytoplasmic organelles that organize the interphase cytoskeleton and contribute to bipolar spindle formation during mitotic cell division. In addition, we demonstrated that ATM is required for p53 localization at centrosome. These findings allowed us to identify a particular phenotype of peripheral blood lymphocytes (PBL) from AT patients or their parents, the obligate ATM-htz that should allow to easily discriminate the ATM-htz carries in the general population with a very high statistical significance (P&amp;lt;0.001). We have employed this new test to assess the contribution of ATM heterozygosity in the risk to develop breast cancer. Results: The frequency of ATM-htz in breast cancer patients and in healthy individuals was assessed by our new test and compared with the Italian theoretical frequency (between 1.69 and 3.43%; Italian AT Registry). At present, we have found 6 ATM-htz out of 80 breast cancer patients (7.5%) and 1 ATM-htz out of 70 healthy donors (1.4%) supporting the proposed link between single copy ATM gene mutation and increased breast cancer risk. In addition, an initial individual evaluation of the 6 ATM-Htz patients showed the following common characteristics: 1) early onset of tumor appearance (37-42 years of age); 2) ER and PgR positive and HER negative breast cancers. Discussion: Beside an accurate assessment of the relative contribution of ATM heterozygosity to tumor susceptibility, this study will open the possibility of performing large screening for ATM-htz carries in the general population for different clinical aspects, such as IR sensitivity, genetic counseling, cancer predisposition, or selection for specific targeted therapies. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-06-01.</jats:p

HIPK2 phosphorylates Delta Np63\u3b1 and promotes its degradation in response to DNA damage

Homeodomain-interacting protein kinase 2 (HIPK2) is an emerging player in cell response to genotoxic agents that senses damage intensity and contributes to the cell's choice between cell cycle arrest and apoptosis. Phosphorylation of p53 at S46, an apoptosis-specific p53 posttranslational modification, is the most characterized HIPK2 function in response to lethal doses of ultraviolet (UV), ionizing radiation or different anticancer drugs, such as cisplatin, roscovitine and doxorubicin (DOX). Indeed, like p53, HIPK2 has been shown to contribute to the effectiveness of these treatments. Interestingly, p53-independent mechanisms of HIPK2-induced apoptosis were described for UV and tumor growth factor-\u392 treatments; however, it is unknown whether these mechanisms are relevant for the responses to anticancer drugs. Because of the importance of the so-called 'p53-independent apoptosis and drug response' in human cancer chemotherapy, we asked whether p53-independent factor(s) might be involved in HIPK2-mediated chemosensitivity. Here, we show that HIPK2 depletion by RNA interference induces resistance to different anticancer drugs even in p53-null cells, suggesting the involvement of HIPK2 targets other than p53 in response to chemotherapy. In particular, we found that HIPK2 phosphorylates and promotes proteasomal degradation of \u394Np63\u3b1, a prosurvival \u394N isoform of the p53 family member, p63. Indeed, effective cell response to different genotoxic agents was shown to require phosphorylation-induced proteasomal degradation of \u394Np63\u3b1. In DOX-treated cells, we show that HIPK2 depletion interferes with \u394Np63\u3b1 degradation, and expression of a HIPK2-resistant \u394Np63\u3b1-\u394390 mutant induces chemoresistance. We identify T397 as the \u394Np63\u3b1 residue phosphorylated by HIPK2, and show that the non-phosphorylatable \u394Np63\u3b1-T397A mutant is not degraded in the face of either HIPK2 overexpression or DOX treatment. These results indicate \u394Np63\u3b1 as a novel target of HIPK2 in response to genotoxic drugs

Abl interconnects oncogenic Met and p53 core pathways in cancer cells

The simplicity of BCR-ABL 'oncogene addiction' characterizing leukemia contrasts with the complexity of solid tumors where multiple 'core pathways', including receptor tyrosine kinases (RTKs) and p53, are often altered. This discrepancy illustrates the limited success of RTK antagonists in solid tumor treatment compared with the impact of Imatinib in BCR-ABL-dependent leukemia. Here, we identified c-Abl as a signaling node interconnecting Met-RTK and p53 core pathways, and showed that its inhibition impairs Met-dependent tumorigenesis. Met ensures cell survival through a new path in which c-Abl and p38-MAPK are employed to elicit p53 phosphorylation on Ser(392) and Mdm2 upregulation. We found a clinical correlation between activated Met, phospho-p53, and Mdm2 levels in human tumors, supporting the role of this path in tumorigenesis. Our findings introduce the concept that RTK-driven tumors may be therapeutically treated by hitting signaling nodes interconnecting core pathways. Moreover, they underline the importance of evaluating the relevance of c-Abl antagonists for combined therapies, based on the tumor signaling signature.Cell Death and Differentiation advance online publication, 1 April 2011; doi:10.1038/cdd.2011.23

ATM-depletion in breast cancer cells confers sensitivity to PARP inhibition

Mutations in the DNA damage response (DDR) factors, breast cancer 1 (BRCA1) and BRCA2, sensitize tumor cells to poly(ADP-ribose) polymerase (PARP) inhibitors. The ataxia telangiectasia mutated (ATM) kinase is a key DDR protein whose heterozygous germline mutation is a moderate-risk factor for developing breast cancer. In this study, we examined whether ATM inactivation in breast cancer cell lines confers sensitivity to PARP inhibitors