Oncogenic functions of hMDMX in in vitro transformation of primary human fibroblasts and embryonic retinoblasts

<p>Abstract</p> <p>Background</p> <p>In around 50% of all human cancers the tumor suppressor p53 is mutated. It is generally assumed that in the remaining tumors the wild-type p53 protein is functionally impaired. The two main inhibitors of p53, hMDM2 (MDM2) and hMDMX (MDMX/MDM4) are frequently overexpressed in wild-type p53 tumors. Whereas the main activity of hMDM2 is to degrade p53 protein, its close homolog hMDMX does not degrade p53, but it represses its transcriptional activity. Here we study the role of hMDMX in the neoplastic transformation of human fibroblasts and embryonic retinoblasts, since a high number of retinoblastomas contain elevated hMDMX levels.</p> <p>Methods</p> <p>We made use of an <it>in vitro </it>transformation model using a retroviral system of RNA interference and gene overexpression in primary human fibroblasts and embryonic retinoblasts. Consecutive knockdown of RB and p53, overexpression of SV40-small t, oncogenic HRasV12 and HA-hMDMX resulted in a number of stable cell lines representing different stages of the transformation process, enabling a comparison between loss of p53 and hMDMX overexpression. The cell lines were tested in various assays to assess their oncogenic potential.</p> <p>Results</p> <p>Both p53-knockdown and hMDMX overexpression accelerated proliferation and prevented growth suppression induced by introduction of oncogenic Ras, which was required for anchorage-independent growth and the ability to form tumors <it>in vivo</it>. Furthermore, we found that hMDMX overexpression represses basal p53 activity to some extent. Transformed fibroblasts with very high levels of hMDMX became largely resistant to the p53 reactivating drug Nutlin-3. The Nutlin-3 response of hMDMX transformed retinoblasts was intact and resembled that of retinoblastoma cell lines.</p> <p>Conclusions</p> <p>Our studies show that hMDMX has the essential properties of an oncogene. Its constitutive expression contributes to the oncogenic phenotype of transformed human cells. Its main function appears to be p53 inactivation. Therefore, developing new drugs targeting hMDMX is a valid approach to obtain new treatments for a subset of human tumors expressing wild-type p53.</p

Overexpression of EZH2 in conjunctival melanoma offers a new therapeutic target

Malignant melanoma of the conjunctiva (CM) is an uncommon but potentially deadly disorder. Many malignancies show an increased activity of the epigenetic modifier enhancer of zeste homolog 2 (EZH2). We studied whether EZH2 is expressed in CM, and whether it may be a target for therapy in this malignancy. Immunohistochemical analysis showed that EZH2 protein expression was absent in normal conjunctival melanocytes and primary acquired melanosis, while EZH2 was highly expressed in 13 (50%) of 26 primary CM and seven (88%) of eight lymph node metastases. Increased expression was positively associated with tumour thickness (p =0.03). Next, we targeted EZH2 with specific inhibitors (GSK503 and UNC1999) or depleted EZH2 by stable shRNA knockdown in three primary CM cell lines. Both pharmacological and genetic inactivation of EZH2 inhibited cell growth and colony formation and influenced EZH2-mediated gene transcription and cell cycle profile in vitro. The tumour suppressor gene p21/CDKN1A was especially upregulated in CM cells after EZH2 knockdown in CM cells. Additionally, the potency of GSK503 against CM cells was monitored in zebrafish xenografts. GSK503 profoundly attenuated tumour growth in CM xenografts at a well-tolerated concentration. Our results indicate that elevated levels of EZH2 are relevant to CM tumourigenesis and progression, and that EZH2 may become a potential therapeutic target for patients with CM. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

MDM4 is a rational target for treating breast cancers with mutant p53

Mutation of the key tumour suppressor p53 defines a transition in the progression towards aggressive and metastatic breast cancer (BC) with the poorest outcome. Specifically, the p53 mutation frequency exceeds 50% in triple-negative BC. Key regulators of mutant p53 that facilitate its oncogenic functions are potential therapeutic targets. We report here that the MDM4 protein is frequently abundant in the context of mutant p53 in basal-like BC samples. Importantly, we show that MDM4 plays a critical role in the proliferation of these BC cells. We demonstrate that conditional knockdown (KD) of MDM4 provokes growth inhibition across a range of BC subtypes with mutant p53, including luminal, Her2(+) and triple-negative BCs. In vivo, MDM4 was shown to be crucial for the establishment and progression of tumours. This growth inhibition was mediated, at least in part, by the cell cycle inhibitor p27. Depletion of p27 together with MDM4 KD led to recovery of the proliferative capacity of cells that were growth-inhibited by MDM4 KD alone. Consistently, we identified low levels of p27 expression in basal-like tumours corresponding to high levels of MDM4 and p53. This predicts a signature for a subset of tumours that may be amenable to therapies targeted towards MDM4 and mutant p53. The therapeutic potential of MDM4 as a target in BC with mutant p53 was shown in vitro by use of a small-molecule inhibitor. Overall, our study supports MDM4 as a novel therapeutic target for BC expressing mutant p53. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.status: publishe