miR-26a/b调控p53/MDM2通路对胃癌细胞凋亡的影响

目的 探讨miR-26a/b调控p53/MDM2通路对胃癌细胞凋亡的影响。方法 实时荧光定量PCR检测胃癌细胞系MGC803、MKN-45和MKN-28中miR-26a/b的表达水平,荧光素酶报告系统分析miR-26a/b与MDM2 3’非翻译区（3’UTR）的结合情况;将化学合成miR-26a/b前体分子mimics和无关序列转染胃癌细胞MKN-45,化学合成miR-26a/b inhibitor转染胃上皮细胞GES-1后,Western blotting检测MDM2、p53及其下游分子p21和Bcl-2的表达,MTT法检测转染24、48、72 和96 h的细胞增殖情况;通过Annexin Ⅴ/PI双染检测miR-26a/b对MKN-45细胞凋亡情况。结果 与永生化胃上皮细胞GES-1相比,miR-26a/b在肿瘤细胞系MGC803、MKN-45和MKN-28中的表达均下调,在MKN-45中表达水平最低;荧光素酶活性检测显示,miR-26a/b过表达抑制MDM2 3’UTR报告载体（Wild）的荧光素酶活性,而对3’UTR突变型报告载体（Mutation）荧光素酶活性无明显影响。miR-26a/b抑制MKN-45细胞中MDM2表达并增强p53及下游分子表达,而在GES-1细胞中抑制miR-26a/b可以增强MDM2表达,降低p53及下游分子表达。MTT结果显示,miR-26a/b抑制MKN-45细胞的增殖,miR-26a/b inhibitor则明显促进GES-1细胞的增殖。通过AnnexinⅤ/PI检测细胞凋亡发现,miR-26a/b过表达的MKN-45细胞的凋亡率高于对照细胞（P＜0.01）。结论 miR-26a/b 能与MDM2 3’UTR 特异结合,调控p53/MDM2通路影响胃癌细胞的增殖及凋亡。     

Activation of p53 by roscovitine-mediated suppression of MDM2 expression.

The p53 tumor suppressor is regulated by the MDM2 oncoprotein. Overexpression of MDM2 maintains p53 at low levels and contributes to the functional inactivation of p53 in a subset of tumors. We found that treatment with roscovitine and olomoucin, which were originally developed as cyclin-dependent kinase (CDK) inhibitors, can efficiently stabilize and activate nuclear p53 in tumor cells with MDM2 amplification or cytoplasmic p53. These inhibitors block the degradation of p53 without affecting p53-MDM2 binding and the nuclear shuttling function of p53 and MDM2. Roscovitine also induces stabilization of the p53 Ala-315 mutant, indicating that it does not act by regulating the CDK phosphorylation of serine 315. Roscovitine induces down-regulation of MDM2 expression at both protein and mRNA levels. Ectopic expression of MDM2 can abrogate the ability of roscovitine to induce p53 stabilization. Low concentrations of roscovitine cooperate with the DNA-damaging agent camptothecin to activate p53 in a synergistic fashion. These results show that the small molecule CDK inhibitors can be used to activate p53 through their potent inhibitory effect on MDM2 expression and may be useful as sensitizing agents for other DNA-damaging drugs.     

Down Regulation of miR-34a and miR-143 May Indirectly Inhibit p53 in Oral Squamous Cell Carcinoma: a Pilot Study

Background: Aberrant microRNA expression has been associated with the pathogenesis of a variety of human malignancies including oral squamous cell carcinoma (SCC). In this study, we examined primary oral SCCs for the expression of 6 candidate miRNAs, of which five (miR-34a, miR-143, miR-373, miR-380-5p, and miR- 504) regulate the tumor suppressor TP53 and one (miR-99a) is involved in AKT/mTOR signaling. Materials and Methods: Tumor tissues (punch biopsies) were collected from 52 oral cancer patients and as a control, 8 independent adjacent normal tissue samples were also obtained. After RNA isolation, we assessed the mature miRNA levels of the 6 selected candidates against RNU44 and RNU48 as endogenous controls, using specific TaqMan miRNA assays. Results: miR-34a, miR-99a, miR-143 and miR-380-5p were significantly down-regulated in tumors compared to controls. Moreover, high levels of miR-34a were associated with alcohol consumption while those of miR-99a and miR-143 were associated with advanced tumor size. No significant difference was observed in the levels of miR-504 between the tumors and controls whereas miR-373 was below the detection level in all but two tumor samples. Conclusions: Low levels of miR-380-5p and miR-504 that directly target the 3’UTR of TP53 suggest that p53 may not be repressed by these two miRNAs in OSCC. On the other hand, low levels of miR-34a or miR-143 may relieve MDM4 and SIRT1 or MDM2 respectively, which will sequester p53 indicating an indirect mode of p53 suppression in oral tumors.     

Pharmacologic activation of p53-dependent and p53-independent apoptotic pathways in Hodgkin/Reed-Sternberg cells.

The status of the p53 pathway in classical Hodgkin lymphoma (cHL) remains unclear, and a lack of proven TP53 mutations contrasts with often high expression levels of p53 protein. In this study, we demonstrate that pharmacologic activation of the p53 pathway with the murine double minute 2 (MDM2) antagonist nutlin-3 in Hodgkin lymphoma-derived cell lines leads to effective apoptosis induction and sensitizes the cells to other anticancer drugs. Cells with mutant p53 are resistant to nutlin-3, but sensitive to geldanamycin, a pharmacologic inhibitor of heat shock 90 kDa protein (HSP90), indicating that HSP90 inhibition can induce apoptosis in a p53-independent manner. Conversely, cells with defects in the HSP90/nuclear factor-kappa B pathway expressing wild-type p53 are more resistant to geldanamycin, but still sensitive to nutlin-3. Our results suggest that selective activation of p53 by MDM2 antagonists as a single agent or in combination with conventional chemotherapeutics and/or inhibitors of p53-independent survival pathways may offer effective treatment options for patients with cHL. Importantly, because nutlins and HSP90 inhibitors are non-genotoxic agents, their use might offer a means to reduce the genotoxic burden of current chemotherapeutic regimens.     

The initial evaluation of non-peptidic small-molecule HDM2 inhibitors based on p53-HDM2 complex structure

Peptidic Mouse Double Minute (MDM2) inhibitors have been demonstrated to effectively inhibit the interaction between p53 and MDM2, thus providing a therapeutic strategy for some tumors. However, there is no report on non-peptidic inhibitors. In this study non-peptidic HDM2 (the human homologue of MDM2) inhibitors were obtained by computer-aided design and subsequently synthesized by chemical method. Bio-evaluation showed that some of these inhibitors have affinity with HDM2, and can cause death of some tumor cells which express wild-type p53. Cellular assays showed that one of these compounds, syc-7, can activate the p53 pathway in some of these tumor cell lines, and further induce apoptosis. The results suggest that developing non-peptidic small-molecule HDM2 inhibitors is a promising way for new antitumor drug discovery.     

Effects of p53 mutants derived from lung carcinomas on the p53-responsive element (p53RE) of the MDM2 gene.

The present study represents a continuation of previous works in which we observed that lung carcinomas co-expressing MDM2 protein and p53 mutants (mt p53) exhibited more aggressive behaviour. In the above studies, we suggested a ''gain of function'' mechanism of mt p53 proteins based on the fact that the MDM2 gene possesses a p53-responsive element (MDM2-p53RE). In this study, to prove our hypothesis, we selected 12 cases from a series of 51 bronchogenic carcinomas. In these 12 cases, we examined the ability of the expressed mt p53 to bind the MDM2-p53RE and correlated the findings with MDM2 expression. Furthermore, we constructed four of these p53 mutants and studied their transactivation properties by co-transfecting them with a reporter plasmid carrying MDM2-p53RE in the p53 null non-small-cell lung carcinoma cell line (NSCLC) H1299. We observed mutant p53 protein DNA-binding activity, which depended on the nature and the position of the amino acid substitution. The fact that the cases with DNA-binding activity were accompanied with MDM2 protein isoforms'' overexpression is indicative of a ''gain of function'' phenotype. This hypothesis was enforced by the findings of the transfection experiments, which revealed that certain p53 mutants enhanced the expression of the luciferase reporter gene either directly or indirectly via a dominant positive effect on the wild-type p53. In conclusion, this work is one first attempt to examine if the deregulation of the p53/MDM2 autoregulatory feedback loop is due to novel properties of certain p53 mutants in the specific environment of a subset of bronchogenic carcinomas.     

MDM2 inhibitor MI-319 in combination with cisplatin is an effective treatment for pancreatic cancer independent of p53 function

Small molecule inhibitors (SMIs) of murine double minute 2 (MDM2) are known to restore the apoptotic and cell cycle regulatory functions of p53 by disrupting the MDM2–p53 interaction. In principle, these SMIs are not effective against tumours with mutation in the tumour suppressor p53 (mut-p53), which is known to be present in approximately 50% of all cancers. In this study we are reporting, for the first time, that MI-319 in combination with cisplatin induced cell growth inhibition and apoptosis in pancreatic cancer (PC) cells irrespective of their p53 mutational status. MI-319–cisplatin combination synergistically suppressed cell growth (MTT Combination Index [CI] < 1) and colony formation (clonogenic assay) and induced apoptosis. Western blot analysis and siRNA silencing studies in mutant as well as p53 null cells highlighted a mechanism involving p73 which is also known to be under the regulation of MDM2, and unlike p53, it is rarely mutated in PC. Down-regulating MDM2 using siRNA enhanced p73 reactivation and increased cell death. Further, the combination effectively reduced tumour growth in both wt-p53 and mut-p53 tumour xenograft models (50% Capan-2 animals were tumour free). Consistent with our in vitro results, remnant tumour tissue analysis showed up-regulation of p73 and the cell cycle regulator p21. In conclusion, this study highlights a new role of MDM2 inhibitors in combination with cisplatin, and thus warrants further clinical investigation in human pancreatic tumours containing both wt-p53 and mut-p53.     

Expression of p14ARF overcomes tumor resistance to p53

Tumors without p53 mutation are often resistant to p53 gene therapy. We examined the mechanism using p53-resistant A549 cells and p53-sensitive H1299 cells. We found that p53 delivered by adenovirus is poorly expressed in A549 (ARF-null) cells but efficiently expressed in H1299 cells (ARF-positive). Strong p53 expression and apoptosis can be achieved in A549 cells using a p53 mutant resistant to degradation by MDM2 or by coexpression of ARF. The results suggest that enhanced MDM2 activity attributable to loss of ARF contributes to p53 resistance. Surprisingly, tumor cell lines with MDM2 gene amplification are still deficient for ARF expression, suggesting that MDM2 amplification does not substitute for ARF inactivation during tumor development.     

The guardian's little helper: microRNAs in the p53 tumor suppressor network

Several microRNAs (miRNAs) have been implicated in tumor development based on both changes in their expression patterns and gene structural alterations in human tumors. However, we are only now beginning to see how miRNAs interact with classic oncogene and tumor suppressor mechanisms. Several recent studies have implicated the miR-34 family of miRNAs in the p53 tumor suppressor network. The expression of miR-34a, miR-34b, and miR-34c is robustly induced by DNA damage and oncogenic stress in a p53-dependent manner. When overexpressed, miR-34 leads to apoptosis or cellular senescence, whereas reduction of miR-34 function attenuates p53-mediated cell death. These findings, together with the fact that miR-34 is down-regulated in several types of human cancer, show that miRNAs can affect tumorigenesis by working within the confines of well-known tumor suppressor pathways.     

Translating p53 into the clinic

Mutations in the TP53 gene are a feature of 50% of all reported cancer cases. In the other 50% of cases, the TP53 gene itself is not mutated but the p53 pathway is often partially inactivated. Cancer therapies that target specific mutant genes are proving to be highly active and trials assessing agents that exploit the p53 system are ongoing. Many trials are aimed at stratifying patients on the basis of TP53 status. In another approach, TP53 is delivered as a gene therapy; this is the only currently approved p53-based treatment. The p53 protein is overexpressed in many cancers and p53-based vaccines are undergoing trials. Processed cell-surface p53 is being exploited as a target for protein-drug conjugates, and small-molecule drugs that inhibit the activity of MDM2, the E3 ligase that regulates p53 levels, have been developed by several companies. The first MDM2 inhibitors are being trialed in both hematologic and solid malignancies. Finally, the first agent found to restore the active function of mutant TP53 has just entered the clinic. Here we discuss the basis of these trials and the future of p53-based therapy.     

miR-192, miR-194, miR-215, miR-200c and miR-141 are downregulated and their common target ACVR2B is strongly expressed in renal childhood neoplasms

Micro RNAs (miRNAs) play an important role during renal development and show a tissue-specific enrichment in the kidney. Nephroblastomas, embryonal renal neoplasms of childhood, are considered to develop from nephrogenic rests (NRs) and resemble morphologically and genetically developing kidney. We therefore investigated the role of kidney-enriched miRNAs in the pathogenesis of nephroblastomas. miR-192, miR-215 and miR-194 had a significantly lower expression in nephroblastomas regardless of the subtype compared with mature kidney measured by quantitative real-time-PCR. miR-141 and miR-200c showed a significantly lower expression in blastema-type and mixed-type tumors. In comparison with NRs, a significantly lower expression of miR-192, miR-194 and miR-215 was identified in blastema-type, mixed-type and stroma-type nephroblastomas and of miR-141 and miR-200c in blastema-type tumors. Kidney parenchyma had a significantly higher expression of miR-192, miR-194, miR-215 and miR-200c compared with NRs. In this study, the activin receptor type 2B (ACVR2B), a member of the transforming growth factor (TGF)-β pathway, was identified as single common target gene for miR-192, miR-215, miR-194, miR-141 and miR-200c in silico for the first time. The interaction between all five miRNAs and ACVR2B was also verified by an in vitro assay. Additionally, a distinct protein expression of ACVR2B was detected in 53 of 55 nephroblastomas paralleled by an upregulation of ACVR2B messenger RNA demonstrated in 25 nephroblastomas of all subtypes. A differential regulation of ACVR2B by miRNAs in NRs and nephroblastomas appears to be an important step in the pathogenesis of nephroblastomas implicating for the first time the TGF-β pathway in this process.     

The guardians of the genome (p53, TA-p73, and TA-p63) are regulators of tumor suppressor miRNAs network

The tumor suppressor p53 homologues, TA-p73, and p63 have been shown to function as tumor suppressors. However, how they function as tumor suppressors remains elusive. Here, I propose a number of tumor suppressor pathways that illustrate how the TA-p73 and p63 could function as negative regulators of invasion, metastasis, and cancer stem cells (CSCs) proliferation. Furthermore, I provide molecular insights into how TA-p73 and p63 could function as tumor suppressors. Remarkably, the guardians—p53, p73, and p63—of the genome are in control of most of the known tumor suppressor miRNAs, tumor suppressor genes, and metastasis suppressors by suppressing c-myc through miR-145/let-7/miR-34/TRIM32/PTEN/FBXW7. In particular, p53 and TA-p73/p63 appear to upregulate the expression of (1) tumor suppressor miRNAs, such as let-7, miR-34, miR-15/16a, miR-145, miR-29, miR-26, miR-30, and miR-146a; (2) tumor suppressor genes, such as PTEN, RBs, CDKN1a/b/c, and CDKN2a/b/c/d; (3) metastasis suppressors, such as Raf kinase inhibitory protein, CycG2, and DEC2, and thereby they enlarge their tumor suppressor network to inhibit tumorigenesis, invasion, angiogenesis, migration, metastasis, and CSCs proliferation.