Augmented antitumor activity of 5‐fluorouracil by double knockdown of MDM4 and MDM2 in colon and gastric cancer cells

Inactivation of the TP53 tumor suppressor gene is essential during cancer development and progression. Mutations of TP53 are often missense and occur in various human cancers. In some fraction of wild‐type (wt) TP53 tumors, p53 is inactivated by upregulated murine double minute homolog 2 (MDM2) and MDM4. We previously reported that simultaneous knockdown of MDM4 and MDM2 using synthetic DNA‐modified siRNAs revived p53 activity and synergistically inhibited in vitro cell growth in cancer cells with wt TP53 and high MDM4 expression (wtTP53/highMDM4). In the present study, MDM4/MDM2 double knockdown with the siRNAs enhanced 5‐fluorouracil (5‐FU)‐induced p53 activation, arrested the cell cycle at G₁ phase, and potentiated the antitumor effect of 5‐FU in wtTP53/highMDM4 human colon (HCT116 and LoVo) and gastric (SNU‐1 and NUGC‐4) cancer cells. Exposure to 5‐FU alone induced MDM2 as well as p21 and PUMA by p53 activation. As p53‐MDM2 forms a negative feedback loop, enhancement of the antitumor effect of 5‐FU by MDM4/MDM2 double knockdown could be attributed to blocking of the feedback mechanism in addition to direct suppression of these p53 antagonists. Intratumor injection of the MDM4/MDM2 siRNAs suppressed in vivo tumor growth and boosted the antitumor effect of 5‐FU in an athymic mouse xenograft model using HCT116 cells. These results suggest that a combination of MDM4/MDM2 knockdown and conventional cytotoxic drugs could be a promising treatment strategy for wtTP53/highMDM4 gastrointestinal cancers.     

MDM4 alternative splicing and implication in MDM4 targeted cancer therapies

The oncogenic MDM4, initially named MDMX, has been identified as a p53-interacting protein and a key upstream negative regulator of the tumor suppressor p53. Accumulating evidence indicates that MDM4 plays critical roles in the initiation and progression of multiple human cancers. MDM4 is frequently amplified and upregulated in human cancers, contributing to overgrowth and apoptosis inhibition by blocking the expression of downstream target genes of p53 pathway. Disruptors for MDM4-p53 interaction have been shown to restore the anti-tumor activity of p53 in cancer cells. MDM4 possesses multiple splicing isoforms whose expressions are driven by the presence of oncogenes in cancer cells. Some of the MDM4 splicing isoforms lack p53 binding domain and may exhibit p53-independent oncogenic functions. These features render MDM4 to be an attractive therapeutic target for cancer therapy. In the present review, we primarily focus on the detailed molecular structure of MDM4 splicing isoforms, candidate regulators for initiating MDM4 splicing, deregulation of MDM4 isoforms in cancer and potential therapy strategies by targeting splicing isoforms of MDM4.     

Epstein‐Barr virus‐encoded EBNA‐5 forms trimolecular protein complexes with MDM2 and p53 and inhibits the transactivating function of p53

We report that MDM2, a negative regulator of p53, can bind to EBNA‐5. Using GST pull‐down assay, immunoprecipitation, surface plasmon resonance and immunostaining of lymphoblastoid cells, we found that trimolecular complexes are formed between EBNA‐5, MDM2 and p53, where MDM2 serves as a bridge. The EBNA‐5 binding to MDM2 counteracted destabilizing effect of the latter on the p53. In ubiquitination and degradation assays in vitro, EBNA‐5 inhibited p53 polyubiquitination (but not monoubiquitination) in a concentration‐dependent manner. This resembles the effect of p14ARF on p53. Moreover, EBNA‐5 was found to inhibit the degradation of p53 in vitro. High levels of p53 expression were maintained in LCLs. The binding of EBNA‐5 to MDM2 also could impair the functional activity of p53. The p53‐dependent genes P21 and VDR were not induced in EBV‐infected, in contrast to mitogen‐activated cells. This may explain the tolerance of established LCLs to high levels of p53 without undergoing apoptosis.     

S‐MDM4 mRNA overexpression indicates a poor prognosis and marks a potential therapeutic target in chronic lymphocytic leukemia

The purpose of the present study was to investigate the prognostic significance of murine double minute 4 (MDM4) in chronic lymphocytic leukemia (CLL) and to characterize the role of MDM4 in the p53 pathway. Full‐length MDM4 (FL‐MDM4), a splicing variant of MDM4 (S‐MDM4) and murine double minute 2 (MDM2) mRNA expressions were detected by quantitative PCR in 140 Chinese patients with CLL, and primary CLL cells were treated in vitro with either fludarabine or Nutlin‐3 to explore the interaction between p53 status and MDM4 or MDM2 expression. A marked increase of FL‐MDM4 and S‐MDM4 expressions were observed in the CLL patients with p53 aberrations (deletion and/or mutation) (P = 0.024, P < 0.001). A high level of S‐MDM4 mRNA expression was associated with short treatment free survival (TFS) (P = 0.004). FL‐MDM4 expression was significantly decreased after fludarabine treatment (P = 0.001) but increased after Nutlin‐3 treatment (P = 0.008) of primary CLL cells without p53 aberrations. Both S‐MDM4 and MDM2 expressions were significantly increased after fludarabine treatment of CLL cells without p53 aberrations (P = 0.013 and P = 0.030). MDM2 overexpression also occurred in CLL cells with p53 wild type after Nutlin‐3 treatment (P = 0.018). FL‐MDM4 and S‐MDM4 overexpression are indicators of p53 aberrations in CLL patients, suggesting that those patients have a poor prognosis. FL‐MDM4 inhibitory effects on p53 can be removed by MDM2‐p53 and saved by Nutlin‐3.     

Long noncoding RNA actin filament‐associated protein 1 antisense RNA 1 promotes malignant phenotype through binding with lysine‐specific demethylase 1 and repressing HMG box‐containing protein 1 in non‐small‐cell lung cancer

The number of documented long noncoding RNAs (lncRNAs) has dramatically increased, and their biological functions and underlying mechanisms in pathological processes, especially cancer, remain to be elucidated. Actin filament‐associated protein 1 antisense RNA 1 (AFAP1‐AS1) is a 6810‐nt lncRNA located on chromosome 4p16.1 that was first reported to be upregulated in esophageal adenocarcinoma tissues and cell lines. Here we reported that AFAP1‐AS1, recruiting and binding to lysine‐specific demethylase 1 (LSD1), was generally overexpressed in human non‐small‐cell lung cancer (NSCLC) tissues using quantitative real‐time PCR. Higher AFAP1‐AS1 expression was significantly correlated with larger tumor size (P = .008), lymph node metastasis (P = .025), higher TNM stage (P = .024), and worse overall survival in NSCLC patients. In vitro experiments revealed that AFAP1‐AS1 downregulation inhibited cell migration and induced apoptosis; AFAP1‐AS1 knockdown also hindered tumorigenesis in vivo. Moreover, mechanistic investigations including RNA immunoprecipitation and ChIP assays validated that AFAP1‐AS1 repressed HMG box‐containing protein 1 (HBP1) expression by recruiting LSD1 to the HBP1 promoter regions in PC‐9 and H1975 cells. Furthermore, HBP1 functions as a tumor suppressor, and its ectopic expression hindered cell proliferation. Rescue assays determined that the oncogenic effect of AFAP1‐AS1 is partially dependent on the epigenetic silencing of HBP1. In conclusion, our results indicate that AFAP1‐AS1 is carcinogenic and that the AFAP1‐AS1/LSD1/HBP1 axis could constitute a new therapeutic direction for NSCLC.     

Involvement of p53 in oroxylin A‐induced apoptosis in cancer cells

Oroxylin A, a naturally occurring monoflavonoid extracted from Scutellariae radix, exhibits anticancer activity and induces apoptosis in human hepatocellular carcinoma HepG2 cells according to our previous data. In this study, we investigate whether p53 is involved in oroxylin A‐triggered viability inhibition and apoptosis induction in cancer cells. In a panel of different cancer cell lines, more potent inhibitory effects of oroxylin A were observed in wtp53 cells than those in mtp53 or p53‐null cells. Moreover, p53‐siRNA‐transfected HepG2 cells showed lower levels of apoptosis induced by oroxylin A than control‐siRNA‐transfected cells. Likewise, after oroxylin A treatment, p53‐null K‐562 cells displayed promoted apoptosis rate when transfected with wtp53 plasmid. Western blot and real‐time RT‐PCR assay revealed that oroxylin A markedly upregulated p53 protein expression in HepG2 and p53‐overexpressing K‐562 cells, but had no influence on p53 mRNA synthesis. Furthermore, after co‐treatment with cycloheximide, oroxylin A still exerted a little effect on p53 expression. The negative regulator of p53, MDM2 protein was detected, and downregulated expression was observed. In the presence of MG132, an inhibitor of proteasome‐mediated proteolysis, no change in p53 expression was obtained. Additionally, the antioxidant N‐acetyl‐L‐cysteine could obviously abrogate p53 stabilization triggered by oroxylin A. Therefore, it is summarized that oroxylin A stabilized p53 expression and induced apoptosis at the posttranslational level via downregulating MDM2 expression and interfering MDM2‐modulated proteasome‐related p53 degradation. This indicated that oroxylin A could be served as a potential, novel agent candidate for cancer therapy. © 2009 Wiley‐Liss, Inc.     

DN‐R175H p53 mutation is more effective than p53 interference in inducing epithelial disorganization and activation of proliferation signals in human carcinoma cells: Role of E‐cadherin

One of the hallmarks of carcinomas is epithelial disorganization, linked to overexpression of matrix metalloproteases (MMP) like MMP‐9, loss of intercellular E‐cadherin and activation of epidermal growth receptor (EGFR/erbB1). Since the p53 tumor suppressor pathway is inactivated in most human cancers due to gene mutations or defective wt p53 signaling, we now investigated in human wt p53 breast carcinoma MCF‐7 cells, whether single treatment with the p53 transactivation pharmacological inhibitor pifithrin‐α, transient p53 siRNA interference or stable insertion of a dominant‐negative (DN) R175H p53 mutant increase: (i) EGFR/erbB1 activation, (ii) MMP‐9 expression and (iii) loss of surface E‐cadherin. Transient abrogation of wt p53 function increased phosphorylation of EGFR/erbB1 and MMP‐9 expression. However, all these effects were much more pronounced in cells stably transduced with the dominant negative–Arg‐175His mutant p53 (DN‐R175H mutant p53), which also showed loss of epithelial cytoarchitecture and extensive E‐cadherin downregulation. Collectively, these results support the notion that the DN‐R175H mutant p53 exerts a gain of oncogenic function by promoting disruption of E‐cadherin intercellular contacts and activation of proliferation signals. Our data suggests that epithelial shape and growth control are unequally affected depending on how wt p53 function is impaired and whether partial or full tumor suppressor activity is lost. © 2009 UICC     

Inhibitors of differentiation‐1 promotes nitrosopyrrolidine‐induced transformation of HPV 16‐immortalized cervical epithelial cell

Our previous study implied a correlation between inhibitors of differentiation‐1 (Id‐1) and cervical cancer development. However, how Id‐1 contributes to cervical carcinogenesis is unknown. In the present study, we used an in vitro transformation model to investigate the role of Id‐1 in the transformation of cervical cells. Human papillomavirus (HPV)‐immortalized cervical epithelial cells (H8) were successfully transformed by exposure to the carcinogen N‐nitrosopyrrolidine (NPYR). The expression of both Id‐1 RNA and protein was significantly increased in transformed H8 cells, suggesting a possible role of Id‐1 in cervical cell transformation. Ectopic expression of Id‐1 in H8 cells potentiated NPYR‐induced cell transformation. In contrast, silencing of Id‐1 suppressed NPYR‐induced H8 cell transformation. In addition, the expression of HPV E6 and E7 oncoproteins was upregulated while that of the tumor suppressors p53 and pRb was suppressed after H8 cell transformation. Our results suggest that Id‐1 plays an oncogenic role in HPV‐related cervical carcinogenesis, which sheds light on cervical cancer development mechanisms and implies that Id‐1 is a potential target for cervical cancer prevention and therapy.     

Mouse p10, an alternative spliced form of p15INK4b, inhibits cell cycle progression and malignant transformation

The INK4 family of proteins negatively regulates cell cycle progression at the G(1)-S transition by inhibiting cyclin-dependent kinases. Two of these cell cycle inhibitors, p16(INK4A) and p15(INK4B), have tumor suppressor activities and are inactivated in human cancer. Interestingly, both INK4 genes express alternative splicing variants. In addition to p16(INK4A), the INK4A locus encodes a splice variant, termed p12--specifically expressed in human pancreas--and ARF, a protein encoded by an alternative reading frame that acts as a tumor suppressor through the p53 pathway. Similarly, the human INK4B locus encodes the p15(INK4B) tumor suppressor and one alternatively spliced form, termed as p10. We show here that p10, which arises from the use of an alternative splice donor site within intron 1, is conserved in the mouse genome and is widely expressed in mouse tissues. Similarly to mouse p15(INK4B), p10 expression is also induced by oncogenic insults and transforming growth factor-beta treatment and acts as a cell cycle inhibitor. Importantly, we show that mouse p10 is able to induce cell cycle arrest in a p53-dependent manner. We also show that mouse p10 is able to inhibit foci formation and anchorage-independent growth in wild-type mouse embryonic fibroblasts, and that these antitransforming properties of mouse p10 are also p53-dependent. These results indicate that the INK4B locus, similarly to INK4A-ARF, harbors two different splicing variants that can be involved in the regulation of both the p53 and retinoblastoma pathways, the two major molecular pathways in tumor suppression.     

Li–Fraumeni and Li–Fraumeni‐like syndrome mutations in p53 are associated with exonic methylation and splicing regulatory elements

Mutations in codon 133 of p53, which cause the loss of the Δ133 isoform(s) expression, are very frequent in the Li–Fraumeni (LF) and Li–Fraumeni‐like (LFL) syndromes. In sporadic cancers, silent p53 mutations are correlated with exonic splicing enhancers (ESEs) and exonic methylated sites. The present study shows that mutations in splice sites are also very frequent in LF/LFL syndromes, while missense mutations are less common compared to other familial or sporadic cancers (P = 0 in both cases). Furthermore, it is shown that the codons at which LF/LFL germline missense mutations occur, correlate with CpG‐containing ESEs (r = 0.181, P = 0.014) which are all methylated in p53. While both silent and LF/LFL missense mutations correlate with SC35 motifs, only the latter are associated with SRp55. On the contrary, only silent mutations in sporadic cancers correlate with SF2/ASF motifs in p53. Moreover, 12.1% of LF/LFL missense mutations involve the formation of potential splice sites of considerable splicing scores. Finally, mutations that are not at, or adjacent to CpGs (±1 codon, 34% of all LF/LFL mutated sites), introduce considerable changes of the ESE scores (>1.3 score change). The above data verify that LF/LFL missense mutations probably result also in splicing deregulation, in addition to any changes of the protein function and are mostly associated with alterations of the exonic methylation landscape. Some of the ESEs affected in LF/LFL syndromes are also genetically unstable in sporadic cancers but non‐CpG cytosine instability, which is predominantly associated with specific ESEs, is only common in sporadic cancers. Mol. Carcinog. © 2009 Wiley‐Liss, Inc.     

Alternative splicing related genetic variants contribute to bladder cancer risk

Emerging evidence has shown that aberrant alternative splicing (AS) events are involved in the carcinogenesis. The association between genetic variants in AS and bladder cancer susceptibility remains to be fully elucidated. We searched for single nucleotide polymorphisms (SNPs) which are located in splicing quantitative trait loci (sQTLs) in bladder cancer through CancerSplicingQTL database and the 1000 Genomes Project. A case-control study including 580 cases and 1,101 controls was conducted to assess the association between the functional genetic variants and bladder cancer risk. Next, we used GTEx, TCGA, and GEO databases conducting sQTL analysis and gene expression differences analysis to evaluate the potential biological function of the candidate SNPs and related genes. We found that SNP rs4383 C>G was remarkably related with the reduced risk of bladder cancer (odds ratio = 0.68, 95% confidence interval = 0.59-0.79, P = 3.91 × 10⁻⁷). Similar results were obtained in codominant, dominant and recessive model. Stratified analyses revealed that the effect of SNP rs4383 C>G on bladder cancer was more significant in the older subjects (age > 65), female and nonsmokers. sQTL analysis showed that SNP rs4383 was associated with the AS events of its downstream gene MAFF with a splicing event of alternative 5′ splice site. The messenger RNA expression of MAFF in bladder tumor tissues was lowered compared with normal tissues. Patients with high expression of MAFF had higher survival rates. These findings indicated that SNP rs4383 related with the AS events of MAFF was associated with bladder cancer risk and could represent a possible biomarker for bladder cancer susceptibility.