ATM and Chk2 kinase target the p53 cofactor Strap

The p53 cofactor Strap (stress responsive activator of p300) is directly targeted by the DNA damage signalling pathway where phosphorylation by ATM (ataxia telangiectasia mutated) kinase facilitates nuclear accumulation. Here, we show that Strap regulation reflects the coordinated interplay between different DNA damage-activated protein kinases, ATM and Chk2 (Checkpoint kinase 2), where phosphorylation by each kinase provides a distinct functional consequence on the activity of Strap. ATM phosphorylation prompts nuclear accumulation, which we show occurs by impeding nuclear export, whereas Chk2 phosphorylation augments protein stability once Strap has attained a nuclear location. These results highlight the various functional roles undertaken by the DNA damage signalling kinases in Strap control and, more generally, shed light on the pathways that contribute to the regulation of the p53 response.     

HDM2 negatively affects the Chk2-mediated phosphorylation of p53

By GST pull downs and co-immunoprecipitation analyses we found that recombinant Chk2 and HDM2 can form stable complexes in vitro. Chk2/HDM2 complexes were also detected in transfected Cos-1 cells over-expressing both proteins. Furthermore, we show that HDM2, as would be expected, severely affects the Chk2-catalyzed phosphorylation of p53. HDM2 itself is only slightly phosphorylated by Chk2. However, whereas HDM2 inhibits the Chk2-catalyzed p53 phosphorylation, HDM2 phosphorylation by Chk2 doubles in the presence of p53. The significance of the HDM2 phosphorylation is unknown, but it is possible that it might influence the stability of the HDM2/p53 complex.     

Low ATM protein expression and depletion of p53 correlates with olaparib sensitivity in gastric cancer cell lines

Small-molecule inhibitors of poly (ADP-ribose) polymerase (PARP) have shown considerable promise in the treatment of homologous recombination (HR)-defective tumors, such as BRCA1- and BRCA2-deficient breast and ovarian cancers. We previously reported that mantle cell lymphoma cells with deficiency in ataxia telangiectasia mutated (ATM) are sensitive to PARP-1 inhibitors in vitro and in vivo. Here, we report that PARP inhibitors can potentially target ATM deficiency arising in a solid malignancy. We show that ATM protein expression varies between gastric cancer cell lines, with NUGC4 having significantly reduced protein levels. Significant correlation was found between ATM protein expression and sensitivity to the PARP inhibitor olaparib, with NUGC4 being the most sensitive. Moreover, reducing ATM kinase activity using a small-molecule inhibitor (KU55933) or shRNA-mediated depletion of ATM protein enhanced olaparib sensitivity in gastric cancer cell lines with depletion or inactivation of p53. Our results demonstrate that ATM is a potential predictive biomarker for PARP-1 inhibitor activity in gastric cancer harboring disruption of p53, and that combined inhibition of ATM and PARP-1 is a rational strategy for expanding the utility of PARP-1 inhibitors to gastric cancer with p53 disruption.     

Resveratrol- induced apoptosis is mediated by p53-dependent pathway in Hep G2 cells

Resveratrol, a phytoalexin found in many plants, has been reported to possess a wide range of pharmacological properties and is one of the promising chemopreventive agents for cancer. Here, we examined the antiproliferation effect of resveratrol in two human liver cancer cell lines, Hep G2 and Hep 3B. Our results showed that resveratrol inhibited cell growth in p53-positive Hep G2 cells only. This anticancer effect was a result of cellular apoptotic death induced by resveratrol via the p53-dependent pathway. Here we demonstrated that the resveratrol-treated cells were arrested in G1 phase and were associated with the increase of p21 expression. In addition, we also illustrated that the resveratrol-treated cells had enhanced Bax expression but they were not involved in Fas/APO-1 apoptotic signal pathway. In contrast, the p53-negative Hep 3B cells treated with resveratrol did not show the antiproliferation effect neither did they show significant changes in p21 nor Fas/APO-1 levels. In summary, our study demonstrated that the resveratrol effectively inhibited cell growth and induced programmed cell death in Hepatoma cells on a molecular basis. Furthermore, these results implied that resveratrol might also be a new potent chemopreventive drug candidate for liver cancer as it played an important role to trigger p53-mediated molecules involved in the mechanism of p53-dependent apoptotic signal pathway.     

Kaempferol induces apoptosis in human HCT116 colon cancer cells via the Ataxia-Telangiectasia Mutated-p53 pathway with the involvement of p53 Upregulated Modulator of Apoptosis

Dietary flavonols have been found to possess preventive and therapeutic potential against several kinds of cancers. This study is conducted to investigate the anti-proliferation effects of kaempferol, a major component of food flavonols, against colon cancer cells. In the human HCT116 colon cancer cell line, kaempferol induced p53-dependent growth inhibition and apoptosis. Furthermore, kaempferol was found to induce cytochrome c release from mitochondria and activate caspase-3 cleavage. The Bcl-2 family proteins including PUMA were involved in this process. Kaempferol also induced ATM and H2AX phosphorylation in HCT116 cells, inhibition of ATM by a chemical inhibitor resulted in abrogation of the downstream apoptotic cascades. These findings suggest kaempferol could be a potent candidate for colorectal cancer management.     

Modulation of Janus kinase 2 by p53 in ovarian cancer cells

The constitutive activation of the Janus kinase 2 (JAK2) and mutation of the p53 tumor suppressor are both detected in human cancer. We examined the potential regulation of JAK2 phosphorylation by wild-type (wt) p53 in human ovarian cancer cell lines, Caov-3 and MDAH2774, which harbor mutant form of p53 tumor suppressor gene and high levels of phosphorylated JAK2. The wt p53 gene was re-introduced into the cells using an adenovirus vector. In addition to wt p53, mutant p53 22/23, mutant p53-175, and NCV (negative control virus) were introduced into the cells in the control groups. Expression of wt p53, but not that of p53-175 mutant, diminished JAK2 tyrosine phosphorylation in MDAH2774 and Caov-3 cell lines. Expression of wt p53 or p53 22/23 mutant did not cause a reduction in the phosphorylation of unrelated protein kinases, ERK1 and ERK2 (ERK1/2). The inhibition of JAK2 tyrosine phosphorylation can be reversed by tyrosine phosphatase inhibitor, sodium orthovanadate. Protein tyrosine phosphatase 1-B levels increased with introduction of wt p53 and may be involved in the dephosphorylation of JAK2. These findings present a possible p53-dependent cellular process of modulating JAK2 tyrosine phosphorylation in ovarian cancer cell lines.     

The p53/miRNAs/Ccna2 pathway serves as a novel regulator of cellular senescence: Complement of the canonical p53/p21 pathway

Aging is a multifactorial process characterized by the progressive deterioration of physiological functions. Among the multiple molecular mechanisms, microRNAs (miRNAs) have increasingly been implicated in the regulation of Aging process. However, the contribution of miRNAs to physiological Aging and the underlying mechanisms remain elusive. We herein performed high‐throughput analysis using miRNA and mRNA microarray in the physiological Aging mouse, attempted to deepen into the understanding of the effects of miRNAs on Aging process at the “network” level. The data showed that various p53 responsive miRNAs, including miR‐124, miR‐34a and miR‐29a/b/c, were up‐regulated in Aging mouse compared with that in Young mouse. Further investigation unraveled that similar as miR‐34a and miR‐29, miR‐124 significantly promoted cellular senescence. As expected, mRNA microarray and gene co‐expression network analysis unveiled that the most down‐regulated mRNAs were enriched in the regulatory pathways of cell proliferation. Fascinatingly, among these down‐regulated mRNAs, Ccna2 stood out as a common target of several p53 responsive miRNAs (miR‐124 and miR‐29), which functioned as the antagonist of p21 in cell cycle regulation. Silencing of Ccna2 remarkably triggered the cellular senescence, while Ccna2 overexpression delayed cellular senescence and significantly reversed the senescence‐induction effect of miR‐124 and miR‐29. Moreover, these p53 responsive miRNAs were significantly up‐regulated during the senescence process of p21‐deficient cells; overexpression of p53 responsive miRNAs or knockdown of Ccna2 evidently accelerated the cellular senescence in the absence of p21. Taken together, our data suggested that the p53/miRNAs/Ccna2 pathway might serve as a novel senescence modulator independent of p53/p21 pathway.     

Molecular interaction map of the p53 and Mdm2 logic elements, which control the Off-On switch of p53 in response to DNA damage

The molecular network that controls responses to genotoxic stress is centered at p53 and Mdm2. Recent findings have shown this network to be more complex than previously envisioned. Using a notation specifically designed for circuit diagram-like representations of bioregulatory networks, we have prepared an updated molecular interaction map of the immediate connections of p53 and Mdm2, which are described as logic elements of the network. We use the map as the basis for a comprehensive review of current concepts of signal processing by these logic elements (an interactive version of the maps-eMIMs can be examined at ). We also used molecular interaction maps to propose a p53 Off-On switch in response to DNA damage.     

Wip1 cooperates with KPNA2 to modulate the cell proliferation and migration of colorectal cancer via a p53-dependent manner

Due to the increasing incidence and mortality, the early diagnosis, specific targeted therapies, and prognosis for colorectal cancer (CRC) attract more and more attention. Wild-type p53-induced phosphatase 1 (Wip1) and karyopherin α2 (KPNA2) have been regarded as oncogenes in many cancers, including CRC. Wip1 dephosphorylates p53 to inactivate it. TP53 activator and Wip1 inhibitor downregulate KPNA2 expression. Therefore, we speculate that Wip1 may co-operate with KPNA2 to modulate CRC progression in a p53-dependent manner. Here, Wip1 and KPNA2 messenger RNA expression and protein levels are significantly increased in CRC tissues and cell lines and are positively correlated with each other. Wip1 silence increases p53 phosphorylation while decreases KPNA2 protein. Wip1 knockdown remarkably suppresses CRC cell proliferation and migration while KPNA2 overexpression exerts an opposing effect. KPNA2 overexpression could partially rescue Wip1 silence-inhibited CRC cell proliferation and migration. Finally, Wip1 interacts with KPNA2 to modulate the activation of AKT/GSK-3β signaling and metastasis-related factors. In summary, Wip1 could co-operate with KPNA2 to modulate CRC cell proliferation and migration, possibly via a p53-dependent manner, through downstream AKT/GSK-3β pathway. We provided a novel mechanism of Wip1 interacting with KPNA2, therefore modulating CRC cell proliferation and migration.     

miR-34a in extracellular vesicles from bone marrow mesenchymal stem cells reduces rheumatoid arthritis inflammation via the cyclin I/ATM/ATR/p53 axis

Extracellular vesicles (Evs) participate in the development of rheumatoid arthritis (RA), but the mechanisms remain unclear. This study aimed to determine the mechanism by which microRNA-34a (miR-34a) contained in bone marrow mesenchymal stem cell (BM-MSC)-derived Evs functions in RA fibroblast-like synoviocytes (RA-FLSs). BM-MSC-derived Evs and an Evs inhibitor were extracted. A rat model of RA was established. miR-34a gain- and loss-of-function experiments were performed, and the inflammation in rat synovial fluid and tissues was detected. The role of miR-34a in RA-FLSs was also measured in vitro. The target gene of miR-34a was predicted using the online software TargetScan and identified using a dual-luciferase reporter gene assay, and the activation of the ATM/ATR/p53 signalling pathway was assessed. BM-MSC-derived Evs mainly elevated miR-34a expression, which reduced RA inflammation in vivo and inhibited RA-FLS proliferation and resistance to apoptosis in vitro, while inhibited miR-34a expression enhanced RA development. In addition, miR-34a could target cyclin I to activate the ATM/ATR/p53 signalling pathway, thus inhibiting abnormal RA-FLS growth and RA inflammation. Our study showed that miR-34a contained in BM-MSC-derived Evs could reduce RA inflammation by inhibiting the cyclin I/ATM/ATR/p53 signalling pathway.     

LncRNA MST1P2/miR‐133b axis affects the chemoresistance of bladder cancer to cisplatin‐based therapy via Sirt1/p53 signaling

Although bladder cancer is commonly chemosensitive to standard first‐line therapy, the acquisition of the resistance to cisplatin (DDP)‐based therapeutic regimens remains a huge challenge. Noncoding RNAs (ncRNAs), including long noncoding RNAs (lncRNAs) and microRNAs, have been reported to play a critical role in cancer resistance to DDP. Here, we attempted to provide a novel mechanism by which the resistance of bladder cancer to DDP treatment could be modulated from the perspective of ncRNA regulation. We demonstrated that lncRNA MST1P2 (lnc‐MST1P2) expression was dramatically upregulated, whereas miR‐133b expression was downregulated in DDP‐resistant bladder cancer cell lines, SW 780/DDP and RT4/DDP. Lnc‐MST1P2 and miR‐133b negatively regulated each other via targeting miR‐133b. Both lnc‐MST1P2 silence and miR‐133b overexpression could resensitize DDP‐resistant bladder cancer cells to DDP treatment. More important, miR‐133b could directly target the Sirt1 3′‐untranslated region to inhibit its expression. Inc‐MST1P2/miR‐133b axis affected the resistance of bladder cancer cells to DDP via Sirt1/p53 signaling. In conclusion, MST1P2 serves as a competing endogenous RNA for miR‐133b to counteract miR‐133b‐induced suppression on Sirt1, therefore enhancing the resistance of bladder cancer cells to DDP. MST1P2/miR‐133b axis affects the resistance of bladder cancer cells to DDP via downstream Sirt1/p53 signaling.     

DNAJB1 stabilizes MDM2 and contributes to cancer cell proliferation in a p53-dependent manner

Both MDM2 and MDMX regulate p53, but these proteins play different roles in this process. To clarify the difference, we performed a yeast 2 hybrid (Y2H) screen using the MDM2 acidic domain as bait. DNAJB1 was found to specifically bind to MDM2, but not MDMX, in vitro and in vivo. Further investigation revealed that DNAJB1 stabilizes MDM2 at the post-translational level. The C-terminus of DNAJB1 is essential for its interaction with MDM2 and for MDM2 accumulation. MDM2 was degraded faster by a ubiquitin-mediated pathway when DNAJB1 was depleted. DNAJB1 inhibited the MDM2-mediated ubiquitination and degradation of p53 and contributed to p53 activation in cancer cells. Depletion of DNAJB1 in cancer cells inhibited activity of the p53 pathway, enhanced the activity of the Rb/E2F pathway, and promoted cancer cell growth in vitro and in vivo. This function was p53 dependent, and either human papillomavirus (HPV) E6 protein or siRNA against p53 was able to block the contribution caused by DNAJB1 depletion. In this study, we discovered a new MDM2 interacting protein, DNAJB1, and provided evidence to support its p53-dependent tumor suppressor function.     

Expression of securin promotes colorectal cancer cell death via a p53-independent pathway after radiation

Securin has been shown to regulate genomic stability; nevertheless, the role of securin on the cytotoxicity after radiation is still unclear. Exposure to 1–10 Gy X-ray radiation induced cell death in RKO colorectal cancer cells. The protein levels of securin, p53, and p21 were elevated by radiation. The proteins of phosphorylation of p53 at serine-15, which located on the nuclei of cancer cells, were highly induced by radiation. However, radiation increased securin proteins, which located on both of nuclei and cytoplasma in RKO cells. The p53-wild type colorectal cancer cells were more susceptible on cytotoxicity than the p53-mutant cells following exposure to radiation. Besides, the existence of securin in colorectal cancer cells induced higher apoptosis than the securin-null after radiation. Securin proteins were elevated by radiation in the p53-wild type and -mutant cells; furthermore, radiation raised the p53 protein expression in both the securin-wild type and -null cells. As a whole, these findings suggest that the existence of securin promotes apoptosis via a p53-indpendent pathway after radiation in human colorectal cancer cells.     

Chloride ions control the G1/S cell-cycle checkpoint by regulating the expression of p21 through a p53-independent pathway in human gastric cancer cells

The aim of the present study is to investigate whether the chloride affects cell growth and cell-cycle progression of cancer cells. In human gastric cancer MKN28 cells, the culture in the Cl⁻-replaced medium (replacement of Cl⁻ by NO₃⁻) decreased the intracellular chloride concentration ([Cl⁻]_i) and inhibited cell growth. The inhibition of cell growth was due to cell-cycle arrest at the G₀/G₁ phase caused by diminution of CDK2 and phosphorylated Rb. The culture of cells in the Cl⁻-replaced medium significantly increased expressions of p21 mRNA and protein without any effects on p53. These observations indicate that chloride ions play important roles in cell-cycle progression by regulating the expression of p21 through a p53-independent pathway in human gastric cancer cells, leading to a novel, unique therapeutic strategy for gastric cancer treatment via control of [Cl⁻]_i.