Novel Implications of DNA Damage Response in Drug Resistance of Malignant Cancers Obtained from the Functional Interaction between p53 Family and RUNX2

During the lifespan of cells, their genomic DNA is continuously exposed to theendogenous and exogenous DNA insults. Thus, the appropriate cellular response to DNAdamage plays a pivotal role in maintaining genomic integrity and also acts as a molecularbarrier towards DNA legion-mediated carcinogenesis. The tumor suppressor p53 participatesin an integral part of proper regulation of DNA damage response (DDR). p53 is frequentlymutated in a variety of human cancers. Since mutant p53 displays a dominant-negative behavioragainst wild-type p53, cancers expressing mutant p53 sometimes acquire drug-resistantphenotype, suggesting that mutant p53 prohibits the p53-dependent cell death pathwayfollowing DNA damage, and thereby contributing to the acquisition and/or maintenance ofdrug resistance of malignant cancers. Intriguingly, we have recently found that silencing ofpro-oncogenic RUNX2 enhances drug sensitivity of aggressive cancer cells regardless of p53status. Meanwhile, cancer stem cells (CSCs) have stem cell properties such as drug resistance.Therefore, the precise understanding of the biology of CSCs is quite important to overcometheir drug resistance. In this review, we focus on molecular mechanisms behind DDR as wellas the serious drug resistance of malignant cancers and discuss some attractive approachesto improving the outcomes of patients bearing drug-resistant cancers

CD133 prevents colon cancer cell death induced by serum deprivation through activation of Akt‐mediated protein synthesis and inhibition of apoptosis

During the early phase of tumorigenesis, primary malignant cells survive within a low nutrition environment caused by a poorly organized vascular system. Here, we sought to determine the functional significance of CD133 in the survival of cancer cells under nutrient-poor conditions. Knockdown and overexpression experiments demonstrated that CD133 suppresses colon cancer cell death induced by serum deprivation through activation of Akt-mediated anti-apoptosis and protein synthesis pathways. Furthermore, serum deprivation increased the amount of endogenous CD133 protein, which was regulated at least in part by phosphoinositide 3-kinase. Thus, it is highly likely that CD133 contributes to the acquisition/maintenance of the resistance to stress arising from nutrient deficiency in early avascular tumor tissues

Fas Ligand DNA Enhances a Vaccination Effect by Coadministered DNA Encoding a Tumor Antigen through Augmenting Production of Antibody against the Tumor Antigen

Interaction of Fas and Fas ligand (FasL) plays an important role in the regulation of immune responses by inducing apoptosis of activated cells; however, a possible role of FasL in DNA vaccination has not been well understood. We examined whether administration of DNA encoding FasL gene enhanced antitumor effects in mice that were vaccinated with DNA expressing a putative tumor antigen gene, β-galactosidase (β-gal). Growth of β-gal-positive Colon 26 tumors was retarded in the syngeneic mice immunized with β-gal and FasL DNA compared with those vaccinated with β-gal or FasL DNA. We did not detect increased numbers of β-gal-specific CD8+ T cells in lymph node of mice that received combination of β-gal and FasL DNA, but amounts of anti-β-gal antibody increased with the combination but not with β-gal or FasL DNA injection alone. Subtype analysis of anti-β-gal antibody produced by the combination of β-gal and FasL DNA or β-gal DNA injection showed that IgG2a amounts were greater in mice injected with both DNA than those with β-gal DNA alone, but IgG2b amounts were lower in both DNA-injected than β-gal DNA-injected mice. These data suggest that FasL is involved in boosting humoral immunity against a gene product encoded by coinjected DNA and enhances the vaccination effects

Depletion of runt-related transcription factor 2 (RUNX2) enhances SAHA sensitivity of <i>p53</i>-mutated pancreatic cancer cells through the regulation of mutant p53 and TAp63

<div><p>Suberoylanilide hydroxamic acid (SAHA) represents one of the new class of anti-cancer drugs. However, multiple lines of clinical evidence indicate that SAHA might be sometimes ineffective on certain solid tumors including pancreatic cancer. In this study, we have found for the first time that RUNX2/mutant p53/TAp63-regulatory axis has a pivotal role in the determination of SAHA sensitivity of <i>p53</i>-mutated pancreatic cancer MiaPaCa-2 cells. According to our present results, MiaPaCa-2 cells responded poorly to SAHA. Forced depletion of mutant <i>p53</i> stimulated SAHA-mediated cell death of MiaPaCa-2 cells, which was accomapanied by a further accumulation of γH2AX and cleaved PARP. Under these experimental conditions, pro-oncogenic RUNX2 was strongly down-regulated in mutant <i>p53</i>-depleted MiaPaCa-2 cells. Surprisingly, <i>RUNX2</i> silencing augmented SAHA-dependent cell death of MiaPaCa-2 cells and caused a significant reduction of mutant p53. Consistent with these observations, overexpression of RUNX2 in MiaPaCa-2 cells restored SAHA-mediated decrease in cell viability and increased the amount of mutant p53. Thus, it is suggestive that there exists a positive auto-regulatory loop between RUNX2 and mutant p53, which might amplify their pro-oncogenic signals. Intriguingly, knockdown of mutant <i>p53</i> or <i>RUNX2</i> potentiated SAHA-induced up-regulation of TAp63. Indeed, SAHA-stimulated cell death of MiaPaCa-2 cells was partially attenuated by <i>p63</i> depletion. Collectively, our present observations strongly suggest that RUNX2/mutant p53/TAp63-regulatory axis is one of the key determinants of SAHA sensitivity of <i>p53</i>-mutated pancreatic cancer cells.</p></div

Silencing of <i>RUNX2</i> stimulates SAHA-induced accumulation of TAp63 and reduction of mutant p53.

<p>MiaPaCa-2 cells were transfected and treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179884#pone.0179884.g006" target="_blank">Fig 6A</a>. Forty-eight hours after treatment, cell lysates and total RNA were extracted and processed for immunoblotting (A) and RT-PCR (B), respectively. Actin and <i>GAPDH</i> were used as a loading and an internal control, respectively.</p

Forced depletion of mutant <i>p53</i> augments SAHA-mediated accumulation of TAp63 and reduction of RUNX2.

<p>MiaPaCa-2 cells were transfected and treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179884#pone.0179884.g004" target="_blank">Fig 4A</a>. Forty-eight hours post-treatment, cell lysates and total RNA were prepared and analyzed by immunoblotting (A) and RT-PCR (B), respectively. Actin and <i>GAPDH</i> were used as a loading and an internal control, respectively.</p

<i>p53</i>-mutated human pancreatic cancer MiaPaCa-2 and Panc-1 cells respond poorly to SAHA.

<p>MiaPaCa-2 (solid boxes), Panc-1 (grey boxes) and <i>p53</i>-proficient human breast cancer MCF-7 (open boxes) cells were exposed to DMSO, the indicated concentrations of SAHA or left untreated (W/O). Twenty-four (A) and 48 h (B) after treatment, cell viability was examined by standard WST cell survival assay.</p