Kedar Hastak

Ablation of either p21 or Bax prevents p53-dependent apoptosis induced by green tea polyphenol epigallocatechin-3-gallate

Treatment with epigallocatechin‐3‐gallate (EGCG), a polyphenolic compound of green tea, results in activation of p53 and induction of apoptosis in prostate cancer LnCaP cells. However, no direct evidence has delineated the role of p53 and p53‐dependent pathways in EGCG‐mediated apoptosis. To understand the mechanism of negative growth regulation of prostate cancer cells by EGCG we undertook a genetic approach and generated an isogenic pair of prostate carcinoma cells PC3 (p53−/−) by stably introducing a cDNA encoding wild‐type p53. Treatment of the resultant cells, PC3‐p53, with EGCG led to, as reported earlier in LnCaP cells, an increase in p53 protein, which exacerbated both G1 arrest and apoptosis. This response was accompanied by an increase in the levels of p21 and Bax. The cells lacking p53 continued to cycle and did not undergo apoptosis upon treatment with similar concentrations of EGCG, thus establishing the action of EGCG in a p53‐dependent manner. This observation was revalidated in another prostate cancer LNCaP cells harboring wild‐type p53. Inactivation of p53 using small interfering RNA (siRNA) rendered these cells resistant to EGCG‐mediated apoptosis. Because p53 activation led to increase in p21 and Bax, we investigated whether these two proteins are important in this process. Ablation of p21 protein by siRNA prevented G1 arrest and apoptosis in PC3‐p53 cells. The p53‐dependent increase in Bax expression altered the Bax/Bcl‐2 ratio and paralleled the activation of caspase 9 and 3 and cleavage of PARP. Transfection of cells with Bax siRNA abolished these effects and inhibited apoptosis but did not affect the accumulation of the cells in G1. In summary, using isogenic cell lines and siRNA, we have clearly demonstrated that EGCG activates growth arrest and apoptosis primarily via p53‐dependent pathway that involves the function of both p21 and Bax such that down‐regulation of either molecule confers a growth advantage to the cells.

Synergistic Chemosensitivity of Triple-Negative Breast Cancer Cell Lines to Poly(ADP-Ribose) Polymerase Inhibition, Gemcitabine, and Cisplatin

The basal-like subtype of breast cancer is characterized by a triple-negative (TN) phenotype (estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2/neu negative). TN breast cancers share similar gene expression profiles and DNA repair deficiencies with BRCA1-associated breast cancers. BRCA1-mutant cells exhibit sensitivity to gemcitabine, cisplatin, and poly(ADP-ribose) polymerase (PARP) inhibition; therefore, we hypothesized that TN cancer cells may also exhibit sensitivity to these drugs. In this study, we report that TN breast cancer cells are more sensitive to these drugs compared with non-TN breast cancer cells. Moreover, combination treatments indicated that PARP inhibition by the small-molecule inhibitor PJ34 or siRNA knockdown synergized with gemcitabine and cisplatin in TN cells but not in luminal cancer cells. TN cells exhibited reduced repair of UV-induced cyclobutane pyrimidine dimers after PARP inhibition, suggesting that the synergistic effect of PJ34 and gemcitabine or cisplatin reflected inefficient nucleotide excision repair. Mechanistic investigations revealed that in TN cells, PJ34 reduced the levels of ΔNp63α with a concurrent increase in p73 and its downstream target p21. Thus, the sensitivity to combination treatment seemed to be mediated by sustained DNA damage and inefficient DNA repair triggering p63/p73-mediated apoptosis. Our results suggest a novel therapeutic strategy to treat women with TN breast cancer, an aggressive disease that presently lacks effective treatment options.

Macrophage inhibitory cytokine 1 mediates a p53-dependent protective arrest in S phase in response to starvation for DNA precursors

p53 is essential for the cellular responses to DNA damage that help to maintain genomic stability. Protective p53-dependent cell-cycle checkpoints are activated in response to a wide variety of stresses, including not only DNA damage but also arrest of DNA synthesis and of mitosis. In addition to its role in activating the G1 and G2 checkpoints, p53 also helps to protect cells in S phase when they are starved for DNA precursors by treatment with the specific aspartate transcarbamylase inhibitor N-phosphonacetyl-l-aspartate (PALA), which blocks the synthesis of pyrimidine nucleotides. Even though p53 is activated, PALA-treated cells expressing low levels of p53 or lacking expression of p21 do not arrest in G1 or G2 but are blocked in S phase instead. In the complete absence of p53, PALA-treated cells continue to synthesize DNA slowly and eventually progress through S phase, suffering severe DNA damage that in turn triggers apoptosis. Expression of the secreted protein macrophage inhibitory cytokine 1 (MIC-1), a member of the TGF-β superfamily, increases substantially after PALA treatment, and application of exogenous MIC-1 or its constitutive expression from a cDNA provides remarkable protection of p53-null cells from PALA-mediated apoptosis, arguing that the p53-dependent secretion of MIC-1 provides a major part of such protection. Stimulation of MIC-1-dependent S phase arrest in normal gut epithelial cells might help to revitalize the clinical use of PALA, which has been limited by gut toxicity.

p53-dependent p21-mediated growth arrest pre-empts and protects HCT116 cells from PUMA-mediated apoptosis induced by EGCG

The tumor suppressor protein p53 plays a key role in regulation of negative cellular growth in response to EGCG. To further explore the role of p53 signaling and elucidate the molecular mechanism, we employed colon cancer HCT116 cell line and its derivatives in which a specific transcriptional target of p53 is knocked down by homologous recombination. Cells expressing p53 and p21 accumulate in G1 upon treatment with EGCG. In contrast, same cells lacking p21 traverse through the cell cycle and eventually undergo apoptosis as revealed by TUNEL staining. Treatment with EGCG leads to induction of p53, p21 and PUMA in p21 wild-type, and p53 and PUMA in p21(-/-) cells. Ablation of p53 by RNAi protects p21(-/-) cells, thus indicating a p53-dependent apoptosis by EGCG. Furthermore, analysis of cells lacking PUMA or Bax with or without p21 but with p53 reveals that all the cells expressing p53 and p21 survived after EGCG treatment. More interestingly, cells lacking both PUMA and p21 survived ECGC treatment whereas those lacking p21 and Bax did not. Taken together, our results present a novel concept wherein p21-dependent growth arrest pre-empts and protects cells from otherwise, in its absence, apoptosis which is mediated by activation of pro-apoptotic protein PUMA. Furthermore, we find that p53-dependent activation of PUMA in response to EGCG directly leads to apoptosis with out requiring Bax as is the case in response to agents that induce DNA damage. p21, thus can be used as a molecular switch for therapeutic intervention of colon cancer.

DNA synthesis from unbalanced nucleotide pools causes limited DNA damage that triggers ATR-CHK1-dependent p53 activation

p53-dependent G1 and G2 cell cycle checkpoints are activated in response DNA damage that help to maintain genomic stability. p53 also helps to protect cells from damage that occurs during S phase, for example, when the cells are starved for DNA precursors or irradiated with a low dose of UV. p53 is activated in normal cells starved for pyrimidine nucleotides by treatment with N-(phosphonacetyl)-l-aspartate (PALA). The treated cells progress through a first S phase with kinetics similar to those of untreated cells. However, the DNA of the treated cells begins to become damaged rapidly, within 12 h, as revealed by a comet assay, which detects broken DNA, and by staining for phosphorylated histone H2AX, which accumulates at sites of DNA damage. Because the cells survive, the damage must be reversible, suggesting single-strand breaks or gaps as the most likely possibility. The transiently damaged DNA stimulates activation of ATR and CHK1, which in turn catalyze the phosphorylation and accumulation of p53. Although PALA-induced DNA damage occurs only in dividing cells, the p53 that is activated is only competent to transcribe genes such as p21 and macrophage inhibitory cytokine 1 (whose products regulate G2 and G1 or S phase checkpoints, respectively) after the cells have exited the S phase during which damage occurs. We propose that p53 is activated by stimulation of mismatch repair in response to the misincorporation of deoxynucleotides into newly synthesized DNA, long before the lack of pyrimidine nucleoside triphosphates causes the rate of DNA synthesis to slow appreciably.

Identification of a Functional In Vivo p53 Response Element in the Coding Sequence of the Xeroderma Pigmentosum Group C Gene

The protein product of the xeroderma pigmentosum group C (XPC) gene is a DNA damage recognition factor that functions early in the process of global genomic nucleotide excision repair. Regulation of XPC expression is governed in part by p53 at the transcriptional level. To identify the regulatory elements involved in the p53-dependent control of XPC expression, we performed a quantitative PCR tiling experiment using multiple regularly spaced primer pairs over an 11-kb region centered around the XPC transcriptional start site. p53 chromatin immunoprecipitation was performed following ultraviolet irradiation, and DNA was analyzed for enrichment at each of 48 amplicons covering this region. A segment just upstream of the XPC translational initiation site was significantly enriched, whereas no enrichment of any other region was noted. In vitro promoter reporter assays and gel retardation assays were used to confirm the p53 responsiveness of this region and to define the minimal region with stimulating activity. We identified a p53 response element that has significant similarity to a consensus sequence, with 3 mismatches. This response element is unique in that part of the p53 binding site included the coding sequence for the first 2 amino acids in the XPC protein.

Poly (ADP-ribose) polymerase inhibitor, an effective radiosensitizer in lung and pancreatic cancers

The development of stereotactic body radiation therapy (SBRT) has revolutionized radiation therapy for lung cancers and is an emerging treatment option for pancreatic cancers. However, there are many questions on how to optimize its use in chemoradiotherapy. The most relevant addition to radiotherapy regimens are inhibitors of DNA repair and DNA damage response pathways. One such class of agents are inhibitors of poly (ADP-ribose) polymerase (PARP). In this study we examined the effects of the PARP inhibitor LT626 in combination with ionizing radiation in lung and pancreatic cancers. Our study demonstrated that combination treatment with LT626 and radiation effectively inhibited growth in lung and pancreatic cancer cell lines, better than individual treatment alone. Combination treatment also increased expression of γH2AX and 53BP1 foci and upregulated expression of phosphorylated ATM, ATR and their respective kinases. Using in vivo lung cancer xenograft models we demonstrated that LT626 functioned as an effective radiosensitizer during fractionated radiation treatment, leading to significant decrease in tumor burden and doubling the median survival compared to control group. Overall our in vitro and in vivo studies showed that PARP inhibitor LT626 acted synergistically with radiation in lung and pancreatic cancers.

Abstract 5490: Poly (ADP-ribose) polymerase inhibitor LT-626 sensitizes lung and pancreatic cancers to fractionated radiation therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The development of stereotactic body radiation therapy (SBRT) has revolutionized radiation therapy for lung cancers and is an emerging treatment option for pancreatic cancers. However, there are many questions on how to optimize its use in chemoradiotherapy. The most relevant addition to radiotherapy regimens are inhibitors of DNA repair and DNA damage response pathways. One such class of agents are small-molecule inhibitors of poly (ADP-ribose) polymerase (PARP). PARP inhibitors target DNA base excision repair and radiosensitize cells through impaired DNA repair. In this study we examined the effects of the PARP inhibitor LT-626 (BioMarin) together with ionizing radiation in lung and pancreatic cancers, in vitro and in vivo. Clonogenic assays showed that lung cancer cells H1299 and H460 and pancreatic cancer cells Miapaca2 and PDA were sensitive to LT-626 (IC50 from 0.1-5.8 µM) and irradiation (IC50 from 2.2-5.9 Gy). Next, we found the combination of LT-626 and irradiation effective for inhibiting growth in lung and pancreatic cancer cells. Our study showed that both 1 and 10 µM concentrations of LT-626 were highly synergistic with 2, 4 and 6 Gy of irradiation in lung and pancreatic cell lines. Furthermore, in a fractionated radiation regimen study pretreatment with LT-626 followed by irradiation for three days significantly decreased cell survival as studied by clonogenic assay compared to LT-626 or radiation alone. Lung and pancreatic cancer cells treated with LT-626 and irradiation also exhibited DNA damage as evident by increased γH2AX and Rad51 foci formation. However, DNA damage caused by irradiation peaked by 2-4 h following treatment compared with LT-626 treated cells which exhibited maximum DNA damage around 24 h after treatment. Our in vitro study clearly demonstrated that PARP inhibition enhanced the efficacy of irradiation; we therefore decided to study the efficacy LT-626 and radiation therapy in vivo, using two lung xenograft (H1299 and H460) cancer models. After tumor implantation mice were treated with either drug alone (10mg/kg or 20 mg/kg) or radiation alone (2 Gy) or a combination of drug and radiation for five consecutive days after which they were followed until the end of the study. We found that combination of radiation plus LT-626 significantly decreased tumor burden in both H1299 and H460 xenograft models compared to drug alone or radiation alone group. Moreover, the median survival in combination treated animals more than doubled compared to vehicle treated group. Overall our in vitro and in vivo studies proved that PARP inhibitor LT-626 acts synergistically with fractionated irradiation in lung and pancreatic cancers. Citation Format: Kedar Hastak, Lisa McPherson, Yuqiao Shen, Renate Parry, James M. Ford. Poly (ADP-ribose) polymerase inhibitor LT-626 sensitizes lung and pancreatic cancers to fractionated radiation therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5490. doi:10.1158/1538-7445.AM2014-5490