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Array-based genome-wide RNAi screening to identify shRNAs that enhance p53-related apoptosis in human cancer cells.

Idogawa M, Ohashi T, Sugisaka J, Sasaki Y, Suzuki H, Tokino T - Oncotarget (2014)

Bottom Line: We screened a genome-wide lentiviral shRNA library in liver cancer Huh-7 and pancreatic cancer Panc-1 cells, both of which resist p53-induced apoptosis.We identified shRNAs that were significantly decreased in p53-infected cells compared with control cells.Furthermore, the enhanced apoptotic response by shRNA-58335 was also confirmed by treatment with PRIMA-1, which reactivates mutant p53, instead of adenoviral p53 transduction.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genome Sciences, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan. Contributed equally to this work.

ABSTRACT
p53 transduction is a potentially effective cancer therapy but does not result in a good therapeutic response in all human cancers due to resistance to apoptosis. To discover factors that overcome resistance to p53-induced apoptosis, we attempted to identify RNAi sequences that enhance p53-induced apoptosis. We screened a genome-wide lentiviral shRNA library in liver cancer Huh-7 and pancreatic cancer Panc-1 cells, both of which resist p53-induced apoptosis. After the infection of adenovirus expressing p53 or LacZ as a control, shRNA-treated populations were analyzed by microarray. We identified shRNAs that were significantly decreased in p53-infected cells compared with control cells. Among these shRNAs, shRNA-58335 was markedly decreased in both cancer cell lines tested. shRNA-58335 enhanced p53-related apoptosis in vitro and augmented the inhibitory effect of adenoviral p53 transduction on tumor growth in vivo. Furthermore, the enhanced apoptotic response by shRNA-58335 was also confirmed by treatment with PRIMA-1, which reactivates mutant p53, instead of adenoviral p53 transduction. We found that shRNA-58335 evokes the apoptotic response following p53 transduction or functional restoration of p53 with a small molecule drug in cancer cells resistant to p53-induced apoptosis. The combination of p53 restoration and RNAi-based drugs is expected to be a promising novel cancer therapy.

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Effect of shRNA-58335 on apoptosis with the functional restoration of p53 with PRIMA-1(A) Huh-7 cells stably infected with lentivirus expressing shRNA-58335 or control sequences were treated with PRIMA-1 (200 μM) and/or adriamycin (0.5 μg/ml). Seventy-two hours after treatment, the cells were analyzed by flow cytometry. The percentage of cells in the sub-G1 phase is indicated. (B) Representative flow cytometry data in Huh-7 cells treated with PRIMA-1 and adriamycin. The percentage of cells in the sub-G1 phase is indicated. (C) Panc-1 cells were stably infected with lentivirus expressing shRNA-58335 or a control sequence. These cells were treated with VP-16 (300 μM) in the presence or absence of PRIMA-1 (200 μM). Forty-eight hours after treatment, the cells were analyzed by flow cytometry. The percentage of cells in the sub-G1 phase is indicated. In (A) and (C), error bars indicate the S.E. * indicates a p value < 0.05 by t-test.
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Figure 5: Effect of shRNA-58335 on apoptosis with the functional restoration of p53 with PRIMA-1(A) Huh-7 cells stably infected with lentivirus expressing shRNA-58335 or control sequences were treated with PRIMA-1 (200 μM) and/or adriamycin (0.5 μg/ml). Seventy-two hours after treatment, the cells were analyzed by flow cytometry. The percentage of cells in the sub-G1 phase is indicated. (B) Representative flow cytometry data in Huh-7 cells treated with PRIMA-1 and adriamycin. The percentage of cells in the sub-G1 phase is indicated. (C) Panc-1 cells were stably infected with lentivirus expressing shRNA-58335 or a control sequence. These cells were treated with VP-16 (300 μM) in the presence or absence of PRIMA-1 (200 μM). Forty-eight hours after treatment, the cells were analyzed by flow cytometry. The percentage of cells in the sub-G1 phase is indicated. In (A) and (C), error bars indicate the S.E. * indicates a p value < 0.05 by t-test.

Mentions: p53 transduction by adenoviral vector is difficult to deliver to cancer tissues in vivo. Previous report indicated that histone deacetylase inhibitors reactivated mutant p53 [12]. Recently, PRIMA-1 was identified as a low-molecular-weight compound that restores the sequence-specific DNA binding and active conformation of mutant p53 proteins [13] and demonstrated positive data in phase 1/2 clinical trials of hematologic malignancies and prostate cancer [14]. Therefore, we replaced adenoviral p53 transduction with PRIMA-1 treatment to activate p53, performed as in Fig. 3A. As observed in Fig. 3A, the combinatorial treatment of PRIMA-1 and adriamycin induced a strong apoptotic response in shRNA-58335-infected cells compared with control-shRNA-infected cells (Fig. 5A, B). Furthermore, we evaluated the apoptotic effect on Panc-1 cells by shRNA-58335 and PRIMA-1. Adriamycin treatment alone did not induce an apoptotic response in Panc-1 cells (data not shown). Therefore, we treated Panc-1 cells with VP-16 (etoposide) and quantified apoptosis by evaluating the sub-G1 population (Fig. 5C). The combinatorial treatment of PRIMA-1 and VP-16 significantly enhanced the apoptotic response in shRNA-58335-infected cells compared with control-shRNA-infected cells. These results demonstrated that shRNA-58335 effectively enhanced the p53-induced apoptotic response not only by p53 transduction but also by the functional restoration of p53 with small molecule treatment.


Array-based genome-wide RNAi screening to identify shRNAs that enhance p53-related apoptosis in human cancer cells.

Idogawa M, Ohashi T, Sugisaka J, Sasaki Y, Suzuki H, Tokino T - Oncotarget (2014)

Effect of shRNA-58335 on apoptosis with the functional restoration of p53 with PRIMA-1(A) Huh-7 cells stably infected with lentivirus expressing shRNA-58335 or control sequences were treated with PRIMA-1 (200 μM) and/or adriamycin (0.5 μg/ml). Seventy-two hours after treatment, the cells were analyzed by flow cytometry. The percentage of cells in the sub-G1 phase is indicated. (B) Representative flow cytometry data in Huh-7 cells treated with PRIMA-1 and adriamycin. The percentage of cells in the sub-G1 phase is indicated. (C) Panc-1 cells were stably infected with lentivirus expressing shRNA-58335 or a control sequence. These cells were treated with VP-16 (300 μM) in the presence or absence of PRIMA-1 (200 μM). Forty-eight hours after treatment, the cells were analyzed by flow cytometry. The percentage of cells in the sub-G1 phase is indicated. In (A) and (C), error bars indicate the S.E. * indicates a p value < 0.05 by t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4202142&req=5

Figure 5: Effect of shRNA-58335 on apoptosis with the functional restoration of p53 with PRIMA-1(A) Huh-7 cells stably infected with lentivirus expressing shRNA-58335 or control sequences were treated with PRIMA-1 (200 μM) and/or adriamycin (0.5 μg/ml). Seventy-two hours after treatment, the cells were analyzed by flow cytometry. The percentage of cells in the sub-G1 phase is indicated. (B) Representative flow cytometry data in Huh-7 cells treated with PRIMA-1 and adriamycin. The percentage of cells in the sub-G1 phase is indicated. (C) Panc-1 cells were stably infected with lentivirus expressing shRNA-58335 or a control sequence. These cells were treated with VP-16 (300 μM) in the presence or absence of PRIMA-1 (200 μM). Forty-eight hours after treatment, the cells were analyzed by flow cytometry. The percentage of cells in the sub-G1 phase is indicated. In (A) and (C), error bars indicate the S.E. * indicates a p value < 0.05 by t-test.
Mentions: p53 transduction by adenoviral vector is difficult to deliver to cancer tissues in vivo. Previous report indicated that histone deacetylase inhibitors reactivated mutant p53 [12]. Recently, PRIMA-1 was identified as a low-molecular-weight compound that restores the sequence-specific DNA binding and active conformation of mutant p53 proteins [13] and demonstrated positive data in phase 1/2 clinical trials of hematologic malignancies and prostate cancer [14]. Therefore, we replaced adenoviral p53 transduction with PRIMA-1 treatment to activate p53, performed as in Fig. 3A. As observed in Fig. 3A, the combinatorial treatment of PRIMA-1 and adriamycin induced a strong apoptotic response in shRNA-58335-infected cells compared with control-shRNA-infected cells (Fig. 5A, B). Furthermore, we evaluated the apoptotic effect on Panc-1 cells by shRNA-58335 and PRIMA-1. Adriamycin treatment alone did not induce an apoptotic response in Panc-1 cells (data not shown). Therefore, we treated Panc-1 cells with VP-16 (etoposide) and quantified apoptosis by evaluating the sub-G1 population (Fig. 5C). The combinatorial treatment of PRIMA-1 and VP-16 significantly enhanced the apoptotic response in shRNA-58335-infected cells compared with control-shRNA-infected cells. These results demonstrated that shRNA-58335 effectively enhanced the p53-induced apoptotic response not only by p53 transduction but also by the functional restoration of p53 with small molecule treatment.

Bottom Line: We screened a genome-wide lentiviral shRNA library in liver cancer Huh-7 and pancreatic cancer Panc-1 cells, both of which resist p53-induced apoptosis.We identified shRNAs that were significantly decreased in p53-infected cells compared with control cells.Furthermore, the enhanced apoptotic response by shRNA-58335 was also confirmed by treatment with PRIMA-1, which reactivates mutant p53, instead of adenoviral p53 transduction.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genome Sciences, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan. Contributed equally to this work.

ABSTRACT
p53 transduction is a potentially effective cancer therapy but does not result in a good therapeutic response in all human cancers due to resistance to apoptosis. To discover factors that overcome resistance to p53-induced apoptosis, we attempted to identify RNAi sequences that enhance p53-induced apoptosis. We screened a genome-wide lentiviral shRNA library in liver cancer Huh-7 and pancreatic cancer Panc-1 cells, both of which resist p53-induced apoptosis. After the infection of adenovirus expressing p53 or LacZ as a control, shRNA-treated populations were analyzed by microarray. We identified shRNAs that were significantly decreased in p53-infected cells compared with control cells. Among these shRNAs, shRNA-58335 was markedly decreased in both cancer cell lines tested. shRNA-58335 enhanced p53-related apoptosis in vitro and augmented the inhibitory effect of adenoviral p53 transduction on tumor growth in vivo. Furthermore, the enhanced apoptotic response by shRNA-58335 was also confirmed by treatment with PRIMA-1, which reactivates mutant p53, instead of adenoviral p53 transduction. We found that shRNA-58335 evokes the apoptotic response following p53 transduction or functional restoration of p53 with a small molecule drug in cancer cells resistant to p53-induced apoptosis. The combination of p53 restoration and RNAi-based drugs is expected to be a promising novel cancer therapy.

Show MeSH
Related in: MedlinePlus