Abstract
TP53 (p53) is a tumor suppressor whose functions are lost or altered in most malignancies. p53 homozygous knockout (p53−/−) mice uniformly die of spontaneous malignancy, typically T-cell lymphoma. RALBP1 (RLIP76, Rlip) is a stress-protective, mercapturic acid pathway transporter protein that also functions as a Ral effector involved in clathrin-dependent endocytosis. In stark contrast to p53−/− mice, Rlip−/− mice are highly resistant to carcinogenesis. We report here that partial Rlip deficiency induced by weekly administration of an Rlip-specific phosphorothioate antisense oligonucleotide, R508, strongly inhibited spontaneous as well as benzo(a)pyrene-induced carcinogenesis in p53−/− mice. This treatment effectively prevented large-scale methylomic and transcriptomic abnormalities suggestive of inflammation found in cancer-bearing p53−/− mice. The remarkable efficiency with which Rlip deficiency suppresses spontaneous malignancy in p53−/− mice has not been observed with any previously reported pharmacologic or genetic intervention. These findings are supported by cross-breeding experiments demonstrating that hemizygous Rlip deficiency also reduces the spontaneous malignancy phenotype of p53+/− mice. Rlip is found on the cell surface, and antibodies directed against Rlip were found to inhibit growth and promote apoptosis of cell lines as effectively as Rlip siRNA. The work presented here investigates several features, including oxidative DNA damage of the Rlip-p53 association in malignant transformation, and offers a paradigm for the mechanisms of tumor suppression by p53 and the prospects of suppressing spontaneous malignancy in hereditary cancer syndromes such as Li-Fraumeni.
Original language | English (US) |
---|---|
Pages (from-to) | 3918-3923 |
Number of pages | 6 |
Journal | Proceedings of the National Academy of Sciences of the United States of America |
Volume | 115 |
Issue number | 15 |
DOIs | |
State | Published - 2018 |
Externally published | Yes |
Keywords
- Cancer prevention
- Cancer signalling
- Cytokine
- RALBP1
- TP53
ASJC Scopus subject areas
- General
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Rlip depletion prevents spontaneous neoplasia in TP53 null mice. / Awasthi, Sanjay; Tompkins, Joshua; Singhal, Jyotsana et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 115, No. 15, 2018, p. 3918-3923.Research output: Contribution to journal › Article › peer-review
}
TY - JOUR
T1 - Rlip depletion prevents spontaneous neoplasia in TP53 null mice
AU - Awasthi, Sanjay
AU - Tompkins, Joshua
AU - Singhal, Jyotsana
AU - Riggs, Arthur D.
AU - Yadav, Sushma
AU - Wu, Xiwei
AU - Singh, Sharda
AU - Warden, Charles
AU - Liu, Zheng
AU - Wang, Jinhui
AU - Slavin, Thomas P.
AU - Weitzel, Jeffrey N.
AU - Yuan, Yate Ching
AU - Awasthi, Meenakshi
AU - Srivastava, Satish K.
AU - Awasthi, Yogesh C.
AU - Singhal, Sharad S.
N1 - Funding Information: ACKNOWLEDGMENTS. We thank the personnel in the Animal Research Center, Pathology, Genomics, and Bioinformatics Cores of the City of Hope for their invaluable assistance. The authors are grateful to Dr. Brian Armstrong (Microscope Core Lab, City of Hope) and Lucy Brown (Analytical Cytometry Core, City of Hope) for technical assistance in the fluorescence microscope and flow cytometry analyses, respectively. A.D.R. holds the Samuel Rahbar Chair in Diabetes and Drug Discovery. This work was supported by NIH-R01-CA77495 (to S.A.), NIH/ National Cancer Institute Grant CA129383 (to S.K.S.), the City of Hope Cancer Center Support Grant P30 CA33572, the Perricone Foundation, Texas Tech University Health Sciences Center Ethel S. Neely & Emma S. Treadwell Endowment (S.A.), and the University Medical Center Chair of Excellence. Funding from the Department of Defense Grant W81XWH-16-1-0641 (to S.S.S.) is also acknowledged. Funding Information: 11. Awasthi S, et al. (2001) Functional reassembly of ATP-dependent xenobiotic transport by the Nand C-terminal domains of RLIP76 and identification of ATP binding se-quences. Biochemistry 40:4159–4168. Methods To study suppression of spontaneous carcinogenesis by R508 (phosphorothioate antisense oligonucleotide; 508GGCTCCTGAATTGGCTTTTTC529) or CAS (control scrambled phosphorothioate oligonucleotide, CATCGAAATCGTTG-CAGTTAC), p53−/− mice were treated with 200 μg R508 or CAS by i.p. injection starting at 8 wk of age until killing. To study suppression of spontaneous carcinogenesis cross-bred mice, p53−/−Rlip+/+, p53−/−Rlip+/−, p53−/−Rlip−/−, and p53+/−Rlip+/− mice were monitored three times per week for distress or overt malignancy, and all surviving mice were killed at age 48 wk. Chemical carcinogenesis was studied in 10–15 mice of each genotype per treatment group. Mice were administered 3 mg B[a]P in 0.1 mL corn oil or corn oil alone by gavage at the age of 8 and 12 wk. Raji and human LCL lymphoma cell lines were grown in RPMI1640 medium and MEFs in DMEM containing 10% FBS and 1% penicillin/ streptomycin at 37 °C in 5% CO2. Cytotoxicity, signaling, and endocytosis studies were performed as described in SI Appendix, Methods. RNA-Seq studies, whole-genome bisulfite sequencing, and validation studies were conducted in triplicate for each biological replicate in p53−/−CAS or R508-treated mice liver. The RNA-sequencing run was performed in a Illumina HiSEq. 2500 platform with HiSeq SBS V4 Kits, and reads were aligned using Tophat v2.0 to mouse reference genome mm9 (66, 67). To validate RNA-Seq results, qRT-PCR was conducted with primers, prevalidated from BioRad (PrimePCR, cat. 10025636), for the following genes: Cib3, Dlk1, Cyp4a32, Fzd10, Gpr3, Tff1, and Six3. For the whole-genome bisulfite sequencing, 200 ng DNA was sonicated to get 200-bp-size DNA (68), and library templates were prepared for sequencing. Reads were aligned to in silico bisulfite-converted mm9 genome using Bismark aligner (69), using the default settings. Quantitative validation was carried out for the PTPN6 and HOXA5 gene promoters by conventional bisulfite sequencing (70). Statistical methods used for RNA-Seq and WGBS analyses are given in SI Appendix. The statistical significance of differences between control and treatment groups was determined by ANOVA followed by Bonferroni correction and Benjamini-Hochberg procedure with a false-discovery rate <0.05. The heat map of the P values of top differentially expressed genes by Euclidean distance and an average linkage strategy for the four groups (wt, PBS-p53−/−, CAS-p53−/−, and R508-p53−/−) are presented. Changes in tumor size and body weight during the experiments were visualized by scatter plot. Animal experiments were approved by the Institutional Animal Care and Use Committee at City of Hope and performed under approved protocol no. 11016. ACKNOWLEDGMENTS. We thank the personnel in the Animal Research Center, Pathology, Genomics, and Bioinformatics Cores of the City of Hope for their invaluable assistance. The authors are grateful to Dr. Brian Armstrong (Microscope Core Lab, City of Hope) and Lucy Brown (Analytical Cytometry Core, City of Hope) for technical assistance in the fluorescence microscope and flow cytometry analyses, respectively. A.D.R. holds the Samuel Rahbar Chair in Diabetes and Drug Discovery. This work was supported by NIH-R01-CA77495 (to S.A.), NIH/ National Cancer Institute Grant CA129383 (to S.K.S.), the City of Hope Cancer Center Support Grant P30 CA33572, the Perricone Foundation, Texas Tech University Health Sciences Center Ethel S. Neely & Emma S. Treadwell Endowment (S.A.), and the University Medical Center Chair of Excellence. Funding from the Department of Defense Grant W81XWH-16-1-0641 (to S.S.S.) is also acknowledged. 12. Awasthi S, et al. (1998) ATP-dependent human erythrocyte glutathione-conjugate transporter. II. Functional reconstitution of transport activity. Biochemistry 37:5239–5248. 13. Awasthi S, et al. (1998) ATP-dependent human erythrocyte glutathione-conjugate transporter. I. Purification, photoaffinity labeling, and kinetic characteristics of ATPase activity. Biochemistry 37:5231–5238. 14. Awasthi S, et al. (1994) Adenosine triphosphate-dependent transport of doxorubicin, daunomycin, and vinblastine in human tissues by a mechanism distinct from the P-glycoprotein. J Clin Invest 93:958–965. 15. Awasthi S, et al. (2005) RLIP76 is a major determinant of radiation sensitivity. Cancer Res 65:6022–6028. 16. Awasthi S, et al. (2010) A central role of RLIP76 in regulation of glycemic control. Diabetes 59:714–725. 17. Cheng JZ, et al. (2001) Accelerated metabolism and exclusion of 4-hydroxynonenal through induction of RLIP76 and hGST5.8 is an early adaptive response of cells to heat and oxidative stress. 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Radiat Res 184:235–248. 47. Singhal J, Nagaprashantha L, Vatsyayan R, Awasthi S, Singhal SS (2011) RLIP76, a glutathione-conjugate transporter, plays a major role in the pathogenesis of meta-bolic syndrome. PLoS One 6:e24688. 48. Morris SM, et al. (2008) Effect of p53 genotype on gene expression profiles in murine liver. Mutat Res 640:54–73. 49. Uren AG, et al. (2008) Large-scale mutagenesis in p19(ARF)-and p53-deficient mice identifies cancer genes and their collaborative networks. Cell 133:727–741. 50. Goding CR, Pei D, Lu X (2014) Cancer: Pathological nuclear reprogramming? Nat Rev Cancer 14:568–573. 51. Suzuki H, Yamamoto E, Maruyama R, Niinuma T, Kai M (2014) Biological significance of the CpG island methylator phenotype. Biochem Biophys Res Commun 455:35–42. 52. Kulis M, Esteller M (2010) DNA methylation and cancer. Adv Genet 70:27–56. 53. Singhal J, et al. (2017) 2′-Hydroxyflavanone: A novel strategy for targeting breast cancer. Oncotarget 8:75025–75037. 54. Ames BN, Shigenaga MK, Gold LS (1993) DNA lesions, inducible DNA repair, and cell division: Three key factors in mutagenesis and carcinogenesis. Environ Health Perspect 101:35–44. 55. Deferme L, et al. (2016) Dynamic interplay between the transcriptome and methylome in response to oxidative and alkylating stress. Chem Res Toxicol 29:1428–1438. 56. Esterbauer H, Schaur RJ, Zollner H (1991) Chemistry and biochemistry of 4-hydrox-ynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med 11:81–128. 57. Shigenaga MK, Gimeno CJ, Ames BN (1989) Urinary 8-hydroxy-2′-deoxyguanosine as a biological marker of in vivo oxidative DNA damage. Proc Natl Acad Sci USA 86: 9697–9701. 58. Li Q, Martinez JD (2011) P53 is transported into the nucleus via an Hsf1-dependent nuclear localization mechanism. Mol Carcinog 50:143–152. 59. Hu Y, Mivechi NF (2003) HSF-1 interacts with Ral-binding protein 1 in a stress-responsive, multiprotein complex with HSP90 in vivo. J Biol Chem 278:17299–17306. 60. Min JN, Huang L, Zimonjic DB, Moskophidis D, Mivechi NF (2007) Selective suppression of lymphomas by functional loss of Hsf1 in a p53-deficient mouse model for spon-taneous tumors. Oncogene 26:5086–5097. 61. Morinaka K, et al. (1999) Epsin binds to the EH domain of POB1 and regulates receptor-mediated endocytosis. Oncogene 18:5915–5922. 62. Nakashima S, et al. (1999) Small G protein Ral and its downstream molecules regulate endocytosis of EGF and insulin receptors. EMBO J 18:3629–3642. 63. Yamaguchi A, Urano T, Goi T, Feig LA (1997) An Eps homology (EH) domain protein that binds to the Ral-GTPase target, RalBP1. J Biol Chem 272:31230–31234. 64. Enari M, Ohmori K, Kitabayashi I, Taya Y (2006) Requirement of clathrin heavy chain for p53-mediated transcription. Genes Dev 20:1087–1099. 65. Endo Y, et al. (2008) Regulation of clathrin-mediated endocytosis by p53. Genes Cells 13:375–386. 66. Li H, Ruan J, Durbin R (2008) Mapping short DNA sequencing reads and calling var-iants using mapping quality scores. Genome Res 18:1851–1858. 67. Li R, Li Y, Kristiansen K, Wang J (2008) SOAP: Short oligonucleotide alignment pro-gram. Bioinformatics 24:713–714. 68. Hahn MA, Li AX, Wu X, Pfeifer GP (2015) Cancer Epigenetics. Methods Mol Biol 1238: 273–287. 69. Krueger F, Andrews SR (2011) Bismark: A flexible aligner and methylation caller for bisulfite-seq applications. Bioinformatics 27:1571–1572. 70. Li LC, Dahiya R (2002) MethPrimer: Designing primers for methylation PCRs. Bioinformatics 18:1427–1431. MEDICAL SCIENCES Funding Information: We thank the personnel in the Animal Research Center, Pathology, Genomics, and Bioinformatics Cores of the City of Hope for their invaluable assistance. The authors are grateful to Dr. Brian Armstrong (Microscope Core Lab, City of Hope) and Lucy Brown (Analytical Cytometry Core, City of Hope) for technical assistance in the fluorescence microscope and flow cytometry analyses, respectively. A.D.R. holds the Samuel Rahbar Chair in Diabetes and Drug Discovery. This work was supported by NIH-R01-CA77495 (to S.A.), NIH/ National Cancer Institute Grant CA129383 (to S.K.S.), the City of Hope Cancer Center Support Grant P30 CA33572, the Perricone Foundation, Texas Tech University Health Sciences Center Ethel S. Neely & Emma S. Treadwell Endowment (S.A.), and the University Medical Center Chair of Excellence. Funding from the Department of Defense Grant W81XWH-16-1-0641 (to S.S.S.) is also acknowledged. Publisher Copyright: © 2018 National Academy of Sciences. All Rights Reserved.
PY - 2018
Y1 - 2018
N2 - TP53 (p53) is a tumor suppressor whose functions are lost or altered in most malignancies. p53 homozygous knockout (p53−/−) mice uniformly die of spontaneous malignancy, typically T-cell lymphoma. RALBP1 (RLIP76, Rlip) is a stress-protective, mercapturic acid pathway transporter protein that also functions as a Ral effector involved in clathrin-dependent endocytosis. In stark contrast to p53−/− mice, Rlip−/− mice are highly resistant to carcinogenesis. We report here that partial Rlip deficiency induced by weekly administration of an Rlip-specific phosphorothioate antisense oligonucleotide, R508, strongly inhibited spontaneous as well as benzo(a)pyrene-induced carcinogenesis in p53−/− mice. This treatment effectively prevented large-scale methylomic and transcriptomic abnormalities suggestive of inflammation found in cancer-bearing p53−/− mice. The remarkable efficiency with which Rlip deficiency suppresses spontaneous malignancy in p53−/− mice has not been observed with any previously reported pharmacologic or genetic intervention. These findings are supported by cross-breeding experiments demonstrating that hemizygous Rlip deficiency also reduces the spontaneous malignancy phenotype of p53+/− mice. Rlip is found on the cell surface, and antibodies directed against Rlip were found to inhibit growth and promote apoptosis of cell lines as effectively as Rlip siRNA. The work presented here investigates several features, including oxidative DNA damage of the Rlip-p53 association in malignant transformation, and offers a paradigm for the mechanisms of tumor suppression by p53 and the prospects of suppressing spontaneous malignancy in hereditary cancer syndromes such as Li-Fraumeni.
AB - TP53 (p53) is a tumor suppressor whose functions are lost or altered in most malignancies. p53 homozygous knockout (p53−/−) mice uniformly die of spontaneous malignancy, typically T-cell lymphoma. RALBP1 (RLIP76, Rlip) is a stress-protective, mercapturic acid pathway transporter protein that also functions as a Ral effector involved in clathrin-dependent endocytosis. In stark contrast to p53−/− mice, Rlip−/− mice are highly resistant to carcinogenesis. We report here that partial Rlip deficiency induced by weekly administration of an Rlip-specific phosphorothioate antisense oligonucleotide, R508, strongly inhibited spontaneous as well as benzo(a)pyrene-induced carcinogenesis in p53−/− mice. This treatment effectively prevented large-scale methylomic and transcriptomic abnormalities suggestive of inflammation found in cancer-bearing p53−/− mice. The remarkable efficiency with which Rlip deficiency suppresses spontaneous malignancy in p53−/− mice has not been observed with any previously reported pharmacologic or genetic intervention. These findings are supported by cross-breeding experiments demonstrating that hemizygous Rlip deficiency also reduces the spontaneous malignancy phenotype of p53+/− mice. Rlip is found on the cell surface, and antibodies directed against Rlip were found to inhibit growth and promote apoptosis of cell lines as effectively as Rlip siRNA. The work presented here investigates several features, including oxidative DNA damage of the Rlip-p53 association in malignant transformation, and offers a paradigm for the mechanisms of tumor suppression by p53 and the prospects of suppressing spontaneous malignancy in hereditary cancer syndromes such as Li-Fraumeni.
KW - Cancer prevention
KW - Cancer signalling
KW - Cytokine
KW - RALBP1
KW - TP53
UR - http://www.scopus.com/inward/record.url?scp=85045082604&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85045082604&partnerID=8YFLogxK
U2 - 10.1073/pnas.1719586115
DO - 10.1073/pnas.1719586115
M3 - Article
C2 - 29572430
AN - SCOPUS:85045082604
VL - 115
SP - 3918
EP - 3923
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 15
ER -