p53 and Tumor Suppression: It Takes a Network
2021; Elsevier BV; Volume: 31; Issue: 4 Linguagem: Inglês
10.1016/j.tcb.2020.12.011
ISSN1879-3088
AutoresAnthony M. Boutelle, Laura D. Attardi,
Tópico(s)Epigenetics and DNA Methylation
ResumoIn vivo shRNA and CRISPR/Cas9 screens identify p53 target genes such as Zmat3 and Mlh1 as important for tumor suppression.Emerging p53 functions potentially important for tumor suppression include activation of DNA damage repair, opposition to metabolic rewiring, stimulation of ferroptosis, inhibition of cellular plasticity, and maintenance of an anticancer immune tumor microenvironment.Through modulation of metabolism and redox biology, p53 can promote cell survival and homeostasis in addition to promoting cell death, raising the possibility that, in some contexts, p53 might support tumor growth.p53 can concurrently regulate many cellular processes in a given biological setting, suggesting that tumor suppression relies on coordinated effects on cellular biology.New evidence suggests that p53 suppresses tumorigenesis by modulating a target gene network comprising cooperative and redundant effectors. The TP53 tumor suppressor is the most frequently mutated gene in human cancer. p53 suppresses tumorigenesis by transcriptionally regulating a network of target genes that play roles in various cellular processes. Though originally characterized as a critical regulator for responses to acute DNA damage (activation of apoptosis and cell cycle arrest), recent studies have highlighted new pathways and transcriptional targets downstream of p53 regulating genomic integrity, metabolism, redox biology, stemness, and non-cell autonomous signaling in tumor suppression. Here, we summarize our current understanding of p53-mediated tumor suppression, situating recent findings from mouse models and unbiased screens in the context of previous studies and arguing for the importance of the pleiotropic effects of the p53 transcriptional network in inhibiting cancer. The TP53 tumor suppressor is the most frequently mutated gene in human cancer. p53 suppresses tumorigenesis by transcriptionally regulating a network of target genes that play roles in various cellular processes. Though originally characterized as a critical regulator for responses to acute DNA damage (activation of apoptosis and cell cycle arrest), recent studies have highlighted new pathways and transcriptional targets downstream of p53 regulating genomic integrity, metabolism, redox biology, stemness, and non-cell autonomous signaling in tumor suppression. Here, we summarize our current understanding of p53-mediated tumor suppression, situating recent findings from mouse models and unbiased screens in the context of previous studies and arguing for the importance of the pleiotropic effects of the p53 transcriptional network in inhibiting cancer. The TP53 gene encodes a transcription factor that is a critical barrier to carcinogenesis. Inactivation of TP53 is the most common mutation in sporadic human cancers, suggesting a strong selection against p53 function during tumorigenesis [1.Kandoth C. et al.Mutational landscape and significance across 12 major cancer types.Nature. 2013; 502: 333-339Crossref PubMed Scopus (2557) Google Scholar]. The inheritance of a mutant TP53 allele is observed in Li-Fraumeni syndrome, predisposing patients to early onset cancer development, further underscoring the role of p53 in tumor suppression. The importance of p53 as a tumor suppressor is cemented by experimental evidence from p53-/- mice, which develop cancer, mostly thymic lymphomas, with 100% penetrance [2.Kaiser A.M. Attardi L.D. Deconstructing networks of p53-mediated tumor suppression in vivo.Cell Death Differ. 2018; 25: 93-103Crossref PubMed Scopus (98) Google Scholar]. Despite this unequivocal characterization of the fundamental role for p53 in tumor suppression, a comprehensive mechanistic understanding of p53 tumor suppressor function is lacking. Understanding the underpinnings of p53 tumor suppressor function is of critical importance to elucidating cancer etiology and developing therapeutic strategies. p53 is thought to act as a tumor suppressor by serving as a cellular stress sensor (Figure 1). In unstressed cells, p53 is targeted by the E3 ubiquitin ligase MDM2 for degradation, keeping p53 at low levels. A variety of stress signals, including DNA damage, oncogene expression, and hypoxia, relieve p53 from MDM2 inhibition. p53 binds DNA in a sequence-specific manner and recruits transcriptional machinery components to activate expression of a network of target genes. Transactivation of p53 target genes is compromised by mutations found in human cancers, which occur predominantly in the sequence-specific DNA binding domain. These mutations disable DNA binding capacity by perturbing residues involved in direct contact of DNA or disrupting p53 protein structure, although oncogenic gain-of-function activity has also been reported for some TP53 mutations (reviewed in [3.Zhang Y. Lozano G. p53: multiple facets of a Rubik's Cube.Annu. Rev. Cancer Biol. 2017; 1: 185-201Crossref PubMed Scopus (9) Google Scholar]). The importance of transcriptional activation for p53-mediated tumor suppression is supported by analyses of mice expressing a transactivation-dead mutant, with alterations in TAD1 and TAD2, p5325,26,53,54. In mouse models for a range of cancers, including B- and T-cell lymphomas, lung adenocarcinoma (LUAD), and pancreatic ductal adenocarcinoma (PDAC), this mutant behaves indistinguishably from p53 deletion, suggesting the importance of transcriptional activation for p53-mediated tumor suppression [4.Brady C.A. et al.Distinct p53 transcriptional programs dictate acute DNA damage responses and tumor suppression.Cell. 2011; 145: 571-583Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 5.Jiang D. et al.Full p53 transcriptional activation potential is dispensable for tumor suppression in diverse lineages.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 17123-17128Crossref PubMed Scopus (57) Google Scholar, 6.Mello S.S. et al.A p53 super-tumor suppressor reveals a tumor suppressive p53-Ptpn14-Yap Axis in pancreatic cancer.Cancer Cell. 2017; 32: 460-473.e6Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar]. Although we focus in this review primarily on transcriptional programs underlying p53 tumor suppression, roles for p53 independent of transcriptional activation have also been reported (Box 1).Box 1Transactivation-Independent p53 Functions add Another Dimension to Tumor SuppressionAlthough most investigation of the mechanisms of p53-mediated tumor suppression has focused on transcriptional activation (transactivation) of p53 target genes, transactivation-independent functions of p53 have also been reported. The best characterized transactivation-independent p53 function is induction of apoptosis at the mitochondria. p53 can bind antiapoptotic BCL-2 family members to displace them from proapoptotic BCL-2 family members or directly activate BAX and BAK to trigger mitochondrial outer matrix permeabilization and apoptosis [75.Ho T. et al.How the other half lives: what p53 does when it is not being a transcription factor.Int. J. Mol. Sci. 2019; 21: 13Crossref Scopus (19) Google Scholar]. p53 has other described transactivation-independent functions both in the cytoplasm and the nucleus that could contribute to tumor suppression. For example, in the cytoplasm, p53 can bind and inhibit the rate-limiting enzyme of the pentose phosphate pathway, G6PD, thus restricting this tumor-supporting metabolic pathway [76.Jiang P. et al.p53 regulates biosynthesis through direct inactivation of glucose-6-phosphate dehydrogenase.Nat. Cell Biol. 2011; 13: 310-316Crossref PubMed Scopus (455) Google Scholar]. In the nucleus, p53 can promote genomic integrity through different mechanisms, such as by restraining the mobility of transposons and other classes of repetitive elements and by binding the homologous recombination protein RAD51 to prevent excessive or inaccurate recombination events [75.Ho T. et al.How the other half lives: what p53 does when it is not being a transcription factor.Int. J. Mol. Sci. 2019; 21: 13Crossref Scopus (19) Google Scholar]. Recent GEMM studies have highlighted transactivation-independent roles for p53 in vivo. In one study using mouse models of Wnt-driven intestinal cancer, resulting from either Csnk1a1 deletion or ApcMin/+ mutation in the proximal gut, cancer was suppressed in the presence p53R172H, a DNA binding domain mutant deficient for target gene transactivation, relative to the p53-/- model [77.Kadosh E. et al.The gut microbiome switches mutant p53 from tumour-suppressive to oncogenic.Nature. 2020; 586: 133-138Crossref PubMed Scopus (66) Google Scholar]. This activity, related to suppressing the Wnt pathway through inhibition of TCF4 chromatin binding, suggested p53 transactivation-independent tumor suppression, although further experiments are necessary to clarify whether the activity of this mutant is neomorphic or present in wild-type p53 [77.Kadosh E. et al.The gut microbiome switches mutant p53 from tumour-suppressive to oncogenic.Nature. 2020; 586: 133-138Crossref PubMed Scopus (66) Google Scholar]. In another study, analysis of knock-in mice expressing the p53R178E mutant, which is also deficient for p53 target gene activation, has revealed that p53R178E behaves like a p53 null allele in both spontaneous and Eμ-Myc-driven mouse tumorigenesis [78.Timofeev O. et al.Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses.EMBO J. 2019; 38e102096Crossref PubMed Scopus (11) Google Scholar]. However, this mutant retains apoptotic activity in vivo both in apoptosis triggered by Mdm2 deficiency in developing embryos and in chemotherapeutic treatment of Eμ-Myc mouse lymphomas [78.Timofeev O. et al.Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses.EMBO J. 2019; 38e102096Crossref PubMed Scopus (11) Google Scholar]. p53R178E can localize to the mitochondria, providing a potential mechanism for p53R178E-triggered transactivation-independent apoptosis [78.Timofeev O. et al.Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses.EMBO J. 2019; 38e102096Crossref PubMed Scopus (11) Google Scholar]. These observations raise the possibility that this transactivation-independent p53 function might have significance in tumor suppression in some settings. Future experiments designed to test the significance of specific transactivation-independent functions of p53 for tumor suppression in vivo will shed more light on the importance of these phenomena for understanding cancer. Although most investigation of the mechanisms of p53-mediated tumor suppression has focused on transcriptional activation (transactivation) of p53 target genes, transactivation-independent functions of p53 have also been reported. The best characterized transactivation-independent p53 function is induction of apoptosis at the mitochondria. p53 can bind antiapoptotic BCL-2 family members to displace them from proapoptotic BCL-2 family members or directly activate BAX and BAK to trigger mitochondrial outer matrix permeabilization and apoptosis [75.Ho T. et al.How the other half lives: what p53 does when it is not being a transcription factor.Int. J. Mol. Sci. 2019; 21: 13Crossref Scopus (19) Google Scholar]. p53 has other described transactivation-independent functions both in the cytoplasm and the nucleus that could contribute to tumor suppression. For example, in the cytoplasm, p53 can bind and inhibit the rate-limiting enzyme of the pentose phosphate pathway, G6PD, thus restricting this tumor-supporting metabolic pathway [76.Jiang P. et al.p53 regulates biosynthesis through direct inactivation of glucose-6-phosphate dehydrogenase.Nat. Cell Biol. 2011; 13: 310-316Crossref PubMed Scopus (455) Google Scholar]. In the nucleus, p53 can promote genomic integrity through different mechanisms, such as by restraining the mobility of transposons and other classes of repetitive elements and by binding the homologous recombination protein RAD51 to prevent excessive or inaccurate recombination events [75.Ho T. et al.How the other half lives: what p53 does when it is not being a transcription factor.Int. J. Mol. Sci. 2019; 21: 13Crossref Scopus (19) Google Scholar]. Recent GEMM studies have highlighted transactivation-independent roles for p53 in vivo. In one study using mouse models of Wnt-driven intestinal cancer, resulting from either Csnk1a1 deletion or ApcMin/+ mutation in the proximal gut, cancer was suppressed in the presence p53R172H, a DNA binding domain mutant deficient for target gene transactivation, relative to the p53-/- model [77.Kadosh E. et al.The gut microbiome switches mutant p53 from tumour-suppressive to oncogenic.Nature. 2020; 586: 133-138Crossref PubMed Scopus (66) Google Scholar]. This activity, related to suppressing the Wnt pathway through inhibition of TCF4 chromatin binding, suggested p53 transactivation-independent tumor suppression, although further experiments are necessary to clarify whether the activity of this mutant is neomorphic or present in wild-type p53 [77.Kadosh E. et al.The gut microbiome switches mutant p53 from tumour-suppressive to oncogenic.Nature. 2020; 586: 133-138Crossref PubMed Scopus (66) Google Scholar]. In another study, analysis of knock-in mice expressing the p53R178E mutant, which is also deficient for p53 target gene activation, has revealed that p53R178E behaves like a p53 null allele in both spontaneous and Eμ-Myc-driven mouse tumorigenesis [78.Timofeev O. et al.Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses.EMBO J. 2019; 38e102096Crossref PubMed Scopus (11) Google Scholar]. However, this mutant retains apoptotic activity in vivo both in apoptosis triggered by Mdm2 deficiency in developing embryos and in chemotherapeutic treatment of Eμ-Myc mouse lymphomas [78.Timofeev O. et al.Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses.EMBO J. 2019; 38e102096Crossref PubMed Scopus (11) Google Scholar]. p53R178E can localize to the mitochondria, providing a potential mechanism for p53R178E-triggered transactivation-independent apoptosis [78.Timofeev O. et al.Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses.EMBO J. 2019; 38e102096Crossref PubMed Scopus (11) Google Scholar]. These observations raise the possibility that this transactivation-independent p53 function might have significance in tumor suppression in some settings. Future experiments designed to test the significance of specific transactivation-independent functions of p53 for tumor suppression in vivo will shed more light on the importance of these phenomena for understanding cancer. The best characterized p53 function is in response to acute DNA damage, when p53 either promotes G1 cell cycle arrest, facilitating DNA repair, or apoptosis, to eliminate damaged cells. Genetic studies in mouse models have suggested, however, that responding to acute DNA damage is dispensable for p53-mediated tumor suppression (Box 2), or perhaps that other responses can compensate when these pathways are compromised. The surprising retention of p53-mediated tumor suppression observed in acute DNA damage response-deficient p5325,26, p533KR, and p21–/–;Puma–/–;Noxa–/– mutant mouse strains (Box 2) emphasizes a need to rethink how the pathways downstream of p53 inhibit tumorigenesis. Recent in vivo studies both highlight new and elaborate on previously described p53 functions, calling for integration and analysis of the expansive catalog of functions ascribed to p53 in tumor suppression. In this review, we will summarize these recent findings, focusing on genetically engineered mouse models (GEMMs), and will propose a model that integrates the many functions of p53 in tumor suppression.Box 2Acute DNA Damage Responses Are Dispensable for Tumor SuppressionEarly work on p53 focused on transactivation of target genes like the CDK inhibitor, p21, and the proapoptotic BCL-2 family members Bax, Puma, and Noxa, which were shown in genetic experiments to be critical for the acute DNA damage responses of G1 cell cycle arrest and apoptosis, respectively [74.Aubrey B.J. et al.How does p53 induce apoptosis and how does this relate to p53-mediated tumour suppression?.Cell Death Differ. 2018; 25: 104-113Crossref PubMed Scopus (368) Google Scholar,79.el-Deiry W.S. et al.WAF1, a potential mediator of p53 tumor suppression.Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7798) Google Scholar]. These pathways delineated by studying acute DNA damage-induced cell cycle arrest and apoptosis were envisioned to sufficiently explain p53-mediated tumor suppression. Since these studies, however, multiple studies in mouse models suggested that p53-dependent acute DNA damage responses are dispensable for tumor suppression. Using mice expressing temporally-regulatable p53 alleles, two independent studies demonstrated that the presence of p53 during treatment with acute DNA damage is dispensable for tumor suppression, proposing instead that p53 activation by oncogenic signaling is more important for tumor suppression [80.Christophorou M.A. et al.The pathological response to DNA damage does not contribute to p53-mediated tumour suppression.Nature. 2006; 443: 214-217Crossref PubMed Scopus (326) Google Scholar,81.Hinkal G. et al.Timed somatic deletion of p53 in mice reveals age-associated differences in tumor progression.PLoS ONE. 2009; 4e6654Crossref PubMed Scopus (51) Google Scholar]. Building on these initial observations, subsequent studies provided deeper mechanistic insight into the issue. The first such study used knock-in mice with inactivating mutations in TAD1 of p53 (p5325,26). Functional analysis revealed that cells expressing p5325,26 fail to undergo cell cycle arrest or apoptosis in response to acute DNA damage, and genome-wide transcriptomics revealed that p5325,26 activates only a subset of target genes activated by wild-type p53. p5325,26 cannot robustly activate canonical targets including p21, Puma, and Noxa, yet it retains the ability to suppress both spontaneous cancer (T-cell lymphoma) and oncogene-driven cancers (KrasG12D-driven LUAD and PDAC, Myc-driven B-cell lymphoma, and Patched inactivation-driven medulloblastoma) in GEMMs [4.Brady C.A. et al.Distinct p53 transcriptional programs dictate acute DNA damage responses and tumor suppression.Cell. 2011; 145: 571-583Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar,5.Jiang D. et al.Full p53 transcriptional activation potential is dispensable for tumor suppression in diverse lineages.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 17123-17128Crossref PubMed Scopus (57) Google Scholar], suggesting that the robust transactivation of canonical p53 targets is dispensable for tumor suppression. These findings are supported by p21–/–;Puma–/–;Noxa–/– triple knockout mice and mice expressing p533KR, an acetylation site mutant that cannot activate canonical p53 target genes or acute DNA damage responses, both of which are resistant to the spontaneous cancer development observed in p53-/- mice [13.Valente L.J. et al.p53 efficiently suppresses tumor development in the complete absence of its cell-cycle inhibitory and proapoptotic effectors p21, Puma, and Noxa.Cell Rep. 2013; 3: 1339-1345Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar,21.Li T. et al.Tumor suppression in the absence of p53-mediated cell-cycle arrest, apoptosis, and senescence.Cell. 2012; 149: 1269-1283Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar]. Furthermore, although there is an enhancement of tumorigenesis observed in Eμ-Myc;p21–/–;Puma–/– mice relative to Eμ-Myc counterparts, this effect is not as potent as the acceleration of lymphomagenesis in Eμ-Myc;p53+/– mice [82.Valente L.J. et al.Combined loss of PUMA and p21 accelerates c-MYC-driven lymphoma development considerably less than loss of one allele of p53.Oncogene. 2016; 35: 3866-3871Crossref PubMed Scopus (24) Google Scholar]. Collectively, these studies have highlighted the need to investigate additional p53-regulated pathways that might compensate for p21, Puma, and Noxa or their associated functions to maintain tumor suppression in their absence. These studies do not strictly rule out a tumor-suppressive role for cell cycle arrest and apoptosis responses activated by other stresses that p53 responds to, such as chronic DNA damage, oncogenic signaling, hypoxia, and nutrient deprivation, amongst others. Early work on p53 focused on transactivation of target genes like the CDK inhibitor, p21, and the proapoptotic BCL-2 family members Bax, Puma, and Noxa, which were shown in genetic experiments to be critical for the acute DNA damage responses of G1 cell cycle arrest and apoptosis, respectively [74.Aubrey B.J. et al.How does p53 induce apoptosis and how does this relate to p53-mediated tumour suppression?.Cell Death Differ. 2018; 25: 104-113Crossref PubMed Scopus (368) Google Scholar,79.el-Deiry W.S. et al.WAF1, a potential mediator of p53 tumor suppression.Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7798) Google Scholar]. These pathways delineated by studying acute DNA damage-induced cell cycle arrest and apoptosis were envisioned to sufficiently explain p53-mediated tumor suppression. Since these studies, however, multiple studies in mouse models suggested that p53-dependent acute DNA damage responses are dispensable for tumor suppression. Using mice expressing temporally-regulatable p53 alleles, two independent studies demonstrated that the presence of p53 during treatment with acute DNA damage is dispensable for tumor suppression, proposing instead that p53 activation by oncogenic signaling is more important for tumor suppression [80.Christophorou M.A. et al.The pathological response to DNA damage does not contribute to p53-mediated tumour suppression.Nature. 2006; 443: 214-217Crossref PubMed Scopus (326) Google Scholar,81.Hinkal G. et al.Timed somatic deletion of p53 in mice reveals age-associated differences in tumor progression.PLoS ONE. 2009; 4e6654Crossref PubMed Scopus (51) Google Scholar]. Building on these initial observations, subsequent studies provided deeper mechanistic insight into the issue. The first such study used knock-in mice with inactivating mutations in TAD1 of p53 (p5325,26). Functional analysis revealed that cells expressing p5325,26 fail to undergo cell cycle arrest or apoptosis in response to acute DNA damage, and genome-wide transcriptomics revealed that p5325,26 activates only a subset of target genes activated by wild-type p53. p5325,26 cannot robustly activate canonical targets including p21, Puma, and Noxa, yet it retains the ability to suppress both spontaneous cancer (T-cell lymphoma) and oncogene-driven cancers (KrasG12D-driven LUAD and PDAC, Myc-driven B-cell lymphoma, and Patched inactivation-driven medulloblastoma) in GEMMs [4.Brady C.A. et al.Distinct p53 transcriptional programs dictate acute DNA damage responses and tumor suppression.Cell. 2011; 145: 571-583Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar,5.Jiang D. et al.Full p53 transcriptional activation potential is dispensable for tumor suppression in diverse lineages.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 17123-17128Crossref PubMed Scopus (57) Google Scholar], suggesting that the robust transactivation of canonical p53 targets is dispensable for tumor suppression. These findings are supported by p21–/–;Puma–/–;Noxa–/– triple knockout mice and mice expressing p533KR, an acetylation site mutant that cannot activate canonical p53 target genes or acute DNA damage responses, both of which are resistant to the spontaneous cancer development observed in p53-/- mice [13.Valente L.J. et al.p53 efficiently suppresses tumor development in the complete absence of its cell-cycle inhibitory and proapoptotic effectors p21, Puma, and Noxa.Cell Rep. 2013; 3: 1339-1345Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar,21.Li T. et al.Tumor suppression in the absence of p53-mediated cell-cycle arrest, apoptosis, and senescence.Cell. 2012; 149: 1269-1283Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar]. Furthermore, although there is an enhancement of tumorigenesis observed in Eμ-Myc;p21–/–;Puma–/– mice relative to Eμ-Myc counterparts, this effect is not as potent as the acceleration of lymphomagenesis in Eμ-Myc;p53+/– mice [82.Valente L.J. et al.Combined loss of PUMA and p21 accelerates c-MYC-driven lymphoma development considerably less than loss of one allele of p53.Oncogene. 2016; 35: 3866-3871Crossref PubMed Scopus (24) Google Scholar]. Collectively, these studies have highlighted the need to investigate additional p53-regulated pathways that might compensate for p21, Puma, and Noxa or their associated functions to maintain tumor suppression in their absence. These studies do not strictly rule out a tumor-suppressive role for cell cycle arrest and apoptosis responses activated by other stresses that p53 responds to, such as chronic DNA damage, oncogenic signaling, hypoxia, and nutrient deprivation, amongst others. The finding that p5325,26 activates only a small subset of p53 target genes yet remains an active tumor suppressor presented an opportunity to interrogate genes downstream of p53 involved in tumor suppression in a focused manner [4.Brady C.A. et al.Distinct p53 transcriptional programs dictate acute DNA damage responses and tumor suppression.Cell. 2011; 145: 571-583Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar]. Gene expression analysis identified 87 genes activated in both oncogene-expressing p53+/+ and p5325,26/25,26 murine embryonic fibroblasts (MEFs) [7.Bieging-Rolett K.T. et al.Zmat3 is a key splicing regulator in the p53 tumor suppression program.Mol. Cell. 2020; 80: 452-469.e9Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar]. In an unbiased approach to probe tumor suppressor activity in vivo, oncogene-expressing MEFs expressing shRNA or sgRNA libraries targeting these 87 genes were each transplanted subcutaneously into mice to seed tumors. Enrichment of sh/sgRNA elements was sought in the resulting tumors to identify tumor suppressors. The top hit identified by both the shRNA and sgRNA screens was the p53 target Zmat3, which encodes an RNA-binding protein, suggesting that Zmat3 is a potent tumor suppressor. The shRNA screen also confirmed the tumor suppressor status of Ptpn14, a p53-inducible gene recently linked with suppression of PDAC as an inhibitor of the Yap protein [6.Mello S.S. et al.A p53 super-tumor suppressor reveals a tumor suppressive p53-Ptpn14-Yap Axis in pancreatic cancer.Cancer Cell. 2017; 32: 460-473.e6Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar]. Follow-up studies confirmed Zmat3 tumor suppressor function in Kras-driven LUAD and hepatocellular carcinoma (HCC) GEMMs [7.Bieging-Rolett K.T. et al.Zmat3 is a key splicing regulator in the p53 tumor suppression program.Mol. Cell. 2020; 80: 452-469.e9Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar]. Combined CLIP-seq and RNA-seq analyses revealed that Zmat3 regulates an alternative splicing program by binding RNAs upstream of 3′ splice sites of specific introns and regulating exon skipping, leading to changes in the transcriptome. These findings, along with a recent study that found that ZMAT3 inhibits clonogenicity of colon cancer cells by controlling the splicing of CD44, suggest a link between p53-mediated tumor suppression and splicing [7.Bieging-Rolett K.T. et al.Zmat3 is a key splicing regulator in the p53 tumor suppression program.Mol. Cell. 2020; 80: 452-469.e9Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar,8.Muys B.R. et al.The p53-induced RNA-binding protein ZMAT3 is a splicing regulator that inhibits the splicing of oncogenic CD44 variants in colorectal carcinoma.Genes Dev. 2020; (Published online December 17, 2020. https://doi.org/10.1101/gad.342634.120)PubMed Google Scholar]. Zmat3 deficiency did not promote tumor growth to the extent of p53 loss, suggesting that Zmat3 is one of multiple tumor suppression effectors downstream of p53. The significance of ZMAT3 to cancer suppression in humans is supported by recent meta-analyses of CRISPR/Cas9 and shRNA screens in human cell lines, which revealed that ZMAT3 depletion significantly promotes proliferation in cells with functional p53 but not with p53-deficiency [7.Bieging-Rolett K.T. et al.Zmat3 is a key splicing regulator in the p53 tumor suppression program.Mol. Cell. 2020; 80: 452-469.e9Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar,9.Giacomelli A.O. et al.Mutational processes shape the landscape of TP53 mutations in human cancer.Nat. Genet. 2018; 50: 1381-1387Crossref PubMed Scopus (128) Google Scholar]. ZMAT3 and p21 were the two p53 target gene effectors to reach the threshold of significant enrichment in wild-type versus p53-deficient cells set in this 'Cancer Dependency Map' (DepMap) meta-analysis, suggesting a centrality of these two target genes to the p53 tumor suppression network [9.Giacomelli A.O. et al.Mutational processes shape the landscape of TP53 mutations in human cancer.Nat. Genet. 2018; 50: 1381-1387Crossref PubMed Scopus (128) Google Scholar]. The surprising lack of spontaneous tumorigenesis in p21–/–;Puma–/–;Noxa–/– mice spurred efforts to identify p53 target genes that might play redundant or cooperative roles in tumor suppression. Toward this end, functional genetic screens using an shRNA library directed against 166 p53 target genes were conducted in mouse leukemia/lymphoma models in vivo [10.Janic A. et al.DNA repair processes are critical mediators of p53-dependent tumor suppression.Nat. Med. 2018; 24: 947-953Crossref PubMed Scopus (57) Google Scholar]. These screens were performed in sensitized backgrounds, using Eμ-Myc;Puma-/- or
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