The occurrence and development of tumors require the metabolic reprogramming of cancer cells, namely the alteration of flux in an autonomous manner via various metabolic pathways to meet increased bioenergetic and biosynthetic demands. Tumor cells consume large quantities of nutrients and produce related metabolites via their metabolism; this leads to the remodeling of the tumor microenvironment (TME) to better support tumor growth. During TME remodeling, the immune cell metabolism and antitumor immune activity are affected. This further leads to the escape of tumor cells from immune surveillance and therefore to abnormal proliferation. This review summarizes the regulatory functions associated with the abnormal biosynthesis and activity of metabolic signaling molecules during the process of tumor metabolic reprogramming. In addition, we provide a comprehensive description of the competition between immune cells and tumor cells for nutrients in the TME, as well as the metabolites required for tumor metabolism, the metabolic signaling pathways involved, and the functionality of the immune cells. Finally, we summarize current research targeted at the development of tumor immunotherapy. We aim to provide new concepts for future investigations of the mechanisms underlying the metabolic reprogramming of tumors and explore the association of these mechanisms with tumor immunity.
Homologous recombination (HR) is an error-free DNA double-strand break (DSB) repair pathway, which safeguards genome integrity and cell viability. Human C-terminal binding protein (CtBP)-interacting protein (CtIP) is a central regulator of the pathway which initiates the DNA end resection in HR. Ubiquitination modification of CtIP is known in some cases to control DNA resection and promote HR. However, it remains unclear how cells restrain CtIP activity in unstressed cells. We show that the ubiquitin E3 ligase PPIL2 is recruited to DNA damage sites through interactions with an HR-related protein ZNF830, implying PPIL2's involvement in DNA repair. We found that PPIL2 interacts with and ubiquitinates CtIP at the K426 site, representing a hereunto unknown ubiquitination site. Ubiquitination of CtIP by PPIL2 suppresses HR and DNA resection. This inhibition of PPIL2 is also modulated by phosphorylation at multiple sites by PLK1, which reduces PPIL2 ubiquitination of CtIP. Our findings reveal new regulatory complexity in CtIP ubiquitination in DSB repair. We propose that the PPIL2-dependent CtIP ubiquitination prevents CtIP from interacting with DNA, thereby inhibiting HR.
In the challenging tumor microenvironment (TME), tumors coexist with diverse stromal cell types. During tumor progression and metastasis, a reciprocal interaction occurs between cancer cells and their environment. These interactions involve ongoing and evolving paracrine and proximal signaling. Intrinsic signal transduction in tumors drives processes such as malignant transformation, epithelial-mesenchymal transition, immune evasion, and tumor cell metastasis. In addition, cancer cells embedded in the tumor microenvironment undergo metabolic reprogramming. Their metabolites, serving as signaling molecules, engage in metabolic communication with diverse matrix components. These metabolites act as direct regulators of carcinogenic pathways, thereby activating signaling cascades that contribute to cancer progression. Hence, gaining insights into the intrinsic signal transduction of tumors and the signaling communication between tumor cells and various matrix components within the tumor microenvironment may reveal novel therapeutic targets. In this review, we initially examine the development of the tumor microenvironment. Subsequently, we delineate the oncogenic signaling pathways within tumor cells and elucidate the reciprocal communication between these pathways and the tumor microenvironment. Finally, we give an overview of the effect of signal transduction within the tumor microenvironment on tumor metabolism and tumor immunity.
Abstract The reversible post-translational modification (PTM) of proteins plays an important role in many cellular processes. Lysine crotonylation (Kcr) is a newly identified PTM, but its functional significance remains unclear. Here, we found that Kcr is involved in the replication stress response. We show that crotonylation of histone H2A at lysine 119 (H2AK119) and ubiquitination of H2AK119 are reversibly regulated by replication stress. Decrotonylation of H2AK119 by SIRT1 is a prerequisite for subsequent ubiquitination of H2AK119 by BMI1. Accumulation of ubiquitinated H2AK119 at reversed replication forks leads to the release of RNA Polymerase II and transcription repression in the vicinity of stalled replication forks. These effects attenuate transcription–replication conflicts (TRCs) and TRC-associated R-loop formation and DNA double-strand breaks. These findings suggest that decrotonylation and ubiquitination of H2A at lysine 119 act together to resolve replication stress-induced TRCs and protect genome stability.
Following a DNA double strand break (DSB), several nucleases and helicases coordinate to generate single-stranded DNA (ssDNA) with 3' free ends, facilitating precise DNA repair by homologous recombination (HR). The same nucleases can act on stalled replication forks, promoting nascent DNA degradation and fork instability. Interestingly, some HR factors, such as CtIP and BRCA1, have opposite regulatory effects on the two processes, promoting end resection at DSB but inhibiting the degradation of nascent DNA on stalled forks. However, the reason why nuclease actions are regulated by different mechanisms in two DNA metabolism is poorly understood. We show that human HELQ acts as a DNA end resection regulator, with opposing activities on DNA end resection at DSBs and on stalled forks as seen for other regulators. Mechanistically, HELQ helicase activity is required for EXO1-mediated DSB end resection, while ssDNA-binding capacity of HELQ is required for its recruitment to stalled forks, facilitating fork protection and preventing chromosome aberrations caused by replication stress. Here, HELQ synergizes with CtIP but not BRCA1 or BRCA2 to protect stalled forks. These findings reveal an unanticipated role of HELQ in regulating DNA end resection at DSB and stalled forks, which is important for maintaining genome stability.
Genetic mutations arising from various internal and external factors drive cells to become cancerous. Cancerous cells undergo numerous changes, including metabolic reprogramming and epigenetic modifications, to support their abnormal proliferation. This metabolic reprogramming leads to the altered expression of many metabolic enzymes and the accumulation of metabolites. Recent studies have shown that these enzymes and metabolites can serve as substrates or cofactors for chromatin-modifying enzymes, thereby participating in epigenetic modifications and promoting carcinogenesis. Additionally, epigenetic modifications play a role in the metabolic reprogramming and immune evasion of cancer cells, influencing cancer progression. This review focuses on the origins of cancer, particularly the metabolic reprogramming of cancer cells and changes in epigenetic modifications. We discuss how metabolites in cancer cells contribute to epigenetic remodeling, including lactylation, acetylation, succinylation, and crotonylation. Finally, we review the impact of epigenetic modifications on tumor immunity and the latest advancements in cancer therapies targeting these modifications.
Genome instability often arises at common fragile sites (CFSs) leading to cancer-associated chromosomal rearrangements. However, the underlying mechanisms of how CFS protection is achieved is not well understood. We demonstrate that BLM plays an important role in the maintenance of genome stability of structure-forming AT-rich sequences derived from CFSs (CFS-AT). BLM deficiency leads to increased DSB formation and hyper mitotic recombination at CFS-AT and induces instability of the plasmids containing CFS-AT. We further showed that BLM is required for suppression of CFS breakage upon oncogene expression. Both helicase activity and ATR-mediated phosphorylation of BLM are important for preventing genetic instability at CFS-AT sequences. Furthermore, the role of BLM in protecting CFS-AT is not epistatic to that of FANCM, a translocase that is involved in preserving CFS stability. Loss of BLM helicase activity leads to drastic decrease of cell viability in FANCM deficient cells. We propose that BLM and FANCM utilize different mechanisms to remove DNA secondary structures forming at CFS-AT on replication forks, thereby preventing DSB formation and maintaining CFS stability.
miRNAs are important regulators of eukaryotic gene expression. The post-transcriptional maturation of miRNAs is controlled by the Drosha-DiGeorge syndrome critical region gene 8 (DGCR8) microprocessor. Dysregulation of miRNA biogenesis has been implicated in the pathogenesis of human diseases, including cancers. C-terminal–binding protein–interacting protein (CtIP) is a well-known DNA repair factor that promotes the processing of DNA double-strand break (DSB) to initiate homologous recombination–mediated DSB repair. However, it was unclear whether CtIP has other unknown cellular functions. Here, we aimed to uncover the roles of CtIP in miRNA maturation and cancer cell metastasis. We found that CtIP is a potential regulatory factor that suppresses the processing of miRNA primary transcripts (pri-miRNA). CtIP directly bound to both DGCR8 and pri-miRNAs through a conserved Sae2-like domain, reduced the binding of Drosha to DGCR8 and pri-miRNA substrate, and inhibited processing activity of Drosha complex. CtIP depletion significantly increased the expression levels of a subset of mature miRNAs, including miR-302 family members that are associated with tumor progression and metastasis in several cancer types. We also found that CtIP-inhibited miRNAs, such as miR-302 family members, are not crucial for DSB repair. However, increase of miR-302b levels or loss of CtIP function severely suppressed human colon cancer cell line tumor cell metastasis in a mouse xenograft model. These studies reveal a previously unrecognized mechanism of CtIP in miRNA processing and tumor metastasis that represents a new function of CtIP in cancer. miRNAs are important regulators of eukaryotic gene expression. The post-transcriptional maturation of miRNAs is controlled by the Drosha-DiGeorge syndrome critical region gene 8 (DGCR8) microprocessor. Dysregulation of miRNA biogenesis has been implicated in the pathogenesis of human diseases, including cancers. C-terminal–binding protein–interacting protein (CtIP) is a well-known DNA repair factor that promotes the processing of DNA double-strand break (DSB) to initiate homologous recombination–mediated DSB repair. However, it was unclear whether CtIP has other unknown cellular functions. Here, we aimed to uncover the roles of CtIP in miRNA maturation and cancer cell metastasis. We found that CtIP is a potential regulatory factor that suppresses the processing of miRNA primary transcripts (pri-miRNA). CtIP directly bound to both DGCR8 and pri-miRNAs through a conserved Sae2-like domain, reduced the binding of Drosha to DGCR8 and pri-miRNA substrate, and inhibited processing activity of Drosha complex. CtIP depletion significantly increased the expression levels of a subset of mature miRNAs, including miR-302 family members that are associated with tumor progression and metastasis in several cancer types. We also found that CtIP-inhibited miRNAs, such as miR-302 family members, are not crucial for DSB repair. However, increase of miR-302b levels or loss of CtIP function severely suppressed human colon cancer cell line tumor cell metastasis in a mouse xenograft model. These studies reveal a previously unrecognized mechanism of CtIP in miRNA processing and tumor metastasis that represents a new function of CtIP in cancer. miRNAs are a major class of short noncoding RNA molecules that post-transcriptionally modulate gene expression by repressing the translation and/or promoting the degradation of target messenger RNAs (1Ghildiyal M. Zamore P.D. Small silencing RNAs: An expanding universe.Nat. Rev. Genet. 2009; 10: 94-108Crossref PubMed Scopus (1676) Google Scholar, 2Bartel D.P. MicroRNAs: Target recognition and regulatory functions.Cell. 2009; 136: 215-233Abstract Full Text Full Text PDF PubMed Scopus (14311) Google Scholar, 3Huntzinger E. Izaurralde E. Gene silencing by microRNAs: Contributions of translational repression and mRNA decay.Nat. Rev. Genet. 2011; 12: 99-110Crossref PubMed Scopus (1554) Google Scholar). MiRNAs are involved in a wide range of physiological and pathological processes. Generally, miRNAs are encoded as individual genes or within clusters comprising several different miRNAs. The biogenesis of miRNAs starts through the transcription of miRNA genes by RNA polymerase II, which produces miRNA primary transcripts (pri-miRNA) containing a stem–loop hairpin structure (4Kim V.N. Han J. Siomi M.C. Biogenesis of small RNAs in animals.Nat. Rev. Mol. Cell Biol. 2009; 10: 126-139Crossref PubMed Scopus (2404) Google Scholar, 5Lee Y. Kim M. Han J. Yeom K.H. Lee S. Baek S.H. Kim V.N. MicroRNA genes are transcribed by RNA polymerase II.EMBO J. 2004; 23: 4051-4060Crossref PubMed Scopus (2987) Google Scholar). In the nucleus, pri-miRNAs are cleaved by the microprocessor complex containing the ribonuclease (RNase) III enzyme Drosha and its cofactor DiGeorge syndrome critical region gene 8 (DGCR8) into precursor miRNAs (pre-miRNAs) with 70 to 100 nucleotide-long hairpin structures (6Gregory R.I. Yan K.P. Amuthan G. Chendrimada T. Doratotaj B. Cooch N. Shiekhattar R. The Microprocessor complex mediates the genesis of microRNAs.Nature. 2004; 432: 235-240Crossref PubMed Scopus (1954) Google Scholar, 7Lee Y. Ahn C. Han J. Choi H. Kim J. Yim J. Lee J. Provost P. Radmark O. Kim S. Kim V.N. The nuclear RNase III Drosha initiates microRNA processing.Nature. 2003; 425: 415-419Crossref PubMed Scopus (3710) Google Scholar). The pre-miRNAs are then exported to the cytoplasm by exportin-5, where they are further processed into mature miRNAs by cytoplasmic Dicer RNase III to yield an approximately 22-nucleotide miRNA duplex (8Yi R. Qin Y. Macara I.G. Cullen B.R. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs.Genes Dev. 2003; 17: 3011-3016Crossref PubMed Scopus (2058) Google Scholar, 9Lund E. Guttinger S. Calado A. Dahlberg J.E. Kutay U. Nuclear export of microRNA precursors.Science. 2004; 303: 95-98Crossref PubMed Scopus (1939) Google Scholar, 10Bernstein E. Caudy A.A. Hammond S.M. Hannon G.J. Role for a bidentate ribonuclease in the initiation step of RNA interference.Nature. 2001; 409: 363-366Crossref PubMed Scopus (3562) Google Scholar, 11Ketting R.F. Fischer S.E. Bernstein E. Sijen T. Hannon G.J. Plasterk R.H. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans.Genes Dev. 2001; 15: 2654-2659Crossref PubMed Scopus (1371) Google Scholar). One strand of the miRNA duplex is subsequently incorporated into the RNA-induced silencing complex. Base pairing between the miRNA and target mRNA transcripts at their 3′ UTRs guides the RNA-induced silencing complex to induce the mRNA degradation or translation inhibition (12Khvorova A. Reynolds A. Jayasena S.D. Functional siRNAs and miRNAs exhibit strand bias.Cell. 2003; 115: 209-216Abstract Full Text Full Text PDF PubMed Scopus (1909) Google Scholar, 13Schwarz D.S. Hutvagner G. Du T. Xu Z. Aronin N. Zamore P.D. Asymmetry in the assembly of the RNAi enzyme complex.Cell. 2003; 115: 199-208Abstract Full Text Full Text PDF PubMed Scopus (2093) Google Scholar). The production and maturation of miRNA must be strictly regulated, as any disruption of the control mechanisms may lead to the development of various kinds of diseases, including cancer. As noted previously, miRNA expression involves the transcription of miRNA genes and the maturation of primary transcripts. Thus, miRNA levels may be regulated in both a transcription-dependent and transcription-independent manner. However, in many cases, the levels of mature miRNAs are determined by post-transcriptional maturation rather than transcriptional regulation. The level of expression and activity of core components of the miRNA biogenesis machinery are often found to be dysregulated in cancer. For instance, the expression levels of Drosha and Dicer are either increased or decreased in various types of cancers, and they are inversely correlated with advanced tumor stages and poor clinical outcomes (14Hata A. Kashima R. Dysregulation of microRNA biogenesis machinery in cancer.Crit. Rev. Biochem. Mol. Biol. 2016; 51: 121-134Crossref PubMed Scopus (92) Google Scholar). Somatic and germline mutations of Drosha or DGCR8 have also been frequently found in some cancers (15Davalos V. Esteller M. MicroRNAs and cancer epigenetics: A macrorevolution.Curr. Opin. Oncol. 2010; 22: 35-45Crossref PubMed Scopus (123) Google Scholar, 16Merritt W.M. Lin Y.G. Han L.Y. Kamat A.A. Spannuth W.A. Schmandt R. Urbauer D. Pennacchio L.A. Cheng J.F. Nick A.M. Deavers M.T. Mourad-Zeidan A. Wang H. Mueller P. Lenburg M.E. et al.Dicer, Drosha, and outcomes in patients with ovarian cancer.New Engl. J. Med. 2008; 359: 2641-2650Crossref PubMed Scopus (565) Google Scholar). In particular, the activity of the Drosha microprocessor is modulated by different nuclear proteins in a manner that usually affects the processing of only a small subset of miRNAs. For example, the RNA-editing enzyme adenosine deaminase acting on RNA 1 interacts with DGCR8 and suppresses microprocessor activity by reducing the availability of DGCR8 to Drosha. The expression of a proportion of miRNAs was upregulated in adenosine deaminase acting on RNA 1-defective cancer cells, which may have facilitated the malignant activity of metastatic melanoma (17Nemlich Y. Greenberg E. Ortenberg R. Besser M.J. Barshack I. Jacob-Hirsch J. Jacoby E. Eyal E. Rivkin L. Prieto V.G. Chakravarti N. Duncan L.M. Kallenberg D.M. Galun E. Bennett D.C. et al.MicroRNA-mediated loss of ADAR1 in metastatic melanoma promotes tumor growth.J. Clin. Invest. 2013; 123: 2703-2718Crossref PubMed Scopus (111) Google Scholar). Smad proteins, the signal transducers of transforming growth factor-β, also modulate Drosha activity in the nucleus. Smads are recruited to the Drosha complex by DEAD-box helicase 5 (DDX5) and promote the pri-miRNA processing of about 20 miRNAs. Among them, miR-21 promotes the metastatic and invasive potential of cancer cells through the inhibition of a large group of tumor suppressor genes (18Davis B.N. Hilyard A.C. Lagna G. Hata A. SMAD proteins control DROSHA-mediated microRNA maturation.Nature. 2008; 454: 56-61Crossref PubMed Scopus (1053) Google Scholar, 19Davis B.N. Hilyard A.C. Nguyen P.H. Lagna G. Hata A. Smad proteins bind a conserved RNA sequence to promote microRNA maturation by Drosha.Mol. Cell. 2010; 39: 373-384Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). In addition, the central tumor suppressor p53 and several RNA-binding proteins, including KH-type splicing regulatory protein, TAR DNA-binding protein-43, DEAD-box 1, heterogeneous nuclear ribonucleoprotein A1, and breast cancer 1 (BRCA1), have also been identified as regulatory proteins that interact with Drosha complexes and modulate the maturation of specific miRNAs (20Guil S. Caceres J.F. The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a.Nat. Struct. Mol. Biol. 2007; 14: 591-596Crossref PubMed Scopus (415) Google Scholar, 21Michlewski G. Caceres J.F. Antagonistic role of hnRNP A1 and KSRP in the regulation of let-7a biogenesis.Nat. Struct. Mol. Biol. 2010; 17: 1011-1018Crossref PubMed Scopus (199) Google Scholar, 22Suzuki H.I. Yamagata K. Sugimoto K. Iwamoto T. Kato S. Miyazono K. Modulation of microRNA processing by p53.Nature. 2009; 460: 529-533Crossref PubMed Scopus (904) Google Scholar, 23Trabucchi M. Briata P. Garcia-Mayoral M. Haase A.D. Filipowicz W. Ramos A. Gherzi R. Rosenfeld M.G. The RNA-binding protein KSRP promotes the biogenesis of a subset of microRNAs.Nature. 2009; 459: 1010-1014Crossref PubMed Scopus (491) Google Scholar, 24Han C. Liu Y. Wan G. Choi H.J. Zhao L. Ivan C. He X. Sood A.K. Zhang X. Lu X. The RNA-binding protein DDX1 promotes primary microRNA maturation and inhibits ovarian tumor progression.Cell Rep. 2014; 8: 1447-1460Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 25Kawahara Y. Mieda-Sato A. TDP-43 promotes microRNA biogenesis as a component of the Drosha and Dicer complexes.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 3347-3352Crossref PubMed Scopus (271) Google Scholar, 26Kawai S. Amano A. BRCA1 regulates microRNA biogenesis via the DROSHA microprocessor complex.J. Cell Biol. 2012; 197: 201-208Crossref PubMed Scopus (132) Google Scholar). It is likely that regulatory components in the core microprocessor complex select specific miRNAs by controlling miRNA processing. Although initially identified as a transcription repressor, C-terminal–binding protein (CtBP)–interacting protein (CtIP) is better known for its functions within DNA double-strand break (DSB) processing. Together with the meiotic recombination 11 (MRE11)–ATP-binding cassette—ATPase (RAD50)–Nijmegen breakage syndrome protein 1 (NBS1) (MRN) complex, CtIP efficiently promotes end resection, which generates 3′-end ssDNA filaments to promote homologous recombination (HR)–mediated DSB repair (27Sartori A.A. Lukas C. Coates J. Mistrik M. Fu S. Bartek J. Baer R. Lukas J. Jackson S.P. Human CtIP promotes DNA end resection.Nature. 2007; 450: 509-514Crossref PubMed Scopus (922) Google Scholar, 28Chen L. Nievera C.J. Lee A.Y. Wu X. Cell cycle-dependent complex formation of BRCA1.CtIP.MRN is important for DNA double-strand break repair.J. Biol. Chem. 2008; 283: 7713-7720Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). CtIP and its yeast functional homolog, Sae2, harbor structure-dependent endonuclease activity, which is required for cleaning "dirty" DSB ends, R-loop processing, and the stabilization of stalled replication forks, but it is dispensable for end resection of regular DSBs (29Makharashvili N. Tubbs A.T. Yang S.H. Wang H. Barton O. Zhou Y. Deshpande R.A. Lee J.H. Lobrich M. Sleckman B.P. Wu X. Paull T.T. Catalytic and noncatalytic roles of the CtIP endonuclease in double-strand break end resection.Mol. Cell. 2014; 54: 1022-1033Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 30Wang H. Li Y. Truong L.N. Shi L.Z. Hwang P.Y. He J. Do J. Cho M.J. Li H. Negrete A. Shiloach J. Berns M.W. Shen B. Chen L. Wu X. CtIP maintains stability at common fragile sites and inverted repeats by end resection-independent endonuclease activity.Mol. Cell. 2014; 54: 1012-1021Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 31Przetocka S. Porro A. Bolck H.A. Walker C. Lezaja A. Trenner A. von Aesch C. Himmels S.F. D'Andrea A.D. Ceccaldi R. Altmeyer M. Sartori A.A. CtIP-mediated fork protection synergizes with BRCA1 to suppress genomic instability upon DNA replication stress.Mol. Cell. 2018; 72: 568-582 e566Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 32Makharashvili N. Arora S. Yin Y. Fu Q. Wen X. Lee J.H. Kao C.H. Leung J.W. Miller K.M. Paull T.T. Sae2/CtIP prevents R-loop accumulation in eukaryotic cells.eLife. 2018; 7Crossref PubMed Scopus (26) Google Scholar, 33Lengsfeld B.M. Rattray A.J. Bhaskara V. Ghirlando R. Paull T.T. Sae2 is an endonuclease that processes hairpin DNA cooperatively with the Mre11/Rad50/Xrs2 complex.Mol. Cell. 2007; 28: 638-651Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). CtIP is a large protein containing several functional domains. The N-terminal region is required for the oligomerization of the protein and interaction with Nbs1 (34Wang H. Shao Z. Shi L.Z. Hwang P.Y. Truong L.N. Berns M.W. Chen D.J. Wu X. CtIP protein dimerization is critical for its recruitment to chromosomal DNA double-stranded breaks.J. Biol. Chem. 2012; 287: 21471-21480Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 35Davies O.R. Forment J.V. Sun M. Belotserkovskaya R. Coates J. Galanty Y. Demir M. Morton C.R. Rzechorzek N.J. Jackson S.P. Pellegrini L. CtIP tetramer assembly is required for DNA-end resection and repair.Nat. Struct. Mol. Biol. 2015; 22: 150-157Crossref PubMed Scopus (47) Google Scholar, 36Yuan J. Chen J. N terminus of CtIP is critical for homologous recombination-mediated double-strand break repair.J. Biol. Chem. 2009; 284: 31746-31752Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The middle part of CtIP is important for its endonuclease activity and interactions with multiple proteins, including BRCA1, CtBP transcriptional repressor, retinoblastoma-associated protein, and proliferating cell nuclear antigen. Phosphorylations at multiple sites of this region facilitate the capacity of CtIP to promote MRN- and DNA2-mediated DSB end resection and end resection–dependent DSB repair (37Wang H. Shi L.Z. Wong C.C. Han X. Hwang P.Y. Truong L.N. Zhu Q. Shao Z. Chen D.J. Berns M.W. Yates 3rd, J.R. Chen L. Wu X. The interaction of CtIP and Nbs1 connects CDK and ATM to regulate HR-mediated double-strand break repair.PLoS Genet. 2013; 9e1003277Crossref PubMed Scopus (136) Google Scholar, 38Anand R. Ranjha L. Cannavo E. Cejka P. Phosphorylated CtIP functions as a co-factor of the MRE11-RAD50-NBS1 endonuclease in DNA end resection.Mol. Cell. 2016; 64: 940-950Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 39Ceppi I. Howard S.M. Kasaciunaite K. Pinto C. Anand R. Seidel R. Cejka P. CtIP promotes the motor activity of DNA2 to accelerate long-range DNA end resection.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 8859-8869Crossref PubMed Scopus (13) Google Scholar, 40Anand R. Jasrotia A. Bundschuh D. Howard S.M. Ranjha L. Stucki M. Cejka P. NBS1 promotes the endonuclease activity of the MRE11-RAD50 complex by sensing CtIP phosphorylation.EMBO J. 2019; 38Crossref Scopus (32) Google Scholar). The C terminus of CtIP shares the most similarity with Sae2 and thus was named the Sae2-like domain (27Sartori A.A. Lukas C. Coates J. Mistrik M. Fu S. Bartek J. Baer R. Lukas J. Jackson S.P. Human CtIP promotes DNA end resection.Nature. 2007; 450: 509-514Crossref PubMed Scopus (922) Google Scholar, 41Limbo O. Chahwan C. Yamada Y. de Bruin R.A. Wittenberg C. Russell P. Ctp1 is a cell-cycle-regulated protein that functions with Mre11 complex to control double-strand break repair by homologous recombination.Mol. Cell. 2007; 28: 134-146Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). This region is critical for the regulation of MRN nuclease activity in vitro and used for end resection of some DSBs in vivo (27Sartori A.A. Lukas C. Coates J. Mistrik M. Fu S. Bartek J. Baer R. Lukas J. Jackson S.P. Human CtIP promotes DNA end resection.Nature. 2007; 450: 509-514Crossref PubMed Scopus (922) Google Scholar, 38Anand R. Ranjha L. Cannavo E. Cejka P. Phosphorylated CtIP functions as a co-factor of the MRE11-RAD50-NBS1 endonuclease in DNA end resection.Mol. Cell. 2016; 64: 940-950Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). However, the mechanisms by which conserved Sae2-like domains contribute to the function of CtIP are still largely unknown. In this study, we aimed to investigate the functions of CtIP in miRNA post-transcription processing, its association with the Drosha-DGCR8 microprocessor, and its effects in cancer cells. Using immunoprecipitation experiments, we assessed the associations CtIP with Drosha-DGCR8 microprocessor complex proteins and pri-miRNA. We then investigated the effects of knocking out the CtIP gene on colorectal cancer cell miRNA expression and metastasis as well as osteosarcoma cell DSB end processing and repair. We found that CtIP is involved in Drosha-DGCR8 microprocessor–mediated pri-miRNA processing through its Sae2-like domain. This domain was shown to directly interact with DGCR8 and pri-miRNAs and suppress the Drosha complex processing of a subset of pri-miRNA substrates, including miR-302 family members. The depletion of CtIP or overexpression of miR-302b strongly inhibited the metastasis of human colon cancer cell line (HCT116) colorectal cancer cells in vitro and in an orthotopic xenograft mouse model. The findings suggest that the classical DSB repair factor CtIP promotes the metastatic capacity of tumor cells through the regulation of miRNA maturation. In order to explore the CtIP-associated proteins that specifically interact with its C-terminal conserved Sae2-like domain, high-purity CtIP-C (Sae2-like domain) protein (Fig. 1A) was obtained and coupled with cyanogen bromide–activated Sepharose 4B, and CtIP-C affinity purification was performed using 293T cell lysate. CtIP-C–associated proteins were then subjected to mass spectrometry analysis (Fig. 1A). We found several known CtIP-associated proteins, including DNA2, proliferating cell nuclear antigen, 3'-5' exonuclease domain-containing protein 2, and CtBP. Surprisingly, several Drosha-DGCR8 microprocessor components, such as DDX5, DEAD-box 1, and KH-type splicing regulatory protein, also were copurified (Fig. 1A). To confirm the association of CtIP with the DGCR8 microprocessor, we performed a regular glutathione-S-transferase (GST) pull-down assay using GST-CtIP-C fusion protein as bait. We again found that CtIP-C interacts with the DSB repair factors Nbs1 and Rad50 and the microprocessor components DGCR8 and DDX5 (Fig. 1B). Furthermore, we performed coimmunoprecipitation experiments to confirm the interactions and found that endogenous Drosha and DGCR8 were present in the CtIP immunocomplex (Fig. 1C). These data indicate that CtIP is a new Drosha-DGCR8–associated protein, and CtIP may have a role in miRNA biogenesis. To determine the effect of CtIP on cancer miRNA biogenesis, we analyzed the miRNA expression profiles in wildtype HCT116 and CtIP-deficient HCT116 cells (CtIP-KO) using a human cancer miRNA quantitative PCR (qPCR) array. The depletion of CtIP in HCT116 cells significantly increased the expression of a subset of 49 miRNAs (cutoff greater than 3-fold; Fig. 2A), which suggests CtIP inhibited the expression of these miRNAs. We prioritized the miRNAs with the most significant changes and performed a regular qPCR assay to confirm the effects of CtIP on these selected miRNAs. Levels of mature miRNAs, including three miR-302 members (miR-302b, miR-302a, and miR-302d) and miR-135a, were upregulated in CtIP-KO cells (Fig. 2B). A similar effect was observed when CtIP expression was suppressed by shRNAs in HCT116 cells (Fig. 2C) and other common tumor cell lines, including Michigan Cancer Foundation-7 (Fig. 2D) and human osteosarcoma cell line (U2OS) (Fig. 2E). Furthermore, CtIP depletion did not alter the primary transcripts of selected miRNAs (Fig. 2F), suggesting that CtIP regulated the expression of specific miRNAs at the post-transcriptional level. As mentioned earlier, after transcription, the pri-miRNAs are processed by nuclear Drosha-DGCR8 and cytoplasmic DICER1. CtIP is only functional in cell nucleus (Fig. S1A). The expression level of DICER1 is not changed in CtIP-KO cells (Fig. S1B). CtIP may not directly or indirectly regulate maturation of miRNA through DICER1. In addition, pre-miR302b level was also upregulated in CtIP-KO cells (Fig. S1C). We thus thought that CtIP should suppress the levels of specific mature miRNAs through modulation of nucleus processing of these miRNAs. To understand how CtIP regulates miRNA processing, we performed GST pull-down assays using purified full-length CtIP from insect cells to examine the interactions between CtIP and components of the nuclear miRNA-processing complex. We found there was a direct interaction between CtIP and DGCR8 (Figs. 3A and S2). The interaction between CtIP and DGCR8 was then characterized. The central region (200–460 and 460–600 fragments) and the C-terminal 770 to 897 fragment (the Sae2-like domain) of CtIP were shown to interact with DGCR8 (Figs. 3B and S2). DGCR8 contains a nuclear localization signal at the N-terminal region, a central RNA-binding heme domain (Rhed), two double-stranded RNA-binding domains, and a C-terminal tail (42Shiohama A. Sasaki T. Noda S. Minoshima S. Shimizu N. Nucleolar localization of DGCR8 and identification of eleven DGCR8-associated proteins.Exp. Cell Res. 2007; 313: 4196-4207Crossref PubMed Scopus (82) Google Scholar, 43Quick-Cleveland J. Jacob J.P. Weitz S.H. Shoffner G. Senturia R. Guo F. The DGCR8 RNA-binding heme domain recognizes primary microRNAs by clamping the hairpin.Cell Rep. 2014; 7: 1994-2005Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 44Sohn S.Y. Bae W.J. Kim J.J. Yeom K.H. Kim V.N. Cho Y. Crystal structure of human DGCR8 core.Nat. Struct. Mol. Biol. 2007; 14: 847-853Crossref PubMed Scopus (91) Google Scholar, 45Han J. Lee Y. Yeom K.H. Nam J.W. Heo I. Rhee J.K. Sohn S.Y. Cho Y. Zhang B.T. Kim V.N. Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex.Cell. 2006; 125: 887-901Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar). Using purified proteins, we found that the GST-fused Rhed domain alone (GST-DG-Rhed) and the nuclear localization signal deletion mutant (GST-DG-△N) bound to CtIP (Fig. 3C). These data suggest that the Rhed domain, but not other domains on DGCR8, is important in the interaction between DGCR8 and CtIP. We next used a bimolecular fluorescence complementation (BiFC) reporter to examine the potential interaction of CtIP and DGCR8 in living cells. This reporter relies on protein interactions bringing together ectopically expressed Venus N-terminal (VN-) and Venus C-terminal (VC-) fragments of fluorescent protein reconstitute fluorescence of YFP protein, thus allowing direct visualization of protein interactions in their normal cellular environment. As expected, cotransfection of 293T cells with either of VN-vector and DGCR8-VC or CtIP-VN and VC-vector constructs generated no fluorescence signal. However, cotransfection of 293T cells with CtIP-VN and DGCR8-VC resulted in obvious fluorescence (Fig. 3D), suggesting a direct interaction between CtIP and DGCR8 in cells. To further confirm this finding, we performed proximity ligation assays (in situ PLAs) using antibodies directed against CtIP and DGCR8. As shown in Figure 3E, PLA foci could be readily detected in the nucleus when both CtIP and DGCR8 antibody were used. The PLA signals were not observed in the absence of one primary antibody. These results suggest that CtIP directly interacts with DGCR8 both in vitro and in the cells. In our previous study, we found that CtIP binds to the hairpin DNA substrate (30Wang H. Li Y. Truong L.N. Shi L.Z. Hwang P.Y. He J. Do J. Cho M.J. Li H. Negrete A. Shiloach J. Berns M.W. Shen B. Chen L. Wu X. CtIP maintains stability at common fragile sites and inverted repeats by end resection-independent endonuclease activity.Mol. Cell. 2014; 54: 1012-1021Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). We thus speculated that CtIP also engages with pri-miRNA, which contains a similar secondary structure with a hairpin DNA substrate. As revealed by RNA chromatin immunoprecipitation (RNA-ChIP), overexpressed CtIP was associated with pri-miR302 transcripts in vivo (Fig. 4A). We further performed an RNA pull-down assay using a purified GST-CtIP fusion protein as bait to test whether CtIP directly interacts with pri-miRNA in vitro. We found that the in vitro–transcribed pri-miR302b was efficiently pulled down by purified GST-CtIP (full-length) in comparison with GST protein alone (Fig. 4B). These data indicate the direct binding of CtIP to pri-miR302. We next performed an electrophoretic mobility shift assay (EMSA) assay using Escherichia coli–produced CtIP fragments to map the determinants of the interaction. Intriguingly, pri-miR302 efficiently bound to a region of CtIP 770 to 897, highlighting the importance of the conserved Sae2-like domain of CtIP for pri-miRNA binding. We further confirmed the interaction between CtIP and pri-miR302 using a microscale thermophoresis (MST) assay, which can quantitatively define the interaction between two molecules. Consistent with the RNA pull-down and EMSA data, both GFP-tagged CtIP (GFP-CtIP) with pri-miR302b (Fig. 4D) and Cyp-labeled pri-miR302b with CtIP-C protein (Fig. 4E) exhibited obvious interactions in vitro. The CtIP-C protein yielded a dissociation constant (Kd) of 2.39 μM, which is comparable to the Kd value obtained for full-length CtIP (GFP-CtIP, 3.94 μM). These data support the notion that the conserved Sae2-like domain of CtIP is a secondary structure containing RNA interactor that mediates the interaction between CtIP and pri-miRNA. The direct interaction of CtIP with DGCR8 and pri-miRNA substrates suggests that CtIP may interfere with the binding of DGCR8 or pri-miRNA substrate to Drosha, which is the core RNase III enzyme in the microprocessor. We compared the capacity of Drosha to bind to DGCR8 and pri-miR302 in the presence or absence of CtIP. Consistent with previous reports, we found that endogenous Drosha or overexpressed GFP-Drosha interacted with DGCR8 in HCT116 cells (Fig. 5, A and B). Interestingly, when CtIP was knocked out, the interactions increased (Fig. 5, A and B), suggesting that endogenous CtIP hinders the interaction between Drosha and its cofactors. In line with these results, ectopic expression of Flag-CtIP reduced the YFP fluorescent intensity reconstituted by cotransfection of Drosha-VN and DGCR8-VC in 293T cells (Fig. 5C). These data support a notion that CtIP may interfere with the binding of Drosha to DGCR8. Next, we performed RNA-ChIP using Flag-Drosha to analyze the relationship between Drosha and RNA substrates. The data indicated that CtIP depletion obviously increased the association between Drosha and pri-miR302 (Fig. 5D). The attenuated binding of Drosha to a substrate might suppress its enzyme activity. Therefore, we used an in vitro pri-miRNA processing assay to assess the effect of CtIP on Drosha activity. Clearly, purified GST-CtIP but not GST alone efficiently inhibited the processing activity of Drosha on pri-miR302b (Fig. 5E). Taken together, our data suggest that CtIP reduces the binding of Drosha to its cofactor DGCR8 and pri-miRNA
The DNA damage response (DDR) system plays an important role in maintaining genome stability and preventing related diseases. The DDR network comprises many proteins and posttranslational modifications (PTMs) to proteins, which work in a coordinated manner to counteract various genotoxic stresses. Lysine crotonylation (Kcr) is a newly identified PTM occurring in both core histone and non-histone proteins in various organisms. This novel PTM is classified as a reversible acylation modification, which is regulated by a variety of acylases and deacylases and the intracellular crotonyl-CoA substrate concentration. Recent studies suggest that Kcr links cellular metabolism with gene regulation and is involved in numerous cellular processes. In this review, we summarize the regulatory mechanisms of Kcr and its functions in DDR, including its involvement in double-strand break (DSB)-induced transcriptional repression, DSB repair, and the DNA replication stress response.
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