General control non-depressible 5 (GCN5) is a crucial catalytic component of a transcriptional regulatory complex that plays important roles in cellular functions from cell cycle regulation to DNA damage repair. Although GCN5 has recently been implicated in certain oncogenic roles, its role in liver cancer progression remains vague.In this study, we report that GCN5 was overexpressed in 17 (54.8 %) of 31 human hepatocellular carcinoma (HCC) specimens. Down-regulation of GCN5 inhibited HCC cell proliferation and xenograft tumor formation. GCN5 knockdown decreased the protein levels of the proliferation marker proliferating cell nuclear antigen (PCNA) and amplified in breast cancer 1 (AIB1), but increased the protein levels of cell cycle inhibitor p21(Cip1/Waf1) in HepG2 cells. GCN5 regulated AIB1 expression, at least in part, by cooperating with E2F1 to enhance AIB1 transcription. Consistently, GCN5 expression was positively correlated with AIB1 expression in human HCC specimens in two GEO profile datasets.Since AIB1 plays a promoting role in HCC progression, our results propose that GCN5 promotes HCC progression at least partially by regulating AIB1 expression. This study implicates that GCN5 might be a potential molecular target for HCC diagnosis and treatment.
// Ming Li 1, 2, 3, 4, * , Wei Wang 1, 3, * , Yuzhen Dan 1 , Zhangwei Tong 1 , Wenbo Chen 1 , Liping Qin 1 , Kun Liu 1, 5 , Wengang Li 2, 4 , Pingli Mo 1 , Chundong Yu 1, 2, 3 1 State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, China 2 Xiamen City Key Laboratory of Biliary Tract Diseases, Chenggong Hospital of Xiamen University, Xiamen, China 3 Engineering Research Center of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, China 4 Department of Hepatobiliary Pancreas and Vessel Surgery, Chenggong Hospital of Xiamen University, Xiamen, China 5 Department of Pathology, Chenggong Hospital of Xiamen University, Xiamen, China * These authors contributed equally to this work Correspondence to: Chundong Yu, e-mail: cdyu@xmu.edu.cn Pingli Mo, e-mail: mop@xmu.edu.cn Keywords: sorafenib, AIB1, HCC, cell death, mRNA translation Received: October 14, 2015 Accepted: March 28, 2016 Published: April 18, 2016 ABSTRACT Multi-kinase inhibitor sorafenib represents a major breakthrough in the therapy of advanced hepatocellular carcinoma (HCC). Amplified in breast cancer 1 (AIB1) is frequently overexpressed in human HCC tissues and promotes HCC progression. In this study, we investigated the effects of sorafenib on AIB1 expression and the role of AIB1 in anti-tumor effects of sorafenib. We found that sorafenib downregulated AIB1 protein expression by inhibiting AIB1 mRNA translation through simultaneously blocking eIF4E and mTOR/p70S6K/RP-S6 signaling. Knockdown of AIB1 significantly promoted sorafenib-induced cell death, whereas overexpression of AIB1 substantially diminished sorafenib-induced cell death. Downregulation of AIB1 contributed to sorafenib-induced cell death at least in part through upregulating the levels of reactive oxygen species in HCC cells. In addition, resistance to sorafenib-induced downregulation of AIB1 protein contributes to the acquired resistance of HCC cells to sorafenib-induced cell death. Collectively, our study implicates that AIB1 is a molecular target of sorafenib and downregulation of AIB1 contributes to the anti-tumor effects of sorafenib.
Chronic hepatitis B virus (HBV) infection is one of the most serious global public health problems. The role of steroid receptor coactivator 3 (SRC-3) in HBV biosynthesis is unknown. The aim of this study is to investigate the function of SRC-3 in regulating HBV biosynthesis both in vitro and in vivo and to identify the underlying mechanism. In this study, we found that knockdown of SRC-3 could increase the levels of HBV mRNA and HBV proteins HBsAg and HBeAg in human liver cancer cell line HepG2 transfected with pHBV1.3 plasmids. In contrast, enforced expression of SRC-3 in SRC-3-knockdown HepG2 cells reduced the levels of HBV mRNA and HBV proteins HBsAg and HBeAg. Knockdown of SRC-3 dampened the Akt signaling, which has been shown to play a negative role in HBV transcription. Ectopic expression of constitutively activated Akt impaired the enhancement of HBV transcription by SRC-3 knockdown, indicating that SRC-3 inhibits HBV transcription by enhancing Akt signaling. Both SRC-3 and constitutively activated Akt could inhibit hepatocyte nuclear factor 4α (HNF4α)-mediated upregulation of HBV core promoter activity by preventing HNF4α nuclear translocation. Consistent with the in vitro results, in an in vivo chronic HBV replication mouse model developed by hydrodynamic injection of pHBV1.3 plasmids into mouse tail vein, enforced expression of SRC-3 in mouse liver reduced the levels of HBV mRNA in the liver and HBV antigens in serum, whereas knockout of SRC-3 in mouse increased the levels of HBV mRNA in the liver and HBV antigens in the serum. Our study suggests that SRC-3 inhibits HBV gene expression by activating Akt signaling to prevent HNF4α nuclear translocation.
Cancer stem-like cells (CSCs) contribute to the high rate of tumor heterogeneity, metastasis, therapeutic resistance, and recurrence. Histone lysine demethylase 4D (KDM4D or JMJD2D) is highly expressed in colon and liver tumors, where it promotes cancer progression; however, the role of JMJD2D in CSCs remains unclear. Here, we show that JMJD2D expression was increased in liver cancer stem-like cells (LCSCs); downregulation of JMJD2D inhibited the self-renewal of LCSCs in vitro and in vivo and inhibited the lung metastasis of LCSCs by reducing the survival and the early lung seeding of circulating LCSCs. Mechanistically, JMJD2D promoted LCSC self-renewal by enhancing the expression of CSC markers EpCAM and Sox9; JMJD2D reduced H3K9me3 levels on the promoters of EpCAM and Sox9 to enhance their transcription via interaction with β-catenin/TCF4 and Notch1 intracellular domain, respectively. Restoration of EpCAM and Sox9 expression in JMJD2D-knockdown liver cancer cells rescued the self-renewal of LCSCs. Pharmacological inhibition of JMJD2D using 5-c-8HQ reduced the self-renewal of LCSCs and liver cancer progression. Collectively, our findings suggest that JMJD2D promotes LCSC self-renewal by enhancing EpCAM and Sox9 expression via Wnt/β-catenin and Notch signaling pathways and is a potential therapeutic target for liver cancer. Cancer stem-like cells (CSCs) contribute to the high rate of tumor heterogeneity, metastasis, therapeutic resistance, and recurrence. Histone lysine demethylase 4D (KDM4D or JMJD2D) is highly expressed in colon and liver tumors, where it promotes cancer progression; however, the role of JMJD2D in CSCs remains unclear. Here, we show that JMJD2D expression was increased in liver cancer stem-like cells (LCSCs); downregulation of JMJD2D inhibited the self-renewal of LCSCs in vitro and in vivo and inhibited the lung metastasis of LCSCs by reducing the survival and the early lung seeding of circulating LCSCs. Mechanistically, JMJD2D promoted LCSC self-renewal by enhancing the expression of CSC markers EpCAM and Sox9; JMJD2D reduced H3K9me3 levels on the promoters of EpCAM and Sox9 to enhance their transcription via interaction with β-catenin/TCF4 and Notch1 intracellular domain, respectively. Restoration of EpCAM and Sox9 expression in JMJD2D-knockdown liver cancer cells rescued the self-renewal of LCSCs. Pharmacological inhibition of JMJD2D using 5-c-8HQ reduced the self-renewal of LCSCs and liver cancer progression. Collectively, our findings suggest that JMJD2D promotes LCSC self-renewal by enhancing EpCAM and Sox9 expression via Wnt/β-catenin and Notch signaling pathways and is a potential therapeutic target for liver cancer. Liver cancer represents the second most common cancer-related cause of death worldwide with most cases being diagnosed at an advanced stage (1Gingold J.A. Zhu D. Lee D.F. Kaseb A. Chen J. Genomic profiling and metabolic homeostasis in primary liver cancers.Trends Mol. Med. 2018; 24: 395-411Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Although there are significant developments in clinical therapies and research, the 5-year survival rate of liver cancer has not been improved well (2El-Serag H.B. Kanwal F. Epidemiology of hepatocellular carcinoma in the United States: where are we? Where do we go?.Hepatology. 2014; 60: 1767-1775Crossref PubMed Scopus (377) Google Scholar). Patients with liver cancer often encounter tumor progression, recurrence, and distant metastasis. A subset of cells with stem/progenitor cell features called cancer stem-like cells (CSCs) are responsible for the tumor initiation, growth, metastasis, recurrence, and therapeutic resistance (3Lobo N.A. Shimono Y. Qian D. Clarke M.F. The biology of cancer stem cells.Annu. Rev. Cell. Dev. Biol. 2007; 23: 675-699Crossref PubMed Scopus (829) Google Scholar, 4Ma S. Chan K.W. Hu L. Lee T.K. Wo J.Y. Ng I.O. Zheng B.J. Guan X.Y. Identification and characterization of tumorigenic liver cancer stem/progenitor cells.Gastroenterology. 2007; 132: 2542-2556Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar). Liver cancer stem-like cells (LCSCs) have been shown to be enriched by several CSCs markers, including CD13 (5Haraguchi N. Ishii H. Mimori K. Tanaka F. Ohkuma M. Kim H.M. Akita H. Takiuchi D. Hatano H. Nagano H. Barnard G.F. Doki Y. Mori M. 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Significance of CD90+ cancer stem cells in human liver cancer.Cancer Cell. 2008; 13: 153-166Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar). Some CSC markers such as EpCAM and Sox9 play essential roles in maintaining the self-renewal of LCSCs. Extensive signaling pathways (such as Wnt/β-catenin, Notch, Hedgehog, transforming growth factor-beta (TGF-β), and IL6/STAT3 pathways) have been reported to be involved in regulating the expression of CSC markers and promoting the self-renewal of CSCs (13Cairo S. Armengol C. De Reynies A. Wei Y. Thomas E. Renard C.A. Goga A. Balakrishnan A. Semeraro M. Gresh L. Pontoglio M. Strick-Marchand H. Levillayer F. Nouet Y. Rickman D. et al.Hepatic stem-like phenotype and interplay of Wnt/beta-catenin and Myc signaling in aggressive childhood liver cancer.Cancer Cell. 2008; 14: 471-484Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 14Takebe N. Miele L. Harris P.J. Jeong W. Bando H. Kahn M. Yang S.X. Ivy S.P. 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JMJD2D, also known as KDM4D, is a histone demethylase that belongs to JMJD2 family, which has five members (JMJD2A−2E) (18Katoh M. Katoh M. Identification and characterization of JMJD2 family genes in silico.Int. J. Oncol. 2004; 24: 1623-1628PubMed Google Scholar). JMJD2D contains a Jumonji C (JmjC) domain that removes methyl groups from lysine 9 on histone 3 (H3K9), lysine 79 on histone 3 (H3K79), and lysine 26 on histone 1.4 (H1.4K26) (19Berry W.L. Janknecht R. KDM4/JMJD2 histone demethylases: epigenetic regulators in cancer cells.Cancer Res. 2013; 73: 2936-2942Crossref PubMed Scopus (252) Google Scholar, 20Jbara M. Guttmann-Raviv N. Maity S.K. Ayoub N. Brik A. Total chemical synthesis of methylated analogues of histone 3 revealed KDM4D as a potential regulator of H3K79me3.Bioorg. Med. Chem. 2017; 25: 4966-4970Crossref PubMed Scopus (14) Google Scholar). JMJD2D has been reported to play important roles in androgen target genes activation, DNA damage repair, DNA replication, somatic cell nuclear transfer (SCNT), colitis, and colorectal, liver, and gastrointestinal stromal cancers (21Shin S. Janknecht R. Activation of androgen receptor by histone demethylases JMJD2A and JMJD2D.Biochem. Biophysical Res. Commun. 2007; 359: 742-746Crossref PubMed Scopus (150) Google Scholar, 22Khoury-Haddad H. Guttmann-Raviv N. Ipenberg I. Huggins D. Jeyasekharan A.D. Ayoub N. PARP1-dependent recruitment of KDM4D histone demethylase to DNA damage sites promotes double-strand break repair.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: E728-E737Crossref PubMed Scopus (74) Google Scholar, 23Wu R. Wang Z. Zhang H. Gan H. Zhang Z. H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication.Nucleic Acids Res. 2017; 45: 169-180Crossref PubMed Scopus (24) Google Scholar, 24Liu X. Wang Y. Gao Y. Su J. Zhang J. Xing X. 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Demethylase-independent function of JMJD2D as a novel antagonist of p53 to promote liver cancer initiation and progression.Theranostics. 2020; 10: 8863-8879Crossref PubMed Scopus (4) Google Scholar, 28Hu F. Li H. Liu L. Xu F. Lai S. Luo X. Hu J. Yang X. Histone demethylase KDM4D promotes gastrointestinal stromal tumor progression through HIF1beta/VEGFA signalling.Mol. Cancer. 2018; 17: 107Crossref PubMed Scopus (12) Google Scholar); however, the role of JMJD2D in CSCs remains unclear. In the present study, we reported that JMJD2D was upregulated in LCSCs and downregulation of JMJD2D markedly inhibited the self-renewal and proliferation of LCSCs in vitro and in vivo. Mechanistically, JMJD2D promoted the self-renewal of LCSC through enhancement of EpCAM and Sox9 expression Wnt/β-catenin and Notch signaling pathways. The JMJD2D inhibitor 5-c-8HQ could attenuate the self-renewal of LCSCs in vitro and in vivo. Our findings indicate that JMJD2D may be a potential therapeutic target against LCSCs. We previously reported that JMJD2D was overexpressed in colorectal and liver cancers and promoted cancer progression (25Zhuo M. Chen W. Shang S. Guo P. Peng K. Li M. Mo P. Zhang Y. Qiu X. Li W. Yu C. Inflammation-induced JMJD2D promotes colitis recovery and colon tumorigenesis by activating Hedgehog signaling.Oncogene. 2020; 39: 3336-3353Crossref PubMed Scopus (6) Google Scholar, 26Peng K. Kou L. Yu L. Bai C. Li M. Mo P. Li W. Yu C. Histone demethylase JMJD2D interacts with beta-catenin to induce transcription and activate colorectal cancer cell proliferation and tumor growth in mice.Gastroenterology. 2018; 156: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 27Ming L. Yuan D. Minghui Z. Hui Z. Xu K. Xiaogang X. Zhaojie S. Qiang C. Peng G. Pingli M. Chundong Y. Wengang L. Demethylase-independent function of JMJD2D as a novel antagonist of p53 to promote liver cancer initiation and progression.Theranostics. 2020; 10: 8863-8879Crossref PubMed Scopus (4) Google Scholar); however,the role of JMJD2D in CSCs remains unclear. Formation of tumorsphere in CSC culture media represents the principal characteristic of CSC self-renewal. To investigate the role of JMJD2D in liver LCSCs, we enriched the LCSCs by inducing hepatoma spheroid formation from human liver cancer cell lines HepG2 and Huh-7 as well as mouse liver cancer cell line Hepa1-6 and then measured the protein and mRNA expression of JMJD2D. As shown in Fig. 1A and Fig. S1A, both the protein and mRNA levels of JMJD2D were upregulated in LCSCs (tumorsphere) compared with non-CSCs (attached cells), suggesting that JMJD2D may promote the self-renewal of LCSCs. To test this hypothesis, we knocked down JMJD2D in HepG2 and Huh-7 using two different JMJD2D shRNAs (sh2D-1 and sh2D-2) and knocked out JMJD2D in Hepa1-6 using CRISPR-Cas9 system (2D-KO) (Fig. 1B) and then performed MTT assay to measure the cell proliferation and tumorsphere formation assay to measure the tumorsphere formation ability. Downregulation of JMJD2D significantly inhibited liver cancer cell proliferation (Fig. S1, B–D) and tumorsphere formation ability as demonstrated by reduced tumorsphere number and size (Fig. 1C), indicating that JMJD2D can promote the proliferation and tumorsphere formation of liver cancer cells. Furthermore, we employed the limiting dilution assay to determine the CSC self-renewal frequency. At 200, 400, 600, and 800 cell levels, downregulation of JMJD2D resulted in a significant decrease in tumorsphere number (Fig. 1D). Serial sphere formation assays validated that the self-renewal capacities of JMJD2D-downregulated cells were decreased as compared with control cells (Fig. 1E). Collectively, these results suggest that downregulation of JMJD2D reduces the self-renewal of LCSCs in vitro. CSCs have a strong ability to form tumors (29Clevers H. The cancer stem cell: premises, promises and challenges.Nat. Med. 2011; 17: 313-319Crossref PubMed Scopus (1315) Google Scholar). To characterize the role of JMJD2D in LCSC-derived tumor initiation and progression, in vivo limiting dilution assay was performed using LCSCs disassociated from cultured spheroids. LCSCs derived from JMJD2D-downregulated spheroids displayed a lower tumorigenicity compared with the cells from control spheroids (Fig. 2, A–B). LCSCs from JMJD2D-downregulated spheroids exhibited decreased subcutaneous graft tumor growth and tumor weight (Fig. 2, C–D). Furthermore, we performed Ki67 staining to determine the effects of JMJD2D downregulation on cell proliferation in vivo. As shown in Fig. 2, E–F, downregulation of JMJD2D dramatically reduced Ki67-positive cell number in subcutaneous graft tumors. Although subcutaneous graft tumor is the most common graft tumor model, orthotopic graft tumor model is representative of natural progression of liver cancer. Therefore, we performed orthotopic graft tumor model to determine the effect of JMJD2D downregulation on the initiation and progression of LCSC-derived tumors in vivo. We found that orthotopic graft tumors derived from JMJD2D-downregulated spheroids grew much slower than control tumors (Fig. 2, G–H). These results indicate that downregulation of JMJD2D inhibits the initiation and progression of LCSC-derived tumor in vivo. Tumor metastasis is the major cause of cancer-associated mortality. Tumor cells invade the surrounding tissue of the primary tumor, intravasate into blood to become circulating tumor cells (CTCs), translocate to distant tissues, and eventually seed, proliferate, and colonize to form metastatic tumors (30Lu W. Kang Y. Epithelial-Mesenchymal plasticity in cancer progression and metastasis.Dev. Cell. 2019; 49: 361-374Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). CSCs are regarded to be the source of CTCs and responsible for tumor metastasis. To evaluate the function of JMJD2D in CTCs, we injected green fluorescent protein (GFP)-labeled LCSCs into mouse tail vein to establish a mouse model of CTCs and then detected GFP-labeled CTCs in mouse peripheral blood by flow cytometry as well as counted the early lung seeding GFP-labeled CTCs under the fluorescence microscope at 36 h after LCSCs injection. Downregulation of JMJD2D significantly reduced the number of CTCs in mouse peripheral blood (Fig. 3, A–B) and the number of the early lung seeding CTCs (Fig. 3, C–D), suggesting that JMJD2D promotes the survival of CTCs in blood and the early seeding of CTCs in the lung. Consequently, the number of metastasizing tumors in the lungs was significantly decreased in mice injected with JMJD2D-downregulated LCSCs as compared with control cells (Fig. 3, E and F). These results suggest that downregulation of JMJD2D inhibits the lung metastasis of LCSCs by reducing the survival and the early lung seeding of circulating LCSCs. To investigate the molecular mechanisms by which JMJD2D promotes the self-renewal of LCSCs, we examined the effects of JMJD2D knockdown on the expression of several LCSCs-related CSC markers, including CD13, CD44, CD133, OCT4, Sox2, KLF4, Nanog, EpCAM, CD90, and Sox9. As shown in Fig. S2A, the mRNA levels of EpCAM and Sox9, but not other CSC markers, were significantly reduced in two JMJD2D-knockdown HepG2 cell lines. Knockdown of either EpCAM or Sox9 significantly inhibited the proliferation and tumorsphere formation ability of HepG2 cells (Fig. S2, B–C), suggesting that downregulation of EpCAM and Sox9 may, at least in part, be responsible for the inhibitory effects of JMJD2D knockdown on the self-renewal of LCSCs. To show that the inhibitory effects of JMJD2D knockdown on EpCAM and Sox9 expression is not just limited to HepG2 cells, we also measured the mRNA levels of EpCAM and Sox9 in JMJD2D-downregulated Huh-7 and Hepa1-6 cells. As shown in Fig. 4A, downregulation of JMJD2D significantly decreased EpCAM and Sox9 mRNA levels in HepG2, Huh-7, and Hepa1-6 cells. Consistent with the mRNA levels, the protein levels of EpCAM and Sox9 were decreased in JMJD2D-downregulated HepG2, Huh7, and Hepa1-6 cells compared with control cells (Fig. 4B). Furthermore, we found that the expression of JMJD2D, EpCAM, and Sox9 was upregulated in human liver cancer specimens in GEO database (Fig. S3A), and the mRNA levels of JMJD2D were positively correlated with EpCAM and Sox9 in TCGA database (Fig. S3B). To determine whether JMJD2D regulates EpCAM and Sox9 expression at the transcriptional level, we transfected EpCAM and Sox9 promoter reporters into JMJD2D-knockdown and control cells, respectively. The results showed that knockdown of JMJD2D decreased the promoter activities of EpCAM and Sox9 (Fig. 4C), suggesting that JMJD2D regulates EpCAM and Sox9 expression at the transcriptional level. It has been reported that EpCAM is a transcriptional target gene of Wnt/β-catenin signaling pathway with two TCF4 binding elements (TBE) on the EpCAM promoter (TBE1 and TBE2) (31Yamashita T. Budhu A. Forgues M. Wang X.W. Activation of hepatic stem cell marker EpCAM by Wnt-beta-catenin signaling in hepatocellular carcinoma.Cancer Res. 2007; 67: 10831-10839Crossref PubMed Scopus (340) Google Scholar), and Sox9 is a transcriptional target gene of Notch signaling pathway with a NICD1 binding site on the Sox9 promoter (32Capaccione K.M. Hong X.H. Morgan K.M. Liu W.Y. Bishop M.J. Liu L.X. Markert E. Deen M. Minerowicz C. Allen T. Pine S.R. Sox9 mediates Notch1-induced mesenchymal features in lung adenocarcinoma.Oncotarget. 2014; 5: 3636-3650Crossref PubMed Scopus (57) Google Scholar). Therefore, we performed promoter reporter assays to determine whether JMJD2D can cooperate with β-catenin/TCF4 and NICD1 to enhance the promoter activities of EpCAM and Sox9, respectively. As shown in Fig. 4D, ectopic expression of JMJD2D and β-catenin/TCF4 as well as JMJD2D and NICD1 synergistically increased the promoter activities of EpCAM and Sox9, respectively, indicating that JMJD2D cooperates with β-catenin/TCF4 and NICD1 to enhance the transcription of EpCAM and Sox9, respectively. Next, we wondered whether JMJD2D and β-catenin/TCF4 can be recruited to the endogenous EpCAM promoter, and JMJD2D and NICD1 can be recruited to the endogenous Sox9 promoter. To this end, chromatin immunoprecipitation (ChIP) assays were conducted. As shown in Fig. 4E, JMJD2D could be recruited to the promoters of EpCAM and Sox9, but JMJD2D knockdown reduced its recruitment as expected. As a histone demethylase, JMJD2D promotes gene transcription through demethylating H3K9me3 on the promoter. The results of ChIP assays showed that the H3K9me3 levels on the promoters of EpCAM and Sox9 were increased in JMJD2D-knockdown cells compared with control cells (Fig. 4F), suggesting that JMJD2D is responsible for demethylating H3K9me3 on the promoters of EpCAM and Sox9. The results of ChIP assays showed that β-catenin/TCF4 could be recruited to the EpCAM promoter and NICD1 could be recruited to the Sox9 promoter as expected, but their recruitment was reduced in JMJD2D-knockdown cells (Fig. 4G), indicating that H3K9me3 demethylation facilitates the recruitment of β-catenin/TCF4 and NICD1 to the promoters of EpCAM and Sox9, respectively. Collectively, these results suggest that JMJD2D demethylates H3K9me3 on the promoters of EpCAM and Sox9 and facilitates the recruitment and transactivation of TCF4/β-catenin and NICD1, respectively. To determine whether the demethylase activity of JMJD2D is required for the transcription of EpCAM and Sox9 to promote the self-renewal of LCSCs, we transfected wild-type JMJD2D and demethylase-defective JMJD2DS200M mutant plasmids into HepG2 cells, respectively. Compared with wild-type JMJD2D, JMJD2DS200M mutant failed to cooperate with β-catenin/TCF4 and NICD1 to enhance the promoter activities of EpCAM and Sox9 (Fig. S4A), failed to induce the expression of EpCAM and Sox9 in HepG2 cells (Fig. S4B), and failed to promote tumorsphere formation of LCSCs (Fig. S4C). These results suggest that the demethylase activity is required for JMJD2D to enhance EpCAM and Sox9 expression to promote the self-renewal of LCSCs. To determine whether JMJD2D could interact with β-catenin/TCF4 and NICD1 in liver cancer cells, we performed Co-IP assays. The results showed that JMJD2D interacted with β-catenin/TCF4 and NICD1 in HepG2, Huh-7, and Hepa1-6, respectively (Fig. 5, A and B). Furthermore, GST pull-down assays showed that JMJD2D interacted with TCF4 at its HMG Box domain and C-terminal domain (Fig. 5C), and TCF4 interacted with JMJD2D at its C-terminal domain (Fig. 5D). We previously reported that the Jmjc domain of JMJD2D interacted with the ARM-3-10 domain of β-catenin (33Peng K. Kou L. Yu L. Bai C. Li M. Mo P. Li W. Yu C. Histone demethylase JMJD2D interacts with beta-catenin to induce transcription and activate colorectal cancer cell proliferation and tumor growth in mice.Gastroenterology. 2019; 156: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Thus, JMJD2D bound to β-catenin and TCF4 via different domains. The N-terminal domain of TCF4 could interact with the ARM-3-10 domain of β-catenin (Fig. S5, A and B). Thus, JMJD2D, β-catenin, and TCF4 interact with each other to form a ternary complex (Fig. 5E). GST pull-down assays also showed that JMJD2D interacted with NICD1 at its ANK domain (Fig. 5F), and NICD1 interacted with JMJD2D at its C-terminal domain (Fig. 5G). Collectively, these results implicate that β-catenin/TCF4 could interact with JMJD2D to recruit it to the EpCAM promoter and NICD1 could interact with JMJD2D to recruit it to the Sox9 promoter to enhance EpCAM and Sox9 expression, respectively. To further confirm that EpCAM and Sox9 mediate the promoting effect of JMJD2D on the self-renewal of LCSCs, we ectopically expressed EpCAM, Sox9, and EpCAM plus Sox9 in JMJD2D-knockdown HepG2 cells, respectively, and then measured the proliferation and tumorsphere formation abilities of cells. Restoration of EpCAM or Sox9 expression alone partially rescued the proliferation (Fig. S6, A and B) and the tumorsphere formation abilities of JMJD2D-knockdown cells (Fig. 6, A and B) Restoration of both EpCAM and Sox9 expression more efficiently rescued the proliferation (Fig. S6C) and the tumorsphere formation abilities of JMJD2D-knockdown cells (Fig. 6C). Consistently, restoration of EpCAM and Sox9 expression in JMJD2D-knockdown LCSCs partially rescued the subcutaneous tumor growth (Fig. 6D). Taken together, these results support the notion that JMJD2D promotes the self-renewal of LCSCs through enhancing the expression of EpCAM and Sox9. Since JMJD2D can activate Wnt and Notch signaling pathways, apart from EpCAM and Sox9, JMJD2D may promote LCSC self-renewal by regulating the expression of other CSC-related genes such as c-Myc and Hes1 via Wnt and Notch signaling pathways, respectively (Fig. S7). Given that downregulation of JMJD2D could inhibit LCSC self-renewal, we wondered whether a JMJD2D inhibitor 5-chloro-8-hydroxyquinoline (5-c-8HQ), which reduces both demythelase activity and protein levels of JMJD2D (26Peng K. Kou L. Yu L. Bai C. Li M. Mo P. Li W. Yu C. Histone demethylase JMJD2D interacts with beta-catenin to induce transcription and activate colorectal cancer cell proliferation and tumor growth in mice.Gastroenterology. 2018; 156: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), could inhibit the self-renewal of LCSCs. To this end, HepG2, Huh-7, and Hepa1-6 cells were treated with 5-c-8HQ (Fig. S8A), and then the self-renewal of LCSCs was measured by tumorsphere formation assay. As shown in Fig. 7, A and B, 5-c-8HQ treatment for 48 h significantly reduced the protein and mRNA levels of EpCAM and Sox9 in a dose-dependent manner. Consistently, 5-c-8HQ treatment markedly reduced the tumorsphere formation abilities of LCSCs in a dose-dependent manner (Fig. 7C). Furthermore, 5-c-8HQ treatment significantly inhibited LCSC orthotopic graft tumor growth (Fig. 7D) and decreased Ki67-positive tumor cells (Fig. 7E). As expected, 5-c-8HQ treatment significantly reduced the protein levels of EpCAM and Sox9 in LCSC orthotopic graft tumors (Fig. 7F). Moreover, JMJD2D inhibitor 5-c-8HQ could also suppress the expression of c-Myc and Hes1 in orthotopic tumors (Fig. S8B). Treatment with 10 mg/kg or 20 mg/kg 5-c-8HQ did not result in obvious adverse effects on mice as demonstrated by no body weight reduction and no toxicity to the major organs after treatment (Fig. S8, C and D). Collectively, these results demonstrate that JMJD2D could be targeted by a chemical inhibitor to reduce the expression of EpCAM, Sox9, c-Myc, and Hes1 and to inhibit the self-renewal and tumorigenesis of LCSCs (Fig. 7G). In addition, transient knockdown of JMJD2D by siRNA reduced the mRNA and protein levels of EpCAM, Sox9, c-Myc, and Hes1 (Fig. S9, A and B), as well as the proliferation and tumorsphere formation of Hepa1-6 cells (Fig. S9, C and D), supporting the notion that inhibition of JMJD2D is an effective way to inhibit the self-renewal of LCSCs for liver cancer therapy. CSCs are believed to be responsible for the initiation, propagation, metastasis, chemoresistance, and heterogeneity of solid tumor (3Lobo N.A. Shimono Y. Qian D. Clarke M.F. The biology of cancer stem cells.Annu. Rev. Cell. Dev. Biol. 2007; 23: 675-699Crossref PubMed Scopus (829) Google Scholar). Nowadays, increasing evidence shows that epigenetic alterations including DNA methylation and histone modifications participate in CSC self-renewal maintenance (34Shukla S. Meeran S.M. Epigenetics of cancer stem cells: pathways and therapeutics.Biochim. Biophys. Acta. 2014; 1840: 3494-3502Crossref PubMed Scopus (92) Google Scholar). Histone-modifying enzymes, such as EZH2, SUV39H1, and LSD1, have been reported to regulate CSC self-renewal (35Onder T.T. Kara N. Cherry A. Sinha A.U. Zhu N. Bernt K.M. Cahan P. Mancarci B.O. Unternaehrer J. Gupta P.B. Chromatin-modifying enzymes as modulators of reprogramming.Nature. 2012; 483: 598-602Crossref PubMed Scopus (457) Google Scholar, 36Wang J. Lu F. Ren Q. Sun H. Zhang H. Novel histone demethylase LSD1 inhibitors selectively target cancer cells with pluripotent stem cell properties.Cancer Res. 2011; 71: 7238-7249Crossref PubMed Scopus (169) Google Scholar). In the present study, we demonstrate that histone demethylase JMJD2D promotes LCSC self-renewal and could be a potential therapeutic target against LCSCs as follows: (1Gingold J.A. Zhu D. Lee D.F. Kaseb A. Chen J. Genomic profiling and metabolic homeostasis in primary liver cancers.Trends Mol. Med. 2018; 24: 395-411Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) JMJD2D is upregulated in LCSCs; (2El-Serag H.B. Kanwal F. Epidemiology of hepatocellular carcinoma in the United States: where are we? Where do we go?.Hepatology. 2014; 60: 1767-1775Crossref PubMed Scopus (377) Google Scholar) downregulation of JMJD2D inhibits the self-renewal of LCSCs in vitro and in vivo; (3Lobo N.A. Shimono Y. Qian D
Abstract SRC‐1 functions as a transcriptional coactivator for steroid receptors and various transcriptional factors. Notably, SRC‐1 has been implicated in oncogenic roles in multiple cancers, including breast cancer and prostate cancer. Previous investigations from our laboratory have established the high expression of SRC‐1 in human HCC specimens, where it accelerates HCC progression by enhancing Wnt/beta‐catenin signalling. In this study, we uncover a previously unknown role of SRC‐1 in HCC metastasis. Our findings reveal that SRC‐1 promotes HCC metastasis through the augmentation of MMP‐9 expression. The knockdown of SRC‐1 effectively mitigated HCC cell metastasis both in vitro and in vivo by suppressing MMP‐9 expression. Furthermore, we observed a positive correlation between SRC‐1 mRNA levels and MMP‐9 mRNA levels in limited and larger cohorts of HCC specimens from GEO database. Mechanistically, SRC‐1 operates as a coactivator for NF‐κB and AP‐1, enhancing MMP‐9 promoter activity in HCC cells. Higher levels of SRC‐1 and MMP‐9 expression are associated with worse overall survival in HCC patients. Treatment with Bufalin, known to inhibit SRC‐1 expression, significantly decreased MMP‐9 expression and inhibited HCC metastasis in both in vitro and in vivo settings. Our results demonstrated the pivotal role of SRC‐1 as a critical modulator in HCC metastasis, presenting a potential therapeutic target for HCC intervention.
Abstract Backgroud: SRC-1 works as a transcriptional coactivator for steroid receptors and other transcrip-tional factors. SRC-1 is shown to play oncogenic roles in many cancers, like breast cancer and prostate cancer. Our lab anteriorly accounted that SRC-1 is highly expressed in human HCC spec-imens. SRC-1 accelerates HCC progression via enhancing Wnt/beta-catenin signaling. However, the role of SRC-1 in HCC metastasis is unknown. Methods: RNA inteference was used to knockdown the expression of SRC-1, and the protein level was detected via Western blot assay. Matrigel invasion assay was performed for assessment of HCC cell metastasis. MMP9 expression was detected via Zymography. Luciferase assays were performed to detect MMP-9 promoter activity. Results: In this study, we report that SRC-1 promotes HCC metastasis through enhancing MMP-9 expression. Knockdown of SRC-1 decreased HCC cell metastasis in vitro and in vivo by inhibiting the expression of MMP-9. SRC-1 mRNA level is found to positively correlated with MMP-9 mRNA level in a limited number cohort of HCC specimens and a larger number cohort of HCC specimen from GEO database. SRC-1 functions as a coactiva-tor for NF-κB and AP-1 to regulate MMP-9 promoter activity in HCC cells. Higher SRC-1 and MMP-9 expression correlates with a worse overall survival in HCC patients. Bufalin treatment, which can inhibit SRC-1 expression, can significantly decreased MMP-9 expression and inhibit HCC metastasis both in vitro and in vivo . Conclusion: Our results demonstrated that SRC-1 is a crucial modulator for HCC metastasis and offered a potential target for HCC therapy.
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