PARP12 (ARTD12) suppresses hepatocellular carcinoma metastasis through interacting with FHL2 and regulating its stability.
ABSTRACT: PARP12 is a mono-ADP-ribosyltransferase, but its function remains largely unknown. Here, we identified four-and-a-half LIM-only protein 2 (FHL2) as a functional partner of PARP12 through protein affinity purification. Although PARP12 did not mono-ADP-ribosylate FHL2 in vitro and in vivo, PARP12 deficiency decreased the protein level of FHL2 by promoting its ubiquitination and increased the expression level of transforming growth factor beta1 (TGF-β1), which is independent of PARP12 enzymatic activity. We also provided evidence that PARP12 deficiency increased the migration and invasion of hepatocellular carcinoma (HCC) cells and promoted HCC metastasis in vivo by regulating the epithelial-mesenchymal transition process. These results indicated that PARP12 is a tumor suppressor that plays an important role in HCC metastasis through the regulation of FHL2 stability and TGF-β1 expression.
Project description:The transcription factor Krüppel-like factor (KLF)8 plays an important role in the formation of several human tumors, including colorectal cancer. We recently identified four-and-a-half LIM protein 2 (FHL2) as a critical inducer of the epithelial-to-mesenchymal transition (EMT) and invasion. However, the molecular mechanism by which KLF8 affects FHL2-mediated tumor proliferation, EMT and metastasis remains unknown. Here, we showed that KLF8 overexpression promoted EMT and metastatic phenotypes. KLF8 expression was stimulated by transforming growth factor (TGF)-β1. Moreover, KLF8 acted as a potential EMT inducer by stimulating vimentin expression and inducing a loss of E-cadherin in stable KLF8-transfected cells. KLF8 overexpression induced a strong increase in FHL2 expression, and a positive correlation between the expression patterns of KLF8 and FHL2 was observed in CRC cells. Promoter reporter and chromatin immunoprecipitation (ChIP) assays demonstrated that KLF8 directly bound to and activated the human FHL2 gene promoter. However, siRNA-mediated repression of FHL2 in KLF8-overexpressing cells reversed the EMT and the proliferative and metastatic phenotypes. In vivo, KLF8 promoted FHL2-mediated proliferation and metastasis via orthotopic implantation. Taken together, this work identified KLF8-induced FHL2 activation as a novel and critical signaling mechanism underlying human breast/colorectal cancer invasion and metastasis.
Project description:Transforming growth factor ?1 (TGF-?1) is a master cytokine in many biological processes, including tissue homeostasis, epithelial-to-mesenchymal transition, and wound repair. Here, we report that four and a half LIM-only protein 2 (FHL2) is a critical regulator of TGF-?1 expression. Devoid of a DNA-binding domain, FHL2 is a transcriptional cofactor that plays the role of coactivator or corepressor, depending on the cell and promoter contexts. We detected association of FHL2 with the TGF-?1 promoter, which showed higher activity in Fhl2-/- cells than in wild-type (WT) cells in a reporter assay. Overexpression of FHL2 abrogates the activation of the TGF-?1 promoter, whereas the upregulation of TGF-?1 gene transcription correlates with reduced occupancy of FHL2 on the promoter. Moreover, ablation of FHL2 facilitates recruitment of RNA polymerase II on the TGF-?1 promoter, suggesting that FHL2 may be involved in chromatin remodeling in the control of TGF-?1 gene transcription. Enhanced expression of TGF-?1 mRNA and cytokine was evidenced in the livers of Fhl2-/- mice. We tested the in vivo impact of Fhl2 loss on hepatic fibrogenesis that involves TGF-?1 activation. Fhl2-/- mice developed more severe fibrosis than their WT counterparts. These results demonstrate the repressive function of FHL2 on TGF-?1 expression and contribute to the understanding of the TGF-?-mediated fibrogenic response.
Project description:BACKGROUND:Pirfenidone, an antifibrotic agent used for the treatment of idiopathic pulmonary fibrosis (IPF), functions by inhibiting myofibroblast differentiation, which is involved in transforming growth factor (TGF)-β1-induced IPF pathogenesis. However, unlike normal lung fibroblasts, the relationship between pirfenidone responses of TGF-β1-induced human fibrotic lung fibroblasts and lung fibrosis has not been elucidated. METHODS:The effects of pirfenidone were evaluated in lung fibroblasts isolated from fibrotic human lung tissues after TGF-β1 exposure. The ability of two new pharmacological targets of pirfenidone, collagen triple helix repeat containing protein 1(CTHRC1) and four-and-a-half LIM domain protein 2 (FHL2), to mediate contraction of collagen gels and migration toward fibronectin were assessed in vitro. RESULTS:Compared to control lung fibroblasts, pirfenidone significantly restored TGF-β1-stimulated fibroblast-mediated collagen gel contraction, migration, and CTHRC1 release in lung fibrotic fibroblasts. Furthermore, pirfenidone attenuated TGF-β1- and CTHRC1-induced fibroblast activity, upregulation of bone morphogenic protein-4(BMP-4)/Gremlin1, and downregulation of α-smooth muscle actin, fibronectin, and FHL2, similar to that observed post-CTHRC1 inhibition. In contrast, FHL2 inhibition suppressed migration and fibronectin expression, but did not downregulate CTHRC1. CONCLUSIONS:Overall, pirfenidone suppressed fibrotic fibroblast-mediated fibrotic processes via inverse regulation of CTHRC1-induced lung fibroblast activity. Thus, CTHRC1 can be used for predicting pirfenidone response and developing new therapeutic targets for lung fibrosis.
Project description:Four and a Half LIM protein 2 (FHL2) is a LIM domain only protein that is able to form various protein complexes and regulate gene transcription. Recent findings showed that FHL2 is a potential tumor suppressor gene that was down-regulated in hepatocellular carcinoma (HCC). Moreover, FHL2 can bind to and activate the TP53 promoter in hepatic cells. In this study, the activity of the two promoters of FHL2, 1a and 1b, were determined in the human embryonic kidney cell line HEK293 and the activation of these two promoters by p53 was investigated. Our results showed that the 1b promoter has a higher activity than the 1a promoter in HEK 293 cells but the 1a promoter is more responsive to the activation by p53 when compared with the 1b promoter. The regulation of FHL2 by p53 was further confirmed in liver cells by the overexpression of p53 in Hep3B cells and the knockdown of p53 in HepG2 cells. Combining promoter activity results of truncated mutants and predictions by bioinformatics tools, a putative p53 binding site was found in the exon 1a of FHL2 from +213 to +232. The binding between the p53 protein and the putative p53 binding site was then validated by the ChIP assay. Furthermore, the expression of FHL2 and TP53 were down-regulated in majority of HCC tumour samples (n?=?41) and significantly correlated (P?=?0.026). Finally, we found that the somatic mutation 747 (G?T), a hot spot mutation of the TP53 gene, is potentially associated with a higher expression of FHL2 in HCC tumour samples. Taken together, this is the first in-depth study about the transcriptional regulation of FHL2 by p53.
Project description:The transcriptional factor Forkhead box k1 (FOXK1) is a member of the FOX family. The abnormal expression of FOXK1 may have an important role in tumour development. Our previous studies showed that four-and-a-half LIM protein 2 (FHL2) is a critical inducer of the epithelial-to-mesenchymal transition (EMT) and invasion. However, the molecular mechanism by which FOXK1 synergizes with FHL2 tumour proliferation, EMT and metastasis is not well defined. We evaluated that messenger RNA (mRNA) and protein expression levels by quantitative RT-PCR, western blot, immunofluorescence and immunohistochemistry (IHC) assays. The migration and invasive abilities of colorectal cancer (CRC) cells were evaluated using short hairpin RNA (shRNA)-mediated inhibition in vitro and in vivo. We showed that FOXK1 expression was upregulated in CRC compared with matched normal tissues. FOXK1 physically interacts with FHL2 in CRC. Moreover, higher expression levels of the two proteins were significantly associated with differentiation, lymph node metastasis, AJCC stage and poorer prognosis. Furthermore, the overexpression of FOXK1 in CRC cells is associated with EMT, invasion and metastasis. However, the siRNA-mediated repression of FHL2 in FOXK1-overexpressing cells reversed EMT and both the proliferative and metastatic phenotypes in vitro and in vivo. These data identified that the co-expression of FOXK1 and FHL2 enhances cell proliferation and metastasis through the induction of EMT. Thus, FOXK1 and FHL2 may serve as putative targets in the combined therapy of CRC.
Project description:Here, we report the biochemical characterization of the mono-ADP-ribosyltransferase 2,3,7,8-tetrachlorodibenzo-p-dioxin poly-ADP-ribose polymerase (TIPARP/ARTD14/PARP7), which is known to repress aryl hydrocarbon receptor (AHR)-dependent transcription. We found that the nuclear localization of TIPARP was dependent on a short N-terminal sequence and its zinc finger domain. Deletion and in vitro ADP-ribosylation studies identified amino acids 400-657 as the minimum catalytically active region, which retained its ability to mono-ADP-ribosylate AHR. However, the ability of TIPARP to ADP-ribosylate and repress AHR in cells was dependent on both its catalytic activity and zinc finger domain. The catalytic activity of TIPARP was resistant to meta-iodobenzylguanidine but sensitive to iodoacetamide and hydroxylamine, implicating cysteines and acidic side chains as ADP-ribosylated target residues. Mass spectrometry identified multiple ADP-ribosylated peptides in TIPARP and AHR. Electron transfer dissociation analysis of the TIPARP peptide 33ITPLKTCFK41 revealed cysteine 39 as a site for mono-ADP-ribosylation. Mutation of cysteine 39 to alanine resulted in a small, but significant, reduction in TIPARP autoribosylation activity, suggesting that additional amino acid residues are modified, but loss of cysteine 39 did not prevent its ability to repress AHR. Our findings characterize the subcellular localization and mono-ADP-ribosyltransferase activity of TIPARP, identify cysteine as a mono-ADP-ribosylated residue targeted by this enzyme, and confirm the TIPARP-dependent mono-ADP-ribosylation of other protein targets, such as AHR.
Project description:Poly-ADP-ribose-polymerases (PARPs) 1 and 2 are nuclear enzymes that catalyze the poly-ADP-ribosylation of nuclear proteins transferring poly-ADP-ribose (PAR) polymers to specific residues. PARPs and PAR intervene in diverse functions, including DNA repair in the nucleus and stress granule assembly in the cytoplasm. Stress granules contribute to the regulation of translation by clustering and stabilizing mRNAs as well as several cytosolic PARPs and signaling proteins to modulate cell metabolism and survival. Our study is focused on one of these PARPs, PARP12, a Golgi-localized mono-ADP-ribosyltransferase that under stress challenge reversibly translocates from the Golgi complex to stress granules. PARP1 activation and release of nuclear PAR drive this translocation by direct PAR binding to the PARP12-WWE domain. Thus, PAR formation functionally links the activity of the nuclear and cytosolic PARPs during stress response, determining the release of PARP12 from the Golgi complex and the disassembly of the Golgi membranes, followed by a block in anterograde-membrane traffic. Notably, these functions can be rescued by reverting the stress condition (by drug wash-out). Altogether these data point at a novel, reversible nuclear signaling that senses stress to then act on cytosolic PARP12, which in turn converts the stress response into a reversible block in intracellular-membrane traffic.
Project description:The interactions of cancer cells with neighboring non-malignant cells in the microenvironment play an important role for progressive neoplastic development and metastasis. Long-term direct co-culture of human MDA-MB-231cherry breast cancer cells with benign human mesenchymal stroma/stem-like cells (MSC) MSC544GFP stably expressing mCherry and eGFP fluorescence proteins, respectively, was associated with the formation of three-dimensional (3D) tumor spheroids in vitro. The quantification of the breast tumor marker urokinase plasminogen activator (uPA) in mono-cultured MDA-MB-231 cells revealed an approximately 14-fold enhanced expression when compared to five different normal human MSC mono-cultures. Moreover, uPA levels in 3D tumor spheroids remained elevated 9.4-fold above the average of five different human MSC cultures. In contrast, the expression of the corresponding plasminogen activator inhibitor type-1 (PAI-1) declined by 2.6-fold in the breast cancer cells and was even further reduced by 3.2-fold in the MDA-MB-231cherry/MSC544GFP 3D co-culture spheroids when compared to the various MSC populations. The supportive data were obtained for the production of TGF-β1, which is an important growth factor in the regulation of tumor growth and metastasis formation. Whereas, TGF-β1 release in MDA-MB-231cherry/MSC544GFP co-cultures was elevated by 1.56-fold as compared to MSC544 mono-cultures after 24 h; this ratio further increased to 2.19-fold after 72 h. Quantitative PCR analyses in MSC544 and MDA-MB-231 cells revealed that MSC, rather than the breast cancer cells, are responsible for TGF-β1 synthesis and that TGF-β1 contributes to its own synthesis in these cells. These findings suggested potential synergistic effects in the expression/secretion of uPA, PAI-1, and TGF-β during the co-culture of breast cancer cells with MSC.
Project description:BACKGROUND:DEPTOR is an endogenous inhibitor of mTORC1 and mTORC2 that plays a vital role in the progression of human malignances. However, the biological function of DEPTOR in HCC metastasis and the underlying molecular mechanisms are still unclear. METHODS:Western blot analysis and immunohistochemistry(IHC) were employed to examine DEPTOR expression in HCC cell lines and tissues. A series of in vivo and in vitro assays were performed to determine the function of DEPTOR and the possible mechanisms underlying its role in HCC metastasis. RESULTS:We found that DEPTOR was frequently overexpressed in HCC tissues, and its high expression was associated with high serum AFP levels, increased tumor size, vascular invasion and more advanced TMN and BCLC stage, as well as an overall poor prognosis. Functional experiments demonstrated that DEPTOR silencing inhibited the proliferation and mobility of HCC cells in vitro and suppressed tumor growth and metastasis of HCC cells in vivo. Accordingly, DEPTOR overexpression promoted the invasion and metastasis of HCC cells in vitro and in vivo, but had no effect on cell proliferation in vitro. Overexpression of DEPTOR induced EMT by snail induction. Conversely, knockdown of snail expression impaired the DEPTOR-induced migration, invasion and EMT of HCC cells. Furthermore, we found that the increase of snail expression by DEPTOR overexpression was due to an activation of TGF-β1-smad3/smad4 signaling possibly through feedback inhibition of mTOR. CONCLUSION:DEPTOR promotes the EMT and metastasis of HCC cells by activating the TGF-β1-smad3/smad4-snail pathway via mTOR inhibition. Therefore, targeting DEPTOR may be an ideal treatment strategy for inhibiting the growth and metastasis of HCC.
Project description:Although increasing evidence indicated that deregulation of microRNAs (miRNAs) contributed to tumor initiation and progression, but little is known about the biological role of miR-340 in ovarian cancer (OC). In this study, we found that miR-340 expression was downregulated in OC tissues compared with its expression in normal ovarian epithelium and endometrium, and treatment with 5-aza-2'-deoxycytidine (5-Aza-dC) or trichostatin A (TSA) increased miR-340 expression in OC cells. In addition, ectopic miR-340 expression inhibited OC cell growth and metastasis in vitro and in vivo. Four and a half LIM domains protein 2 (FHL2) was confirmed as a direct target of miR-340 and silencing FHL2 mimicked the effects of miR-340 in OC cells. Further mechanistic study showed that miR-340 inhibited the Wnt/?-catenin pathway by targeting FHL2, as well as downstream cell cycle and epithelial-to-mesenchymal transition (EMT) signals in OC cells. Moreover, the greatest association between miR-340 and FHL2 was found in 481 ovarian serous cystadenocarcinoma tissues via pan-cancer analysis. Finally, we revealed that lower miR-340 or higher FHL2 was associated with poor OC patient outcomes. Our findings indicate that the miR-340-FHL2 axis regulates Wnt/?-catenin signaling and is involved in tumorigenesis in OC. Therefore, manipulating the expression of miR-340 or its target genes is a potential strategy in OC therapy.