The pediatric liver cancers, hepatoblastoma and hepatocellular carcinoma, are dangerous cancers which often spread to the lungs. Although treatments with cisplatin significantly improve outcomes, cisplatin may not eliminate metastasis-initiating cells. Our group has recently shown that the metastatic microenvironments of hepatoblastoma contain Cancer Associated Fibroblasts (CAFs) and neuron-like cells, which initiate cancer spread from liver to lungs. In this study, we found that these cells express high levels of HDAC1; therefore, we examined if histone deacetylase inhibition improves cisplatin anti-proliferative effects and reduces the formation of tumor clusters in pediatric liver cancer metastatic microenvironments.
METHODS: New cell lines were generated from primary hepatoblastoma liver tumors (hbl) and lung metastases (LM) of HBL patients. In addition, cell lines were generated from hepatocellular neoplasm, not otherwise specified (HCN-NOS) tumor samples, and hcc cell lines. Hbl, LM and hcc cells were treated with cisplatin, SAHA or in combination. The effect of these drugs on the number of cells, formation of tumor clusters and HDAC1-Sp5-p21 axis were examined.
RESULTS: Both HBL and HCC tissue specimens have increased HDAC1-Sp5 pathway activation, recapitulated in cell lines generated from the tumors. HDAC inhibition with vorinostat (SAHA) increases cisplatin efficacy to eliminate CAFs in hbl and in hcc cell lines. Although the neuron-like cells survive the combined treatments, proliferation was inhibited. Notably, combining SAHA with cisplatin overcame cisplatin resistance in an LM cell line from an aggressive case with multiple metastases. Underlying mechanisms of this enhanced inhibition include suppression of the HDAC1-Sp5 pathway and elevation of an inhibitor of proliferation p21. Similar findings were found with gemcitabine treatments suggesting that elimination of proliferative CAFs cells is a key event in the inhibition of mitotic microenvironment.
CONCLUSIONS: Our studies demonstrate the synergistic benefits of HDAC inhibition and cisplatin to eliminate metastasis-initiating cells in pediatric liver cancers.

Succinylation modification involves in the progression of human cancers. The present study aimed to investigate the role of CPT1A, which is a succinyltransferase in the progression of prostate cancer (PCa). CCK-8 was used to detect the cell viability. Seahorse was performed to evaluate the cell glycolysis. Luciferase assay was used to detect the transcriptional regulation. ChIP was performed to assess the binding between transcriptional factors with the promoters. Co-IP was used to assess the binding between proteins. We found that CPT1A was highly expressed in PCa tissues and cell lines. Silencing of CPT1A inhibited the viability and glycolysis of PCa cells. Mechanistically, CPT1A promoted the succinylation of SP5, which strengthened the binding between SP5 and the promoter of PDPK1. SP5 activated PDPK1 transcription and PDPK1 activated the AKT/mTOR signal pathway. These findings might provide novel targets for the diagnosis or therapy of prostate cancer.

Liver metastasis of colorectal cancer (CRC) is a major cause of cancer morbidity and mortality. Circular RNAs (circRNAs) have been widely reported to be implicated in cancer metastasis. This study aims to investigate the effect of circSP5 (has_circ_0057010) on liver metastasis of CRC. Quantitative real-time PCR (RT-qPCR) analysis was performed to detect gene expression. The level of proteins was measured by western blot. The migration and invasion of CRC cells were assessed by wound healing assay and transwell assay. In vivo assays were performed after the construction of the CRC xenograft model and CRC model with liver metastasis. Mechanism analyses were performed via RNA-binding protein immunoprecipitation (RIP), RNA pulldown, luciferase reporter, chromatin immunoprecipitation (ChIP), and DNA pulldown assays. We found that circSP5 is significantly overexpressed in CRC with liver metastasis and its depletion suppresses the progression of CRC with liver metastasis in vitro and in vivo. Moreover, circSP5 enhances the expression of Sp5 transcription factor (SP5) via competitively sponging microRNA (miR)-1249-3p and could regulate BMP and activin membrane-bound inhibitor (BAMBI) via transcriptional activation. CircSP5 promotes the migration, invasion, and epithelial-mesenchymal transition (EMT) of CRC cells via BAMBI. In sum, circSP5 promotes liver metastasis of CRC by up-regulating SP5-mediated BAMBI transcription.

Cell-based therapy has been recognized as a novel technique for the management of diabetic foot ulcers, and cell-sheet engineering leads to improved efficacy in cell transplantation. This study aims to explore the possible molecular mechanism of the rat adipose-derived stem cell (ASC) sheet loaded with exosomal interferon regulatory factor 1 (IRF1) in foot wound healing.
Rats were rendered diabetic with streptozotocin, followed by measurement of miR-16-5p expression in wound tissues. Relationship between IRF1, microRNA (miR)-16-5p, and trans-acting transcription factor 5 (SP5) was analyzed using luciferase activity, RNA pull-down, and chromatin immunoprecipitation assays. IRF1 was overexpressed in rat ASCs (rASCs) or loaded onto the rASC sheet, and then exosomes were extracted from rASCs. Accordingly, we assessed the effects of IRF1-exosome or IRF1-rASC sheet on the proliferation and migration of the fibroblasts along with endothelial cell angiogenesis.
miR-16-5p was poorly expressed in the wound tissues of diabetic rats. Overexpression of miR-16-5p promoted fibroblast proliferation and migration as well as endothelial cell angiogenesis, thus expediting wound healing. IRF1 was an upstream transcription factor that could bind to the miR-16-5p promoter and increase its expression. In addition, SP5 was a downstream target gene of miR-16-5p. IRF1-exosome from rASCs or the IRF1-rASC sheet facilitated the foot wound healing in diabetic rats through miR-16-5p-dependent inhibition of SP5.
The present study demonstrates that exosomal IRF1-loaded rASC sheet regulates miR-16-5p/SP5 axis to facilitate wound healing in diabetic rats, which aids in development of stem cell-based therapeutic strategies for diabetic foot wounds.

OBJECTIVE: Epigenetic regulation of gene expression plays a critical role in the development of liver cancer; however, the molecular mechanisms of epigenetic-driven liver cancers are not well understood. The aims of this study were to examine molecular mechanisms that cause the dedifferentiation of hepatocytes into cancer cells in aggressive hepatoblastoma and test if the inhibition of these mechanisms inhibits tumor growth.
METHODS: We have analyzed CCAAT/Enhancer Binding Protein alpha (C/EBPα), Transcription factor Sp5, and histone deacetylase (HDAC)1 pathways from a large biobank of fresh hepatoblastoma (HBL) samples using high-pressure liquid chromatography-based examination of protein-protein complexes and have examined chromatin remodeling on the promoters of markers of hepatocytes and p21. The HDAC1 activity was inhibited in patient-derived xenograft models of HBL and in cultured hepatoblastoma cells and expression of HDAC1-dependent markers of hepatocytes was examined.
RESULTS: Analyses of a biobank showed that a significant portion of HBL patients have increased levels of an oncogenic de-phosphorylated-S190-C/EBPα, Sp5, and HDAC1 compared with amounts of these proteins in adjacent regions. We found that the oncogenic de-phosphorylated-S190-C/EBPα is created in aggressive HBL by protein phosphatase 2A, which is increased within the nucleus and dephosphorylates C/EBPα at Ser190. C/EBPα-HDAC1 and Sp5-HDAC1 complexes are abundant in hepatocytes, which dedifferentiate into cancer cells. Studies in HBL cells have shown that C/EBPα-HDAC1 and Sp5-HDAC1 complexes reduce markers of hepatocytes and p21 via repression of their promoters. Pharmacologic inhibition of C/EBPα-HDAC1 and Sp5-HDAC1 complexes by Suberoylanilide hydroxamic acid (SAHA) and small interfering RNA-mediated inhibition of HDAC1 increase expression of hepatocyte markers, p21, and inhibit proliferation of cancer cells.
CONCLUSIONS: HDAC1-mediated repression of markers of hepatocytes is an essential step for the development of HBL, providing background for generation of therapies for aggressive HBL by targeting HDAC1 activities.

The Sp-family genes encode important transcription factors in animal development. Here we investigate the embryonic expression patterns of the complete set of Sp-genes in the velvet worm Euperipatoides kanangrensis (Onychophora), with a special focus on the Sp6-9 ortholog. In arthropods, Sp6-9, the ortholog of the Drosophila melanogaster D-Sp1 gene plays a conserved role in appendage development. Our data show that the expression of Sp6-9 during the development of the velvet worm is conserved, suggesting that the key function of the Sp6-9 gene dates back to at least the last common ancestor of arthropods and onychophorans and thus likely the last common ancestor of Panarthropoda.

EMMPRIN (extracellular matrix metalloproteinase inducer, EMN, CD147) is a member of the immunoglobulin superfamily expressed in numerous cell types both as a soluble and a membrane-spanning glycoprotein. It is involved in many physiological processes, as well as in cancer. This study addresses mechanisms of crosstalk between EMN-driven cancer-related cellular responses and the canonical Wnt-pathway in MCF-7 breast carcinoma cells. Genetic knockdown of EMN in MCF-7 resulted in characteristic changes in cellular shape, organization of the actin cytoskeleton and malignancy profile, indicating that EMN expression represses cell motility, but, in contrast, exerts a stimulatory effect on cell proliferation and invasive properties. Increased invasiveness coincided with elevated expression of Wnt-target genes and established invasion driver matrix metalloproteinase MMP14. Activation of the downstream Wnt-pathway by means of heterologous β-catenin and/or TCF-4 expression, through inhibition of GSK-3β by LiCl treatment, or by cell stimulation with insulin-like growth factor-1 (IGF-1) resulted in increased EMN expression. EMN over-expression raised the ratio of the two opposing Wnt pathway-driven transcription factors Sp1 and Sp5, leading to stimulation of the EMN promoter. Furthermore, the EMN promoter was activated by a feed-forward circuit involving an EMN-dependent drop in expression of the repressive signal transducer and activator of transcription 1 (STAT1). Taken together, we show that the influence of EMMPRIN on malignancy-related properties of breast cancer cells is functionally connected to both Wnt- and JAK/STAT pathways.

The Wnt signaling pathway is aberrantly activated in the majority of human colorectal tumors. β-catenin, a key component of the Wnt signaling pathway, interacts with the T-cell factor/lymphoid enhancer-binding factor family of transcription factors and activates transcription of Wnt target genes. Sp5 is one of the Wnt target genes, and its expression is commonly upregulated in colon cancer cells. The present study demonstrates that the expression of Sp5 is not upregulated in the colon cancer cell line HCT116, in which Wnt signaling is constitutively activated. Furthermore, the results demonstrate that Sp5 has the potential to inhibit cell proliferation through upregulation of the cell cycle inhibitor p27. These findings suggest that HCT116 cells downregulate Sp5 to avoid p27-mediated growth arrest.

Activation of leukemia inhibitor factor (LIF)-Stat3 or Wnt/β-catenin signaling promotes mouse embryonic stem cell (mESC) self-renewal. A myriad of downstream targets have been identified in the individual signal pathways, but their common targets remain largely elusive. In this study, we found that the LIF-Stat3 and Wnt/β-catenin signaling pathways converge on Sp5 to promote mESC self-renewal. Forced Sp5 expression can reproduce partial effects of Wnt/β-catenin signaling but mimics most features of LIF-Stat3 signaling to maintain undifferentiated mESCs. Moreover, Sp5 is able to convert mouse epiblast stem cells into a naïve pluripotent state. Thus, Sp5 is an important component of the regulatory network governing mESC naïve pluripotency.

Prolyl carboxypeptidase (PRCP), a serine protease, is widely expressed in the body including liver, lung, kidney and brain, with a variety of known substrates such as plasma prekallikrein, bradykinin, angiotensins II and III, and α-MSH, suggesting its role in the processing of tissue-specific substrates. In the brain, PRCP has been shown to inactivate hypothalamic α-MSH, thus modulating melanocortin signaling in the control of energy metabolism. While its expression pattern has been reported in the hypothalamus, little is known on the distribution of PRCP throughout the mouse brain. This study was undertaken to determine PRCP expression in the mouse brain. Radioactive in situ hybridization was performed to determine endogenous PRCP mRNA expression. In addition, using a gene-trap mouse model for PRCP deletion, X-gal staining was performed to further determine PRCP distribution. Results from both approaches showed that PRCP gene is broadly expressed in the brain.