Actin cytoskeleton remodeling drives epithelial-mesenchymal transition for hepatoma invasion and metastasis in mice.

High invasiveness is a hallmark of human hepatocellular carcinoma (HCC). Large tumors predict invasion and metastasis. Epithelial-mesenchymal transition (EMT) is crucial for cancer invasion and metastasis. However, the mechanisms whereby large tumors tend to undergo EMT remain unclear. We conducted a subgenome-wide screen and identified KLHL23 as an HCC invasion suppressor by inhibiting EMT. KLHL23 binds to actin and suppresses actin polymerization. KLHL23 silencing induced filopodium and lamellipodium formation. Moreover, EMT was suppressed by KLHL23 through its action on actin dynamics. Traditionally, actin cytoskeleton remodeling is downstream of EMT reprogramming. It is therefore intriguing to ask why and how KLHL23 inversely regulates EMT. Activation of actin cytoskeleton remodeling by either KLHL23 silencing or treatment with actin cytoskeleton modulators augmented cellular hypoxic responses in a cell-density-dependent manner, resulting in hypoxia-inducible factor (HIF) and Notch signals and subsequent EMT. Environmental hypoxia did not induce EMT unless actin cytoskeleton remodeling was simultaneously activated and only when cells were at high density. The resulting EMT was reversed by either adenosine 5'-triphosphate supplementation or actin polymerization inhibitors. Down-regulation of KLHL23 was associated with invasion, metastasis, and poor prognosis of HCC and pancreatic cancer. Correlations of tumor size with EMT and inverse association of expression of KLHL23 with HIF/Notch signals were further validated in patient-derived xenograft HCCs in mice. CONCLUSION: Simultaneously activation of actin cytoskeleton remodeling by intrinsic (such as KLHL23 down-regulation) or microenvironment cues is crucial for cell-density-dependent and hypoxia-mediated EMT, providing a mechanistic link between large tumor size and invasion/metastasis. Our findings provide a means of developing the prevention and treatment strategies for tumor invasion and metastasis. (Hepatology 2018;67:2226-2243).

Genetic variants in the circadian rhythm pathway as indicators of prostate cancer progression.

BACKGROUND: To determine the association between circadian pathway genetic variants and the risk of prostate cancer progression. METHODS: We systematically evaluated 79 germline variants in nine circadian pathway genes in a cohort of 458 patients with localized prostate cancer as the discovery phase. We then replicated the significant findings in another cohort of 324 men with more advanced disease. The association of each variant with prostate cancer progression was evaluated by a log-rank test and Cox regression. RESULTS: A single nucleotide polymorphism of the neuronal PAS domain protein 2 (NPAS2) gene (rs6542993 A>T) was found to be associated with a significantly higher risk of disease progression in both localized (P = 0.001) and advanced (P = 0.039) prostate cancer cases. In silico analysis revealed decreased expression levels of NPAS2 in carriers of the T allele of rs6542993 compared with those carrying the A allele. Consistently, downregulation of NPAS2 expression was associated with more aggressive prostate cancer and poor progression-free survival (log-rank P = 0.002). CONCLUSIONS: The NPAS2 rs6542993 polymorphism may be a promising biomarker, and may shed light on the pathways that govern prostate cancer progression.

Functional genomics identified a novel protein tyrosine phosphatase receptor type F-mediated growth inhibition in hepatocarcinogenesis.

UNLABELLED: It is unclear how proliferating cells elicit suppression on cell proliferation and how cancer cells evade this growth suppression. Using a loss-of-function screening of the human kinome and phosphatome to identify genes suppressing tumor initiation in human hepatocellular carcinoma (HCC), we identified 19 genes and characterized one of the top-scoring tumor suppressor candidates, protein tyrosine phosphatase receptor type F (PTPRF). We found that PTPRF was induced during cell proliferation by cell-cell contact. Ectopic expression of wild-type PTPRF, but not the phosphatase-inactive mutant, suppressed cell proliferation and colony formation in soft-agar assays. In contrast, PTPRF silencing led to cell hyperproliferation, enhanced tumor colony formation in soft agar, and increased xenograft tumor growth in nude mice. Mechanistically, PTPRF silencing showed aberrant ERK-dependent signaling including the phosphorylation/stabilization of v-myc avian myelocytomatosis viral oncogene homolog (MYC) through the direct activation of v-src avian sarcoma viral oncogene homolog (SRC) and suppression of PP2A. This PTPRF-mediated growth suppression during cell proliferation functioned independently of the Hippo-Yap pathway. Clinically, PTPRF was down-regulated in 42% HCC (37/89), 67% gastric cancer (27/40), and 100% colorectal cancer (40/40). PTPRF up-regulation was found in 24% HCC (21/89) and associated with better clinical outcomes. CONCLUSION: A novel PTPRF-mediated growth suppression pathway was identified by way of a functional genomics screening in human hepatoma cells. Induction of PTPRF by cell-cell contact during cell proliferation quenched the activated ERK-dependent proliferation signaling to prevent cell hyperproliferation and tumor initiation. PTPRF down-regulation in HCC facilitated tumor development. Our findings shed light on how cancer cells can evade growth suppression and open a new avenue for future development of anticancer therapies.

Combination of mesenchymal stem cell-conditioned medium and botulinum toxin type A for treating human hypertrophic scars.

BACKGROUND: Both mesenchymal stem cell-conditioned medium (MSC CM) and Botox have demonstrated therapeutic effects for hypertrophic scar (HS). It is unclear whether a synergistic effect occurs when these treatments are used in combination. We aimed to investigate the therapeutic effects of MSC CM and Botox alone when compared with those of a combined regimen on HS. METHODS: Fibroblasts from human HS were isolated and treated with Dulbecco's modified Eagle's medium (DMEM), MSCCM, or Botox alone or a combination of MSCCM and Botox. We also used an in vivo HS-buried null mice model to investigate the efficacy of combination treatment. RESULTS: The results demonstrated that the combination of MSC CM and Botox downregulated both mRNA and protein levels of type I collagen, type III collagen, and alpha-smooth muscle actin (α-SMA) in HS fibroblasts. The combined regimen also suppressed fibroblast proliferative activity, increased apoptosis, and displayed significant inhibitory effects on the contractile ability of HS fibroblasts compared to MSC CM, Botox, or DMEM alone. Using an in vivo HS-buried null mice model, significant scar weight reduction, cell apoptosis, and less α-SMA expression were observed from the combined regimen of MSC CM and Botox compared to those from the other groups. The combined regimen also significantly improved arrangement and deposition of collagen fibers. CONCLUSIONS: This study demonstrates that a combination of MSC CM and Botox exhibited a significant therapeutic effect compared to monotherapy. Clinical translation of this therapy should be further considered.

Comparative analyses of coding and noncoding DNA regions indicate that Acropora (Anthozoa: Scleractina) possesses a similar evolutionary tempo of nuclear vs. mitochondrial genomes as in plants.

Evidence suggests that the mitochondrial (mt)DNA of anthozoans is evolving at a slower tempo than their nuclear DNA; however, parallel surveys of nuclear and mitochondrial variations and calibrated rates of both synonymous and nonsynonymous substitutions across taxa are needed in order to support this scenario. We examined species of the scleractinian coral genus Acropora, including previously unstudied species, for molecular variations in protein-coding genes and noncoding regions of both nuclear and mt genomes. DNA sequences of a calmodulin (CaM)-encoding gene region containing three exons, two introns and a 411-bp mt intergenic spacer (IGS) spanning the cytochrome b (cytb) and NADH 2 genes, were obtained from 49 Acropora species. The molecular evolutionary rates of coding and noncoding regions in nuclear and mt genomes were compared in conjunction with published data, including mt cytochrome b, the control region, and nuclear Pax-C introns. Direct sequencing of the mtIGS revealed an average interspecific variation comparable to that seen in published data for mt cytb. The average interspecific variation of the nuclear genome was two to five times greater than that of the mt genome. Based on the calibration of the closure of Panama Isthmus (3.0 mya) and closure of the Tethy Seaway (12 mya), synonymous substitution rates ranged from 0.367% to 1.467% Ma(-1) for nuclear CaM, which is about 4.8 times faster than those of mt cytb (0.076-0.303% Ma(-1)). This is similar to the findings in plant genomes that the nuclear genome is evolving at least five times faster than those of mitochondrial counterparts.

Bone marrow concentrate-induced mesenchymal stem cell conditioned medium facilitates wound healing and prevents hypertrophic scar formation in a rabbit ear model.

BACKGROUND: Hypertrophic scars (HSs) are formed via an aberrant response to the wound healing process. HSs can be cosmetic or can result in functional problems. Prolonged proliferation and remodeling phases disrupt wound healing, leading to excessive collagen production and HS formation. However, there are currently no satisfactory drugs to prevent HS formation. Mesenchymal stem cell (MSC) conditioned medium (CM) has therapeutic effects on wound healing and preventing HS formation. Bone marrow concentrate (BMC) contains various growth factors and cytokines that are crucial for regeneration and has been applied in the clinical setting. In this study, we evaluated the effects of BMC-induced MSC CM on HS formation in a rabbit ear model. METHODS: We established a rabbit ear wound model by generating full-thickness wounds in the ears of rabbits (n = 12) and treated wounds with MSC CM, BMC CM, or BMC-induced MSC CM. Dermal fibroblasts from human hypertrophic scar were stimulated with transforming growth factor beta 1 (TGF-ß1) for 24 h and cultured in each culture medium for 72 h. We measured the hypertrophic scar (HS) formation during the skin regeneration by measuring the expression of several remodeling molecules and the effect of these conditioned media on active human HS fibroblasts. RESULTS: Our results showed that BMC-induced MSC CM had greater antifibrotic effects than MSC CM and BMC CM significantly attenuated HS formation in rabbits. BMC-induced MSC CM accelerated wound re-epithelization by increasing cell proliferation. Additionally, BMC-induced MSC CM also inhibited fibrosis by decreasing profibrotic gene and protein expression, promoting extracellular matrix turnover, inhibiting fibroblast contraction, and reversing myofibroblast activation. CONCLUSIONS: BMC-induced MSC CM modulated the proliferation and remodeling phases of wound healing, representing a potential wound healing agent and approach for preventing HS formation.

Unique mitogenomic features in the scleractinian family pocilloporidae (scleractinia: astrocoeniina).

The complete DNA sequences of three mitochondrial (mt) genomes were obtained from the scleractinian corals, Stylophora pistillata, Pocillopora damicornis, and Madracis mirabilis, and were compared to the published mt genomes to elucidate phylogenetically unique features of the family Pocilloporidae. The entire mt genomes of pocilloporid corals ranged from 16,951 to 17,425 bp with the A+T contents of their sense strands ranging from 68.4% to 70.2%. The gene order of protein-coding genes was identical to those of other scleractinian corals. The novel atp8 gene, first described in confamilial Seriatopora corals, was also confirmed using reverse transcription-polymerase chain reaction (RT-PCR), Northern blot, and sequence analyses in other genera of the Pocilloporidae. The intergenic spacer between atp6 and nad4, containing distinct repeated elements, conserved sequence blocks and domains, and functional structures, possesses typical characteristics of a putative control region for the four coral genera. A duplicated trnW, detected in the region close to the cox1 gene and which shares the highly conserved primary and secondary structures of its original counterpart, was discovered in both Seriatopora and Stylophora. These molecular characteristics are unique and provide the phylogenetic information for future evaluation of the status of the family Pocilloporidae in the evolutionary history of scleractinian corals.

The complete mitochondrial genomes of needle corals, Seriatopora spp. (Scleractinia: Pocilloporidae): an idiosyncratic atp8, duplicated trnW gene, and hypervariable regions used to determine species phylogenies and recently diverged populations.

Complete DNA sequences were determined for the mitochondrial (mt) genomes of the needle corals, Seriatopora caliendrum (17,011bp) and S. hystrix (17,060bp). Gene arrangement of the Seriatopora mt genomes is similar to the 14 currently published scleractinian mitogenomes with three unusual features, including an idiosyncratic atp8, a duplicated trnW (tRNA(TRP)), and a putative control region located between atp6 and nad4. Atp8, located between duplicate trnW genes, showed relatively low amino acid similarity (25.6-34.6%) with those of published scleractinian corals. A reverse-transcription polymerase chain reaction confirmed the transcription of this novel atp8 gene in Seriatopora. A duplicated trnW was detected in the region close to the cox1 gene and shares the highly conserved primary and secondary structure of its original counterpart. The intergenic spacer between atp6 and nad4, which contains several distinct repeated elements, is being designated as the putative control region in the Seriatopora mt genomes. Evaluation of the molecular evolution of several protein-coding genes and intergenic spacers showed 3- to 4-fold higher divergence rates among populations or between species than those published for scleractinian mt genomes. This study not only successfully revealed the phylogenies of S. hystrix and S. caliendrum from the West Pacific Ocean by mtDNA, but also highlighted the potential utilities of mt hypervariable regions in phylogenetic construction below the species level for Seriatopora.

Analysis of Acropora muricata calmodulin (CaM) indicates that scleractinian corals possess the ancestral exon/intron organization of the eumetazoan CaM gene.

Calmodulin (CaM), belonging to the tropinin C (TnC) superfamily, is one of the calcium-binding proteins that are highly conserved in their protein and gene structure. Based on the structure comparison among published vertebrate and invertebrate CaM, it is proposed that the ancestral form of eumetazoan CaM genes should have five exons and four introns (four-intron hypothesis). In this study, we determined the gene structure of CaM in the coral Acropora muricata, an anthozoan cnidarian representing the basal position in animal evolution. A CaM clone was isolated from a cDNA library constructed from the spawned eggs of A. muricata. This clone was composed of 908 nucleotides, including 162 base pairs (bp) of 5'-untranslated region (UTR), 296 bp of 3'-UTR, and an open reading frame 450 bp in length. The deduced amino acid indicated that the Acropora CaM protein is identical to that of the actiniarian, Metridinium senile, and has four putative calcium-binding domains highly similar to those of other vertebrate or invertebrate CaMs. Southern blot analysis revealed that Acropora CaM is a putative single-copy gene in the nuclear genome. Genomic sequencing showed that Acropora CaM was composed of five exons and four introns, with intron II not corresponding to any region in the actiniarian CaM gene, which possesses only four exons and three introns. Our results highlight that the coral CaM gene isolated from A. muricata has four introns at the predicted positions of the early metazoan CaM gene organization, providing the first evidence from the basal eumetazoan phylum to support the four-intron hypothesis.

Identification of a lactose-responsive element upstream of the promoter of Bacillus megaterium beta-galactosidase-encoding gene mbgA.

The Bacillus megaterium mbgA gene encodes a lactose-hydrolyzing beta-galactosidase. An AraC/XylS-type activator BgaR can activate mbgA transcription in response to lactose. In this report, we show by various deletion analyses and point mutagenesis analyses that an inverted repeat centered at position -60.5 relative to the mbgA transcriptional initiation site is the cis-acting element responsible for lactose induction of mbgA expression.

mbgA-dependent lactose utilization by Bacillus megaterium.

The beta-galactosidase-encoding mbgA gene has recently been cloned from Bacillus megaterium. We now report that disruption of the chromosomal mbgA rendered B. megaterium unable to utilize lactose as a sole carbon source. Complementation of the mbgA mutant with a multicopy plasmid carrying intact mbga restored the ability to utilize lactose for cell growth. Crude extracts from the wild-type B. megaterium cells grown in the presence of lactose exhibited a significant level of lactose-hydrolyzing activity, whereas no activity was observed in crude extracts of the mbgA mutant grown under the same condition. The mbgA gene could also confer the ability of lactose utilization on a lacZ deletion mutant of Escherichia coli. Lactose-hydrolyzing activity was also observed in crude extracts of the lacZ deletion mutant carrying mbgA on a multicopy plasmid. In addition, inactivation of the chromosomal mbgA did not affect lactose induction of expression of the mbgA promoter-xylE transcriptional fusion. Taken together, these results suggest that mbgA is essential for lactose utilization by B. megaterium, but is not involved in generation of the intracellular inducer for lactose induction of mbgA expression.