An autophagy-inducing stapled peptide promotes c-MET degradation and overrides adaptive resistance to sorafenib in c-MET+ hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) accounts for approximately 90% of primary liver cancer cases and ranks as the second leading cause of cancer related death. Multiple receptor tyrosine kinases such as EGFR, FGFR and c-MET have been shown to drive tumorigenesis and progression of HCC. However, tyrosine kinase inhibitors (TKIs) that target these kinases, including the FDA-approved sorafenib, only offer limited clinical success. Resistance to sorafenib and other TKIs also readily emerge in HCC patients, further limiting the usage of these drugs. Novel therapeutic strategies are needed to address the urgent unmet medical need for HCC patients. Results: Autophagy is an evolutionally conserved lysosome-dependent degradation process that is also functionally implicated in HCC. We previously developed an autophagy-inducing stapled peptide (Tat-SP4) that induced autophagy and endolysosomal degradation of EGFR in lung cancer and breast cancer cells. Here we present data to show that Tat-SP4 also induced significant autophagic response in multiple HCC cell lines and promoted the endolysosomal degradation of c-MET to attenuate its downstream signaling activities although it didn't affect the intrinsically fast turnover of EGFR. Tat-SP4 also overrode adaptive resistance to sorafenib in c-MET+ HCC cells but employed the distinct mechanism of inducing non-apoptotic cell death. Conclusion: With its distinct mechanism of promoting autophagy and endolysosomal degradation of c-MET, Tat-SP4 may serve as a novel therapeutic agent that complement and synergize with sorafenib to enhance its clinical efficacy in HCC patients.

Long noncoding RNA ELDR promotes cell cycle progression in normal oral keratinocytes through induction of a CTCF-FOXM1-AURKA signaling axis.

Long noncoding RNAs (lncRNAs) have gained widespread attention as a new layer of regulation in biological processes during development and disease. The lncRNA ELDR (EGFR long noncoding downstream RNA) was recently shown to be highly expressed in oral cancers as compared to adjacent nontumor tissue, and we previously reported that ELDR may be an oncogene as inhibition of ELDR reduces tumor growth in oral cancer models. Furthermore, overexpression of ELDR induces proliferation and colony formation in normal oral keratinocytes (NOKs). In this study, we examined in further detail how ELDR drives the neoplastic transformation of normal keratinocytes. We performed RNA-seq analysis on NOKs stably expressing ELDR (NOK-ELDR), which revealed that ELDR enhances the expression of cell cycle-related genes. Expression of Aurora kinase A and its downstream targets Polo-like kinase 1, cell division cycle 25C, cyclin-dependent kinase 1, and cyclin B1 (CCNB1) are significantly increased in NOK-ELDR cells, suggesting induction of G2/M progression. We further identified CCCTC-binding factor (CTCF) as a binding partner of ELDR in NOK-ELDR cells. We show that ELDR stabilizes CTCF and increases its expression. Finally, we demonstrate the ELDR-CTCF axis upregulates transcription factor Forkhead box M1, which induces Aurora kinase A expression and downstream G2/M transition. These findings provide mechanistic insights into the role of the lncRNA ELDR as a potential driver of oral cancer during neoplastic transformation of normal keratinocytes.

RUVBL1/2 Complex Regulates Pro-Inflammatory Responses in Macrophages via Regulating Histone H3K4 Trimethylation.

Macrophages play an important role in the host defense mechanism. In response to infection, macrophages activate a genetic program of pro-inflammatory response to kill any invading pathogen, and initiate an adaptive immune response. We have identified RUVBL2 - an ATP-binding protein belonging to the AAA+ (ATPase associated with diverse cellular activities) superfamily of ATPases - as a novel regulator in pro-inflammatory response of macrophages. Gene knockdown of Ruvbl2, or pharmacological inhibition of RUVBL1/2 activity, compromises type-2 nitric oxide synthase (Nos2) gene expression, nitric oxide production and anti-bacterial activity of mouse macrophages in response to lipopolysaccharides (LPS). RUVBL1/2 inhibitor similarly inhibits pro-inflammatory response in human monocytes, suggesting functional conservation of RUVBL1/2 in humans. Transcriptome analysis further revealed that major LPS-induced pro-inflammatory pathways in macrophages are regulated in a RUVBL1/2-dependent manner. Furthermore, RUVBL1/2 inhibition significantly reduced the level of histone H3K4me3 at the promoter region of Nos2 and Il6, two prototypical pro-inflammatory genes, and diminished the recruitment of NF-kappaB to the corresponding enhancers. Our study reveals RUVBL1/2 as an integral component of macrophage pro-inflammatory responses through epigenetic regulations, and the therapeutic potentials of RUVBL1/2 inhibitors in the treatment of diseases caused by aberrant activation of pro-inflammatory pathways.

Tetrandrine, an Activator of Autophagy, Induces Autophagic Cell Death via PKC-α Inhibition and mTOR-Dependent Mechanisms.

Emerging evidence suggests the therapeutic role of autophagic modulators in cancer therapy. This study aims to identify novel traditional Chinese medicinal herbs as potential anti-tumor agents through autophagic induction, which finally lead to autophagy mediated-cell death in apoptosis-resistant cancer cells. Using bioactivity-guided purification, we identified tetrandrine (Tet) from herbal plant, Radix stephaniae tetrandrae, as an inducer of autophagy. Across a number of cancer cell lines, we found that breast cancer cells treated with tetrandrine show an increase autophagic flux and formation of autophagosomes. In addition, tetrandrine induces cell death in a panel of apoptosis-resistant cell lines that are deficient for caspase 3, caspase 7, caspase 3 and 7, or Bax-Bak respectively. We also showed that tetrandrine-induced cell death is independent of necrotic cell death. Mechanistically, tetrandrine induces autophagy that depends on mTOR inactivation. Furthermore, tetrandrine induces autophagy in a calcium/calmodulin-dependent protein kinase kinase-ß (CaMKK-ß), 5' AMP-activated protein kinase (AMPK) independent manner. Finally, by kinase profiling against 300 WT kinases and computational molecular docking analysis, we showed that tetrandrine is a novel PKC-α inhibitor, which lead to autophagic induction through PKC-α inactivation. This study provides detailed insights into the novel cytotoxic mechanism of an anti-tumor compound originated from the herbal plant, which may be useful in promoting autophagy mediated- cell death in cancer cell that is resistant to apoptosis.

Depletion of sirtuin 1 (SIRT1) leads to epigenetic modifications of telomerase (TERT) gene in hepatocellular carcinoma cells.

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase that is implicated in plethora of biological processes, including metabolism, aging, stress response, and tumorigenesis. Telomerase (TERT) is essential for telomere maintenance. Activation of TERT is considered a crucial step in tumorigenesis, and therefore it is a potential therapeutic target against cancer. We have recently found that SIRT1 expression is highly elevated in hepatocellular carcinoma, and the depletion of SIRT1 leads to substantial reduction in TERT mRNA and protein expression. However, the underlying molecular mechanism of SIRT1-dependent TERT expression remains uncharacterized. Here, we elucidated if SIRT1 regulates TERT expression via transcriptional, epigenetic and post-transcriptional mechanisms. We report that depletion of SIRT1 does not lead to significant change in transcriptional activity and CpG methylation patterns of the TERT promoter, nor does it affect mRNA stability or 3'-UTR regulation of TERT. Intriguingly, depletion of SIRT1 is associated with substantial induction of acetylated histone H3-K9 and reduction of trimethyl H3-K9 at the TERT gene, which are known to be associated with gene activation. Our data revealed that SIRT1 regulates histone acetylation and methylation at the TERT promoter. We postulated that SIRT1 may regulate TERT expression via long-range interaction, or via yet unidentified histone modifications.

Sirtuin 1 regulates hepatitis B virus transcription and replication by targeting transcription factor AP-1.

Chronic hepatitis B virus (HBV) infection is a major risk factor for liver cirrhosis and hepatocellular carcinoma. Nevertheless, the molecular mechanism of HBV replication remains elusive. SIRT1 is a class III histone deacetylase that is a structure component of the HBV cccDNA minichromosome. In this study, we found by using microarray-based gene expression profiling analysis that SIRT1 was upregulated in HBV-expressing cells. Gene silencing of SIRT1 significantly inhibited HBV DNA replicative intermediates, 3.5-kb mRNA, and core protein levels. In contrast, the overexpression of SIRT1 augmented HBV replication. Furthermore, SIRT1 enhanced the activity of HBV core promoter by targeting transcription factor AP-1. The c-Jun subunit of AP-1 was bound to the HBV core promoter region, as demonstrated by using a chromatin immunoprecipitation assay. Mutation of AP-1 binding site or knockdown of AP-1 abolished the effect of SIRT1 on HBV replication. Finally, SIRT1 inhibitor sirtinol also suppressed the HBV DNA replicative intermediate, as well as 3.5-kb mRNA. Our study identified a novel host factor, SIRT1, which may facilitate HBV replication in hepatocytes. These data suggest a rationale for the use of SIRT1 inhibitor in the treatment of HBV infection.

SIRT2 overexpression in hepatocellular carcinoma mediates epithelial to mesenchymal transition by protein kinase B/glycogen synthase kinase-3ß/ß-catenin signaling.

UNLABELLED: Sirtuin 1 (SIRT1) has been implicated in telomere maintenance and the growth of hepatocellular carcinoma (HCC). Nevertheless, the role of other sirtuins in the pathogenesis of HCC remains elusive. We found that sirtuin 2 (SIRT2), another member of the sirtuin family, also contributes to cell motility and invasiveness of HCC. SIRT2 is up-regulated in HCC cell lines and in a subset of human HCC tissues (23/45). Up-regulations of SIRT2 in primary HCC tumors were significantly correlated with the presence of microscopic vascular invasion (P = 0.001), a more advanced tumor stage (P = 0.004), and shorter overall survival (P = 0.0499). Functional studies by short hairpin RNA-mediated suppression of SIRT2 expression in HCC cell lines revealed significant inhibition of motility and invasiveness. Depletion of SIRT2 also led to the regression of epithelial-mesenchymal transition (EMT) phenotypes, whereas the ectopic expression of SIRT2 in the immortalized hepatocyte cell line L02 promoted cell motility and invasiveness. Mechanistic studies revealed that SIRT2 regulates the deacetylation and activation of protein kinase B, which subsequently impinges on the glycogen synthase kinase-3ß/ß-catenin signaling pathway to regulate EMT. CONCLUSIONS: Our findings have uncovered a novel role for SIRT2 in HCC metastasis, and provide a rationale to explore the use of sirtuin inhibitors in HCC therapy. (HEPATOLOGY 2013;).

Sirtuin 1 is upregulated in a subset of hepatocellular carcinomas where it is essential for telomere maintenance and tumor cell growth.

Hepatocellular carcinoma (HCC) is a highly malignant tumor with a poor prognosis. Treatment of HCC is complicated by the fact that the disease is often diagnosed at an advanced stage when it is no longer amenable to curative surgery, and current systemic chemotherapeutics are mostly inefficacious. Sirtuin 1 (SIRT1) is a class III histone deacetylase that is implicated in gene regulations and stress resistance. In this study, we found that SIRT1 is essential for the tumorigenesis of HCC. We showed that although SIRT1 was expressed at very low levels in normal livers, it was overexpressed in HCC cell lines and in a subset of HCC. Tissue microarray analysis of HCC and adjacent nontumoral liver tissues revealed a positive correlation between the expression levels of SIRT1 and advancement in tumor grades. Downregulation of SIRT1 consistently suppressed the proliferation of HCC cells via the induction of cellular senescence or apoptosis. SIRT1 silencing also caused telomere dysfunction-induced foci and nuclear abnormality that were clearly associated with reduced expressions of telomerase reverse transcriptase (TERT), and PTOP, which is a member of the shelter in complex. Ectopic expression of either TERT or PTOP in SIRT1-depleted cells significantly restored cell proliferation. There was also a positive correlation between the level of induction of SIRT1 and TERT [corrected] in human HCC. Finally, SIRT1-silencing sensitized HCC cells to doxorubicin treatment. Together, our findings reveal a novel function for SIRT1 in telomere maintenance of HCC, and they rationalize the clinical exploration of SIRT1 inhibitors for HCC therapy.

Heparin sulphate D-glucosaminyl 3-O-sulfotransferase 3B1 plays a role in HBV replication.

Hepatitis B virus infection is a worldwide epidemic and is closely associated with the development of hepatocellular carcinoma. Nevertheless, the molecular mechanisms of HBV infection and carcinogenesis remain elusive. Using a hepatocyte model of HBV infection and comparing the gene expression profiling analysis we found that heparan sulfate D-glucosaminyl 3-O-sulfotransferase 3 B1 (HS3ST3B1,3-OST3-B) is down-regulated in the hepatocytes of chronic HBV infection model. HS3ST3B1 showed potent inhibitory effect on HBV replication. The inhibitory effect of HS3ST3B1 overexpression was lost upon gene silencing of HS3ST3B1 or when a catalytic inactive mutant of HS3ST3B1 was expressed. Our study revealed the anti-viral activity of HS3ST3B1 on HBV replication. It is conceivable that possible therapeutic applications of HBV infection could be devised by manipulating HS3ST3B1 activity.

Pseudolaric acids: isolation, bioactivity and synthetic studies.

The pseudolaric acids are diterpenoids isolated from the root bark of Pseudolarix amabilis, or the golden larch. Pseudolaric acids A and B are the major antifungal and anti-angiogenic congeners of this family of compounds. This review presents the results of the isolation, biological and synthetic studies of these natural products. 127 references are cited.

Alisol B, a novel inhibitor of the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase pump, induces autophagy, endoplasmic reticulum stress, and apoptosis.

Emerging evidence suggests that autophagic modulators have therapeutic potential. This study aims to identify novel autophagic inducers from traditional Chinese medicinal herbs as potential antitumor agents. Using an image-based screen and bioactivity-guided purification, we identified alisol B 23-acetate, alisol A 24-acetate, and alisol B from the rhizome of Alisma orientale as novel inducers of autophagy, with alisol B being the most potent natural product. Across several cancer cell lines, we showed that alisol B-treated cells displayed an increase of autophagic flux and formation of autophagosomes, leading to cell cycle arrest at the G(1) phase and cell death. Alisol B induced calcium mobilization from internal stores, leading to autophagy through the activation of the CaMKK-AMPK-mammalian target of rapamycin pathway. Moreover, the disruption of calcium homeostasis induces endoplasmic reticulum stress and unfolded protein responses in alisol B-treated cells, leading to apoptotic cell death. Finally, by computational virtual docking analysis and biochemical assays, we showed that the molecular target of alisol B is the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase. This study provides detailed insights into the cytotoxic mechanism of a novel antitumor compound.

Solution structures, dynamics, and lipid-binding of the sterile alpha-motif domain of the deleted in liver cancer 2.

The deleted in liver cancer 2 (DLC2) is a tumor suppressor gene, frequently found to be underexpressed in hepatocellular carcinoma. DLC2 is a multidomain protein containing a sterile alpha-motif (SAM) domain, a GTPase-activating protein (GAP) domain, and a lipid-binding StAR-related lipid-transfer (START) domain. The SAM domain of DLC2, DLC2-SAM, exhibits a low level of sequence homology (15-30%) with other SAM domains, and appears to be the prototype of a new subfamily of SAM domains found in DLC2-related proteins. In the present study, we have determined the three-dimensional solution structure of DLC2-SAM using NMR methods together with molecular dynamics simulated annealing. In addition, we performed a backbone dynamics study. The DLC2-SAM packed as a unique four alpha-helical bundle stabilized by interhelix hydrophobic interactions. The arrangement of the four helices is distinct from all other known SAM domains. In contrast to some members of the SAM domain family which form either dimers or oligomers, both biochemical analyses and rotational correlation time (tau(c)) measured by backbone 15N relaxation experiments indicated that DLC2-SAM exists as a monomer in solution. The interaction of DLC2-SAM domain with sodium dodecyl sulfate (SDS) micelles and 1,2-dimyristoyl-sn-glycerol-3-phosphatidylglycerol (DMPG) phospholipids was examined by CD and NMR spectroscopic techniques. The DLC2-SAM exhibits membrane binding properties accompanied by minor loss of the secondary structure of the protein. Deletion studies showed that the self-association of DLC2 in vivo does not require SAM domain, instead, a protein domain consisting of residues 120-672 mediates the self-association of DLC2.

Pseudolaric acid B, a novel microtubule-destabilizing agent that circumvents multidrug resistance phenotype and exhibits antitumor activity in vivo.

PURPOSE: Pseudolaric acid B (PAB) is the major bioactive constituent in the root bark of Pseudolarix kaempferi that has been used as an antifungal remedy in traditional Chinese medicine. Previous studies showed that PAB exhibited substantial cytotoxicity. The aims of this study were to elucidate the molecular target of PAB, to examine its mechanism of action, and to evaluate the efficacy of this compound in vivo. EXPERIMENTAL DESIGN: The effect of PAB on cell growth inhibition toward a panel of cancer cell lines was assayed. Cell cycle analysis, Western blotting, immunocytochemistry, and apoptosis analysis were carried out to examine the mechanism of action. Tubulin polymerization assays were conducted to examine the interaction between PAB and tubulin. A P-glycoprotein-overexpressing cell line was used to evaluate the efficacy of PAB toward multidrug-resistant phenotypes. In vivo efficacy of PAB was evaluated by the murine xenograft model. RESULTS: PAB induces cell cycle arrest at G2-M transition, leading to apoptosis. The drug disrupts cellular microtubule networks and inhibits the formation of mitotic spindles. Polymerization of purified bovine brain tubulin was dose-dependently inhibited by PAB. Furthermore, PAB circumvents the multidrug resistance mechanism, displaying notable potency also in P-glycoprotein-overexpressing cells. Finally, we showed that PAB is effective in inhibiting tumor growth in vivo. CONCLUSIONS: We identified the microtubules as the molecular target of PAB. Furthermore, we showed that PAB circumvents P-glycoprotein overexpression-induced drug resistance and is effective in inhibiting tumor growth in vivo. Our work will facilitate the future development of PAB as a cancer therapeutic.

Osmotic response element-binding protein (OREBP) is an essential regulator of the urine concentrating mechanism.

OREBP (osmotic response element-binding protein), also called TonEBP or NFAT5, is thought to induce the expression of genes that increase the accumulation of organic osmolytes to protect cells against a hypertonic environment. To investigate the consequences of lacking OREBP activity, transgenic (Tg) mice that overexpress OREBPdn (dominant negative form of OREBP) specifically in the epithelial cells of the renal collecting tubules were generated. These mice showed impairment in their urine concentrating mechanism, most likely due to reduced expression of the aquaporin AQP2 and the urea transporter UT-A1 and UT-A2 mRNAs. When deprived of water or after the administration of a vasopressin analogue, urine osmolality of the Tg mice was significantly increased but not to the same extent as that of the wild type mice. The expression of AQP2 and UT-A1, but not UT-A2 mRNAs, was increased to the same level as that of the wild type mice in the water deprivation state, indicating that the vasopressin regulatory mechanism was not affected by OREBPdn. These data indicate that in addition to vasopressin, OREBP is another essential regulator of the urine concentrating mechanism. Furthermore, the OREBPdn Tg mice developed progressive hydronephrosis soon after weaning, confirming the osmoprotective function of OREBP implicated by the in vitro experiments.

Fyn and p38 signaling are both required for maximal hypertonic activation of the osmotic response element-binding protein/tonicity-responsive enhancer-binding protein (OREBP/TonEBP).

When cells are challenged by hyperosmotic stress, one of the crucial adaptive responses is the expression of osmoprotective genes that are responsible for raising the intracellular level of compatible osmolytes such as sorbitol, betaine, and myo-inositol. This is achieved by the activation of the transcription factor called OREBP (also known as TonEBP or NFAT5) that specifically binds to the osmotic response element (ORE) or tonicity-responsive enhancer that enhances the transcription of these genes. Here we show that p38, a subgroup of the mitogen-activated kinases activated by hypertonic stress, and Fyn, a shrinkage-activated tyrosine kinase, are both involved in the hypertonic activation of OREBP/TonEBP. Inhibition of p38 by SB203580 or by the dominant negative p38 mutant partially blocked the hypertonic induction of ORE reporter (reporter gene regulated by ORE). Similarly, hypertonic activation of ORE reporter was partially blocked by pharmacological inhibition of Fyn or by a dominant negative Fyn and was attenuated in Fyn-deficient cells. Importantly, inhibiting p38 in Fyn-deficient cells almost completely abolished the hypertonic induction of ORE reporter activity, indicating that p38 and Fyn are the major signaling pathways for the hypertonic activation of OREBP/TonEBP. Further we show that the transactivation domain of OREBP/TonEBP is the target of p38- and Fyn-mediated hypertonic activation. These results indicate a dual control in regulating the expression of the osmoprotective genes in mammalian cells.