Klotho Endows Hepatoma Cells with Resistance to Anoikis via VEGFR2/PAK1 Activation in Hepatocellular Carcinoma

Klotho was originally characterized as an aging suppressor gene that predisposed Klotho-deficient mice to premature aging-like syndrome. Although Klotho was recently reported to exhibit tumor suppressive properties during various malignant transformations, the functional role and molecular mechanism of Klotho in hepatocarcinogenesis remains poorly understood. In our present study, immunohistochemical Klotho staining levels in a clinical follow-up of 52 hepatoma patients were significantly associated with liver cirrhosis, tumor multiplicity and venous invasion. The overall survival rate of hepatoma patients with high Klotho expression was significantly lower than those patients with low Klotho expression. Moreover, Klotho overexpression increased cellular migration, anchorage-independent growth, and anoikis resistance in hepatoma cells. Klotho overexpression elevated p21-activated kinase 1 (PAK1) expression and shRNA-mediated PAK1 knockdown and kinase activity inhibition with kinase dead mutant PAK1 K299R coexpression or allosteric inhibitor IPA3 treatment reversed anoikis resistance in Klotho-overexpressed hepatoma cells. More importantly, the pivotal significance of upregulated VEGFR2 protein levels mediated by Klotho expression was confirmed by VEGFR2 inhibitor Axitinib and blocking antibody treatment in hepatoma cells. Axitinib treatment sensitized anoikis was reversed by constitutive active mutant PAK1 T423E coexpression in Klotho-overexpressed hepatoma cells. Conversely, knockdown of Klotho reduced VEGFR2/PAK1 dependent anoikis resistance, which could be reversed by PAK1 T423E. These results revealed a novel oncogenic function of Klotho in promoting anoikis resistance via activating VEGFR2/PAK1 signaling, thus facilitating tumor migration and invasion during hepatoma progression, which could provide a putative molecular mechanism for tumor metastasis.     

IPA-3 Inhibits the Growth of Liver Cancer Cells By Suppressing PAK1 and NF-κB Activation

Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide and is associated with poor prognosis due to the high incidences of metastasis and tumor recurrence. Our previous study showed that overexpression of p21-activated protein kinase 1 (PAK1) is frequently observed in HCC and is associated with a more aggressive tumor behavior, suggesting that PAK1 is a potential therapeutic target in HCC. In the current study, an allosteric small molecule PAK1 inhibitor, IPA-3, was evaluated for the potential in suppressing hepatocarcinogenesis. Consistent with other reports, inhibition of PAK1 activity was observed in several human HCC cell lines treated with various dosages of IPA-3. Using cell proliferation, colony formation and BrdU incorporation assays, we demonstrated that IPA-3 treatment significantly inhibited the growth of HCC cells. The mechanisms through which IPA-3 treatment suppresses HCC cell growth are enhancement of apoptosis and blockage of activation of NF-κB. Furthermore, our data suggested that IPA-3 not only inhibits the HCC cell growth, but also suppresses the metastatic potential of HCC cells. Nude mouse xenograft assay demonstrated that IPA-3 treatment significantly reduced the tumor growth rate and decreased tumor volume, indicating that IPA-3 can suppress the in vivo tumor growth of HCC cells. Taken together, our demonstration of the potential preclinical efficacy of IPA-3 in HCC provides the rationale for cancer therapy.     

The enhancer of zeste homolog 2 (EZH2), a potential therapeutic target, is regulated by miR-101 in renal cancer cells

We investigated a prognostic significance and the mechanism of aberrant nuclear expression of EZH2, a histone methyltransferase, in human renal cell carcinoma (RCC). We found nuclear EZH2 in 48 of 100 RCCs and it was significantly correlated with worse survival in RCC patients. We detected a decreased expression of miR-101 in 15 of 54 RCCs. We found that re-expression of miR-101 resulted in EZH2 depletion and decreased renal cancer cell proliferation. Our results show nuclear EZH2 as a prognostic marker of worse survival in human RCC, and identify miR-101 as a negative regulator of EZH2 expression and renal cancer cell proliferation.     

p21-activated kinase 1 determines stem-like phenotype and sunitinib resistance via NF-κB/IL-6 activation in renal cell carcinoma

The p21-activated kinase 1 (PAK1), a serine/threonine kinase that orchestrates cytoskeletal remodeling and cell motility, has been shown to function as downstream node for various oncogenic signaling pathways to promote cell proliferation, regulate apoptosis and accelerate mitotic abnormalities, resulting in tumor formation and invasiveness. Although alterations in PAK1 expression and activity have been detected in various human malignancies, its potential biological and clinical significance in renal cell carcinoma (RCC) remains obscure. In this study, we found increased PAK1 and phosphorylated PAK1 levels in tumor tissues according to TNM stage progression. Elevated phosphorylated PAK1 levels associated with progressive features and indicated unfavorable overall survival (OS) as an independent adverse prognosticator for patients with RCC. Moreover, PAK1 kinase activation with constitutive active PAK1 mutant T423E promoted growth, colony formation, migration, invasion and stem-like phenotype of RCC cells, and vice versa, in PAK1 inhibition by PAK1 kinase inactivation with specific PAK1 shRNA, dead kinase PAK1 mutant K299R or allosteric inhibitor IPA3. Stem-like phenotype due to sunitinib administration via increased PAK1 kinase activation could be ameliorated by PAK1 shRNA, PAK1 mutant K299R and IPA3. Furthermore, nuclear factor-κB (NF-κB)/interleukin-6 (IL-6) activation was found to be responsible for PAK1-mediated stem-like phenotype following sunitinib treatment. Both IL-6 neutralizing antibody and IPA3 administration enhanced tumor growth inhibition effect of sunitinib treatment on RCC cells in vitro and in vivo. Our results unraveled that oncogenic activation of PAK1 defines an important mechanism for maintaining stem-like phenotype and sunitinib resistance through NF-κB/IL-6 activation in RCC, lending PAK1-mediated NF-κB/IL-6 activation considerable appeal as novel pharmacological therapeutic targets against sunitinib resistance.Arising from the renal tubular epithelial cells, renal cell carcinoma (RCC) accounts for ∼4% of all malignant diseases and 90% of renal malignancies in adults.^{1, 2} Although most RCCs are detected incidentally by the widespread use of abdominal imaging examinations for unrelated symptoms, ∼25–30% of patients are still diagnosed with metastatic disease.³ In addition, 20% of patients with localized RCC undergoing radical surgery experience relapse and develop metastatic RCC (mRCC) during follow-up.^{4, 5} Unfortunately, mRCC is refractory chemotherapy and radiotherapy with a 5-year survival rate of ^<10%.⁶ Immunotherapy with interleukin 2 (IL-2) and/or interferon-α (IFN-α) is the standard treatment for mRCC and has limited efficacy by substantial number of adverse effects.⁷ Despite the significant improvement in mRCC treatment with antiangiogenesis drugs such as sunitinib and sorafinib, its duration of therapeutic effect is often short.⁸ Clearly, this dire situation mandates better understanding of the molecular mechanism of RCC carcinogenesis so that novel targets could be identified for effective therapies.The p21-activated kinases (PAKs) are a family of conversed nonreceptor serine/threonine kinases that function as key regulators of pleiotropic physiological processes including cytoskeleton dynamics and cell polarity, motility, invasion and survival.⁹ Currently, 6 PAKs have been classified into group I PAKs (PAK1–3) and group II PAKs (PAK4–6) on the basis of structural and functional similarities.¹⁰ As the best-characterized member of the PAK family, PAK1 was identified as a protein that interacts with cell division cycle 42 (CDC42) and RAC1.¹¹ In addition to CDC42 and RAC1, other signaling including PI3K/Akt can also lead to the activation of PAK1.¹² PAK1 phosphorylation at threonine-423 (T423) by upstream signaling has been linked to its activation, as substitution of the acidic residue glutamic acid (E) at this site yields a constitutively active PAK1 T423E enzyme.¹³ Activation and localization of PAK1 lead to mediated physiological effects of downstream signaling via activating additional kinases and other effectors by phosphorylating them at specific serine and threonine residues or through protein–protein interaction.⁹PAK1 expression and activity are upregulated in different human tumors, such as breast, lung, colorectal, liver and kidney cancers,^{14, 15, 16} and are associated with tumor invasiveness, metastasis and poor prognosis. Besides, PAK1 is also a component of various signaling pathways, including mitogen-activated protein kinase (MAPK), JUN N-terminal Kinase (JNK) and nuclear factor-κB (NF-κB) pathways, all of which are believed to be important in carcinogenesis.⁹ Moreover, PAK1 has been found to play critical roles in anoikis resistance that facilitates metastasis by allowing tumor cells to survive following detachment from the matrix in original tissue and travelling to distant sites. Resistance to anoikis program represents a molecular basis for cancer progression and drug resistance.^{15, 17} The regulation of phosphorylation and function of Snail by PAK1 signaling kinase may contribute to the process of epithelial–mesenchymal transition (EMT) that plays a pivotal role in the conversion of early-stage tumors into invasive malignancies.¹⁸ EMT induction in cancer cells results in the acquisition of stem-like phenotype and drug resistance trait.^{19, 20}Based on these previous findings, we hypothesized that PAK1-mediated stem-like phenotype might induce sunitinib resistance and involve in RCC tumor progression. Our present study revealed that upregulation of PAK1 kinase activity conferred stem-like phenotype via NF-κB/IL-6 activation in vitro and in vivo that defines a novel potential mechanism underlying tumor metastasis and sunitinib resistance in RCC patients.     

Suppression of tumorigenicity by MicroRNA-138 through inhibition of EZH2-CDK4/6-pRb-E2F1 signal loop in glioblastoma multiforme

Deregulation of microRNAs (miRNAs) is implicated in tumor progression. We attempt to indentify the tumor suppressive miRNA not only down-regulated in glioblastoma multiforme (GBM) but also potent to inhibit the oncogene EZH2, and then investigate the biological function and pathophysiologic role of the candidate miRNA in GBM. In this study, we show that miRNA-138 is reduced in both GBM clinical specimens and cell lines, and is effective to inhibit EZH2 expression. Moreover, high levels of miR-138 are associated with long overall and progression-free survival of GBM patients from The Cancer Genome Atlas dataset (TCGA) data portal. Ectopic expression of miRNA-138 effectively inhibits GBM cell proliferation in vitro and tumorigenicity in vivo through inducing cell cycles G1/S arrest. Mechanism investigation reveals that miRNA-138 acquires tumor inhibition through directly targeting EZH2, CDK6, E2F2 and E2F3. Moreover, an EZH2-mediated signal loop, EZH2-CDK4/6-pRb-E2F1, is probably involved in GBM tumorigenicity, and this loop can be blocked by miRNA-138. Additionally, miRNA-138 negatively correlates to mRNA levels of EZH2 and CDK6 among GBM clinical samples from both TCGA and our small amount datasets. In conclusion, our data demonstrate a tumor suppressive role of miRNA-138 in GBM tumorigenicity, suggesting a potential application in GBM therapy.     

p21-activated Kinase 6 (PAK6) Inhibits Prostate Cancer Growth via Phosphorylation of Androgen Receptor and Tumorigenic E3 Ligase Murine Double Minute-2 (Mdm2)

The androgen receptor (AR) signaling pathway plays a crucial role in the development and growth of prostate malignancies. Regulation of AR homeostasis in prostate tumorigenesis has not yet been fully characterized. In this study, we demonstrate that p21-activated kinase 6 (PAK6) inhibits prostate tumorigenesis by regulating AR homeostasis. First, we demonstrated that in normal prostate epithelium, AR co-localizes with PAK6 in the cytoplasm and translocates into the nucleus in malignant prostate. Furthermore, AR phosphorylation at Ser-578 by PAK6 promotes AR-E3 ligase murine double minute-2 (Mdm2) association, causing AR degradation upon androgen stimuli. We also showed that PAK6 phosphorylates Mdm2 on Thr-158 and Ser-186, which is critical for AR ubiquitin-mediated degradation. Moreover, we found that Thr-158 collaborates with Ser-186 for AR-Mdm2 association and AR ubiquitin-mediated degradation as it facilitates PAK6-mediated AR homeostasis. PAK6 knockdown promotes prostate tumor growth in vivo. Interestingly, we found a strong inverse correlation between PAK6 and AR expression in the cytoplasm of prostate cancer cells. These observations indicate that PAK6 may be important for the maintenance of androgen-induced AR signaling homeostasis and in prostate malignancy, as well as being a possible new therapeutic target for AR-positive and hormone-sensitive prostate cancer.     

MicroRNA‐214 suppresses cell proliferation and migration and cell metabolism by targeting PDK2 and PHF6 in hepatocellular carcinoma

MiR‐214 has been reported to act as a tumor suppressor or oncogene involved in various malignancies. However, the biological functions and molecular mechanisms of miR‐214 in hepatocellular carcinoma (HCC) still remain unclear. Previous studies suggest that pyruvate dehydrogenase kinase 2 (PDK2) and plant homeodomain finger protein 6 (PHF6) may be involved in some tumor cell proliferation and migration. Therefore, we studied the relationship between PDK2/PHF6 and miR‐214. The expression of miR‐214, PDK2, and PHF6 was determined by quantitative real‐time polymerase chain reaction in HCC tissues and cell lines. The Luciferase reporter assay was used to confirm the interaction between miR‐214 and PDK2/PHF6. Cell proliferation, apoptosis, and migration were evaluated by cell counting kit‐8 assay, flow cytometry, and transwell assay, respectively. The expressions levels of α‐smooth muscle actin (α‐SMA) and E‐cadherin were detected via immunofluorescence assay. Here, we found that the expression of miR‐214 decreased in HCC and was negatively correlated with PDK2 and PHF6. Moreover, PDK2 and PHF6 were the direct targets of miR‐214 in HCC cells. Functional analysis showed that knockdown of PDK2 or PHF6 as well as miR‐214 overexpression significantly suppressed cell proliferation and migration in HCC cells. Furthermore, we found that the suppression of cell proliferation and migration through PDK2 or PHF6 knockdown could be partially reversed by miR‐214 down‐regulation. Moreover, we demonstrated a decrease of mesenchymal cell marker α‐SMA and increase of the epithelial marker E‐cadherin after miR‐214 overexpression, PDK2 knockdown or PHF6 knockdown, respectively, which also suggested that cell proliferation and migration were suppressed. Additionally, lactate and pyruvic acid production experiments confirmed miR‐214 could suppress the HCC cell lactate and pyruvic acid levels by down‐regulating PDK2/PHF6. In conclusion, MiR‐214 may act as a tumor suppressor gene, presenting its suppressive role in cell proliferation and migration of HCC cells by targeting PDK2 and PHF6, and might provide a potential therapy target for patients with HCC.     

PAK1 promotes proliferation,migration and invasion of hepatocellular carcinoma by facilitating EMT via directly up-regulating Snail

BackgroundHepatocellular carcinoma (HCC) is a primary cause of cancer mortality. PAK1 plays key roles in many types of cancers. However, the role of PAK1 in HCC is not clear.MethodsqRT-PCR and Western blotting were used to determine expressions of PAK1, Snail and epithelial mesenchymal transition (EMT)-related proteins. Luciferase reporter assay was used to measure the interaction between PAK1 and Snail. Wound healing, transwell, colony formation assays and flow cytometry were used to assess cell migration, invasion, proliferation and apoptosis. Mouse tumor xenograft model was used to determine the effect of PAK1 on tumor growth in vivo.ResultsPAK1 and Snail were up-regulated in HCC cells. PAK1 knockdown suppressed cell proliferation, migration and invasion, and increased apoptosis of HCC cells. PAK1 knockdown also inhibited tumor growth in vivo. Mechanistically, PAK1 promoted EMT by targeting Snail. Knockdown of PAK1 could up-regulate pro-apoptotic proteins but down-regulate proliferation-related proteins via suppressing β-catenin signaling pathway.ConclusionPAK1 promotes EMT process by increasing Snail, and facilitates progression of HCC by activating β-catenin pathway.     

Aberrant Upregulation of 14-3-3σ and EZH2 Expression Serves as an Inferior Prognostic Biomarker for Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common malignancy in the world. It is of important significance to find biomarkers for the prognostic monitoring of HCC. The 14-3-3σ and EZH2 proteins are involved in cell cycle regulation and epigenetic silencing. We herein examined the significance of 14-3-3 σ and EZH2 in HCC (n = 167) by immunohistochemistry, RT-PCR and qRT-PCR. The correlation between 14-3-3σ and EZH2 expression and patients'' clinicopathologic features were examined, as was the correlation between 14-3-3σ and EZH2 expression and the prognosis of HCC patients. We found that 14-3-3σ and EZH2 were highly expressed in HCC (71% and 90%), the expression of EZH2, but not 14-3-3σ, is associated with vascular invasion and tumor differentiation (p<0.01). The coexistence of 14-3-3σ and EZH2 overexpression is associated with a relatively unfavorable prognosis (p<0.01), suggesting that aberrant upregulation of 14-3-3σ and EZH2 expression serves as an inferior prognostic biomarker for HCC.     

Overexpression of cyclin D1 is associated with elevated levels of MAP kinases, Akt and Pak1 during diethylnitrosamine-induced progressive liver carcinogenesis

Hepatocellular carcinoma (HCC) is a multi-factorial and multi-step process. However, the molecular mechanisms, which play a pivotal role during progressive development of HCC, are not known. Accordingly Sprague-Dawley rats were administered diethylnitrosamine (DEN) for one to three months in order to understand the molecular alterations during progressive development of liver tumor. In this study involvement of G1/S regulatory proteins, MAP kinases and cell survival factors were analyzed using RT-PCR, western blotting and in vitro kinase assays. The data showed overexpression of cyclin D1 and increased expression and activation of ERK1/2, p38 kinase and JNK1/2 with progression of tumor suggesting that MAP kinases play an important role during tumorigenesis. These molecular alterations were supported by Akt upregulation and increase in the levels of inactive GSK3beta with progression of liver tumor. Further, p21-actived kinase1 (Pak1) was found to be upregulated with tumor progression, which is a novel observation during progressive liver carcinogenesis. These results indicate that elevated levels of all the three MAP kinases (ERK1/2, p38 and JNK1/2), Akt/GSK3beta and Pak1 are associated with cyclin D1 upregulation, which helps in the disruption of the G1/S regulatory point of the cell cycle and leads to abnormal cell proliferation during progressive hepatocarcinogenesis.     

Targeting p21-activated kinase 4 (PAK4) with pyrazolo[3,4-d]pyrimidine derivative SPA7012 attenuates hepatic ischaemia-reperfusion injury in mice

p21-Activated kinase 4 (PAK4), one of the serine/threonine kinases activated by Rho-family GTPases, has been widely studied as an oncogenic protein that is overexpressed in many types of cancers. In our recent study, PAK4 upregulation was observed in mice exhibiting hepatic ischaemia-reperfusion (I/R) and in liver transplantation patients. Liver I/R injury was also attenuated in Pak4 KO mice. Herein, we report a novel series of pyrazolo[3,4-d]pyrimidine derivatives of type I ½ PAK4 inhibitors. The most potent compound SPA7012 was evaluated to determine the pharmacological potential of PAK4 inhibitor in I/R injury in mice. Mice with I/R injury showed typical patterns of liver damage, as demonstrated by increases in serum levels of aminotransferases and proinflammatory cytokines, hepatocellular necrosis and apoptosis, and inflammatory cell infiltration, relative to sham mice. Conversely, intraperitoneal administration of SPA7012 dramatically attenuated biochemical and histopathologic changes. Mechanistically, stabilisation of nuclear factor-erythroid 2-related factor 2 (Nrf2), a master regulator of anti-oxidative response, was observed following SPA7012 treatment. SPA7012 treatment in primary hepatocytes also attenuated hypoxia-reoxygenation-induced apoptotic cell death and inflammation. Together, these results provide experimental evidence supporting the use of PAK4 inhibitors for alleviation of I/R-induced liver damage.     

microRNA-489 Plays an Anti-Metastatic Role in Human Hepatocellular Carcinoma by Targeting Matrix Metalloproteinase-7

Dysregulation of microRNAs (miRNAs) is actively involved in the pathogenesis and tumorigenicity of hepatocellular carcinoma (HCC). miR-489 was found to play either oncogenic or tumor suppressive roles in human cancers. Recent study reported that the levels of miR-489 in late recurrent HCC patients were evidently higher than that in early recurrent cases, suggesting that miR-489 may function as a tumor suppressive miRNA in HCC. Yet, the clinical value and biological function of miR-489 remain rarely known in HCC. Here, we presented that miR-489 level in HCC tissues was notably reduced compared to matched non-cancerous specimens. Its decreased level was evidently correlated with adverse clinical parameters and poor prognosis of HCC patients. Accordingly, the levels of miR-489 were obviously down-regulated in HCC cells. Ectopic expression of miR-489 in HCCLM3 and MHCC97H cells prominently inhibits the migration and invasion of tumor cells and reduced lung metastases in vivo, while miR-489 knockdown increased these behaviors of HepG2 and MHCC97L cells. Mechanically, miR-489 negatively regulated matrix metalloproteinase-7 (MMP7) abundance in HCC cells. Herein, MMP7 was found to be a downstream molecule of miR-489 in HCC. An inversely correlation between miR-489 and MMP7 was confirmed in HCC specimens. MMP7 knockdown prohibited cell migration and invasion while MMP7 overexpression showed opposite effects on HCC cells. Furthermore, restoration of MMP7 expression could abrogate the anti-metastatic effects of miR-489 on HCCLM3 cells with enhanced cell migration and invasion. Altogether, miR-489 potentially acts as a prognostic predictor and a drug-target for HCC patients.     

PAK1 and PAK2 have different roles in HGF-induced morphological responses

Hepatocyte growth factor (HGF) stimulates dissociation of epithelial cells (scattering) and cell migration. Several Rho GTPases are required for HGF-induced scattering. PAK1 and PAK2 are members of the p21-activated kinase (PAK) family of serine/threonine kinases, and are activated by the Rho GTPases Rac and Cdc42. Here we investigate the contributions of PAK1 and PAK2 to HGF-induced motile response. HGF stimulates phosphorylation of PAK1 and PAK2. Knockdown of PAK1 inhibits HGF-stimulated migration and loss of cell–cell junctions in DU145 prostate carcinoma cells, whereas knockdown of PAK2 enhances loss of cell–cell junctions and increases lamellipodium extension but does not affect migration speed. On the other hand, in PC3 prostate carcinoma cells, which lack cell–cell junctions, knockdown of PAK1 or PAK2 reduces HGF-stimulated migration. PAK2 knockdown increases phosphorylation of PAK1, indicating that PAK2 provides a negative feedback on PAK1. We hypothesise that PAK2 acts in part via PAK1 to regulate HGF-induced scattering.     

Elevation of highly up-regulated in liver cancer (HULC) by hepatitis B virus X protein promotes hepatoma cell proliferation via down-regulating p18

Long noncoding RNAs (lncRNAs) play crucial roles in human cancers. It has been reported that lncRNA highly up-regulated in liver cancer (HULC) is dramatically up-regulated in hepatocellular carcinoma (HCC). Hepatitis B virus X protein (HBx) contributes importantly to the development of HCC. However, the function of HULC in HCC mediated by HBx remains unclear. Here, we report that HULC is involved in HBx-mediated hepatocarcinogenesis. We found that the expression levels of HULC were positively correlated with those of HBx in clinical HCC tissues. Moreover, we revealed that HBx up-regulated HULC in human immortalized normal liver L-O2 cells and hepatoma HepG2 cells. Luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) assay showed that HBx activated the HULC promoter via cAMP-responsive element-binding protein. We further demonstrated that HULC promoted cell proliferation by methyl thiazolyl tetrazolium, 5-ethynyl-2'-deoxyuridine, colony formation assay, and tumorigenicity assay. Next, we hypothesized that HULC might function through regulating a tumor suppressor gene p18 located near HULC in the same chromosome. We found that the mRNA levels of p18 were inversely correlated with those of HULC in the above clinical HCC specimens. Then, we validated that HULC down-regulated p18, which was involved in the HULC-enhanced cell proliferation in vitro and in vivo. Furthermore, we observed that knockdown of HULC could abolish the HBx-enhanced cell proliferation through up-regulating p18. Thus, we conclude that the up-regulated HULC by HBx promotes proliferation of hepatoma cells through suppressing p18. This finding provides new insight into the roles of lncRNAs in HBx-related hepatocarcinogenesis.     

Inhibition of EZH2 Attenuates Sorafenib Resistance by Targeting NOTCH1 Activation-Dependent Liver Cancer Stem Cells via NOTCH1-Related MicroRNAs in Hepatocellular Carcinoma

Acquired resistance and intrinsic to sorafenib therapy represents a major hurdle in improving the management of advanced hepatocellular carcinoma (HCC), which has been recently shown to be associated with the emergence of liver cancer stem cells (CSCs). However, it remains largely unknown whether and how histone posttranslational modifications, especially H3K27me3, are causally linked to the maintenance of self-renewal ability in sorafenib-resistant HCC. Here, we found that NOTCH1 signaling was activated in sorafenib-resistant HCC cells and NOTCH1 activation conferred hepatoma cells sorafenib resistance through enhanced self-renewal and tumorigenecity. Besides, the overexpression of EZH2 was required for the emergence of cancer stem cells following prolonged sorafenib treatment. As such, modulating EZH2 expression or activity suppressed activation of NOTCH1 pathway by elevating the expression of NOTCH1-related microRNAs, hsa-miR-21-5p and has-miR-26a-1-5p, via H3K27me3, and consequently weakened self-renewal ability and tumorigenecity and restored the anti-tumor effects of sorafenib. Overall, our results highlight the role of EZH2/NICD1 axis, and also suggest that EZH2 and NOTCH1 pathway are rational targets for therapeutic intervention in sorafenib-resistant HCC.     

Hepatitis B virus X protein mutant HBxΔ127 promotes proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT

The mutant of virus is a frequent event. Hepatitis B virus X protein (HBx) plays a vital role in the development of hepatocellular carcinoma (HCC). Therefore, the identification of potent mutant of HBx in hepatocarcinogenesis is significant. Previously, we identified a natural mutant of the HBx gene (termed HBxΔ127). Relative to wild type HBx, HBxΔ127 strongly enhanced cell proliferation and migration in HCC. In this study, we aim to explore the mechanism of HBxΔ127 in promotion of proliferation of hepatoma cells. Our data showed that both wild type HBx and HBxΔ127 could increase the expression of miR-215 in hepatoma HepG2 and H7402 cells. However, HBxΔ127 was able to significantly increase miR-215 expression relative to wild type HBx in the cells. We identified that protein tyrosine phosphatase, receptor type T (PTPRT) was one of the target genes of miR-215 through targeting 3′UTR of PTPRT mRNA. In function, miR-215 was able to promote the proliferation of hepatoma cells. Meanwhile anti-miR-215 could partially abolish the enhancement of cell proliferation mediated by HBxΔ127 in vitro. Knockdown of PTPRT by siRNA could distinctly suppress the decrease of cell proliferation mediated by anti-miR-215 in HepG2-XΔ127/H7402-XΔ127 cells. Moreover, we found that anti-miR-215 remarkably inhibited the tumor growth of hepatoma cells in nude mice. Collectively, relative to wild type HBx, HBxΔ127 strongly enhances proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Our finding provides new insights into the mechanism of HBx mutant HBxΔ127 in promotion of proliferation of hepatoma cells.     

Strategy and validation of a structure-based method for the discovery of selective inhibitors of PAK isoforms and the evaluation of their anti-cancer activity

p21 activated kinase 4 (PAK4), which belongs to the serine/threonine (Ser/Thr) protein kinase family, is a representative member of the PAK family and plays a significant role in multiple processes associated with cancer development. In this study, structure-based virtual screening was performed to discover novel and selective small molecule scaffolds, and a 6-hydroxy-2-mercapto-3-phenylpyrimidin-4(3H)-one-based compound (SPU-106, 14#) was identified as an effective PAK4 inhibitor. By combining both a molecular docking study and molecular dynamics (MD) simulation strategies, the binding mode was determined in the PAK4 site. The SPU-106 compound could efficiently and selectively bind to the PAK4 kinase domain at an IC₅₀ of 21.36 μM according to the kinase analysis. The designed molecular probe demonstrated that SPU-106 binds to the kinase domain in the C-terminus of PAK4. Further investigation revealed that the SPU-106 had a strong inhibitory effect on the invasion of SGC7901 cells but without any cytotoxicity. The western blot analysis indicated that the compound potently inhibited the PAK4/LIMK1/cofilin and PAK4/SCG10 signaling pathways. Thus, our work shows the successful application of computational strategies for the discovery of selective hits, and SPU-106 may be an effective PAK4 inhibitor for further development as an antitumor agent.