Acidic extracellular pH induces autophagy to promote anoikis resistance of hepatocellular carcinoma cells via downregulation of miR-3663-3p.

Metastasis is the major reason for poor prognosis and high fatality in hepatocellular carcinoma (HCC). Due to the "Warburg effect", an acidic tumor microenvironment (TME) exists in solid tumors and plays a critical role in cancer metastasis. Thus, clarifying the mechanism underlying the acidic TME in tumor metastasis could facilitate the development of new therapeutic strategies for HCC. Anoikis resistance is one of the most important events in the early stage of cancer metastasis. Here, we report that acidic extracellular pH (pHe) promotes autophagy of HCC cells via the AMPK/mTOR pathway. We found that autophagy induced by acidity enhances anoikis resistance of HCC cells, which could be reversed by autophagy inhibitors. Furthermore, miR-3663-3p was downregulated by acidity, and overexpression of miR-3663-3p abolished acidic pHe-induced autophagy and anoikis resistance. In summary, acidic pHe enhances anoikis resistance of HCC cells by inducing autophagy, which is regulated by miR-3663-3p. Our findings provide new insight into how the acidic TME is involved in HCC progression.

Self-Assembled and Self-Monitored Sorafenib/Indocyanine Green Nanodrug with Synergistic Antitumor Activity Mediated by Hyperthermia and Reactive Oxygen Species-Induced Apoptosis.

Liver cancer is a leading cause of cancer morbidity and mortality worldwide, especially in China. Sorafenib (SRF) is currently the most commonly used systemic agent against advanced hepatocellular carcinoma (HCC), which is the most common type of liver cancer. However, HCC patients have only limited benefit and suffer a serious side effect from SRF. Therefore, new approaches are urgently needed to improve the therapeutic effectiveness of SRF and reduce its side effect. In our current study, we developed a self-imaging and self-delivered nanodrug with SRF and indocyanine (ICG) to improve the therapeutic effect of sorafenib against HCC. With the π-π stacking effect between SRF and ICG, a one-step nanoprecipitation method was designed to obtain the SRF/ICG nanoparticles (SINP) via self-assembly. Pluronic F127 was used to shield the SINP to further improve the stability in an aqueous environment. The stability, photothermal effect, cell uptake, ROS production, cytotoxicity, tumor imaging, and tumor-targeting and tumor-killing efficacy of the SINP were evaluated in vitro and in vivo by using an HCC cell line Huh7 and its xenograft tumor model. We found that our designed SINP showed monodisperse stability and efficient photothermal effect both in vitro and in vivo. SINP could rapidly enter Huh7 cells and achieve potent cytotoxicity under near-infrared (NIR) laser irradiation partly by producing a great amount of reactive oxygen species (ROS). SINP had significantly improved stability and blood half-life, and could specifically target tumor via the enhanced permeability and retention (EPR) effect in vivo. In addition, SINP showed improved cytotoxicity in both subcutaneous and orthotopic HCC implantation models in vivo. Overall, this rationally designed sorafenib delivery system with a very high loading capacity (33%) has considerably improved antitumor efficiency in vitro and could completely eliminate subcutaneous tumors without any regrowth in vivo. In conclusion, our self-imaging and self-delivered nanodrug could improve the efficacy of SRF and might be a potential therapy for HCC patients.

Downregulation of PDK4 Increases Lipogenesis and Associates with Poor Prognosis in Hepatocellular Carcinoma.

Alterations in cellular metabolism are one of the characteristics in cancer. They are not only the result of tumor progression but also the cause of cancer initiation. Pyruvate dehydrogenase kinase 4 (PDK4) is a key metabolic enzyme, which regulates cell metabolism by inhibiting pyruvate dehydrogenase (PDH). However, the function and regulating mechanism of PDK4 in HCC remain unclear. Here, we found that the expression of PDK4 was significantly decreased in HCC tissues, and its downregulation could predict poor prognosis of HCC patients. Silencing PDK4 significantly facilitated proliferation and migration of HCC cells. Knockdown of PDK4 didn't influence the oxidative phosphorylation and glycolysis capacity of HCC cells in vitro. However, knockdown of PDK4 increased expression of key lipogenic enzymes, fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD), which finally induced lipogenesis. These data suggest that PDK4 inhibits proliferation and migration of HCC cells probably via suppressing lipogenesis.

Liver-specific deletion of LASS2 delayed regeneration of mouse liver after partial hepatectomy.

The capacity of liver regeneration is critical for patients with liver diseases. However, cellular and molecular mechanisms of liver regeneration are still incompletely defined. Here, we assessed roles of LASS2 in liver regeneration following partial hepatectomy (PHx) in mice. Our results showed that protein level of LASS2 remarkably increased during liver regeneration after PHx in wildtype (WT) mice. Comparing to WT mice, liver regeneration index after PHx was significantly decreased from day 1 to day 5 in liver-specific LASS2 knockout (LASS2-LKO) mice. Interestingly, liver mass of LASS2-LKO mice could sufficiently recover at day 14 after PHx. Immunohistochemistry (IHC) and western blot analyses revealed that proliferation markers, such as PCNA and Ki67, were potently reduced during liver regeneration in LASS2-LKO mice. In addition, several cell cycle related molecules, such as cyclin A, CDK2 and p-Rb, were decreased in LASS2-LKO mice after PHx. Co-immunoprecipitation assay further revealed a decreased formation of CDK4/cyclin D1 complex after PHx in LASS2-LKO mice. However, phosphorylation of Akt was significantly activated from day 2 after PHx in LASS2-LKO mice when compared with that in WT mice, which may explain the recovery of liver mass at the late stage of liver regeneration in LASS2-LKO mice. Taken together, we conclude that LASS2 plays an important role in efficient liver regeneration in response to PHx.

Regulator of Calcineurin 1 Gene Isoform 4, Down-regulated in Hepatocellular Carcinoma, Prevents Proliferation, Migration, and Invasive Activity of Cancer Cells and Metastasis of Orthotopic Tumors by Inhibiting Nuclear Translocation of NFAT1.

BACKGROUND & AIMS: Individuals with Down syndrome have a low risk for many solid tumors, prompting the search for tumor suppressor genes on human chromosome 21 (HSA21). We aimed to identify and explore potential mechanisms of tumor suppressors on HSA21 in hepatocellular carcinoma (HCC). METHODS: We compared expression of HSA21 genes in 14 pairs of primary HCC and adjacent noncancer liver tissues using the Affymetrix HG-U133 Plus 2.0 array (Affymetrix, Santa Clara, CA). HCC tissues and adjacent normal liver tissues were collected from 108 patients at a hospital in China for real-time polymerase chain reaction and immunohistochemical analyses; expression levels of regulator of calcineurin 1 (RCAN1) isoform 4 (RCAN1.4) were associated with clinical features. We overexpressed RCAN1.4 from lentiviral vectors in MHCC97H and HCCLM3 cells and knocked expression down using small interfering RNAs in SMMC7721 and Huh7 cells. Cells were analyzed in proliferation, migration, and invasion assays. HCC cells that overexpressed RCAN1.4 or with RCAN1.4 knockdown were injected into livers or tail veins of nude mice; tumor growth and numbers of lung metastases were quantified. We performed bisulfite pyrosequencing and methylation-specific polymerase chain reaction analyses to analyze CpG island methylation. We measured phosphatase activity of calcineurin in HCC cells. RESULTS: RCAN1.4 mRNA and protein levels were significantly decreased in primary HCC compared with adjacent noncancer liver tissues. Reduced levels of RCAN1.4 mRNA were significantly associated with advanced tumor stages, poor differentiation, larger tumor size, and vascular invasion. Kaplan-Meier survival analysis showed that patients with HCCs with lower levels of RCAN1.4 mRNA had shorter time of overall survival and time to recurrence than patients whose tumors had high levels of RCAN1.4 mRNA. In HCC cell lines, expression of RCAN1.4 significantly reduced proliferation, migration, and invasive activity. HCC cells that overexpressed RCAN1.4 formed smaller xenograft tumors, with fewer metastases and blood vessels, than control HCC cells. In HCC cells, RCAN1.4 inhibited expression of insulin-like growth factor 1 and vascular endothelial growth factor A by reducing calcineurin activity and blocking nuclear translocation of nuclear factor of activated T cells (NFAT1). HCC cells incubated with the calcineurin inhibitor cyclosporin A had decreased nuclear level of NFAT1. HCC cells had hypermethylation of a CpG island in the 5' regulatory region of RCAN1.4, which reduced its expression. CONCLUSIONS: RCAN1.4 is down-regulated in HCC tissues, compared with non-tumor liver tissues. RCAN1.4 prevents cell proliferation, migration, and invasion in vitro; overexpressed RCAN1.4 in HCC cells prevents growth, angiogenesis, and metastases of xenograft tumors by inhibiting calcineurin activity and nuclear translocation of NFAT1.

Long noncoding RNA SchLAH suppresses metastasis of hepatocellular carcinoma through interacting with fused in sarcoma.

Emerging evidence has indicated that deregulation of long non-coding RNAs (lncRNAs) can contribute to the progression and metastasis of human cancer, including hepatocellular carcinoma (HCC). However, the roles of most lncRNAs in HCC remain largely unknown. Here we found a long noncoding RNA termed SchLAH (seven chromosome locus associated with HCC; also called BC035072) was generally downregulated in HCC. Low expression of SchLAH was significantly correlated with shorter overall survival of HCC patients. In vitro and in vivo assays indicated that overexpression of SchLAH inhibited the migration and lung metastasis of HCC cells. Knockdown of SchLAH by siRNA pool promoted the migration of HCC cells. RNA pull-down and RNA immunoprecipitation assays demonstrated SchLAH physically interacted with fused in sarcoma (FUS). PCR array analysis showed that RhoA and Rac1 were the downstream effector molecules of SchLAH during HCC metastasis. Knockdown of FUS rescued the mRNA levels of RhoA and Rac1 that were repressed by SchLAH. These results suggest that SchLAH may suppress the metastasis of HCC cells by interacting with FUS, which indicates potential of SchLAH for the prognosis and treatment of HCC.

Co-expression of LASS2 and TGF-ß1 predicts poor prognosis in hepatocellular carcinoma.

Longevity assurance homolog 2 of yeast LAG1 (LASS2) has been reported to act as an important tumor suppressor in the development of human cancers. However, little is known about the prognostic value of LASS2 in hepatocellular carcinoma (HCC) . In the present study, we analyzed correlation between LASS2 and TGF-ß1 levels, and evaluated their prognostic values in HCC patients. We first analyzed the expression of LASS2 and TGF-ß1 in two independent cohorts (test cohort: 184 HCC patients; validation cohort: 118 HCC patients) using immunohistochemistry (IHC). Kaplan-Meier survival and Cox regression analyses were executed to evaluate the prognosis of HCC. The results of IHC analysis revealed a positive correlation between the expression of LASS2 and TGF-ß1. HCC Patients with low expression of LASS2 and TGF-ß1 had shorter overall survival (OS) and time to recurrence (TTR) than patients with high expression of LASS2 and TGF-ß1. Furthermore, combination of LASS2 and TGF-ß1 was an independent and significant risk factor for OS and TTR. In conclusion, low expression of LASS2 and TGF-ß1 contributes to the aggressiveness and poor prognosis of HCC, and may represent a novel prognostic biomarker for HCC patients.

The asialoglycoprotein receptor suppresses the metastasis of hepatocellular carcinoma via LASS2-mediated inhibition of V-ATPase activity.

The asialoglycoprotein receptor (ASGR), which is expressed mainly in hepatocytes, is downregulated in poorly differentiated hepatocellular carcinoma (HCC). Here we investigated the role of ASGR1 in HCC metastasis as well as the possible underlying molecular mechanisms. We found that ASGR1 was downregulated in HCC tissue compared with adjacent non-tumorous liver tissue and that lower ASGR1 expression was associated with higher TNM stage and poorer prognosis in HCC patients. ASGR1 overexpression inhibited hepatoma cell migration and invasion in vitro and in vivo, while ASGR1 knockdown had the opposite effects. Furthermore, ASGR1 interacted directly with human longevity assurance homolog 2 of yeast LAG1 (LASS2). Knockdown of LASS2 attenuated the inhibitory effects of ASGR1 on hepatoma cell migration and invasion in vitro. ASGR1 decreased V-ATPase activity in hepatoma cells, and this was reversed by LASS2 knockdown. Finally, HCC patients with low LASS2 levels had poor prognosis, while those with high ASGR1 and LASS2 levels had better prognosis. Thus, ASGR1 may act as a potential metastasis suppressor in HCC, and the combination of ASGR1 and LASS2 may help predict the prognosis of HCC patients.

Gas6/Axl Axis Contributes to Chemoresistance and Metastasis in Breast Cancer through Akt/GSK-3ß/ß-catenin Signaling.

Chemoresistance in breast cancer has been of great interest in past studies. However, the development of rational therapeutic strategies targeting chemoresistant cells is still a challenge in clinical oncology. By integrating data from global differences of gene expression and phospho-receptor tyrosine kinases between sensitive parental cells (MCF-7) and doxorubicin-resistant cells (MCF-7/ADR), we identified Axl as a potential target for chemoresistance and metastasis in multidrug resistant breast cancer cells. We analyzed Axl expression in 57 breast cancer cell lines and detected a dramatic increase in its expression level in mesenchymal breast cancer cell lines. Axl silencing suppressed invasive and metastatic potentials of chemoresistant breast cancer cells as well as increased elimination of cancer cells when combined with doxorubicin. Furthermore, in preclinical assays, an Axl inhibitor R428 showed increased cell death upon doxorubicin treatment. Additionally, using phospho-kinase array based proteomic analysis, we identified that Akt/GSK-3ß/ß-catenin cascade was responsible for Axl-induced cell invasion. Nuclear translocation of ß-catenin then induced transcriptional upregulation of ZEB1, which in turn regulated DNA damage repair and doxorubicin-resistance in breast cancer cells. Most importantly, Axl was correlated with its downstream targets in tumor samples and was associated with poor prognosis in breast cancer patients. These results demonstrate that Gas6/Axl axis confers aggressiveness in breast cancer and may represent a therapeutic target for chemoresistance and metastasis.

KDM6B Elicits Cell Apoptosis by Promoting Nuclear Translocation of FOXO1 in Non-Small Cell Lung Cancer.

BACKGROUND/AIMS: Non-small cell lung carcinoma (NSCLC) is the most common type of lung cancer and the cause of most cancer-related deaths. The molecular mechanisms that are involved in NSCLC development are currently not well understood. Accumulating evidence shows that histone demethylases play important roles in the regulation of pathological developmental processes in many diseases, including various types of cancers. METHODS: Mitochondrial membrane potential assays, migration and invasion assays, caspase-3 and caspase-9 activity assays and western blot analysis were used in this research. RESULTS: We found that overexpression of KDM6B, a demethylase that acts on histone H3 at lysine 27 (H3K27), inhibited cell growth by initiating mitochondria-dependent apoptosis and by attenuating the invasion-metastasis cascade in NSCLC cells. Moreover, our results showed that KDM6B directly interacted with FOXO1 and that overexpression of KDM6B promoted nuclear accumulation of FOXO1. The effects of KDM6B on cell apoptosis and metastasis were weakened by knockdown of FOXO1 expression. On the contrary, knocking down expression of KDM6B inhibited cell apoptosis and promoted cell growth by mitigating the nuclear translocation of FOXO1 in NSCLC cells. CONCLUSIONS: These findings suggest that KDM6B may act in a pro-apoptotic role in NSCLC by causing the nuclear translocation of FOXO1.

LIFR functions as a metastasis suppressor in hepatocellular carcinoma by negatively regulating phosphoinositide 3-kinase/AKT pathway.

Hepatocellular carcinoma (HCC) is one of the leading causes for cancer related mortality worldwide. Poor prognosis of HCC patients is mainly due to frequent metastasis and recurrence. Deregulation of metastasis suppressors in malignant cells plays critical roles during cancer metastasis. Thus, novel metastasis suppressors are urgently needed to be uncovered to shed new light on molecular mechanisms driving HCC metastasis. In the present study, decreased expression of leukemia inhibitory factor receptor (LIFR) was demonstrated in HCC, and its expression levels were even lower in HCC with metastasis. Downregulated LIFR expression predicted poor prognosis in HCC patients. LIFR was an independent and significant risk factor for their recurrence and survival. Silencing LIFR resulted in forced metastasis of HCC cells, whereas ectopic overexpression of LIFR attenuated migration and invasion of HCC cells in vitro and in vivo. Moreover, LIFR knockdown could activate phosphoinositide 3-kinase/V-akt Murine Thymoma Viral Oncogene Homolog (PI3K/AKT) signaling through enhancing phosphorylation of Janus kinase 1 (JAK1), which successively promoted matrix metalloproteinase 13 (MMP13) expression and HCC metastasis. Combination of LIFR and p-AKT or MMP13 was a more powerful predictor of poor prognosis for HCC patients. Together, these findings conclude that LIFR functions as a novel metastasis suppressor in HCC and may serve as a prognostic biomarker for HCC patients.

Prognostic significance of kynurenine 3-monooxygenase and effects on proliferation, migration, and invasion of human hepatocellular carcinoma.

Kynurenine 3-monooxygenase (KMO) is a pivotal enzyme in the kynurenine pathway of tryptophan degradation and plays a critical role in Huntington's and Alzheimer's diseases. This study aimed to examine the expression of KMO in human hepatocellular carcinoma (HCC) and investigate the relationship between its expression and prognosis of HCC patients. We first analyzed KMO expression in 120 paired HCC samples (HCC tissues vs matched adjacent non-cancerous liver tissues), and 205 clinical HCC specimens using immunohistochemistry (IHC). Kaplan-Meier survival and Cox regression analyses were executed to evaluate the prognosis of HCC. The results of IHC analysis showed that KMO expression was significantly higher in HCC tissues than that in normal liver tissues (all p < 0.05). Survival and recurrence analyses showed that KMO was an independent prognostic factor for overall survival (OS) and time to recurrence (TTR) (both p<0.01). And in vitro studies revealed that KMO positively regulated proliferation, migration, and invasion of HCC cells. These results suggest that KMO exhibits tumor-promoting effects towards HCC and it may serve as a novel prognostic marker in HCC.

Leukemia inhibitory factor receptor is a novel immunomarker in distinction of well-differentiated HCC from dysplastic nodules.

Differential diagnosis of well-differentiated hepatocellular carcinoma (WD-HCC) and high-grade dysplastic nodules (HGDNs) represents a challenge for pathologists. Several immunohistochemistry markers have been identified to distinguish hepatocellular carcinoma (HCC) from HGDNs. However, sensitivity or specificity of the individual marker is still limited. In this study, we analyzed dynamic alteration of leukemia inhibitory factor receptor (LIFR) and CD34 during hepatocarcinogenesis from dysplastic nodules to small HCC. The diagnostic performance of LIFR and CD34 combination in WD-HCC and HGDNs was investigated by logistic regression models and validated in an independent validation cohort. LIFR was decreased and CD34 was increased along with stepwise progression of hepatocarcinogenesis from low-grade dysplastic nodules (LGDNs) to small HCC. The sensitivity and specificity of the LIFR and CD34 combination for WD-HCC detection were 93.5% and 90.5%, respectively. In addition, colony formation assay was used to explore the role of LIFR in tumorigenesis. Silencing of LIFR could significantly promote colony formation of HCC cells, whereas ectopic overexpression of LIFR resulted in impaired ability of colony formation of HCC cells. These findings indicate that LIFR and CD34 combination may be used as an available differential diagnostic model for WD-HCC from HGDNs in clinical practice.

Clusterin facilitates metastasis by EIF3I/Akt/MMP13 signaling in hepatocellular carcinoma.

Clusterin (CLU) is a stress-induced chaperone that confers proliferative and survival advantages to cancer cells. However, effects and molecular mechanisms of CLU in hepatocellular carcinoma (HCC) metastasis are still unknown. In this study, HCC tissue array (n = 198) was utilized to investigate correlation between CLU expression and clinicopathological features. Overexpression of CLU in HCC tissues was correlated with shorter overall survival and higher tumor recurrence. In vitro and in vivo assays demonstrated that silencing CLU attenuated the invasion and metastasis of HCC cells, whereas ectopic overexpression of CLU resulted in the forced metastasis of HCC cells. We also revealed that CLU activated Akt signaling through complexing with eukaryotic translation initiation factor 3 subunit I (EIF3I), which in turn promoted matrix metalloproteinase 13 (MMP13) expression and HCC metastasis. Positive correlations between CLU and MMP13, p-Akt, or EIF3I were found in HCC tissues. We further observed that CLU knockdown using the CLU inhibitor OGX-011 significantly suppressed HCC metastasis in two metastatic models through inhibiting EIF3I/Akt/MMP13 signaling. These findings indicate that CLU is an independent predictive factor for prognosis of HCC and it facilitates metastasis through EIF3I/Akt/MMP13 signaling. CLU suppression using OGX-011 may represent a promising therapeutic option for suppressing HCC metastasis.