Guangzhi Jin

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.

Loss of FBP1 facilitates aggressive features of hepatocellular carcinoma cells through the Warburg effect

Reprogrammed metabolism has been identified as an emerging hallmark in cancer cells. It has been demonstrated that fructose-1, 6-bisphosphatase 1 (FBP1) as a rate-limiting enzyme in gluconeogenesis plays critical roles in tumor initiation and progression in several cancer types. However, function of FBP1 in hepatocellular carcinoma (HCC) is still not clear. In this study, we observed that the expression of FBP1 was obviously downregulated in the cell lines and tissues of HCC. Downregulation of FBP1 in HCC tissues was correlated with a lower overall survival rate and had a relatively higher tendency of tumor recurrence (n = 224). Silencing FBP1 could significantly promote colony formation, proliferation and metastasis of HCC cells, while ectopic overexpression of FBP1 resulted in impaired abilities of colony formation, proliferation and metastasis in vitro and in vivo. Mechanistically, silencing FBP1 facilitated glycolysis in HCC cell lines, which may be responsible for aggressiveness of HCC cells. We further found that targeting the Warburg effect using the specific inhibitor FX11 could suppress the aggressiveness of HCC cells which was mediated by loss of FBP1. These findings indicate that FBP1 appears to be a tumor suppressor in HCC. Strategies to restore the levels and activities of FBP1 might be developed to treat patients with HCC.

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.

A Targetable Molecular Chaperone Hsp27 Confers Aggressiveness in Hepatocellular Carcinoma

Heat shock protein 27 (Hsp27) is an ATP-independent molecular chaperone and confers survival advantages and resistance to cancer cells under stress conditions. The effects and molecular mechanisms of Hsp27 in HCC invasion and metastasis are still unclear. In this study, hepatocellular carcinoma (HCC) tissue array (n = 167) was used to investigate the expression and prognostic relevance of Hsp27 in HCC patients. HCC patients with high expression of Hsp27 exhibited poor prognosis. Overexpression of Hsp27 led to the forced invasion of HCC cells, whereas silencing Hsp27 attenuated invasion and metastasis of HCC cells in vitro and in vivo. We revealed that Hsp27 activated Akt signaling, which in turn promoted MMP2 and ITGA7 expression and HCC metastasis. We further observed that targeting Hsp27 using OGX-427 obviously suppressed HCC metastasis in two metastatic models. These findings indicate that Hsp27 is a useful predictive factor for prognosis of HCC and it facilitates HCC metastasis through Akt signaling. Targeting Hsp27 with OGX-427 may represent an attractive therapeutic option for suppressing HCC metastasis.

A CRISPR screen identifies CDK7 as a therapeutic target in hepatocellular carcinoma

Dear Editor, Liver cancer is one of the most frequent malignancies and the second leading cause of cancer-related mortality worldwide. In the last decade, our understanding of the genetic landscape of hepatocellular carcinoma (HCC) has improved significantly through whole-exome sequencing studies. However, unlike EFGR mutation in lung cancer and BRAF mutation in melanoma, the most frequent mutations in HCC are currently undruggable. Sorafenib, a small-molecule RAF kinase and VEGF receptor kinase inhibitor, was approved for the treatment of advanced HCC patients. However, it only provides limited survival benefit for HCC patients: 2.8 months overall survival benefit in the SHARP trial and 2.3 months overall survival benefit for Asia-Pacific patients. It is therefore urgent to identify new therapeutic approaches for HCC. CRISPR/Cas9-based functional genetic screens represent a powerful tool to identify cancer-relevant genes. To systematically probe kinase-driven pathways required for cell viability in HCC, we screened a gRNA library representing the full complement of human kinases in two HCC cell lines (Supplementary information, Table S1). Hep3B and Huh7 cells were infected with the lentiviral kinome gRNA collection and cultured for 14 days. Changes in library representation were determined after 14 days of culture by next-generation sequencing of the gRNA inserts in three biological replicates (Fig. 1a and Supplementary information, Figure S1a). To identify essential genes in HCC, we focused followup experiments on the significantly depleted gRNAs identified in both cell lines. We identified a set of cyclin-dependent kinases (CDKs), including CDK7, CDK9, CDK12 and CDK13 as hits in our screen (Fig. 1b, Supplementary information, Figure S1b and Table S2). These CDKs play important roles in transcription initiation and elongation. We evaluated the expression levels of these CDKs in the GSE14520 cohort (n= 213) and the TCGA database (n= 50), which provide data on gene expression for both paired non-tumor and HCC tissues. Of the identified hits, only CDK7 expression was selectively upregulated in tumor tissue in both cohorts (paired t-test, P < 0.001, Fig.1c and Supplementary information, Figure S1c). Next, we analyzed CDK7 expression using a tissue microarray (TMA) containing 80 HCC specimens by immunochemistry analysis. Kaplan–Meier analysis indicated that HCC patients with high expression of CDK7 exhibited worse overall survival as compared to patients with low expression of CDK7 (n= 80, P= 0.048, log-rank test, Fig. 1d). We therefore selected CDK7 for more detailed analysis. We infected Hep3B and Huh7 cells with two CDK7 shRNAs, both of which reduced CDK7 levels (Fig. 1e). As expected, inhibition of CDK7 impaired proliferation of Hep3B and Huh7 cells in both short-term IncuCyte® cell proliferation assays and long-term colony formation assays (Fig. 1f, g). We also analyzed the growth inhibitory effects of shCDK7 in five additional cell lines (SNU398, JHH1, SNU449, PLC/ PRF/5 and MHCC97H). We found that SNU398 and JHH1 are also very sensitive to CDK7 knockdown, whereas SNU449, PLC/PRF/5 and MHCC97H cells are resistant to CDK7 knockdown (Supplementary information, Figure S2a, b). Next, we focused our attention on THZ1, a covalent CDK7 inhibitor. Recent data indicate that THZ1 has a therapeutic effect in several types of cancer, including T cell acute lymphoblastic leukemia, MYCN-amplified neuroblastoma, small cell lung cancer and triple-negative breast cancer. To test the sensitivity of HCC cells to THZ1, we treated the panel of 10 HCC cell lines with increasing concentrations of THZ1 for about 2 weeks in colony formation assays. As shown in Fig. 1h, the panel of HCC cell lines exhibited differential responses to CDK7 inhibition. Comparable results were observed in IncuCyte® short-term cell proliferation assays (Supplementary information, Figure S3a, b). CDK7 inhibition induced apoptosis in the sensitive cells (Hep3B, Huh7, HepG2, JHH1 and SNU398), as indicated by the IncuCyte® caspase-3/7 apoptosis assay, but not in the insensitive cell lines (SNU387, SNU449, SNU475, PLC/PRF/5 and MHCC97H) (Fig. 1i). THZ1 treatment also failed to induce apoptosis in non-transformed human BJ fibroblasts and RPE-1 cell lines (Supplementary information, Figure S3c). Together, our data indicate that there may be a subgroup of liver cancer cells with a profound dependence on CDK7 for survival and CDK7 may represent a novel therapeutic target in this subgroup. CDK7 regulates transcriptional processing through phosphorylating RNAPII C-terminal domain (RNAPII CTD). We observed that RNAPII CTD phosphorylation was equally suppressed by THZ1 in both sensitive and insensitive cell lines (Supplementary information, Figure S4a, b). These results indicate that there is no correlation between cell fate induced by CDK7 inhibitor and the activity of CDK7 on direct downstream targets. Recent evidence indicates that the MYC transcriptional network has an important role in the response to CDK7 inhibition in MYCNamplified neuroblastoma and small cell lung cancer. In our HCC cell line panel, we also observed that the presence of high level of MYC protein was associated with high sensitivity to CDK7 inhibition (Fig. 1j, k). Nuclear MYC protein levels showed a similar correlation in the panel of liver cancer cell lines, with the exception of HepG2 cells (Supplementary information, Figure S4c). THZ1 efficiently suppressed the high expression of MYC in the sensitive cells (Hep3B and Huh7), but at best modest changes in MYC expression were observed in the insensitive cell lines having lower intrinsic MYC levels (PLC/PRF/5 and MHCC97H) (Supplementary information, Figure S4d). Interestingly, HCC cell lines also showed a similar response to MYC knockdown as was seen with CDK7 inhibition (Fig. 1l). Ectopic expression of MYC in PLC/PRF/5 cells led to a greater sensitivity to THZ1 as compared to vector control-transfected cells (Supplementary information, Figure S4e). These data suggest that MYC level may serve as a useful biomarker to predict the response to CDK7 inhibition.

Co-expression of LASS2 and TGF-β1 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-β1 levels, and evaluated their prognostic values in HCC patients. We first analyzed the expression of LASS2 and TGF-β1 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-β1. HCC Patients with low expression of LASS2 and TGF-β1 had shorter overall survival (OS) and time to recurrence (TTR) than patients with high expression of LASS2 and TGF-β1. Furthermore, combination of LASS2 and TGF-β1 was an independent and significant risk factor for OS and TTR. In conclusion, low expression of LASS2 and TGF-β1 contributes to the aggressiveness and poor prognosis of HCC, and may represent a novel prognostic biomarker for HCC patients.