Jie Wang

Genomic and Epigenomic Heterogeneity of Hepatocellular Carcinoma.

Understanding the intratumoral heterogeneity of hepatocellular carcinoma is instructive for developing personalized therapy and identifying molecular biomarkers. Here we applied whole-exome sequencing to 69 samples from 11 patients to resolve the genetic architecture of subclonal diversification. Spatial genomic diversity was found in all 11 hepatocellular carcinoma cases, with 29% of driver mutations being heterogeneous, including TERT, ARID1A, NOTCH2, and STAG2. Similar with other cancer types, TP53 mutations were always shared between all tumor regions, that is, located on the trunk of the evolutionary tree. In addition, we found that variants within several drug targets such as KIT, SYK, and PIK3CA were mutated in a fully clonal manner, indicating their therapeutic potentials for hepatocellular carcinoma. Temporal dissection of mutational signatures suggested that mutagenic processes associated with exposure to aristolochic acid and aflatoxin might play a more important role in early, as opposed to late, stages of hepatocellular carcinoma development. Moreover, we observed extensive intratumoral epigenetic heterogeneity in hepatocellular carcinoma based on multiple independent analytical methods and showed that intratumoral methylation heterogeneity might play important roles in the biology of hepatocellular carcinoma cells. Our results also demonstrated prominent heterogeneity of intratumoral methylation even in a stable hepatocellular carcinoma genome. Together, these findings highlight widespread intratumoral heterogeneity at both the genomic and epigenomic levels in hepatocellular carcinoma and provide an important molecular foundation for better understanding the pathogenesis of this malignancy. Cancer Res; 77(9); 2255-65. ©2017 AACR.

CCL18/PITPNM3 enhances migration, invasion, and EMT through the NF-κB signaling pathway in hepatocellular carcinoma

Chemokine ligand 18 (CCL18) has been associated with hepatocellular carcinoma (HCC) metastasis. Here, we demonstrated a novel mechanism through which CCL18 enhances cell migration, invasion, and epithelial–mesenchymal transition (EMT) in HCC. (1) Using immunohistochemistry, we analyzed the expression of PITPNM3, a molecule that correlated with CCL18 signaling, in 149 HCC tissue specimens. The results showed that PITPNM3 expression is highly associated with tumor metastasis and differentiation; (2) in vitro experiments showed that CCL18 enhances cell migration, invasion, and EMT in PITPNM3(+) HCC cells but not in PITPNM3(-) cells. Silencing of PITPNM3 by short interfering RNA (siRNA) inhibited the induction of cell migration, invasion, and EMT by CCL18; (3) Cell migration, invasion, and EMT induced by CCL18 accompanied with the phosphorylation of IKK and IKBα as well as p65 nuclear translocation in PITPNM3(+) HCC cells, but not in the cells that PITPNM3 is silenced with siRNA, implying that the activation of NF-κB signaling is involved in the action of CCL18/PITPNM3. These results suggest that CCL18 enhances HCC cell migration, invasion, and EMT through the expression of PITPNM3 and the activation of the NF-κB signaling pathway.

Expression of CCN family members correlates with the clinical features of hepatocellular carcinoma.

Studies have reported that the CCN family of proteins plays an important role in stimulating tumorigenesis. However, the relationship between the CCN protein family members and the features of hepatocellular carcinoma (HCC) remains unclear. The objective of this study was to determine the relationship between the expression levels of CCN protein family members and the features of HCC. Expression levels of the CCN family of proteins in 80-paired primary HCC samples and 11 normal liver samples were determined by a quantitative real-time PCR assay. Enhanced expression of nephroblastoma overexpressed protein (NOV) and decreased expression of Wnt-induced secreted protein 1 (WISP1), cysteine-rich protein 61 (CYR61) and connective tissue growth factor (CTGF) were found in HCC samples when compared to levels in matched non-cancerous tissues. No significant difference in WISP2 was found between matched-pair samples; only a few samples showed WISP3 expression. Furthermore, the expression levels of NOV, WISP1 and CYR61 were closely correlated with certain clinical features, including venous invasion, cellular differentiation, pTNM stage, disease-free survival and overall survival. Our results suggest that HCC progression may be enhanced by NOV and suppressed by WISP1 and CYR61. Our statistical analysis suggests that these proteins may be valuable in determining the prognosis of this deadly disease and directs attention to modulating the levels of these proteins as a potential mode of therapy.

Safety and efficacy of dehydrated ethanol soaking of the operative field in the treatment of spontaneous hepatocellular carcinoma rupture

BackgroundThe aim of our study was to evaluate the clinical safety and value of ethanol surgical field infiltration (ESFI), combined with distilled water peritoneal lavage (DWPL), after hepatectomy in patients with hepatocellular carcinoma (HCC) rupture.MethodsRat liver tissue samples were soaked in dehydrated ethanol for different soaking times, and 18 rats were assigned to three groups that underwent different soaking methods of the hepatectomy cut surface. We retrospectively reviewed 45 patients who underwent hepatectomy for treatment of ruptured HCC. Among these, EFSI combined with DWPL was used in 21 patients (DAW group), with only DWPL used in the other 24 patients (DW group). Clinical outcomes were compared between the two groups.ResultsFor in vitro experiments, the depth of coagulation degeneration and necrosis increased with the duration of soaking. For in vivo experiments, rats in all three groups survived until postoperative day 7 without significant postoperative complication. In patients, the rate of post-operation complication was comparable between the two groups (Pu2009=u20090.398), with no between-group differences in liver function levels. The incidence of peritoneal dissemination was significantly higher for DW than DAW group (Pu2009=u20090.037). Kaplan–Meier test identified dehydrated ethanol treatment as a significant factor of disease-free survival (DFS) (Pu2009=u20090.036). On univariate analysis, dehydrated ethanol treatment was associated with better prognostic outcomes, although it was not retained as an independent factor of patient outcome.ConclusionsDehydrated ethanol soaking of the cut surface of the hepatectomy could potentially lower the risk of metastasis and improve the effect of hepatectomy for ruptured HCC as well as showed potential therapeutic value for intraoperative iatrogenic rupture of HCC.

PRMT9 promotes hepatocellular carcinoma invasion and metastasis via activating PI3K/Akt/GSK-3β/Snail signaling

Protein arginine methyltransferases (PRMT) catalyze protein arginine methylation and play an important role in many biological processes. Aberrant PRMT expression in tumor cells has been documented in several common cancer types; however, its precise contribution to hepatocellular carcinoma (HCC) cell invasion and metastasis is not fully understood. In this study, we identified a new oncogene, PRMT9, whose overexpression strongly promotes HCC invasion and metastasis. PRMT9 expression was detected more frequently in HCC tissues than in adjacent noncancerous tissues. PRMT9 overexpression was significantly correlated with hepatitis B virus antigen (HBsAg) status, vascular invasion, poor tumor differentiation and advanced TNM stage. Patients with higher PRMT9 expression had a shorter survival time and higher recurrence rate. PRMT9 expression was an independent and significant risk factor for survival after curative resection. Functional studies demonstrated that PRMT9 increased HCC cell invasion and lung metastasis. Knocking down PRMT9 with short hairpin RNA (shRNA) inhibited HCC cell invasion. Further investigations found that PRMT9 increased cell migration and invasion through epithelial‐mesenchymal transition (EMT) by regulating Snail expression via activation of the PI3K/Akt/GSK‐3β/Snail signaling pathway. In clinical HCC samples, PRMT9 expression was positively associated with Snail expression and was negatively associated with E‐cadherin expression. In conclusion, our study demonstrated that PRMT9 is an oncogene that plays an important role in HCC invasion and metastasis through EMT by regulating Snail expression via activation of the PI3K/Akt/GSK‐3β/Snail signaling pathway. Thus, PRMT9 may serve as a candidate prognostic biomarker and a potential therapeutic target.

Therapeutic significance and indications of pulmonary metastasectomy for hepatocellular carcinoma following liver resection

BACKGROUNDnTo explore the therapeutic significance and indications of pulmonary metastasectomy (PMT) in hepatocellular carcinoma (HCC) patients with pulmonary metastasis (PM) following liver resection (LR).nnnPATIENTS AND METHODSnPM-HCC patients who underwent LR were retrospectively enrolled, and survival outcomes and prognostic factors were analyzed. Patients were divided into PMT and non-PMT group, and propensity score matching (PSM) analysis was used for survival comparison. Prognostic analysis and survival comparisons were performed specifically in PMT patients.nnnRESULTSnNinety-seven patients were enrolled, among which twenty-six underwent PMT while seventy-one did not. Survival outcome was superior in PMT group compared to non-PMT group (33.5 vs. 10.5 months) (pxa0=xa00.003), while no statistical difference was found after PSM analysis (33.5 vs. 11.2 months) (pxa0=xa00.138). Synchronous PM-HCC, serum alpha fetal protein≥400xa0ng/ml at PM diagnosis, no intrahepatic treatments (LR, ablation or transarterial chemoembolization) after LR, intrahepatic recurrence or metastasis at repeated PM diagnosis were inferior independent prognostic factors in PMT patients (pxa0<xa00.05). Superior survival outcomes were seen in candidate PMT patients when corresponding indications were satisfied (pxa0=xa00.014, pxa0=xa00.005).nnnCONCLUSIONnPMT might provide potential survival benefits in well selected PM-HCC patients who underwent LR. Well designed, multi-institutional studies with larger patient number were still to be required.

Aggressive intrahepatic therapies for synchronous hepatocellular carcinoma with pulmonary metastasis

PurposePrognosis of synchronous hepatocellular carcinoma (HCC) patients with pulmonary metastasis (PM) was poor, while aggressive intrahepatic therapies remained controversial. This study aimed to investigate the significance of aggressive intrahepatic therapies for synchronous PM–HCC.MethodsSynchronous PM–HCC patients were retrospectively enrolled from Sun Yat-sen Memorial Hospital of Sun Yat-sen University during January 2000 and December 2015. Univariate and multivariate analysis were performed to investigate the prognostic factors. Patients were grouped according to different HCC treatment modalities including liver resection (LR), ablation, transarterial chemoembolization (TACE), systemic therapy (ST, systemic chemotherapy or sorafenib) and supportive care (SC). Case control studies were achieved using propensity score matching (PSM) analysis to further investigate the significance of LR, ablation and TACE.ResultsEighty-one patients were enrolled, and the median overall survival (OS) was 4.5xa0months. Serum alpha fetal protein (AFP)xa0≥xa0400xa0ng/ml, multiple HCC lesions and no intrahepatic therapies (LR/Ablation/TACE) were inferior independent prognostic factors. Patients were divided into LR group (nxa0=xa09), Ablation/TACE group (nxa0=xa024) and ST/SC group (nxa0=xa048). After PSM analysis, survival outcome was superior in LR group compared to Ablation/TACE group (19.6 vs. 6.9xa0months) (pxa0=xa00.023) or ST/SC group (19.6 vs. 2.8xa0months) (pxa0=xa00.034), while no significant difference was found between -Ablation/TACE and ST/SC group (5.1 vs. 3.2xa0months) (pxa0=xa00.338).ConclusionsPrognosis of synchronous PM–HCC patients was poor. Serum AFPxa0≥xa0400xa0ng/ml, multiple HCC lesions and no aggressive intrahepatic therapies were inferior prognostic factors. LR might provide survival benefits in well-selected patients, while the significance of ablation or TACE remained to be further investigated.

CCL18 secreted from M2 macrophages promotes migration and invasion via the PI3K/Akt pathway in gallbladder cancer

PurposeThe presence of M2 macrophages within primary tumors has been correlated with a poor prognosis for many types of cancer. However, little is known about the role of M2 macrophages in gallbladder cancer (GBC).MethodsThe number of M2 macrophages in 78 GBC and 16 normal gallbladder tissue samples was assessed by immunohistochemistry. The THP-1 monocyte cell line was differentiated into M2 macrophages and co-cultured with GBC-derived cell lines. The effect of M2 macrophages on promoting GBC cell migration and invasion was analyzed using migration, invasion and scratch wound healing assays. Western blotting and real-time PCR were used to assess the expression of epithelial-mesenchymal transition (EMT) markers and the activation status of the PI3K/Akt signaling pathway in GBC cells co-cultured with THP-1-derived macrophages.ResultsThe average number of M2 macrophages was found to be significantly higher in GBC tissues than in normal gallbladder tissues. We also found that GBC patients with higher M2 macrophage counts exhibited poorer overall survival rates. Co-culture with M2 macrophages significantly promoted the migration, invasion and EMT of GBC cells. Moreover, we found that CCL18 secreted from M2 macrophages had the same effect on GBC cells as M2 macrophages. Blocking the function of CCL18 with a neutralizing antibody reversed this effect. Finally, we found that M2 macrophages could activate PI3K/Akt signaling in GBC cells, thereby leading to migration, invasion and EMT of these cells.ConclusionsOur findings contribute to our understanding of the role of chronic inflammation in GBC development and progression, and may offer potential therapeutic targets for GBC.

PRMT5 promotes cell proliferation by inhibiting BTG2 expression via the ERK signaling pathway in hepatocellular carcinoma

Increasing evidence suggests that PRMT5, a protein arginine methyltransferase, has roles in cell growth regulation and cancer development. However, the role of PRMT5 in hepatocellular carcinoma (HCC) progression remains unclear. Here, we showed that PRMT5 expression was frequently upregulated in HCC tissues, and its expression was inversely correlated with overall survival in HCC patients. PRMT5 knockdown markedly inhibited in vitro HCC proliferation and in vivo tumorigenesis. We revealed that the mechanism of PRMT5‐induced proliferation was partially mediated by BTG downregulation, leading to cell cycle arrest during the G1 phase in HCC cells. Ectopic BTG2 overexpression decreased HCC growth, caused cell cycle arrest at the G1 phase, and downregulated Cyclin D1 and Cyclin E1 protein expression. Furthermore, we found that PRMT5‐induced ERK phosphorylation regulated BTG2 expression in HCC cells, whereas pretreatment with a selective ERK1/2 inhibitor (PD184352) significantly reversed the effect of PRMT5 on BTG2 expression. Our results indicated that PRMT5 promotes HCC proliferation by downregulating BTG2 expression via the ERK pathway.

Effect of FOLFOX4 combined with Brucea javanica emulsion on VEGF in patients with gastric cancer

The aim of the study was to investigate the effect of FOLFOX4 regimen combined with Brucea javanica emulsion on the content of serum vascular endothelial growth factor (VEGF) in patients with gastric cancer, and to evaluate the efficacy of FOLFOX4 regimen combined with Brucea javanica emulsion on gastric cancer. A total of 60 patients with gastric ulcer were selected as the normal group, and another 150 patients with gastric cancer were randomly divided into two groups, of which 75 patients with gastric cancer treated with FOLFOX4 regimen after operation were selected as the control group and another 75 patients with gastric cancer treated with FOLFOX4 regimen combined with Brucea javanica emulsion after operation were selected as the experimental group. The serum VEGF levels of patients in the different groups before operation, after chemotherapy for 3 times and at 1 and 3 months after chemotherapy were compared via enzyme-linked immunosorbent assay. The content of serum VEGF in patients with gastric cancer before operation was significantly higher than that in the normal group (P<0.05). After administration of chemotherapy 3 times, the content of serum VEGF in the control group had no significant difference from that in the experimental group (P>0.05). Additionally, at 1 and 3 months after chemotherapy, the content of serum VEGF of patients in the experimental group were significantly lower than those in the control group (P<0.05). The results showed that FOLFOX4 regimen combined with Brucea javanica emulsion can significantly reduce the level of serum VEGF in patients with gastric cancer, and has a certain effect in reducing the postoperative recurrence rate of gastric cancer and improving the effect of chemotherapy.