PROX1 promotes hepatocellular carcinoma metastasis by way of up-regulating hypoxia-inducible factor 1α expression and protein stability.

UNLABELLED: Hepatocellular carcinoma (HCC) is one of the most common cancers and the third leading cause of death from cancer worldwide. HCC has a very poor prognosis because of tumor invasiveness, frequent intrahepatic spread, and extrahepatic metastasis. The molecular mechanism of HCC invasiveness and metastasis is poorly understood. The homeobox protein PROX1 is required for hepatocyte migration during mouse embryonic liver development. In this study, we show that high PROX1 protein expression in primary HCC tissues is associated with significantly worse survival and early tumor recurrence in postoperative HCC patients. Knockdown of PROX1 expression in HCC cells inhibited cell migration and invasiveness in vitro and HCC metastasis in nude mice while overexpression of PROX1 in HCC cells promoted these processes. PROX1's pro-metastasis activity is most likely attributed to its up-regulation of hypoxia-inducible factor 1α (HIF-1α) transcription and stabilization of HIF-1α protein by recruiting histone deacetylase 1 (HDAC1) to prevent the acetylation of HIF-1α, which subsequently induces an epithelial-mesenchymal transition response in HCC cells. We further demonstrated the prognostic value of using the combination of PROX1 and HDAC1 levels to predict postoperative survival and early recurrence of HCC. CONCLUSION: PROX1 is a critical factor that promotes HCC metastasis.

Radiofrequency ablation following first-line transarterial chemoembolization for patients with unresectable hepatocellular carcinoma beyond the Milan criteria.

BACKGROUND: Recent studies suggest that a combination of radiofrequency ablation (RFA) and transarterial chemoembolization (TACE) may have theoretical advantages over TACE alone for treatment of hepatocellular carcinoma (HCC). The purpose of this study was to evaluate the effectiveness and safety of radiofrequency ablation following first-line TACE treatment in the management of HCC beyond the Milan Criteria. METHODS: Forty-five patients who consecutively underwent RFA following first-line TACE treatment for HCC beyond the Milan criteria were enrolled in this study. RFA was performed within 1-2 months after TACE treatment in patients who had incomplete necrotic tumor nodules. Primary effectiveness, complications, survival rates, and prognostic factors were evaluated retrospectively. RESULTS: Complete ablation was achieved in 76.2% of the lesions according to 1-month follow-up computed tomography/magnetic resonance imaging evaluation. The mean follow-up period was 30.9 months (range 3-94 months). There were no major complications after RFA therapy. The median overall survival was 29 months (range 20-38 months), with 1-, 2-, and 3-year survival of 89%, 61%, and 43%, respectively. Multivariate analysis revealed that tumor diameter (P = 0.045, hazard ratio [HR] = 0.228, 95% confidence interval [CI]: 0.054-0.968) and pretreatment serum alpha-fetoprotein level (P = 0.024, HR = 2.239, 95% CI: 1.114-4.500) were independent predictors for long-term survival. CONCLUSIONS: HCC beyond the Milan criteria can be completely and safely ablated by radiofrequency ablation following first-line TACE treatment with a low rate of complications and favorable survival outcome. Further assessment of the survival benefits of combination treatment for HCCs beyond the Milan Criteria is warranted.

Cirrhotic-extracellular matrix attenuates aPD-1 treatment response by initiating immunosuppressive neutrophil extracellular traps formation in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is closely associatedwith chronic liver diseases, particularly liver cirrhosis, which has an altered extracellular matrix (ECM) composition. The influence and its mechanism of the cirrhotic-ECM on the response of HCC to immune checkpoint inhibitor (ICI) remains less clarified. METHODS: In silico, proteomic and pathological assessment of alteration of cirrhotic-ECM were applied in clinical cohort. Multiple pre-clinical models with ECM manipulation were used to evaluate cirrhotic-ECM's effect on ICI treatment. In silico, flow cytometry and IHC were applied to explore how cirrhotic-ECM affect HCC microenvironment. In vitro and in vivo experiments were carried out to identify the mechanism of how cirrhotic-ECM undermined ICI treatment. RESULTS: We defined "a pro-tumor cirrhotic-ECM" which was featured as the up-regulation of collagen type 1 (Col1). Cirrhotic-ECM/Col1 was closely related to impaired T cell function and limited anti PD-1 (aPD-1) response of HCC patients from the TCGA pan cancer cohort and the authors' institution, as well as in multiple pre-clinical models. Mechanically, cirrhotic-ECM/Col1 orchestrated an immunosuppressive microenvironment (TME) by triggering Col1-DDR1-NFκB-CXCL8 axis, which initiated neutrophil extracellular traps (NETs) formation to shield HCC cells from attacking T cells and impede approaching T cells. Nilotinib, an inhibitor of DDR1, reversed the neutrophils/NETs dominant TME and efficiently enhanced the response of HCC to aPD-1. CONCLUSIONS: Cirrhotic-ECM modulated a NETs enriched TME in HCC, produced an immune suppressive TME and weakened ICI efficiency. Col1 receptor DDR1 could be a potential target synergically used with ICI to overcome ECM mediated ICI resistance. These provide a mechanical insight and novel strategy to overcome the ICI resistance of HCC.

Antiangiogenic therapy promoted metastasis of hepatocellular carcinoma by suppressing host-derived interleukin-12b in mouse models.

Antiangiogenic therapy, specially sorafenib, has become the standard of care for patients with advanced hepatocellular carcinoma (HCC), however, the improvement in survival time is not satisfactory. Previous studies have found that, in some circumstances, antiangiogenic therapy promoted tumor metastasis and the mechanistic studies were mainly focus on cancer-cell-autonomous manners. In two experimental metastasis models with tail-vein injection with hepatoma cells and an orthotopic HCC mouse model, we found that pretreatment with two vascular endothelial growth factor receptor (VEGFR) inhibitors, sunitinib and sorafenib, facilitated tumor cell survival in blood stream and promoted lung metastasis from tumors that were subsequently incubated after drug discontinuation, indicating that host response joined into the pro-metastatic effects. An antibody microarray identified that interleukin (IL)-12b was decreased in the peripheral blood of the mice treated with the two VEGFR inhibitors. IL-12b suppression in macrophages and dendritic cells from host organs was found to play a crucial role in treatment-induced metastasis. Supplement with recombinant mouse IL-12b or restoration of IL-12b expression in the host by zoledronic acid, which was previously reported to enhance IL-12 expression in vitro and in vivo, alleviated the metastasis-promoting effects of sunitinib and sorafenib. These studies suggest that host response to VEGFR inhibitors facilitates HCC metastasis and restoration of IL-12b expression could translate into clinical benefits.

Development and validation of a mutation-annotated prognostic score for intrahepatic cholangiocarcinoma after resection: a retrospective cohort study.

BACKGROUND: The value of existing prognostic models for intrahepatic cholangiocarcinoma is limited. The inclusion of prognostic gene mutations would enhance the predictive efficacy. METHODS: In the screening cohorts, univariable Cox regression analysis was applied to investigate the effect of individual mutant genes on overall survival (OS). In the training set, multivariable analysis was performed to evaluate the independent prognostic roles of the clinicopathological and mutational parameters, and a prognostic model was constructed. Internal and external validations were conducted to evaluate the performance of this model. RESULTS: Among the recurrent mutations, only TP53 and KRASG12 were significantly associated with OS across all three screening cohorts. In the training cohort, TP53 and KRASG12 mutations in combination with seven other clinical parameters (tumor size, tumor number, vascular invasion, lymph node metastasis, adjacent invasion, CA19-9, and CEA), were independent prognostic factors for OS. A mutation-annotated prognostic score (MAPS) was established based on the nine prognosticators. The C-indices of MAPS (0.782 and 0.731 in the internal and external validation cohorts, respectively) were statistically higher than those of other existing models ( P <0.05). Furthermore, the MAPS model also demonstrated significant value in predicting the possible benefits of upfront surgery and adjuvant therapy. CONCLUSIONS: The MAPS model demonstrated good performance in predicting the OS of intrahepatic cholangiocarcinoma patients. It may also help predict the possible benefits of upfront surgery and adjuvant therapy.

Up-regulation of platelet-derived growth factor-A is responsible for the failure of re-initiated interferon alpha treatment in hepatocellular carcinoma.

BACKGROUND: Postoperative interferon-α(IFN-α) treatment delays hepatocellular carcinoma(HCC) recurrence and prolongs patient survival, and may thus be an effective form of adjuvant therapy. However, clinical observations found that HCC recurs in some patients within 8 months of IFN-α treatment being discontinued. We investigated whether HCC regrowth appears after IFN-α is discontinued, whether re-initiated IFN-α is effective, and the underlying mechanisms of IFN-α treatment. METHODS: The human HCC nude mouse model LCI-D20 was used to study the effects of IFN-α treatment, discontinued IFN-α treatment, and re-initiated IFN-α treatment on tumor growth. Tumor weight, microvessel density(MVD), serum vascular endothelial growth factor (VEGF), and tumor cell apoptosis were analyzed. Angiogenesis-related factors were studied using cDNA microarray in different tumor samples and confirmed using reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting assays. Finally, imatinib was added with re-initiated IFN-α treatment to improve efficacy. RESULTS: IFN-α (1.5 × 107 U/kg/day for 20 days) suppressed HCC growth by 60.3% and decreased MVD by 52.2% compared with the control. However, tumor regrowth occurred after IFN-α was discontinued, and re-initiated IFN-α treatment was not effective for inhibiting tumor growth or reducing MVD compared with a saline-treated group. cDNA microarray showed VEGF was down-regulated while platelet-derived growth factor-A (PDGF-A) was up-regulated when IFN-α treatment was re-initiated. These findings were further confirmed with RT-PCR and Western blotting assay. The combination of imatinib with re-initiated IFN-α reduced HCC weight by 30.7% and decreased MVD by 31.1% compared with IFN-α treatment only (P=0.003 and 0.015, respectively). CONCLUSION: Tumor regrowth occurred after IFN-α treatment was discontinued. Re-initiated IFN-α treatment was not effective and was associated with up-regulation of PDGF-A, while the VEGF remained suppressed. The combination of a PDGF-receptor inhibitor with IFN-α improved the effect of the re-initiated treatment.

Neutrophil extracellular traps in hepatocellular carcinoma are enriched in oxidized mitochondrial DNA which is highly pro-inflammatory and pro-metastatic.

Background: Neutrophil extracellular traps (NETs) are net like extracellular structure formed by neutrophils in response to certain stimulation. It works as inflammatory regulator and metastasis promoter in cancer. Mitochondrial-(mt)DNA is a circular, mitochondria derived double strain molecule, which is involved in NETs formation. Its role in NETs induced inflammatory alteration in hepatocellular carcinoma (HCC) remained unexplored. Method: We evaluated the mitochondrial reactive oxygen species (mitoROS) level in peripheral neutrophils from HCC patients and the oxidative level of mtDNA in derived NETs. The association between the NETs and oxidized mtDNA was assessed to reveal their relevance. A function assay was applied to uncover how the oxidation state of mtDNA directed the metastasis promoting inflammation state in HCC cells in a NETs protein dependent manner. Finally, using animal models, we explored the potential of a therapy strategy against NETs-drove metastasis by targeting the oxidized mtDNA with metformin. Results: Neutrophils in HCC patients contained high level of mitoROS level, and formed NETs that were enriched in oxidized mtDNA in a mitoROS dependent manner. NETs and oxidized mtDNA were clinically relevant. Bound with NETs protein, oxidized mtDNA is more capable of triggering the metastasis-promoting inflammatory mediators in HepG2 cells. Targeting the oxidized mtDNA with metformin attenuated the metastasis-promoting inflammatory state and hereby undermine the metastasis capacity of HCC. Conclusion: HCC is capable to stimulate NETs enriched in oxidized mtDNA, which are highly pro-inflammatory and pro-metastatic. Oxidized mtDNA in NETs may serve as a potential anti-metastatic target by metformin therapy.

Cancer-derived exosomal HSPC111 promotes colorectal cancer liver metastasis by reprogramming lipid metabolism in cancer-associated fibroblasts.

Tumor metastasis is a hallmark of cancer. The communication between cancer-derived exosomes and stroma plays an irreplaceable role in facilitating pre-metastatic niche formation and cancer metastasis. However, the mechanisms underlying exosome-mediated pre-metastatic niche formation during colorectal cancer (CRC) liver metastasis remain incompletely understood. Here we identified HSPC111 was the leading upregulated gene in hepatic stellate cells (HSCs) incubated with CRC cell-derived exosomes. In xenograft mouse model, CRC cell-derived exosomal HSPC111 facilitated pre-metastatic niche formation and CRC liver metastases (CRLM). Consistently, CRC patients with liver metastasis had higher level of HSPC111 in serum exosomes, primary tumors and cancer-associated fibroblasts (CAFs) in liver metastasis than those without. Mechanistically, HSPC111 altered lipid metabolism of CAFs by phosphorylating ATP-citrate lyase (ACLY), which upregulated the level of acetyl-CoA. The accumulation of acetyl-CoA further promoted CXCL5 expression and secretion by increasing H3K27 acetylation in CAFs. Moreover, CXCL5-CXCR2 axis reinforced exosomal HSPC111 excretion from CRC cells and promoted liver metastasis. These results uncovered that CRC cell-derived exosomal HSPC111 promotes pre-metastatic niche formation and CRLM via reprogramming lipid metabolism in CAFs, and implicate HSPC111 may be a potential therapeutic target for preventing CRLM.

Cholesterol suppresses GOLM1-dependent selective autophagy of RTKs in hepatocellular carcinoma.

Aberrant activation of receptor tyrosine kinases (RTKs) and the subsequent metabolic reprogramming play critical roles in cancer progression. Our previous study has shown that Golgi membrane protein 1 (GOLM1) promotes hepatocellular carcinoma (HCC) metastasis by enhancing the recycling of RTKs. However, how this RTK recycling process is regulated and coupled with RTK degradation remains poorly defined. Here, we demonstrate that cholesterol suppresses the autophagic degradation of RTKs in a GOLM1-dependent manner. Further mechanistic studies reveal that GOLM1 mediates the selective autophagy of RTKs by interacting with LC3 through an LC3-interacting region (LIR), which is regulated by a cholesterol-mTORC1 axis. Lowering cholesterol by statins improves the efficacy of multiple tyrosine kinase inhibitors (TKIs) in vivo. Our findings indicate that cholesterol serves as a signal to switch GOLM1-RTK degradation to GOLM1-RTK recycling and suggest that lowering cholesterol by statin may be a promising combination strategy to improve the TKI efficiency in HCC.

Driver mutations of intrahepatic cholangiocarcinoma shape clinically relevant genomic clusters with distinct molecular features and therapeutic vulnerabilities.

Purpose: To establish a clinically applicable genomic clustering system, we investigated the interactive landscape of driver mutations in intrahepatic cholangiocarcinoma (ICC). Methods: The genomic data of 1481 ICCs from diverse populations was analyzed to investigate the pair-wise co-occurrences or mutual exclusivities among recurrent driver mutations. Clinicopathological features and outcomes were compared among different clusters. Gene expression and DNA methylation profiling datasets were analyzed to investigate the molecular distinctions among mutational clusters. ICC cell lines with different gene mutation backgrounds were used to evaluate the cluster specific biological behaviors and drug sensitivities. Results: Statistically significant mutation-pairs were identified across 21 combinations of genes. Seven most recurrent driver mutations (TP53, KRAS, SMAD4, IDH1/2, FGFR2-fus and BAP1) showed pair-wise co-occurrences or mutual exclusivities and could aggregate into three genetic clusters: Cluster1: represented by tripartite interaction of KRAS, TP53 and SMAD4 mutations, exhibited large bile duct histological phenotype with high CA19-9 level and dismal prognosis; Cluster2: co-association of IDH/BAP1 or FGFR2-fus/BAP1 mutation, was characterized by small bile duct phenotype, low CA19-9 level and optimal prognosis; Cluster3: mutation-free ICC cases with intermediate clinicopathological features. These clusters showed distinct molecular traits, biological behaviors and responses to therapeutic drugs. Finally, we identified S100P and KRT17 as "cluster-specific", "lineage-dictating" and "prognosis-related" biomarkers, which in combination with CA19-9 could well stratify Cluster3 ICCs into two biologically and clinically distinct subtypes. Conclusions: This clinically applicable clustering system can be instructive to ICC prognostic stratification, molecular classification, and therapeutic optimization.

Expression and prognostic significance of placental growth factor in hepatocellular carcinoma and peritumoral liver tissue.

Vascular endothelial growth factor-targeted therapy is a promising treatment for hepatocellular carcinoma (HCC), but its clinical benefit is often accompanied by acquired resistance. In animal studies, antiplacental growth factor therapy is effective with less resistance. The role of placental growth factor (PlGF) in the progression of HCC is not clear. In our study, we used immunohistochemistry in tissue microarrays to investigate PlGF expression in tumor and peritumoral liver tissues from 105 patients with HCC. Intratumoral and peritumoral PlGF mRNA expression was analyzed in another cohort of 37 patients. Peritumoral PlGF expression was significantly higher than intratumoral PlGF expression (p < 0.001). Intratumoral PlGF expression was not associated with patients' overall survival (OS) or time to recurrence (TTR). However, peritumoral PlGF expression, which was associated with tumor size, presence of intrahepatic metastasis, TNM stage and Barcelona Clinic Liver Cancer stage, was an independent risk factor for OS (p = 0.026) and TTR (p = 0.041). The prognostic value of peritumoral PlGF expression was further validated in a validation cohort (n = 394). We inferred that the elevation of PlGF in peritumoral liver might be induced by hypoxia. We found that peritumoral PlGF expression was associated with hypoxia-inducible factor-1α (p = 0.017). PlGF expression was elevated in L02, a hepatic cell line, under hypoxic conditions in vitro. These findings indicate that high peritumoral PlGF expression is associated with tumor recurrence and survival after resection of HCC. PlGF could be a target of adjuvant therapy and deserves further investigations.

A novel tripeptide, tyroserleutide, inhibits irradiation-induced invasiveness and metastasis of hepatocellular carcinoma in nude mice.

Previous studies have demonstrated that tyroserleutide (YSL) inhibits tumor growth in an animal model of hepatocellular carcinoma (HCC). However, its effects on HCC metastasis are still not fully understood. To examine YSL as a novel agent to prevent HCC metastasis, a metastatic human HCC orthotopic nude mouse model of MHCC97L was used. The antitumor and antimetastasis effects of YSL were also evaluated in combination with radiation. Hypoxia and epithelial-mesenchymal transition (EMT)-related molecules were studied. YSL inhibited MHCC97L cell invasion in vitro with or without irradiation. YSL did not significantly inhibit tumor growth but decreased pulmonary metastasis and prolonged life-span for more than 40 days, which correlated with down-regulation of matrix metalloproteinase-2. Radiotherapy inhibited early-stage tumor growth and promoted tumor hypoxia. The re-implanted tumor volume in the radiotherapy group was not significantly different from the control, in which the incidence of lung metastasis increased after radiotherapy (6/6 versus 3/6, P = 0.046); however, YSL inhibited the growth of re-implanted tumor after radiotherapy. Furthermore, YSL at 160 or 320 µg/kg/day almost completely inhibited lung metastasis induced by irradiation (1/6 versus 6/6, P = 0.002 for both dosages). YSL down-regulated hypoxia-inducible factor 1α (HIF-1α) and transmembrane protease serine 4 (TMPRSS4), and inhibited EMT was associated with the antimetastasis capability of YSL. Our data suggest that YSL inhibits the enhanced invasiveness and metastatic potential of HCC induced by irradiation through down-regulation of HIF-1α and TMPRSS4 and inhibition of EMT. YSL may have potential as a new antimetastasis agent for radiotherapy.

Antiangiogenic effects of pazopanib in xenograft hepatocellular carcinoma models: evaluation by quantitative contrast-enhanced ultrasonography.

BACKGROUND: Antiangiogenesis is a promising therapy for advanced hepatocellular carcinoma (HCC), but the effects are difficult to be evaluated. Pazopanib (GW786034B) is a pan-vascular endothelial growth factor receptor inhibitor, the antitumor effects or antiangiogenic effects haven't been investigated in HCC. METHODS: In vitro direct effects of pazopanib on human HCC cell lines and endothelial cells were evaluated. In vivo antitumor effects were evaluated in three xenograft nude mice models. In the subcutaneous HCCLM3 model, intratumoral blood perfusion was detected by contrast-enhanced ultrasonography (CEUS), and serial quantitative parameters were profiled from the time-intensity curves of ultrasonograms. RESULTS: In vitro proliferation of various HCC cell lines were not inhibited by pazopanib. Pazopanib inhibited migration and invasion and induced apoptosis significantly in two HCC cell lines, HCCLM3 and PLC/PRF/5. Proliferation, migration, and tubule formation of human umbilical vein endothelial cells were inhibited by pazopanib in a dose-dependent manner. In vivo tumor growth was significantly inhibited by pazopanib in HCCLM3, HepG2, and PLC/PRF/5 xenograft models. Various intratumoral perfusion parameters changed over time, and the signal intensity was significantly impaired in the treated tumors before the treatment efficacy on tumor size could be observed. Mean transit time of the contrast media in hotspot areas of the tumors was reversely correlated with intratumoral microvessel density. CONCLUSIONS: Antitumor effects of pazopanib in HCC xenografts may owe to its antiangiogenic effects, and the in vivo antiangiogenic effects could be evaluated by quantitative CEUS.

High expression of macrophage colony-stimulating factor-1 receptor in peritumoral liver tissue is associated with poor outcome in hepatocellular carcinoma after curative resection.

BACKGROUND: Macrophage colony-stimulating factor 1 receptor (CSF-1R) expression in hepatocellular carcinoma (HCC) and its prognostic values are unclear. This study evaluated the prognostic values of the intratumoral and peritumoral expression of CSF-1R in HCC patients after curative resection. METHODS: Tissue microarrays containing material from cohort 1 (105 patients) and cohort 2 (32 patients) were constructed. Immunohistochemistry was performed and prognostic values of these and other clinicopathological data were evaluated. The CSF-1R mRNA level was assessed by quantitative real-time polymerase chain reaction in cohort 3 (52 patients). RESULTS: Both the CSF-1R density and its mRNA level were significantly higher in peritumoral liver tissue than in the corresponding tumor tissue. CSF-1R was distributed in a gradient in the long-distance peritumoral tissue microarray, with its density decreasing as the distance from the tumor margin increased. High peritumoral CSF-1R was significantly associated with more intrahepatic metastases and poorer survival. Peritumoral CSF-1R was an independent prognostic factor for both overall survival and time to recurrence and affected the incidence of early recurrence. However, intratumoral CSF-1R did not correlate with any clinicopathological feature. Peritumoral CSF-1R was also associated with both overall survival and time to recurrence in a subgroup with small HCCs (< or =5 cm). CONCLUSIONS: Peritumoral CSF-1R is associated with intrahepatic metastasis, tumor recurrence, and patient survival after hepatectomy, highlighting the critical role of the peritumoral liver milieu in HCC progression. CSF-1R may become a potential therapeutic target for postoperative adjuvant treatment.

The Prognostic Comparison Between Hepatocellular Carcinoma with Portal Vein Tumor Thrombus and Bile Duct Cancer Thrombus After Liver Resection.

BACKGROUND AND OBJECTIVE: Hepatocellular carcinoma (HCC) often invades the portal vein and its branches to form portal vein tumor thrombus (PVTT), and it rarely spreads into the bile ducts to cause bile duct tumor thrombus (BDTT). However, the clinical prognosis of patients with the two types of tumor thrombus is different. In this manuscript, we plan to compare the prognosis of HCC with PVTT and BDTT for further clinical treatment. PATIENTS AND METHODS: A total of 60 patients including 48 HCC cases with PVTT and 12 HCC cases with BDTT were enrolled in the study. The medical records were collected from participants. The follow-up was performed in 3 years post-hepatectomy. Statistical analysis was performed to explore the relationship between tumor thrombus with clinicopathological characteristics, to determine the significant preoperative factors influencing overall survival (OS) and time to recurrence (TTR), and to establish the survival and recurrent curves. RESULTS: HCC with BDTT or PVTT often combined with viral hepatitis B, accompanied by varying degrees of cirrhosis, and high AFP level (68.3%), complete tumor capsule (76.7%), and larger tumor size (85.0%). Furtherly, patients with HCC and BDTT tended to have higher total bilirubin (TB) and more possibility of lymph node metastases. The multivariate Cox hazard analyses also revealed that both tumor size and tumor thrombus could be taken as independent prognostic indicators of HCC patients. Survival curves showed that the 1-, 2- and 3-year OS or DFS rates of HCC patients with BDTT were significantly lower than those of HCC patients with PVTT, respectively. CONCLUSION: Tumor thrombus is an independent risk factor for poor survival and high recurrence in HCC. HCC patients with BDTT had shorter overall survival and higher tumor recurrence rate compared to HCC patients with PVTT.

Increased neutrophil extracellular traps promote metastasis potential of hepatocellular carcinoma via provoking tumorous inflammatory response.

BACKGROUND: The propensity of the activated neutrophils to form extracellular traps (NETs) is demonstrated in multiple inflammatory conditions. In this study, we investigated the roles of NETs in metastasis of hepatocellular carcinoma (HCC) and further explored the underlying mechanism of how NETs affect metastasis as well as the therapeutic value. METHODS: The neutrophils were isolated from the blood of human HCC patients and used to evaluate the formation of NETs. The expression of NET markers was detected in tumor specimens. A LPS-induced NET model was used to investigate the role of NETs on HCC metastasis. RNA-seq was performed to identify the key molecular event triggered by NETs, and their underlying mechanism and therapeutic significance were explored using both in vitro and in vivo assays. RESULTS: NET formation was enhanced in neutrophils derived from HCC patients, especially those with metastatic HCCs. NETs trapped HCC cells and subsequently induced cell-death resistance and enhanced invasiveness to trigger their metastatic potential, which was mediated by internalization of NETs into trapped HCC cells and activation of Toll-like receptors TLR4/9-COX2 signaling. Inhibition of TLR4/9-COX2 signaling abrogated the NET-aroused metastatic potential. A combination of DNase 1 directly wrecking NETs with anti-inflammation drugs aspirin/hydroxychloroquine effectively reduced HCC metastasis in mice model. CONCLUSIONS: NETs trigger tumorous inflammatory response and fuel HCC metastasis. Targeting NETs rather than neutrophils themselves can be a practice strategy against HCC metastasis.

A novel peptide that selectively binds highly metastatic hepatocellular carcinoma cell surface is related to invasion and metastasis.

Using a phage display approach, we identified AWYPLPP peptide as a specific peptide ligand that binds to the cell surface of highly metastatic human hepatocellular carcinoma (HCC). Moreover, the peptide was able to promote in vitro invasion of highly metastatic HCC cells by activating matrix metalloproteinase-9 and in vivo lung metastasis of HCC tumors. These results indicate that AWYPLPP peptide likely recognizes a novel receptor that is selectively expressed on the cell surface of highly metastatic HCC and mediates cellular activities associated with the invasive phenotype. Identification of the receptor for the AWYPLPP peptide may provide new insights into the molecular mechanism of HCC metastasis.

GOLM1 Modulates EGFR/RTK Cell-Surface Recycling to Drive Hepatocellular Carcinoma Metastasis.

The mechanism of cancer metastasis remains poorly understood. Using gene profiling of hepatocellular carcinoma (HCC) tissues, we have identified GOLM1 as a leading gene relating to HCC metastasis. GOLM1 expression is correlated with early recurrence, metastasis, and poor survival of HCC patients. Both gain- and loss-of-function studies determine that GOLM1 acts as a key oncogene by promoting HCC growth and metastasis. It selectively interacts with epidermal growth factor receptor (EGFR) and serves as a specific cargo adaptor to assist EGFR/RTK anchoring on the trans-Golgi network (TGN) and recycling back to the plasma membrane, leading to prolonged activation of the downstream kinases. These findings reveal the functional role of GOLM1, a Golgi-related protein, in EGFR/RTK recycling and metastatic progression of HCC.

Peritumoral Cbl is a strong independent prognostic marker after curative resection of hepatocellular carcinoma.

Growing evidences support the concept that peritumoral microenvironment gene expression is an important element for physicians to make an accurate prognosis. Nonetheless, the correlation between peritumoral ubiquitin ligases and the hepatocellular carcinoma (HCC) survival remains unclear till this present. The expression of intratumoral and peritumoral Casitas B-lineage Lymphoma (Cbl) and epidermal growth factor receptor (EGFR) in hepatocellular carcinomas (HCCs) followed by curative resection was assessed by tissue microarray-based immune-histochemistry in two independent cohorts (n = 352). Their respective prognostic values and other clinicopathologic factors were then evaluated. The peritumoral Cbl density, much higher than that in intratumoral tissue, was an independent prognostic factor for overall survival (P < 0.001) and time to recurrence (P < 0.001) of HCCs after curative resection. The hazard ratio were 1.587 and 1.689, respectively. However, there was no correlation between intratumoral Cbl and prognosis. The peritumoral Cbl was also associated with prognosis even in HCC subgroups with small tumor size, negative AFP, without microvascular invasion and negative HBeAg. After a thorough analysis pertaining to the key role of Cbl on ubiquitination and degradation of activated receptor tyrosine kinases, we eventually discovered the negative correlation between peritumoral Cbl and EGFR (P = 0.015). Furthermore, the combination of peritumoral Cbl and EGFR serves as a much stronger indicator to make an accurate prognosis, especially during early recurrence (P < 0.001). These findings suggest that low expression of peritumoral Cbl and EGFR were positively associated with tumor size, microvascular invasion and patients survival after hepatectomy, highlighting the key role of peritumoral liver milieu in HCC progression.