Vasculogenic Mimicry In Hepatocellular Carcinoma Research Articles

Nrf2/ASPM axis regulated vasculogenic mimicry formation in hepatocellular carcinoma under hypoxia.

Hypoxic microenvironment is a common feature of most solid tumors including hepatocellular carcinoma (HCC). Vasculogenic mimicry (VM) formation by tumor cells could provide blood supply to tumor cells under hypoxia. NFE2 like basic leucine zipper (bZIP) transcription factor 2 (Nrf2), a regulator of cellular homeostasis, may promote tumor progression in the hypoxic conditions. However, the role and regulatory mechanisms of Nrf2 in HCC are not fully elucidated. Nrf2 and assembly factor for spindle microtubules (ASPM) expression modulations were conducted by lentiviral transfections. Western blot, immunofluorescence, ChIP-qPCR, dual-luciferase reporter gene assay, flow cytometry, RNA sequencing, multiple bioinformatics databases analysis, cell function assays in vitro, mouse model in vivo and human HCC tissues were employed to assess the effect of Nrf2/ASPM axis on HCC progression under hypoxia. Nrf2 and ASPM expression facilitated epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) feature, and VM formation of HCC cells under hypoxia. Furthermore, Nrf2-regulated ASPM expression, via binding directly to the promoter region of ASPM and transcriptionally promoting ASPM expression. ASPM re-expression in Nrf2 knockdown cells or ASPM knockdown in Nrf2 overexpression cells reversed the cellular function caused by Nrf2. Meantime, retinol metabolism pathway was disrupted following abnormal ASPM expression. Nrf2/ASPM axis in murine models accelerated tumor growth and VM, corroborating in vitro findings. All-trans retinoic acid treatment reversed stemness and VM of HCC cells in vitro and in vivo. Clinically, Nrf2 and ASPM expressions were related to poor prognosis of HCC patients. Nrf2 drives EMT, CSCs characteristics and VM in HCC under hypoxia through the modulation of ASPM. Retinol metabolism pathway was dysregulated in HCC cells with ASPM overexpression. Nrf2/ASPM axis and related pathway provided potential therapeutic target for HCC.

COE targets EphA2 to inhibit vasculogenic mimicry formation induced by hypoxia in hepatocellular carcinoma.

Vasculogenic Mimicry (VM) can reduce the efficacy of anti-angiogenesis and promote distant metastasis in hepatocellular carcinoma (HCC). Our previous studies have found that Celastrus orbiculatus extract (COE) can inhibit the VM formation in HCC by reducing EphA2 expression. However the underlying mechanism related to EphA2 in VM formation is unclear. This study aimed to confirm that EphA2 is one of the potential targets of COE, and to explore the effect of EphA2 in VM formation in hypoxia context in HCC. TCM Systems Pharmacology database and proteomics analysis were used to explore the key targets of COE in HCC treatment. CD31-PAS double staining and VE-CAD staining were used to indicate vasculogenic mimicry. The localization of EphA2 and VE-CAD was examined through fluorescent microscopy. CCK8 assay, cell invasion assay, and tube formation assay were used to indicate the formation of VM under hypoxic conditions. The regulatory relationship of EphA2 upstream and downstream molecules were evaluated through COIP and Western Blot. The nude mouse xenograft tumor models were used to observe the VM formation after knocking down or overexpressing EphA2. EphA2 is identified to the target of COE, and the driving gene of HCC. In HCC surgical specimens, EphA2 expression is closely associated with the VM formation of HCC. COE-regulated EphA2 is involved in hypoxia-induced VM formation in HCC cells in vitro. EphA2 is regulated by HIF directly or indirectly by C-MYC. Overexpression of EphA2 can promote the VM formation of HCC in nude mice, while knocking down EphA2 can inhibit the VM formation. EphA2, as a target of COE, plays a crucial regulatory role in the formation of vasculogenic mimicry in HCC, involving upstream HIF/MYC transcriptional promotion and downstream PI3K/FAK/VE-CAD expression regulation.

Vitamin D binding protein (VDBP) hijacks twist1 to inhibit vasculogenic mimicry in hepatocellular carcinoma.

Rationale: Vitamin D (VD) has been suggested to have antitumor effects, however, research on the role of its transporter vitamin D-binding protein (VDBP, gene name as GC) in tumors is limited. In this study, we demonstrated the mechanism underlying the inhibition of vasculogenic mimicry (VM) by VDBP in hepatocellular carcinoma (HCC) and proposed an anti-tumor strategy of combining anti-PD-1 therapy with VD. Methods: Three-dimensional cell culture models and mice with hepatocyte-specific GC deletion were utilized to study the correlation between VDBP expression and VM. A patient-derived tumor xenograft (PDX) model was further applied to validate the therapeutic efficacy of VD in combination with an anti-PD-1 drug. Results: The study revealed that VDBP expression is negatively correlated with VM in HCC patients and elevated VDBP expression is associated with a favorable prognosis. The mechanism studies suggested VDBP hindered the binding of Twist1 on the promoter of VE-cadherin by interacting with its helix-loop-helix DNA binding domain, ultimately leading to the inhibition of VM. Furthermore, VD facilitated the translocation of the vitamin D receptor (VDR) into the nucleus where VDR interacts with Yin Yang 1 (YY1), leading to the transcriptional activation of VDBP. We further demonstrated that the combination of VD and anti-PD-1 led to an improvement in the anti-tumor efficacy of an anti-PD-1 drug. Conclusion: Collectively, we identified VDBP as an important prognostic biomarker in HCC patients and uncovered it as a therapeutic target for enhancing the efficacy of immune therapy.

Multiple Roles of LOXL2 in the Progression of Hepatocellular Carcinoma and Its Potential for Therapeutic Targeting.

LOXL2, a copper-dependent amine oxidase, has emerged as a promising therapeutic target in hepatocellular carcinoma (HCC). Increased LOXL2 expression in HCC has been linked with an aggressive phenotype and represents a poor prognostic factor. Here, we focus on the mechanisms through which LOXL2 orchestrates multiple oncogenic functions in HCC development. We performed a review of the current knowledge on the roles LOXL2 performs in the modulation of the HCC tumor microenvironment, formation of premetastatic niches, and epithelial-mesenchymal transition. We also highlighted the complex interplay between LOXL2 and hypoxia, angiogenesis, and vasculogenic mimicry in HCC. At the end of the review, we summarize the current LOXL2 inhibitors and discuss their potential in HCC precision treatment.

Androgen receptor suppresses vasculogenic mimicry in hepatocellular carcinoma via circRNA7/miRNA7-5p/VE-cadherin/Notch4 signalling.

Androgen receptor (AR) can suppress hepatocellular carcinoma (HCC) invasion and metastasis at an advanced stage. Vasculogenic mimicry (VM), a new vascularization pattern by which tumour tissues nourish themselves, is correlated with tumour progression and metastasis. Here, we investigated the effect of AR on the formation of VM and its mechanism in HCC. The results suggested that AR could down‐regulate circular RNA (circRNA) 7, up‐regulate micro RNA (miRNA) 7‐5p, and suppress the formation of VM in HCC Small hairpin circR7 (ShcircR7) could reverse the impact on VM and expression of VE‐cadherin and Notch4 increased by small interfering AR (shAR) in HCC, while inhibition of miR‐7‐5p blocked the formation of VM and expression of VE‐cadherin and Notch4 decreased by AR overexpression (oeAR) in HCC. Mechanism dissection demonstrated that AR could directly target the circR7 host gene promoter to suppress circR7, and miR‐7‐5p might directly target the VE‐cadherin and Notch4 3′UTR to suppress their expression in HCC. In addition, knockdown of Notch4 and/or VE‐cadherin revealed that shVE‐cadherin or shNotch4 alone could partially reverse the formation of HCC VM, while shVE‐cadherin and shNotch4 together could completely suppress the formation of HCC VM. Those results indicate that AR could suppress the formation of HCC VM by down‐regulating circRNA7/miRNA7‐5p/VE‐Cadherin/Notch4 signals in HCC, which will help in the design of novel therapies against HCC.

RNA m6A methylation promotes the formation of vasculogenic mimicry in hepatocellular carcinoma via Hippo pathway.

Vasculogenic mimicry (VM) formed by aggressive tumor cells to mimic vasculogenic networks plays an important role in the tumor malignancy of HCC. However, the pathogenesis underlying VM is complex and has not been fully defined. m6A is a common mRNA modification and has many biological effects. However, the relationship between m6A and VM remains unclear. In this research, we found that m6A methyltransferase METTL3 in HCC tissues was positively correlated with VM. The m6A level of mRNA significantly increased in 3D cultured cells treated with VEGFa and was related to VM formation. Transcriptome sequencing analysis of 3D cultured cells with knockdown Mettl3 showed that the Hippo pathway was involved in m6A-mediated VM formation. Further mechanism research indicated that the m6A modification of YAP1 mRNA affected the translation of YAP1 mRNA. In conclusion, m6A methylation plays a key role in VM formation in HCC. METTL3 and YAP1 could be potential therapeutic targets via impairing VM formation in anti-metastatic strategies.

Hypoxic induction of vasculogenic mimicry in hepatocellular carcinoma: role of HIF-1 \u03b1, RhoA/ROCK and Rac1/PAK signaling

BackgroundVasculogenic mimicry (VM), defined as a capability of aggressive tumor Cells to mimic embryonic vasculogenic networks, caused poor prognosis in hepatocellular carcinoma (HCC). Rho kinases (ROCK), p21-activated kinase (PAK), hypoxia or epithelial-mesenchymal transition (EMT) contributed to the VM potential. However, the details underlying these biological behaviors have not been completely elucidated.MethodsKaplan-Meier analysis was conducted to predict relationship with hypoxia Inducible factor (HIF-1α), EMT related markers: Vimentin and patient prognosis. CD34/periodic acid-Schiff (PAS) double staining was examined to differentiate VM-positive (VM+) and VM-negative (VM-) samples. Cells were cultured under controlled hypoxic environments (1% O2) or normoxic conditions. The effect of hypoxia on RhoA/ROCK, Rac1/PAK and EMT were evaluated by real time-qPCR and western blot. HIF-1α small interfering RNA (siRNA), overexpressed or short hairpin RNA (shRNA) of ROCK and kinase inhibitors were used to explore the effect of HIF-1α, RhoA/ROCK, Rac1/PAK and Vimentin on VM.ResultsHIF-1α or Vimentin was upregulated in VM+ HCC tissues, compared to non-cancerous tissues (P < 0.01), and patients with high expression of HIF-1α or Vimentin had worse prognosis (P < 0.001). We showed hypoxia induced RhoA/ROCK and Rac1/PAK signaling transduction, and EMT could be repressed by HIF-1α siRNA. Notably, RhoA/ROCK or Rac1/PAK stabilized HIF-1α in hypoxia, whereas HIF-1α did not significantly altered RhoA/ROCK or Rac1/PAK signaling in hypoxia. Moreover, we found distinct roles of ROCK1, ROCK2 and PAK in regulating Vimentin phosphorylation.ConclusionsRhoA/ROCK and Rac/PAK signaling played crucial roles in hypoxia-induced VM via Ser72 and Ser56 Vimentin phosphorylation in HCC.

Function of BMP4 in the Formation of Vasculogenic Mimicry in Hepatocellular Carcinoma.

Vasculogenic mimicry (VM) is linked to vascular invasion of human hepatocellular carcinoma (HCC). BMP4, one BMP family member, is upregulated in several cancers. The purpose of this report is to identify the function of BMP4 in the formation of VM in HCC and the mechanism underling this regulation. In our report, BMP4 up-regulation resulted in an increase in migration, invasion and channel-like structure formation as well as induced epithelial-mesenchymal transition (EMT) process and stem cell-associated proteins OCT4 and SOX2 expression in HCC cells. In addition, The VM-associated proteins, including EphA2, VE-cadherin and MMP2, also could be effectively enhanced by the overexpression of BMP4. Furthermore, according to the TCGA database, higher expression of BMP4 is seen in HCC in contrast to normal liver samples. Immunohistochemistry revealed that BMP4 was positively associated with VM formation, age, histological differentiation, HCC stage, and shorter survival duration. These data demonstrated that BMP4 could promote VM network formation in HCC through induction of stemness in EMT and modulating the EphA2/VE-cadherin/MMP2 signaling pathway.

Anti-angiogenesis triggers exosomes release from endothelial cells to promote tumor vasculogenesis

ABSTRACTAlthough anti-angiogenic therapies (AATs) have some effects against multiple malignancies, they are limited by subsequent tumor vasculogenesis and progression. To investigate the mechanisms by which tumor vasculogenesis and progression following AATs, we transfected microRNA (miR)-9 into human umbilical vein endothelial cells (HUVECs) to mimic the tumor-associated endothelial cells in hepatocellular carcinoma and simulated the AATs in vitro and in vivo. We found that administration of the angiogenesis inhibitor vandetanib completely abolished miR-9-induced angiogenesis and promoted autophagy in HUVECs, but induced the release of vascular endothelial growth factor (VEGF)-enriched exosomes. These VEGF-enriched exosomes significantly promoted the formation of endothelial vessels and vasculogenic mimicry in hepatocellular carcinoma and its progression in mice. Anti-autophagic therapy is proposed to improve the efficacy of AATs. However, similar effects by AATs were observed with the application of anti-autophagy by 3-methyladenine. Our results revealed that tumor vasculogenesis and progression after AATs and anti-autophagic therapies were due to the cross-talk between endothelial and tumor cells via VEGF-enriched exosomes.

Anti‐angiogenesis Triggers VEGF‐enriched Exosomes Release from Endothelial Cells

Although anti‐angiogenic therapies (AATs) have some effects against multiple malignancies, they are limited by subsequent tumor vasculogenesis and progression. To investigate the mechanisms by which tumor vasculogenesis and progression following AATs, we transfected microRNA (miR)‐9 into human umbilical vein endothelial cells (HUVECs) to mimic the tumor‐associated endothelial cells in hepatocellular carcinoma and simulated the AATs in vitro and in vivo. We found that administration of the angiogenesis inhibitor vandetanib completely abolished miR‐9‐induced angiogenesis and promoted autophagy in HUVECs, but induced the release of vascular endothelial growth factor (VEGF)‐enriched exosomes. These VEGF‐enriched exosomes significantly promoted the formation of endothelial vessels and vasculogenic mimicry in hepatocellular carcinoma and its progression in mice. Anti‐autophagic therapy is proposed to improve the efficacy of AATs. However, similar effects by AATs were observed with the application of anti‐autophagy by 3‐methyladenine. Our results revealed that tumor vasculogenesis and progression after AATs and anti‐autophagic therapies were due to the cross‐talk between endothelial cells and tumor cells via VEGF‐enriched exosomes. These findings provide a critical insight into a new interpretation of why AATs have not achieved expected outcomes. They also suggest that control of exosome release or alteration of exosome cargo composition to inhibit tumor vasculogenesis may augment the anti‐angiogenic and anti‐autophagic therapies for tumors.Support or Funding InformationSupported by National Natural Science Foundation of China (Grant no.11402153).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RhoC/ROCK2 promotes vasculogenic mimicry formation primarily through ERK/MMPs in hepatocellular carcinoma

Vasculogenic mimicry (VM) results in the formation of an alternative circulatory system that can improve the blood supply to multiple malignant tumors, including hepatocellular carcinoma (HCC). However, the potential mechanisms of RhoC/ROCK in VM have not yet been investigated in HCC. Here, RhoC expression was upregulated in HCC tissues, especially the VM-positive (VM+) group, compared to noncancerous tissues (P < 0.01), and patients with high expression of RhoC had shorter survival times (P < 0.001). The knockdown of RhoC via short hairpin RNA (shRNA) in SK-Hep-1 cells significantly decreased VM formation and cell motility. In contrast, cell motility and VM formation were remarkably enhanced when RhoC was overexpressed in HepG2 cells. To further assess the potential role of ROCK1 and ROCK2 on VM, we stably knocked down ROCK1 or ROCK2 in MHCC97H cells. Compared to ROCK1 shRNA, ROCK2 shRNA could largely affect VM formation, cell motility and the key VM factors, as well as the epithelial-mesenchymal transition (EMT) markers in vitro and in vivo. Moreover, p-ERK, p-MEK, p-FAK, p-paxillin, MT1-MMP and MMP2 levels were clearly altered following the overexpression of RhoC, but ROCK2 shRNA had little effect on the expression of p-FAK, which indicated that RhoC regulates FAK/paxillin signaling, but not through ROCK2. In conclusion, our results show that RhoC/ROCK2 may have a major effect on VM in HCC via ERK/MMPs signaling and might be a potential therapeutic target for the treatment of HCC.

Thymidine phosphorylase promotes malignant progression in hepatocellular carcinoma through pentose Warburg effect

Tumor progression is dependent on metabolic reprogramming. Metastasis and vasculogenic mimicry (VM) are typical characteristics of tumor progression. The relationship among metastasis, VM, and metabolic reprogramming remains unclear. In this study, we identified the novel role of Twist1, a VM regulator, in the transcriptional regulation of thymidine phosphorylase (TP) expression. TP promoted the extracellular metabolism of thymidine into ATP and amino acids through the pentose Warburg effect by coupling the pentose phosphate pathway and glycolysis. Moreover, Twist1 relied on TP-induced metabolic reprogramming to promote hepatocellular carcinoma (HCC) metastasis and VM formation mediated by VE–Cad, VEGFR1, and VEGFR2 in vitro and in vivo. The TP inhibitor tipiracil reduced the effect of TP on promoting HCC VM formation and metastasis. Hence, TP, when transcriptionally activated by Twist1, promotes HCC VM formation and metastasis through the pentose Warburg effect and contributes to tumor progression.

RhoC/ ROCK2 promotes vasculogenic mimicry formation primarily through ERK/MMPs in hepatocellular carcinoma

Introduction: Our previous study reported that rho-associated coiled-coil containing kinases (ROCKs) were involved in vasculogenic mimicry (VM) formation in hepatocellular carcinoma (HCC). This study focused on clarifying potential mechanisms of RhoC/ROCK on VM formation in hepatocellular carcinoma (HCC). Method: In total, 80 cases of HCC tissues and adjacent non-tumorous liver tissues were used to identify VM in HCC tissues by PAS/CD34 double staining. Kaplan-Meier analysis was conducted to test the predictive relationship between RhoC/ROCK expression and patient prognosis. Furthermore, we stably overexpressed RhoC and inhibited RhoC, ROCK1 or ROCK2 expression to clarify the effect of RhoC/ROCK on VM formation in vitro and in vivo. Result: RhoC expression was up regulated in HCC tissues, especially the VM-positive (VM+) group, compared to non-cancerous tissues (P < 0.01), and patients with high expression of RhoC had shorter survival times (P < 0.001). Cell motility and VM formation significantly decreased after knockdown of RhoC, while overexpressing RhoC led to the opposite result. Compared to ROCK1 shRNA, ROCK2 shRNA could largely affects VM formation, cell motility and the key VM factors and the epithelial-mesenchymal transition (EMT) markers in vitro and in vivo. Moreover, p-ERK, p-MEK, p-FAK and p-paxillin levels clearly altered following the change of RhoC, but ROCK2 little affected expression of p-FAK. Conclusion: RhoC/ROCK2 may have a major effect on VM in HCC via ERK/MMPs signaling and might be a potential therapeutic target for the treatment of HCC.

RhoC/ROCK2 promotes vasculogenic mimicry formation through ERK/MMP and FAK/paxillin signaling pathways in hepatocellular carcinoma

Our previous study reported that rho-associated coiled-coil containing kinases (ROCKs) were involved in Vasculogenic mimicry (VM) formation in hepatocellular carcinoma (HCC). This study focused on whether the potential activator ras homolog family member C (RhoC) plays an essential role in VM via two isoforms of ROCK (ROCK1 and ROCK2). VM was presented in tumor tissues rather than non-cancerous tissues, which was positively correlated with clinical stage (P = 0.006). RhoC expression was up regulated in HCC tissues, especially the VM-positive (VM+) group, compared to non-cancerous tissues (P < 0.01), and patients with high expression of RhoC had shorter survival times (P < 0.001). The knockdown of RhoC via the short hairpin RNA (shRNA) in SK-Hep-1 significantly decreased VM formation and cell motility. On contrary, cell motility and VM formation were remarkably enhanced when overexpressed RhoC in HepG2. Compared to ROCK1 shRNA, ROCK2 shRNA could largely affects VM formation, cell motility and the key VM factors and the epithelial-mesenchymal transition (EMT) markers in vitro and in vivo. Additionally, RhoC/ROCK2 promoted VM via ERK/MMP and FAK/paxillin signaling pathways. Our results show that RhoC/ROCK2 may have a major effect on VM in HCC and might be a potential therapeutic target for the treatment of HCC.

COE inhibits vasculogenic mimicry in hepatocellular carcinoma via suppressing Notch1 signaling

Ethnopharmacological relevanceVasculogenic mimicry (VM) has been suggested to be present in various malignant tumors and associated with tumor nutrition supply and metastasis, leading to poor prognosis of patients. Notch1 has been demonstrated to contribute to VM formation in hepathocellular carcinoma (HCC). Celastrus orbiculatus extract (COE), a mixture of 11 terpenoids isolated from the Chinese Herb Celastrus orbiculatus Vine, has been suggested to be effective in cancer treatment. Aim of the studyIn the current study, experiments were carried out to examine the effect of COE on VM formation and HCC tumor growth both in vitro and in vivo. Materials and methodsCCK-8 assay and Nikon live-work station were used to observe the viability of malignant cells treated with COE. Cell invasion was examined using Transwell. Matrigel was used to establish a 3-D culture condition for VM formation. Changes of mRNA and protein expression were examined by RT-PCR and Western Blot respectively. Tumor growth in vivo was monitored using in vivo fluorescence imaging device. PAS-CD34 dual staining and electron microscopy were used to observe VM formation. Immunohistochemical staining (IHC) was used to examine Notch1 and Hes1 expression in tumor tissues. ResultsResults showed that COE can inhibit HCC cells proliferation and invasion in a concentration-dependent manner. VM formation induced by TGF-β1 was blocked by COE. In mouse xenograft model, COE inhibited tumor growth and VM formation. Both in vitro and in vivo studies showed that COE can downregulate expression of Notch1 and Hes1. ConclusionThe current results indicate that COE can inhibit VM formation and HCC tumor growth by downregulating Notch1 signaling. This study demonstrates that COE is superior to other anti-angiogenesis agents and can be considered as a promising candidate in HCC treatment.

Vasculogenic mimicry in hepatocellular carcinoma contributes to portal vein invasion.

Portal vein invasion (PVI) is common in hepatocellular carcinoma (HCC) and largely contributes to tumor recurrence after radical tumor resection or liver transplantation. Vasculogenic mimicry (VM) was an independent vascular system lined with tumor cells and associated with poor prognosis of HCC. The present study was conducted to evaluate the relationship between VM and portal vein invasion. A total of 44 HCC cases receiving anatomic liver resection were included in the study and were divided into groups with and without PVI. The prevalence of VM in each group was examined by CD34-PAS dual staining. The regulatory molecules of VM formation such as Notch1, Vimentin and matrix metalloproteinases (MMPs) were investigated by immunohistochemical staining. Analysis was performed to explore the association of PVI, VM and the VM regulatory molecules. PVI was found in 40.91% (18/44) cases and VM was found in 38.64% (17/44) cases in total samples. The incidence of VM was 72.22% (13/18) in PVI group while it was 15.38% (4/26) in non-PVI group (P<0.001), VM formation was positively correlated with PVI (r=0.574, P<0.001). The VM forming regulatory molecules such as Notch1, Vimentin, MMP-2 and MMP-9 were found to be correlated with PVI in HCC patients. Taken together, our results suggested that VM formation, alone with its regulatory molecules, is the promoting factor of PVI in hepatocellular carcinoma.

Notch1 promotes vasculogenic mimicry in hepatocellular carcinoma by inducing EMT signaling.

Hypervascularity is one of the main characteristics of hepatocellular carcinoma (HCC). However, the mechanisms of angiogenesis in HCC remain controversial. In this study, we investigate the role of Notch1 in angiogenesis of HCC. We found that Notch1 expression was correlated with formation of vasculogenic mimicry (VM) and expression of biomarkers of epithelial-to-mesenchymal transition (EMT) in the tumor specimens. Two HCC cell lines, HepG2 and MHCC97-H, with low and high Notch1 expression, respectively, were used to study the mechanism of VM formation both in vitro and in vivo. It was found that MHCC97-H cells, but not HepG2 cells form VM when they grow on matrigel in vitro. HepG2 cells gained the power of forming VM when they were overexpressed with Notch1, while knockdown Notch1 expression in MHCC97-H cells led to the loss of VM forming ability of the cells. Similar results were found in in vivo study. High expression of Notch1 in HepG2 promoted xenograft growth in nude mice, with abundant VM formation in the tumor samples. Moreover, we observed Notch1 was associated with the EMT and malignant behavior of hepatocellular carcinoma by analyzing clinical specimens, models for in vitro and in vivo experiments. HepG2 presented EMT phenomenon when induced by TGF-β1, accompanied by Notch1 activation while MHCC97-H with knockdown of Notch1 lost the responsiveness to TGF-β1 induction. Our results suggest that Notch1 promotes HCC progression through activating EMT pathway and forming VM. Our results will guide targeting Notch1 in new drug development.

Doxycycline as an inhibitor of the epithelial-to-mesenchymal transition and vasculogenic mimicry in hepatocellular carcinoma.

This study was conducted to examine the effects of doxycycline on the survival time and proliferation of hepatocellular carcinoma (HCC) in vivo and on the biologic functions of HCC in vitro. This study was also designed to evaluate the effects of doxycycline on epithelial-to-mesenchymal transition (EMT)- and vasculogenic mimicry (VM)-related protein expression and on matrix metalloproteinase (MMP) and DNA methyltransferase (DNMT) activity in vitro. Human MHCC97H cells were injected into BALB/c mice, which were divided into treatment and control groups. Doxycycline treatment prolonged the mouse survival time and partly suppressed the growth of engrafted HCC tumor cells, with an inhibition rate of 43.39%. Higher amounts of VM and endothelium-dependent vessels were found in the control group than the treatment group. IHC indicated that epithelial (E)-cadherin expression was increased in the doxycycline-treated mice compared with the control group. In in vitro experiments, doxycycline promoted HCC cell adhesion but inhibited HCC cell viability, proliferation, migration, and invasion. Western blot analysis, semiquantitative RT-PCR, qRT-PCR, and immunofluorescence demonstrated that doxycycline inhibited the degradation of the epithelial marker E-cadherin and downregulated the expression levels of EMT promoters, the mesenchymal marker vimentin, and the VM-associated marker vascular endothelial (VE)-cadherin. Furthermore, the activities of MMPs and DNMTs were examined in different groups via gelatin zymography and a DNMT activity assay kit. A methylation-specific PCR was performed to assess the promoter methylation of CDH1 (the gene encoding E-cadherin). Doxycycline prolonged the mouse survival time by inhibiting EMT progression and VM formation.

ROCK is involved in vasculogenic mimicry formation in hepatocellular carcinoma cell line.

Ras homolog family member A (RhoA) and Rho-associated coiled coil-containing protein kinases 1 and 2 (ROCK1 and 2) are key regulators of focal adhesion, actomyosin contraction and cell motility. RhoA/ROCK signaling has emerged as an attractive target for the development of new cancer therapeutics. Whether RhoA/ROCK is involved in regulating the formation of tumor cell vasculogenic mimicry (VM) is largely unknown. To confirm this hypothesis, we performed in vitro experiments using hepatocellular carcinoma (HCC) cell lines. Firstly, we demonstrated that HCC cells with higher active RhoA/ROCK expression were prone to form VM channels, as compared with RhoA/ROCK low-expressing cells. Furthermore, Y27632 (a specific inhibitor of ROCK) rather than exoenzyme C3 (a specific inhibitor of RhoA) effectively inhibited the formation of tubular network structures in a dose-dependent manner. To elucidate the possible mechanism of ROCK on VM formation, real-time qPCR, western blot and immunofluorescence were used to detect changes of the key VM-related factors, including VE-cadherin, erythropoietin-producing hepatocellular carcinoma-A2 (EphA2), phosphoinositide 3-kinase (PI3K), matrix metalloproteinase (MMP)14, MMP2, MMP9 and laminin 5γ2-chain (LAMC2), and epithelial-mesenchymal-transition (EMT) markers: E-cadherin and Vimentin. The results showed that all the expression profiles were attenuated by blockage of ROCK. In addition, in vitro cell migration and invasion assays showed that Y27632 inhibited the migration and invasion capacity of HCC cell lines in a dose-dependent manner markedly. These data indicate that ROCK is an important mediator in the formation of tumor cell VM, and suggest that ROCK inhibition may prove useful in the treatment of VM in HCC.

Slug promoted vasculogenic mimicry in hepatocellular carcinoma

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumour cells to mimic the pattern of embryonic vasculogenic networks. Epithelial–mesenchymal transition (EMT) regulator slug have been implicated in the tumour invasion and metastasis of human hepatocellular carcinoma (HCC). However, the relationship between slug and VM formation is not clear. In the study, we demonstrated that slug expression was associated with EMT and cancer stem cell (CSCs) phenotype in HCC patients. Importantly, slug showed statistically correlation with VM formation. We consistently demonstrated that an overexpression of slug in HCC cells significantly increased CSCs subpopulation that was obvious by the increased clone forming efficiency in soft agar and by flowcytometry analysis. Meantime, the VM formation and VM mediator overexpression were also induced by slug induction. Finally, slug overexpression lead to the maintenance of CSCs phenotype and VM formation was demonstrated in vivo. Therefore, the results of this study indicate that slug induced the increase and maintenance of CSCs subpopulation and contributed to VM formation eventually. The related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis.