Desmoglein 2 and desmocollin 2 depletions promote malignancy through distinct mechanisms in triple-negative and luminal breast cancer.

BACKGROUND: Aberrant expressions of desmoglein 2 (Dsg2) and desmocollin 2(Dsc2), the two most widely distributed desmosomal cadherins, have been found to play various roles in cancer in a context-dependent manner. Their specific roles on breast cancer (BC) and the potential mechanisms remain unclear. METHODS: The expressions of Dsg2 and Dsc2 in human BC tissues and cell lines were assessed by using bioinformatics analysis, immunohistochemistry and western blotting assays. Wound-healing and Transwell assays were performed to evaluate the cells' migration and invasion abilities. Plate colony-forming and MTT assays were used to examine the cells' capacity of proliferation. Mechanically, Dsg2 and Dsc2 knockdown-induced malignant behaviors were elucidated using western blotting assay as well as three inhibitors including MK2206 for AKT, PD98059 for ERK, and XAV-939 for ß-catenin. RESULTS: We found reduced expressions of Dsg2 and Dsc2 in human BC tissues and cell lines compared to normal counterparts. Furthermore, shRNA-mediated downregulation of Dsg2 and Dsc2 could significantly enhance cell proliferation, migration and invasion in triple-negative MDA-MB-231 and luminal MCF-7 BC cells. Mechanistically, EGFR activity was decreased but downstream AKT and ERK pathways were both activated maybe through other activated protein tyrosine kinases in shDsg2 and shDsc2 MDA-MB-231 cells since protein tyrosine kinases are key drivers of triple-negative BC survival. Additionally, AKT inhibitor treatment displayed much stronger capacity to abolish shDsg2 and shDsc2 induced progression compared to ERK inhibition, which was due to feedback activation of AKT pathway induced by ERK inhibition. In contrast, all of EGFR, AKT and ERK activities were attenuated, whereas ß-catenin was accumulated in shDsg2 and shDsc2 MCF-7 cells. These results indicate that EGFR-targeted therapy is not a good choice for BC patients with low Dsg2 or Dsc2 expression. Comparatively, AKT inhibitors may be more helpful to triple-negative BC patients with low Dsg2 or Dsc2 expression, while therapies targeting ß-catenin can be considered for luminal BC patients with low Dsg2 or Dsc2 expression. CONCLUSION: Our finding demonstrate that single knockdown of Dsg2 or Dsc2 could promote proliferation, motility and invasion in triple-negative MDA-MB-231 and luminal MCF-7 cells. Nevertheless, the underlying mechanisms were cellular context-specific and distinct.

Exploring the molecular characteristics of the malignant potential of gastric adenocarcinoma with enteroblastic differentiation.

AIMS: Gastric adenocarcinoma with enteroblastic differentiation (GAED) is a rare subset of alpha-fetoprotein (AFP)-producing carcinomas with poor prognosis. However, the molecular features associated with the malignant potential of GEAD remain partially elucidated. METHODS AND RESULTS: In this study, the relationship between clinicopathological parameters and aggressive biological behaviour was analysed in 37 patients with GAED. The results showed that GAED tended to infiltrate the deep layer of the gastric wall and possessed more frequent vascular invasion than conventional gastric adenocarcinoma (CGA) (P < 0.001). All distant metastases were observed in the GAED group, not the CGA group (P < 0.001). High HER2 expression was found in nearly 24.32% of the informative cases, and none showed EBV-encoded RNA positivity or deficient mismatch repair. The most frequently mutated gene in GAED was p53. Programmed cell death-ligand 1 (PD-L1) immunostaining revealed 13 patients with a combined positive score (CPS) ≥ 5 (65%, 13 of 20). Thus, based on these molecular markers (immunostaining, in situ hybridisation and mutation analysis), GAED may be classified as a unique subgroup of the chromosomal instability subtype with HER2+ /EBV- /MSS/TP53+ /PD-L1+ . Next-generation sequencing analyses showed that mutations in the TOPI, ELOA and NOTCH3 genes were found only in GAED, and abnormally expressed genes in GAED were significantly enriched in hepatocellular carcinoma-, gland development-, and gastric cancer-related pathways. CONCLUSION: The HER2+ /EBV- /MSS/TP53+ /PD-L1+ profile and hepatocellular carcinoma-related pathways may be significant in the malignant potential of GAED. In addition to anti-HER2 therapy, immune check-point inhibitors may be an effective treatment option for patients with GAED.

Spatial maps of hepatocellular carcinoma transcriptomes highlight an unexplored landscape of heterogeneity and a novel gene signature for survival.

BACKGROUND: Hepatocellular carcinoma (HCC) often presents with satellite nodules, rendering current curative treatments ineffective in many patients. The heterogeneity of HCC is a major challenge in personalized medicine. The emergence of spatial transcriptomics (ST) provides a powerful strategy for delineating the complex molecular landscapes of tumours. METHODS: In this study, the heterogeneity of tissue-wide gene expression in tumour and adjacent nonneoplastic tissues using ST technology were investigated. The transcriptomes of nearly 10,820 tissue regions and identified the main gene expression clusters and their specific marker genes (differentially expressed genes, DEGs) in patients were analysed. The DEGs were analysed from two perspectives. First, two distinct gene profiles were identified to be associated with satellite nodules and conducted a more comprehensive analysis of both gene profiles. Their clinical relevance in human HCC was validated with Kaplan-Meier (KM) Plotter. Second, DEGs were screened with The Cancer Genome Atlas (TCGA) database to divide the HCC cohort into high- and low-risk groups according to Cox analysis. HCC patients from the International Cancer Genome Consortium (ICGC) cohort were used for validation. KM analysis was used to compare the overall survival (OS) between the high- and low-risk groups. Univariate and multivariate Cox analyses were applied to determine the independent predictors for OS. RESULTS: Novel markers for the prediction of satellite nodules were identified and a tumour clusters-specific marker gene signature model (6 genes) for HCC prognosis was constructed. CONCLUSION: The establishment of marker gene profiles may be an important step towards an unbiased view of HCC, and the 6-gene signature can be used for prognostic prediction in HCC. This analysis will help us to clarify one of the possible sources of HCC heterogeneity and uncover pathogenic mechanisms and novel antitumour drug targets.

TAZ promotes vasculogenic mimicry in gastric cancer through the upregulation of TEAD4.

BACKGROUND AND AIM: Vasculogenic mimicry (VM) is a unique blood supply pattern in malignant tumors that is closely associated with metastasis and poor prognosis. The Hippo signaling effector TAZ is upregulated in several cancers, promoting cancer proliferation and metastasis. This study aimed to identify the function of TAZ and its regulatory mechanism in promoting VM in gastric cancer (GC). METHODS: The expression of TAZ and TEAD4 and their correlations with overall survival and VM-related markers were analyzed in 228 cases of GC. The regulatory mechanism of TAZ and its interaction with TEAD4 in epithelial-mesenchymal transition (EMT) and VM were investigated in vitro and in vivo. RESULTS: TAZ was highly expressed in GC samples and was associated with shorter patient survival time. TAZ expression was positively correlated with TEAD4 and VM in patients with GC. TAZ enhanced the migration and invasion capacity of GC cells through EMT in vitro and upregulated the expression of VM-associated proteins, including VE-cadherin, MMP2, and MMP9, thus promoting VM formation. Overexpression of TAZ accelerated the growth of subcutaneous xenograft and promoted VM formation in vivo. Co-immunoprecipitation assays showed that TAZ can directly bind to TEAD4, and in vitro experiments showed that this binding mediates the function of TAZ in regulating EMT and VM formation in GC. CONCLUSIONS: TAZ promotes GC metastasis and VM by upregulating TEAD4 expression. Our findings expand the role of TAZ in VM and provide new theoretical support for the use of antiangiogenic therapy in the treatment of advanced GC.

High expression of eIF4E is associated with tumor macrophage infiltration and leads to poor prognosis in breast cancer.

BACKGROUND: The expression and activation of eukaryotic translation initiation factor 4E (eIF4E) is associated with cell transformation and tumor initiation, but the functional role and the mechanism whereby it drives immune cell infiltration in breast cancer (BRCA) remain uncertain. METHODS: Oncomine, Timer and UALCAN were used to analyze the expression of eIF4E in various cancers. PrognoScan, Kaplan-Meier plotter, and GEPIA were utilized to analyze the prognostic value of eIF4E in select cancers. In vitro cell experiments were used to verify the role of eIF4E in promoting the progression of BRCA. ImmuCellAI and TIMER database were used to explore the relationship between eIF4E and tumor infiltrating immune cells. The expression of a macrophage marker (CD68+) and an M2-type marker (CD163+) was evaluated using immunohistochemistry in 50 invasive BRCA samples on tissue microarrays. The Human Protein Atlas (HPA) database was used to show the expression of eIF4E and related immune markers. LinkedOmics and NetworkAnalyst were used to build the signaling network. RESULTS: Through multiple dataset mining, we found that the expression of eIF4E in BRCA was higher than that in normal tissues, and patients with increased eIF4E expression had poorer survival and a higher cumulative recurrence rate in BRCA. At the cellular level, BRCA cell migration and invasion were significantly inhibited after eIF4E expression was inhibited by siRNA. Immune infiltration analysis showed that the eIF4E expression level was significantly associated with the tumor purity and immune infiltration levels of different immune cells in BRCA. The results from immunohistochemical (IHC) staining further proved that the expression of CD68+ and CD163+ were significantly increased and correlated with poor prognosis in BRCA patients (P < 0.05). Finally, interaction network and functional enrichment analysis revealed that eIF4E was mainly involved in tumor-related pathways, including the cell adhesion molecule pathway and the JAK-STAT signaling pathway. CONCLUSIONS: Our study has demonstrated that eIF4E expression has prognostic value for BRCA patients. eIF4E may act as an essential regulator of tumor macrophage infiltration and may participate in macrophage M2 polarization.

Twist1 accelerates tumour vasculogenic mimicry by inhibiting Claudin15 expression in triple-negative breast cancer.

The up-regulation of EMT regulator Twist1 has been implicated in vasculogenic mimicry (VM) formation in human triple-negative breast cancer (TNBC). Twist1 targets the Claudin15 promoter in hepatocellular carcinoma cells. Claudin family members are related with TNBC. However, the relationship between Claudin15 and VM formation is not clear. In this study, we first found that Claudin15 expression was frequently down-regulated in human TNBC, and Claudin15 down-regulation was significantly associated with VM and Twist1 nuclear expression. Claudin15 down-regulation correlated with shorter survival compared with high levels. Claudin15 silence significantly enhanced cell motility, invasiveness and VM formation in the non-TNBC MCF-7 cells. Conversely, an up-regulation of Claudin15 remarkably reduced TNBC MDA-MB-231 cell migration, invasion and VM formation. We also showed that down-regulation of Claudin15 was Twist1-dependent, and Twist1 repressed Claudin15 promoter activity. Furthermore, GeneChip analyses of mammary glands of Claudin15-deficient mice indicated that Claudin18 and Jun might be downstream factors of Twist1-Claudin15. Our results suggest that Twist1 induced VM through Claudin15 suppression in TNBC, and Twist1 inhibition of Claudin15 might involve Claudin18 and Jun expression.

Correction: Cao, Z., et al. The Expression and Functional Significance of Runx2 in Hepatocellular Carcinoma: Its Role in Vasculogenic Mimicry and Epithelial-Mesenchymal Transition. Int. J. Mol. Sci. 2017, 18, 500.

The author wishes to make the following corrections to this paper [...].

LOXL2 promotes vasculogenic mimicry and tumour aggressiveness in hepatocellular carcinoma.

Lysyl oxidase-like 2 (LOXL2) has shown to promote metastasis and poor prognosis in hepatocellular carcinoma (HCC). Also, we have previously reported that vasculogenic mimicry (VM) is associated with invasion, metastasis and poor survival in HCC patients. In the present study, we investigated molecular function of LOXL2 in HCC and VM. We used the immunohistochemical and CD31/periodic acid-Schiff double staining to detect the relationship between LOXL2 and VM formation. We performed the gain and loss of function studies and analysed the migratory, invasion and tube formation in HCC cell lines. We analysed the function of LOXL2 in VM formation and HCC metastasis both in vitro and in vivo. We have showed that LOXL2 was overexpression in HCC and was positively correlated with tumour grade, metastasis, VM formation and poor survival in 201 HCC patients. Secondly, our studies have showed that LOXL2 overexpression in HCC cells significantly promoted migration, invasion and tube formation. Finally, we found that LOXL2 may increase SNAIL expression, thereby enabling VM. Our study indicated that LOXL2 may promote VM formation and tumour metastasis by collaborating with SNAIL in HCC. What's more, the overexpression of LOXL2 indicated a poor prognosis in HCC patients.

Epithelial Splicing Regulatory Protein 1 Inhibits the Invasion and Metastasis of Lung Adenocarcinoma.

Despite the development of various treatments, metastasis remains a significant problem with lung adenocarcinoma (ADC). The role and mechanism of epithelial splicing regulatory protein 1 (ESRP1), an epithelial-specific RNA binding protein, on promoting the invasion and metastasis of lung ADC remain to be fully elucidated. Immunohistochemical analysis in 125 human lung ADC tissue samples demonstrated that ESRP1 overexpression was inversely related to the presence of metastases, tumor size, and clinical stage of lung ADC. Impaired ESRP1 expression was also found to stimulate the invasion capacity of lung ADC cells both in vitro and in vivo. Functionally, overexpression of the ZEB1 gene decreased ESRP1 expression, and knockdown of the ZEB1 gene caused increased ESRP1 expression. On the basis of a gene array analysis, the expression of ESRP1 was associated with the regulation of the extracellular matrix. The expression of CD44 and fibroblast growth factor receptor, representatives that interact with the extracellular matrix, was studied. The CD44 subtypes promoted lung ADC cell invasion by regulating matrix metalloproteinase 2 expression. In conclusion, ESRP1 inhibits the invasion and metastasis of lung ADC and plays a role in regulating proteins involved in epithelial-to-mesenchymal transition.

Expression of epithelial-mesenchymal transition regulators TWIST, SLUG and SNAIL in follicular thyroid tumours may relate to widely invasive, poorly differentiated and distant metastasis.

AIMS: To assess the expression of epithelial-mesenchymal transition (EMT) regulators in follicular thyroid tumours. METHODS AND RESULTS: The expression of E-cadherin (E-CAD) and transcription factors TWIST, SLUG and SNAIL in follicular thyroid tumours was examined by immunohistochemistry in tissue samples, including 18 follicular adenomas (FA), 12 minimally invasive follicular thyroid carcinomas (MI-FTC), 16 widely invasive follicular thyroid carcinomas (WI-FTC), 10 poorly differentiated follicular thyroid carcinomas (PDTC) and six anaplastic thyroid carcinomas (ATC). Metastatic tumour tissues from six of these cases were also examined. The results showed an increasing expression trend of EMT regulators in a panel of follicular tumour cases with a spectrum of morphological subtypes from low- to high-risk malignancy. The expression of EMT regulators was higher in the WI-FTC, PDTC and ATC groups but focal and lower in the FA and MI-FTC groups. Different expression intensity of E-CAD and EMT regulators at the tumour centre part and the invasive front (IF) was observed. The loss of E-CAD and expression of EMT regulators was significantly correlated with distant metastasis and vascular invasion (VI) in the well-differentiated follicular carcinoma (WD-FTC), and six tumours of metastatic sites also showed variables positive for EMT regulators. The disease-free survival analysis showed an apparent relationship between the expression of EMT regulators and the tumour disease-free outcomes in WD-FTC. CONCLUSIONS: Our study supported the role of EMT in the development of follicular thyroid carcinoma and indicated that EMT regulatory proteins may play an important role in WD-FTC that are widely invasive and exhibit distant metastasis.

Long noncoding RNA n339260 promotes vasculogenic mimicry and cancer stem cell development in hepatocellular carcinoma.

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. Cancer stem cells (CSC) represent a subpopulation of tumor cells endowed with the capacity for self-renewal and multilineage differentiation. Previous studies have indicated that CSC may participate in the formation of VM. With the advance of high-resolution microarrays and massively parallel sequencing technology, long noncoding RNAs (lncRNAs) are suggested to play a critical role in tumorigenesis and, in particular, the development of human hepatocellular carcinoma (HCC). Currently, no definitive relationship between lncRNA and VM formation has been described. In the current study, we demonstrated that expression of the lncRNA, n339260, is associated with CSC phenotype in HCC, and n339260 level correlated with VM, metastasis, and shorter survival time in an animal model. Overexpression of n339260 in HepG2 cells was associated with a significant increase in CSC. Additionally, the appearance of VM and vascular endothelial (VE)-cadherin, a molecular marker of VM, was also induced by n339260 overexpression. Using a short hairpin RNA approach, n339260 was silenced in tumor cells, and knockdown of n339260 was associated with reduced VM and CSC. The results of this study indicate that n339260 promotes VM, possibly by the development of CSC. The related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis.

MIIP haploinsufficiency induces chromosomal instability and promotes tumour progression in colorectal cancer.

The gene encoding migration and invasion inhibitory protein (MIIP), located on 1p36.22, is a potential tumour suppressor gene in glioma. In this study, we aimed to explore the role and mechanism of action of MIIP in colorectal cancer (CRC). MIIP protein expression gradually decreased along the colorectal adenoma-carcinoma sequence and was negatively correlated with lymph node and distant metastasis in 526 colorectal tissue samples (p < 0.05 for all). Analysis of The Cancer Genome Atlas (TCGA) data showed that decreased MIIP expression was significantly associated with MIIP hemizygous deletion (p = 0.0005), which was detected in 27.7% (52/188) of CRC cases, and associated with lymph node and distant metastasis (p < 0.05 for both). We deleted one copy of the MIIP gene in HCT116 CRC cells using zinc finger nuclease technology and demonstrated that MIIP haploinsufficiency resulted in increased colony formation and cell migration and invasion, which was consistent with the results from siRNA-mediated MIIP knockdown in two CRC cell lines (p < 0.05 for all). Moreover, MIIP haploinsufficiency promoted CRC progression in vivo (p < 0.05). Genomic instability and spectral karyotyping assays demonstrated that MIIP haploinsufficiency induced chromosomal instability (CIN). Besides modulating the downstream proteins of APC/CCdc20 , securin and cyclin B1, MIIP haploinsufficiency inhibited topoisomerase II (Topo II) activity and induced chromosomal missegregation. Therefore, we report that MIIP is a novel potential tumour suppressor gene in CRC. Moreover, we characterized the MIIP gene as a novel CIN suppressor gene, through altering the stability of mitotic checkpoint proteins and disturbing Topo II activity. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Combination of Scoring Criteria and Whole Exome Sequencing Analysis of Synchronous Endometrial and Ovarian Carcinomas.

OBJECTIVES: The purpose of this study was to distinguish synchronous primary endometrial and ovarian carcinomas from single primary tumor with metastasis by clinical pathologic criteria and whole exome sequencing (WES). MATERIAL AND METHODS: Fifty-two patients with synchronous endometrial and ovarian carcinomas (SEOCs) between 2010 and 2017 were reviewed and subjected to WES. RESULTS: On the basis of the Scully criteria, 11 cases were supposed as synchronous primary endometrial and ovarian carcinomas, 38 cases as single primary tumor with metastasis, and the remaining 3 cases (S50-S52) cannot be defined. Through a quantization scoring analysis, 9 cases that were scored 0-1 point were defined as synchronous primary endometrial and ovarian carcinomas, and 42 cases that were scored 3-8 points were defined as single primary tumor with metastasis. Two of the undefined cases were classified into metastatic disease, and another one that scored 2 points (S52) was subjected to WES. S52 was deemed synchronous primary endometrial and ovarian carcinomas, with few shared somatic mutations and overlapping copy number varieties. The finding of a serous component examined from the uterine endometrium samples further illustrated that the case was synchronous primary endometrial and ovarian carcinomas. CONCLUSION: By scoring criterion, SEOCs were divided into 2 groups: synchronous primary endometrial and ovarian carcinoma group and single primary tumor with metastasis group. The analysis of clonality indicated that the case that scored 2 (S52) can be considered as synchronous primary endometrial and ovarian carcinomas. Scoring criteria of clinical pathology, along with the study of the WES, may further identify the classification of SEOCs.

Mutational landscape of gastric adenocarcinoma in Chinese: implications for prognosis and therapy.

Gastric cancer (GC) is a highly heterogeneous disease. To identify potential clinically actionable therapeutic targets that may inform individualized treatment strategies, we performed whole-exome sequencing on 78 GCs of differing histologies and anatomic locations, as well as whole-genome sequencing on two GC cases, each with three primary tumors and two matching lymph node metastases. The data showed two distinct GC subtypes with either high-clonality (HiC) or low-clonality (LoC). The HiC subtype of intratumoral heterogeneity was associated with older age, TP53 (tumor protein P53) mutation, enriched C > G transition, and significantly shorter survival, whereas the LoC subtype was associated with younger age, ARID1A (AT rich interactive domain 1A) mutation, and significantly longer survival. Phylogenetic tree analysis of whole-genome sequencing data from multiple samples of two patients supported the clonal evolution of GC metastasis and revealed the accumulation of genetic defects that necessitate combination therapeutics. The most recurrently mutated genes, which were validated in a separate cohort of 216 cases by targeted sequencing, were members of the homologous recombination DNA repair, Wnt, and PI3K-ERBB pathways. Notably, the drugable NRG1 (neuregulin-1) and ERBB4 (V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 4) ligand-receptor pair were mutated in 10% of GC cases. Mutations of the BRCA2 (breast cancer 2, early onset) gene, found in 8% of our cohort and validated in The Cancer Genome Atlas GC cohort, were associated with significantly longer survivals. These data define distinct clinicogenetic forms of GC in the Chinese population that are characterized by specific mutation sets that can be investigated for efficacy of single and combination therapies.

MMP-2 and MMP-13 affect vasculogenic mimicry formation in large cell lung cancer.

Matrix metalloproteinases (MMPs) have critical functions in tumour vasculogenic mimicry (VM). This study explored the mechanisms underlying MMP-13 and MMP-2 regulation of tumour VM formation in large cell lung cancer (LCLC). In our study, laminin5 (Ln-5) fragments cleaved by MMP-2 promoted tubular structure formation by the LCLC cell lines H460 and H661 in three-dimensional (3D) cultures. Transient up-regulation of MMP-13 or treatment with recombinant MMP-13 protein abrogated tubular structure formation of H460 cells in 3D culture. Treated cells with Ln-5 fragments cleaved by MMP-2 stimulated EGFR and F-actin expression. Ln-5 fragments cleaved by MMP-13 decreased EGFR/F-actin expression and disrupted VM formation. MMP-13 expression was negatively correlated with VM, Ln-5 and EGFR in LCLC tissues and xenograft. In vivo experiments revealed that VM was decreased when the number of endothelium-dependent vessels (EDVs) increased during xenograft tumour growth, whereas MMP-13 expression was progressively increased. In conclusion, MMP-2 promoted and MMP-13 disrupted VM formation in LCLC by cleaving Ln-5 to influence EGFR signal activation. MMP-13 may regulate VM and EDV formation.

Rictor regulates the vasculogenic mimicry of melanoma via the AKT-MMP-2/9 pathway.

Vasculogenic mimicry (VM)-positive melanomas are usually associated with poor prognosis. Rictor, the key component of the rapamycin-insensitive complex of mTOR (mTORC2), is up-regulated in several cancers, especially in melanomas with poor prognosis. The aim of this study was to investigate the role of Rictor in the regulation of VM and the mechanism underlying this possible regulation. VM channels were found in 35 of 81 tested melanoma samples and high Rictor expression correlated with VM structures. Moreover, Kaplan-Meier survival curves indicated that VM structures and high Rictor expression correlated with shorter survival in patients with melanoma. In vitro, Rictor knockdown by short hairpin RNA (shRNA) significantly inhibited the ability of A375 and MUM-2B melanoma cells to form VM structures, as evidenced by most tubes remaining open. Cell cycle analysis revealed that Rictor knockdown blocked cell growth and resulted in the accumulation of cells in G2/M phase, and cell migration and invasion were greatly affected after Rictor down-regulation. Western blotting assays indicated that down-regulating Rictor significantly inhibited the phosphorylation of AKT at Ser473 and Thr308 , which subsequently inhibited the expression and activity of downstream MMP-2/9, as confirmed by real-time PCR and gelatin Zymography. MK-2206, a small-molecule inhibitor of AKT, similarly inhibited the activity of AKT and secretion of MMP-2/9, further supporting that Rictor down-regulation inhibits the phosphorylation of AKT and activity of downstream MMP-2/9 to affect VM formation. In conclusion, Rictor plays an important role in melanoma VM via the Rictor-AKT-MMP-2/9 signalling pathway.

VEGFR2 regulates endothelial differentiation of colon cancer cells.

BACKGROUND: Recent studies suggested that cancer stem-like cells contribute to tumor vasculogenesis by differentiating into endothelial cells. However, such process is governed by still undefined mechanism. METHODS: At varying differentiation levels, three representative colon cancer cells were cultured in endothelial-inducing conditioned medium: human colon cancer cells HCT116 (HCT116) (poorly differentiated), SW480 (moderately differentiated), and HT29 (well differentiated). We tested for expression of endothelial markers (cluster of differentiation (CD) 31, CD34, and vascular endothelial (VE)-cadherin and their ability to form tube-like structures in 3D culture. We also observed VEGF secretion and expressions of endothelial markers and VEGFRs in HCT116 cells under hypoxia to simulate physiological conditions. In in vitro and in xenotransplantation experiments, VE growth factor receptor 2 (VEGFR2) antagonist SKLB1002 was used to test effect of VEGFR2 in endothelial differentiation of HCT116 cells. Expression levels of VEGFR2 and VE-cadherin were assessed by immunohistochemistry of human colon cancer tissues to evaluate clinicopathological significance of VEGFR2. RESULTS: After culturing in endothelial-inducing conditioned medium, poorly differentiated HCT116 cells expressed endothelial markers and formed tube-like structure in vitro. HCT116 cells secreted more endogenous VEGF and expressed higher VEGFR2 under hypoxia. SKLB1002 impaired endothelial differentiation in vitro and xenotransplantation experiments, suggesting a VEGFR2-dependent mechanism. Increased expression of VEGFR2 correlated with differentiation, metastasis/recurrence, and poor prognosis in 203 human colon cancer samples. Positive correlation was observed between VEGFR2 and VE-cadherin expression. CONCLUSIONS: VEGFR2 regulates endothelial differentiation of colon cancer cell and may be potential platform for anti-angiogenesis cancer therapy.

The Expression and Functional Significance of Runx2 in Hepatocellular Carcinoma: Its Role in Vasculogenic Mimicry and Epithelial-Mesenchymal Transition.

The transcription factor Runx2 has been reported to promote epithelial-mesenchymal transition (EMT) in many tumors. Vasculogenic mimicry (VM) is described as the mimicry of endothelial cells by tumor cells to form microvascular tubes in aggressive tumors. Galectin-3 has been reported to regulate cell invasion, migration, and VM formation; it could be regulated by Runx2. However, the relationship between Runx2, Galectin-3, EMT, and VM has not been studied in hepatocellular carcinoma (HCC). We examined Runx2 expression in 89 human HCC samples and found Runx2 expression was associated with VM. Clinical-pathological data analysis revealed that Runx2 expression was associated with a shorter survival period. Overexpression of Runx2 promoted EMT and enhanced cell migration, invasion, and VM formation in HepG2 cells. Conversely, the downregulation of Runx2 inhibited EMT and reduced cell invasion, migration, and VM formation in SMMC7721. Galectin-3 expression declined following the downregulation of Runx2 in HepG2 cells, and increased in SMMC7721 cells after Runx2 knockdown. We consistently demonstrated that the downregulation of LGALS3 in HepG2-Runx2 cells reduced cell migration; invasion and VM formation; while upregulation of LGALS3 in SMMC7721-shRunx2 cells enhanced cell migration, invasion, and VM formation. The results indicate that Runx2 could promote EMT and VM formation in HCC and Galectin-3 might have some function in this process.

Dickkopf-1-promoted vasculogenic mimicry in non-small cell lung cancer is associated with EMT and development of a cancer stem-like cell phenotype.

To characterize the contributions of Dickkopf-1 (DKK1) towards the induction of vasculogenic mimicry (VM) in non-small cell lung cancer (NSCLC), we evaluated cohorts of primary tumours, performed in vitro functional studies and generated xenograft mouse models. Vasculogenic mimicry was observed in 28 of 205 NSCLC tumours, while DKK1 was detected in 133 cases. Notably, DKK1 was positively associated with VM. Statistical analysis showed that VM and DKK1 were both related to aggressive clinical course and thus were indicators of a poor prognosis. Moreover, expression of epithelial-mesenchymal transition (EMT)-related proteins (vimentin, Slug, and Twist), cancer stem-like cell (CSC)-related proteins (nestin and CD44), VM-related proteins (MMP2, MMP9, and vascular endothelial-cadherin), and ß-catenin-nu were all elevated in VM-positive and DKK1-positive tumours, whereas the epithelial marker (E-cadherin) was reduced in the VM-positive and DKK1-positive groups. Non-small cell lung cancer cell lines with overexpressed or silenced DKK1 highlighted its role in the restoration of mesenchymal phenotypes and development of CSC characteristics. Moreover, DKK1 significantly promotes NSCLC tumour cells to migrate, invade and proliferate. In vivo animal studies demonstrated that DKK1 enhances the growth of transplanted human tumours cells, as well as increased VM formation, mesenthymal phenotypes and CSC properties. Our results suggest that DKK1 can promote VM formation via induction of the expression of EMT and CSC-related proteins. As such, we feel that DKK1 may represent a novel target of NSCLC therapy.

Notch4+ cancer stem-like cells promote the metastatic and invasive ability of melanoma.

Sphere formation in conditioned serum-free culture medium supplemented with epidermal growth factor and basic fibroblast growth factor (tumorospheres) is considered useful for the enrichment of cancer stem-like cells, also known as tumor-initiating cells. We used a gene expression microarray to investigate the gene expression profile of melanoma cancer stem-like cells (MCSLCs). The results showed that MCSLCs highly expressed the following Notch signaling pathway molecules: Notch3 (NM_008716), Notch4 (NM_010929), Dtx4 (NM_172442), and JAG2 (NM_010588). Immunofluorescence staining showed tumorosphere cells highly expressed Notch4. Notch4(high) B16F10 cells were isolated by FACS, and Western blotting showed that high Notch4 expression is related to the expression of epithelial-mesenchymal transition (EMT)-associated proteins. Reduced invasive and migratory properties concomitant with the downregulation of the EMT markers Twist1, vimentin, and VE-cadherin and the overexpression of E-cadherin was observed in human melanoma A375 and MUM-2B cells. In these cells, Notch4 was also downregulated, both by Notch4 gene knockdown and by application of the γ-secretase inhibitor, DAPT. Mechanistically, the re-overexpression of Twist1 by the transfection of cells with a Twist1 expression plasmid led to an increase in VE-cadherin expression and a decrease in E-cadherin expression. Immunohistochemical analysis of 120 human melanoma tissues revealed a significant correlation between the high expression of Notch4 and the metastasis of melanoma. Taken together, our findings indicate that Notch4+ MCSLCs trigger EMT and promote the metastasis of melanoma cells.