Expression of L1-CAM and ADAM10 in human colon cancer cells induces metastasis

L1-CAM, a neuronal cell adhesion receptor, is also expressed in a variety of cancer cells. Recent studies identified L1-CAM as a target gene of beta-catenin-T-cell factor (TCF) signaling expressed at the invasive front of human colon cancer tissue. We found that L1-CAM expression in colon cancer cells lacking L1-CAM confers metastatic capacity, and mice injected in their spleen with such cells form liver metastases. We identified ADAM10, a metalloproteinase that cleaves the L1-CAM extracellular domain, as a novel target gene of beta-catenin-TCF signaling. ADAM10 overexpression in colon cancer cells displaying endogenous L1-CAM enhanced L1-CAM cleavage and induced liver metastasis, and ADAM10 also enhanced metastasis in colon cancer cells stably transfected with L1-CAM. DNA microarray analysis of genes induced by L1-CAM in colon cancer cells identified a cluster of genes also elevated in a large set of human colon carcinoma tissue samples. Expression of these genes in normal colon epithelium was low. These results indicate that there is a gene program induced by L1-CAM in colon cancer cells that is also present in colorectal cancer tissue and suggest that L1-CAM can serve as target for colon cancer therapy.     

Fendiline inhibits proliferation and invasion of pancreatic cancer cells by interfering with ADAM10 activation and β-catenin signaling

ADAM10 (A Disintegrin and Metalloprotease Domain 10) affects the pathophysiology of various cancers, and we had shown that inhibition of ADAM10 sensitizes pancreatic cancer cells to gemcitabine. ADAM10 is activated in response to calcium influx, and here we examined if calcium channel blockers (CCB) would impede ADAM10 activation and affect biology of pancreatic cancer cells. We find that the CCB, fendiline, significantly reduces proliferation, migration, invasion, and anchorage independent growth of pancreatic cancer cells. This was associated with ADAM10 inhibition and its localization at the actin-rich membrane protrusions. Further, fendiline-treated cells formed cadherin-catenin positive tight adherens junctions and elicited defective protein trafficking and recycling. Furthermore, the expression of β-catenin target genes, cyclinD1, c-Myc and CD44, were significantly decreased, suggesting that fendiline might prevent cell proliferation and migration by inhibiting ADAM10 function, cadherin proteolysis and stabilization of cadherin-catenin interaction at the plasma membrane. This will subsequently diminish β-catenin intracellular signaling and repress TCF/LEF target gene expression. Supporting this notion, RNAi-directed downregulation of ADAM10 in cancer cells decreased the expression of cyclinD1, c-Myc and CD44. Furthermore, analysis of human pancreatic tumor tissue microarrays and lysates showed elevated levels of ADAM10, suggesting that aberrant activation of ADAM10 plays a fundamental role in growth and metastasis of PDACs and inhibiting this pathway might be a viable strategy to combat PDACs.     

MicroRNA-145 Targets the Metalloprotease ADAM17 and Is Suppressed in Renal Cell Carcinoma Patients

A disintegrin and metalloproteinase 17 (ADAM17) is a metalloprotease that is overexpressed in many cancer types, including renal cancers. However, the regulatory mechanisms of ADAM17 in cancer development and progression are poorly understood. In the present work, we provide evidence using overexpression and inhibition of microRNA 145 (miR-145) that miR-145 negatively regulates ADAM17 expression. Furthermore, we show that ADAM17 negatively regulates miR-145 through tumor necrosis factor-α, resulting in a reciprocal negative feedback loop. In this study, the expression of ADAM17 and miR-145 correlated negatively in renal cancer tumor tissues and cell lines, suggesting an important regulatory mechanism. Additionally, we showed that the regulation of ADAM17 is partly involved in the effects of miR-145 on proliferation and migration, whereas no involvement in chemosensitivity was observed. Importantly, in the healthy kidney, miR-145 was detected in different cell types including tubular cells, which are considered the origin of renal cancer. In renal cancer cell lines, miR-145 expression was strongly suppressed by methylation. In summary, miR-145 is downregulated in renal cancer patients, which leads to the up-regulation of ADAM17 in renal cancer. Importantly, miR-145 and ADAM17 are regulated in a reciprocal negative feedback loop.     

ADAM10 overexpression in human non-small cell lung cancer correlates with cell migration and invasion through the activation of the Notch1 signaling pathway

A disintegrin and metalloproteinase 10 (ADAM10) was identified as a key protease in the ectodomain shedding of various substrates, such as Notch1 protein, ErbB2 and E-cadherin, which are important in the development of non-small cell lung cancer (NSCLC). The aim of this study was to investi-gate the role of ADAM10 in NSCLC metastasis.We characterized the expression of ADAM10 and Notch1 in human NSCLC tissues in?vivo. Immunohistochemical analysis indicated that ADAM10 expression was significantly increased in the NSCLC tissues, particularly in the metastatic tissues. Futhermore, ADAM10 overexpression positively correlated with Notch1 expression in the NSCLC tissues. The in?vitro downregulation of ADAM10 expression using ADAM10 short hairpin RNA (shRNA) reduced the migration and invasion of NSCLC cells. We present further evidence that ADAM10 promotes NSCLC cell migration and invasion via the activation of the Notch1 signaling pathway. Taken together, our results suggest that ADAM10 may serve as a potential target for the therapeutic intervention of NSCLC metastasis. The data provided in this study may aid in the further understanding of the function of ADAM10 in the progression of NSCLC and open new perspectives for the diagnosis and treatment of NSCLC.     

Loss of cancer cell-derived ADAM15 alters the tumor microenvironment in colorectal tumors

Tumor progression and response to treatment are highly affected by interactions between cancer cells and the tumor microenvironment (TME). Many of the soluble factors and signaling receptors involved in this crosstalk are shed by a disintegrin and metalloproteinases (ADAMs). Upregulation of ADAM15 has been linked to worse survival in cancer patients and a tumor-promoting function both in vitro and in murine cancer models. Although ADAM15 has been involved in cell-cell and cell-extracellular matrix interactions, its role in the crosstalk between cancer cells and the TME in vivo remains unexplored. Therefore, we aimed to understand how ADAM15 regulates the cell composition of the TME and how it affects tumor progression. Here, we showed an upregulation of ADAM15 in tumor tissues from rectal cancer patients. Subcutaneous injection of wildtype and ADAM15-knockout CT26 colon cancer cells in syngeneic mice confirmed the protumorigenic role of ADAM15. Profiling of tumors revealed higher immune cell infiltration and cancer cell apoptosis in the ADAM15-deficient tumors. Specifically, loss of ADAM15 led to a reduced number of granulocytes and higher infiltration of antigen-presenting cells, including dendritic cells and macrophages, as well as more T cells. Using in vitro assays, we confirmed the regulatory effect of ADAM15 on macrophage migration and identified ADAM15-derived CYR61 as a potential molecular mediator of this effect. Based on these findings, we speculate that targeting ADAM15 could increase the infiltration of immune cells in colorectal tumors, which is a prerequisite for effective immunotherapy.     

ADAM23 is downregulated in side population and suppresses lung metastasis of lung carcinoma cells

Cancer cells contain a small population of cancer stem cells or cancer initiating cells, which can be enriched in the side population (SP) after fluorescence activated cell sorting. To examine the members of the ADAM, ADAMTS and MMP gene families related to phenotypes of the SP and the main population (MP), we screened the expression of all the members in the propagated SP and MP of A549 lung adenocarcinoma cells, and found that the relative expression ratio of ADAM23 in the MP to the SP is most highly increased, but none of them are increased in the SP. A similar result on the ADAM23 expression was obtained with another cell line, Calu‐3 cells. Overexpression of ADAM23 inhibited colony formation, cell adhesion and migration, and knockdown of ADAM23 by shRNA showed the reverse effects. ADAM23‐mediated suppression of colony formation, cell adhesion and migration was greatly reduced by treatment with neutralizing anti‐ADAM23 antibody, anti‐αvβ3 integrin antibody and/or ADAM23 disintegrin peptide. Expression of cancer stem cell‐related genes, including AKRC1/2, TM4SF1 and NR0B1, was increased by knockdown of ADAM23. In addition, lung metastasis of A549 transfectants with different levels of ADAM23 expression was negatively regulated by the ADAM23 expression levels. Our data provide evidence that ADAM23 plays a role in suppression of cancer cell progression through interaction with αvβ3 integrin, and suggest that downregulation of ADAM23 in SP cells may contribute toward providing a cancer stem cell phenotype by facilitating the activity of integrin αvβ3.     

ADAM10: a new player in breast cancer progression?

Background:

The ADAM proteases are best known for their role in shedding the extracellular domain of transmembrane proteins. Among the transmembrane proteins shed by ADAM10 are notch, HER2, E-cadherin, CD44, L1 and the EGFR ligands, EGF and betacellulin. As cleavage of several of these proteins has been implicated in cancer formation and progression, we hypothesised that ADAM10 is also involved in these processes.

Methods:

ADAM10 expression was decreased by RNA interference and the effects of this on cell numbers, invasion and migration were determined. We also examined the effect of ADAM10 inhibition on breast cancer cell line invasion and migration.

Results:

Using the triple-negative (TN) breast cancer cell lines, BT20, MDA-MB-231 and the non-TN cell line MDA-MB-453, knockdown of ADAM10 expression significantly decreased in vitro migration (P<0.01; for each cell line). Similarly, treatment with the ADAM10-selective inhibitor GI254023X reduced migration in the three cell lines (for BT20, P<0.001; for MDA-MB-231, P=0.005; for MDA-MB-453, P=0.023). In contrast, neither knockdown of ADAM10 nor treatment with the ADAM10-selective inhibitor GI254023X significantly affected cell numbers. Using extracts of primary breast cancers, higher levels of ADAM10 were found more frequently in high-grade vs low-grade tumours (P<0.001) and in oestrogen receptor (ER)-negative compared with ER-positive tumours (P=0.005). Analysis of pooled publicly available data sets found that high levels of ADAM10 mRNA were associated with adverse outcome in patients with the basal subtype of breast cancer.

Conclusions:

Based on our combined cell line and breast cancer extract data, we conclude that ADAM10 is likely to be involved in breast cancer progression, especially in the basal subtype.     

Hypoxia‐induced downregulation of miR‐30c promotes epithelial‐mesenchymal transition in human renal cell carcinoma

MicroRNAs (miRNAs), which negatively regulate protein expression by binding protein‐coding mRNAs, have been integrated into cancer development and progression as either oncogenes or tumor suppressor genes. miR‐30c was reported to be downregulated in several types of cancer. However, its role in human renal cell carcinoma (RCC) remains largely unknown. Here, we show that miR‐30c is significantly downregulated in human RCC tissues and cell lines. We found that miR‐30c downregulation could be induced by hypoxia in RCC cells in a hypoxia‐inducible factors (HIFs) dependent manner. Repression of miR‐30c through its inhibitor resulted in reduction of E‐cadherin production and promotion of epithelial‐mesenchymal transition (EMT), while overexpression of miR‐30c inhibited EMT in RCC cells. We identified Slug as a direct target of miR‐30c in RCC cells. Slug was upregulated in RCC tissues and its expression could be induced by hypoxia, which is consistent with downregulation of miR‐30c by hypoxia. Forced overexpression of Slug in 786‐O cells reduced E‐cadherin production, and promoted EMT as well as cell migration. Moreover, Slug overexpression abrogated the inhibitory role of miR‐30c in regulating EMT and cell migration, indicating miR‐30c regulates EMT through Slug in RCC cells. Our findings propose a model that hypoxia induces EMT in RCC cells through downregulation of miR‐30c, which leads to subsequent increase of Slug expression and repression of E‐cadherin production, and suggest a potential application of miR‐30c in RCC treatment.     

Tumoural CXCL16 expression is a novel prognostic marker of longer survival times in renal cell cancer patients

The aim of our study was to analyse the expression of CXCL16, ADAM10 and CXCR6 in renal cell carcinoma (RCC) tissue and to correlate the expression pattern with clinicopathologic data, including patient survival. Furthermore, we investigated CXCL16, ADAM10 and CXCR6 expressions by FACS, immunofluorescence and ELISA analysis in renal carcinoma cell lines. Our immunohistochemical analysis on tissue microarray of renal cancer samples of 104 patients revealed that ADAM10 correlated significantly with tumour stage, pathological nodal status, M status and lymphangiosis carcinomatosa. CXCL16, CXCR6 and ADAM10 were significantly increased in papillary carcinomas. Importantly, high levels of CXCL16 expression in renal cancer tissue correlated with better survival of patients, and CXCL16 correlated inversely to the tumour stage. In addition, inhibition of CXCL16 induced the migration of renal cancer cells assuming an anti-migratory function of transmembrane CXCL16. Taken together, our data demonstrate that downregulation of CXCL16 plays an important role in renal cancer development and progression, and that CXCL16 in RCC is an independent prognostic marker for better patient survival.     

解整合素金属蛋白酶10通过介导Wnt/β-catenin信号通路促进子宫内膜癌细胞上皮-间质转化

目的:探讨解整合素金属蛋白酶10（ADAM10）对子宫内膜癌（EC）细胞上皮-间质转化（EMT）的影响及其作用机制。方法:收集84例EC患者的新鲜癌组织及癌旁组织标本,采用qRT-PCR检测癌组织及其癌旁组织中ADAM10 mRNA表达水平。以人EC细胞株HEC-1B作为研究对象,采用ADAM10过表达慢病毒感染HEC-1B细胞,并联合Wnt/β-catenin信号通路特异性抑制剂XAV939进行干预,再将细胞分为空白对照组（blank）、慢病毒阴性对照组（Lv-NC）、ADAM10过表达慢病毒组（Lv-ADAM10）和Lv-ADAM10+XAV939组。采用qRT-PCR检测感染后各组细胞中ADAM10 mRNA表达水平;划痕和Transwell实验检测各组细胞的迁移和侵袭能力;Western blot法分析各组细胞中ADAM10及EMT相关蛋白和Wnt/β-catenin信号通路相关蛋白表达水平。结果:EC患者癌组织中ADAM10 mRNA表达水平显著高于癌旁组织（P＜0.05）。与blank组或Lv-NC组比较,Lv-ADAM10组细胞中ADAM10 mRNA和蛋白表达水平以及N-cadherin、Vimentin、β-catenin、Snail、MMP2和MMP9等蛋白表达水平均显著升高（均P＜0.05）,而E-cadherin蛋白表达水平明显降低（P＜0.05）,同时细胞迁移和侵袭能力显著增强（均P＜0.05）。与Lv-ADAM10组比较,Lv-ADAM10+XAV939组细胞中N-cadherin、Vimentin、β-catenin、Snail、MMP2和MMP9等蛋白表达水平显著降低（均P＜0.05）,E-cadherin蛋白表达水平明显升高（P＜0.05）,且细胞迁移和侵袭能力明显降低（均P＜0.05）。结论:ADAM10在EC组织中高表达,其过表达可促进HEC-1B细胞的EMT进程,其作用机制可能与激活Wnt/β-catenin信号通路有关。     

ADAM15 supports prostate cancer metastasis by modulating tumor cell-endothelial cell interaction

Using human tumor and cDNA microarray technology, we have recently shown that the ADAM15 disintegrin is significantly overexpressed during the metastatic progression of human prostate cancer. In the current study, we used lentiviral-based short hairpin RNA (shRNA) technology to down-regulate ADAM15 in the metastatic prostate cancer cell line, PC-3. ADAM15 down-regulation dramatically attenuated many of the malignant characteristics of PC-3 cells in vitro and prevented the s.c. growth of PC-3 cells in severe combined immunodeficient (SCID) mice. By inhibiting the expression of ADAM15 in PC-3 cells, we showed decreased cell migration and adhesion to specific extracellular matrix proteins. This was accompanied by a reduction in the cleavage of N-cadherin by ADAM15 at the cell surface. Fluorescence-activated cell sorting analysis revealed reduced cell surface expression of the metastasis-associated proteins alpha(v) integrin and CD44. Furthermore, matrix metalloproteinase 9 secretion and activity were abrogated in response to ADAM15 reduction. In an in vitro model of vascular invasion, loss of ADAM15 reduced PC-3 adhesion to, and migration through, vascular endothelial cell monolayers. Using an SCID mouse model of human prostate cancer metastasis, we found that the loss of ADAM15 significantly attenuated the metastatic spread of PC-3 cells to bone. Taken together, these data strongly support a functional role for ADAM15 in prostate tumor cell interaction with vascular endothelium and the metastatic progression of human prostate cancer.     

HMGA2 induces transcription factor Slug expression to promote epithelial-to-mesenchymal transition and contributes to colon cancer progression

Epithelial-to-mesenchymal transition (EMT) is considered to play an essential role in progression and metastasis. This study aims to investigate the expression and underlying molecular targets of high-mobility group AT-hook 2 (HMGA2) in the progression of colon cancer. The expression of HMGA2 is upregulated by both active extracellular signal-regulated kinase (ERK)1/2 and TGF-β signaling in colon cancer cells through a series of lentiviral infection and pharmacological assays. HMGA2 knockdown by specific shRNAs attenuates proliferation, motility and invasion of colon cancer cells in vitro and in vivo. Besides, exogenous HMGA2 expression caused EMT in colon cancer cells, which was confirmed by the downregulation of the epithelial markers and the upregulation of the mesenchymal markers. Moreover, HMGA2 positively regulates the Slug expression by directly binding to the regulatory region in Slug promoter. Importantly, the knockdown of Slug could reverse the HMGA2-induced EMT and decrease the migration and invasion ability of colon cancer cells. Taken together, our results reveal a critical role for HMGA2 in promoting EMT, migration, invasion, and proliferation of colon cancer cells, suggesting HMGA2 as a potential molecular target to prevent colon cancer progression.     

Lipocalin 2 negatively regulates cell proliferation and epithelial to mesenchymal transition through changing metabolic gene expression in colorectal cancer

Lipocalin 2 (LCN2), a member of the lipocalin superfamily, plays an important role in oncogenesis and progression in various types of cancer. However, the expression pattern and functional role of LCN2 in colorectal cancer (CRC) is still poorly understood. The purpose of the present study was to investigate whether LCN2 is associated with proliferation and the epithelial–mesenchymal transition (EMT) in CRC and to elucidate the underlying signaling pathways. LCN2 was preferentially expressed in CRC cells compared to normal tissues. However, LCN2 expression was significantly lower in metastatic or advanced‐stage CRC than in non‐metastatic or early stage CRC. Knockdown of LCN2 using small interfering RNA (siRNA) in CRC cells expressing a high level of LCN2 induced cell proliferation and a morphological switch from an epithelial to mesenchymal state. Furthermore, downregulation of LCN2 in CRC cells increased cell migration and invasion involved in the regulation of EMT markers. Knockdown of LCN2 also induced glucose consumption and lactate production, accompanied by an increase in energy metabolism‐related genes. Taken together, our findings indicated that LCN2 negatively modulated proliferation, EMT and energy metabolism in CRC cells. Accordingly, LCN2 may be a candidate metastasis suppressor and potential therapeutic target in CRC.