RNA interference of metastasis-associated gene 1 inhibits metastasis of B16F10 melanoma cells in a C57BL/6 mouse model.

BACKGROUND INFORMATION: MTA1 (metastasis-associated gene 1) has been reported to be overexpressed in cancers with high potential to metastasize. Studies of the molecular mechanisms revealed that MTA1 plays an important role in the process of metastasis of many types of cancer. However, the role of MTA1 in melanoma development is unclear. RESULTS: We have investigated the therapeutic value of MTA1 in the B16F10 melanoma cell line with the C57BL/6 mouse model. Studies in vitro showed that MTA1 promoted the metastatic ability of B16F10 cancer cells. MTA1 down-regulation by RNA interference greatly reversed the malignant phenotypes of cancer cells. Immunohistochemical staining of MTA1 in human melanoma samples confirmed the up-regulation of MTA1 in the process of carcinogenesis. Studies in vivo confirmed down-regulation of MTA1 suppressed the growth and experimental metastasis of B16F10 melanoma cells. CONCLUSIONS: MTA1 plays an important role in melanoma development and metastasis. It has a promising potential as a target for in cancer gene therapy or chemotherapy.     

E‐cadherin determines Caveolin‐1 tumor suppression or metastasis enhancing function in melanoma cells

The role of caveolin‐1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E‐cadherin in CAV1‐dependent tumor suppression. Here, we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E‐cadherin, to unravel how CAV1 affects tumor growth and metastasis and to assess how co‐expression of E‐cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10 (cav‐1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E‐cadherin expression in B16F10 (E‐cad) cells reduces subcutaneous tumor formation and lung metastasis when intravenously injected. Importantly, co‐expression of CAV1 and E‐cadherin in B16F10 (cav‐1/E‐cad) cells abolishes tumor formation, lung metastasis, increased Rac‐1 activity, and cell migration observed with B16F10 (cav‐1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac‐1 activation in these cells.     

Hic‐5 affects proliferation,migration and invasion of B16 murine melanoma cells

Hic‐5 is a shuttling protein between the cell membrane and the nucleus which functions as a focal adhesion adaptor protein and a nuclear receptor coactivator. Although several studies have shown its involvement in other types of cancer, the role of Hic‐5 in melanoma is unknown. Herein, we show for the first time that Hic‐5 is expressed in B16‐F1 murine melanoma cells. To determine its function in melanoma cells, we used shRNA‐mediated RNA interference and established stable clones with down‐regulated Hic‐5 expression. These clones had impaired growth and metastatic potential compared with controls in vivo, which correlated with decreased proliferation, migration and invasion in vitro. Moreover, silencing of Hic‐5 expression in B16‐F1 activated RhoA with an amoeboid phenotypic change, indicating that Hic‐5 is a key regulator of B16‐F1 metastasis in the context of Rho‐dependent motility. These results provide new evidence that Hic‐5 is a possible molecular target for treatment of melanoma.     

Silencing of Diphthamide Synthesis 3 (Dph3) Reduces Metastasis of Murine Melanoma

Melanoma is the most dangerous skin cancer due to its highly metastatic potential and resistance to chemotherapy. Currently, there is no effective treatment for melanoma once it is progressed to metastatic stage. Therefore, further study to elucidate the molecular mechanism underlying the metastasis of melanoma cells is urgently required for the improvement of melanoma treatment. In the present study, we found that diphthamide synthesis 3 (Dph3) is involved in the metastasis of B16F10 murine melanoma cells by insertional mutagenesis. We demonstrated that Dph3 disruption impairs the migration of B16F10 murine melanoma cells. The requirement of Dph3 in the migration of melanoma cells was further confirmed by gene silencing with siRNA in vitro. In corresponding to this result, overexpression of Dph3 significantly promoted the migratory ability of B16F10 and B16F0 melanoma cells. Moreover, down regulation of Dph3 expression in B16F10 melanoma cells strikingly inhibits their cellular invasion and metastasis in vivo. Finally, we found that Dph3 promotes melanoma migration and invasion through the AKT signaling pathway. To conclude, our findings suggest a novel mechanism underlying the metastasis of melanoma cells which might serve as a new intervention target for the treatment of melanoma.     

MTA1 promotes the invasion and migration of pancreatic cancer cells potentially through the HIF-α/VEGF pathway

Abstract

The metastasis-associated gene 1 (MTA1) has previously been recognized as an oncogene, and abnormal MTA1 expression has been related to progression of numerous cancer types to the metastasis stage. However, the function of MTA1 in the regulation of pancreatic cancer progression and metastasis remains unclear. Western blot analysis was adopted to determine the expression of MTA1 in pancreatic cancer tissues and corresponding near normal tissues. Steady clone with MTA1-overexpression and MTA1-inhibitionweregenerated via lentivirus technology in BxPc-3 cells. Transwell assay was carried out for detecting the invasion of pancreatic cancer cells. The migration activity was assessed using the wound scratch assay. The effect of MTA1 in pancreatic cancer was evaluated in the mice xenografts. Western blot analysis was employed to determine the expression of hypoxia inducible factor-α (HIF-α) and vascular endothelial growth factor (VEGF) in vitro and in vivo. We observed that MTA1 overexpression enhanced migration and invasion ability of pancreatic cancer cells in vitro and increased HIF-α and VEGF protein levels in vitro and in vivo. MTA1 inhibition had the opposite effects. MTA1 protein level was positively related to HIF-α and VEGF protein levels. These results indicated that MTA1 potentially promoted pancreatic cancer metastasis via HIF-α/VEGF pathway. This research supplies a new molecular mechanism for MTA1 in the pancreatic cancer progression and metastasis. MTA1 may be an effective therapy target in pancreatic cancer.     

Epigenetic regulation of REG1A and chemosensitivity of cutaneous melanoma

Regenerating gene 1A (REG1A) plays an important role in tissue regeneration and in cell proliferation in epithelium origin tumors; however, its role in melanoma has not been explored in details. The objective of this study was to identify whether REG1A is expressed in cutaneous melanoma and if REG1A expression status can predict prognosis in cutaneous melanoma patients with metastasis. We also determined whether epigenetic regulation of the promoter region regulates REG1A expression. AJCC stage III cutaneous melanoma specimens with clinically well annotated stage III lymph node melanoma metastasis tissue microarray were assessed by IHC. MALDI-TOF-mass spectrometry and HM450K array were used to identify REG1A promoter region CpG site methylation. Chemotherapeutic agent response by melanoma cells as related to REG1A protein expression was assessed. Post-surgery melanoma patients followed by adjuvant chemotherapy with high REG1A expression had a significantly better prognosis (disease-specific survival) compared with patients with low REG1A expression (log rank test; p = 0.0013). The demethylating reagent 5-Aza-2′-deoxycytidine activated REG1A promoter region resulting in enhanced REG1A mRNA and protein expression in melanoma cell lines. Promoter region CpG methylation was shown to regulate REG1A expression in melanoma cells. Moreover, melanoma lines with high REG1A mRNA expression were more susceptible to Dacarbazine and Cisplatin, as compared with those with low REG1A mRNA expression. In conclusion, REG1A expression status may be useful as a biomarker in melanoma patients for sensitivity to these chemotherapeutic agents. The epigenetic regulation of the REG1A promoter region may offer a potential therapeutic approach to improve chemotherapy for metastatic melanoma patients.     

CD147 regulates melanoma metastasis via the NFAT1‐MMP‐9 pathway

Although accumulating evidence had revealed that NFAT1 has oncogenic characteristics, the role of this molecule in melanoma cells remains unclear. Previous studies proved that CD147 plays a crucial function in melanoma cell metastasis and invasion through matrix metalloproteinase 9 (MMP‐9) expression; however, the details of how CD147 regulates MMP‐9 expression remain elusive. In this study, we demonstrated that CD147 and NFAT1 are overexpressed in the tissues of patients with primary and metastatic melanoma, which has shown a positive correlation. Further, we observed that CD147 regulates NFAT1 activation through the [Ca²⁺]i‐calcineurin pathway. Knockdown of NFAT1 significantly suppresses melanoma metastasis, and we demonstrated that CD147 affects melanoma metastasis in an NFAT1‐dependent manner. Moreover, we verified that NFAT1 directly binds to MMP‐9 promoter. Inhibition of CD147 expression significantly abrogates MMP‐9 promoter luciferase gene reporter activity as well as NFAT1 association with MMP‐9 promoter. Taken together, this study demonstrated that CD147 affects MMP‐9 expression through regulating NFAT1 activity and provided a novel mechanism by which NFAT1 contributes to melanoma metastasis through the regulation of MMP‐9.     

Identification of Metastasis-Promoting Sequences in the Mouse Laminin α1 Chain

Laminin-1, a major basement membrane matrix glycoprotein, enhances adhesion, migration, and metastasis of tumor cells. We have screened 208 overlapping synthetic peptides covering the short and long arms of mouse laminin α1 chain for their adhesion activity with B16-F10 mouse melanoma cells. Cell adhesion activity was determined using various amounts of peptides coated on plastic dishes and by measuring cell adhesion on peptide-conjugated Sepharose beads. Nineteen peptides showed B16-F10 cell adhesion activity. Three peptides, designated A-13, -24, and -208, showed the strongest attachment activity in the plate assay, whereas 4 peptides, A-13, -51, -99, and -112, demonstrated the strongest cell adhesion when conjugated to beads. The 19 peptides were tested in vivo for their effect on experimental pulmonary metastasis by B16–F10 cells. Four peptides, A-13, -51, -64, and -119, significantly enhanced metastasis, with A-13 showing the strongest dramatic enhancement. The four metastasis-promoting peptides also stimulated migration of B16-F10 cells in the Boyden chamber assay in vitro with A-13 being the most potent stimulator. In addition, the 4 peptides inhibited laminin-induced cell attachment and migration, which indicates that these four sequences are possible functional B16-F10 cell binding sites in laminin-1. All the four sequences are located on the globular domains of the short arm. Other peptides, including strong adhesion-active peptides, A-24, -99, -112, and a scrambled A-13 peptide, did not stimulate either migration or metastasis. Thus, laminin-1 has multiple active sites in the globular domains of the short arm which promote migration and metastasis of B16-F10 cells.     

Akt Mediates Metastasis-Associated Gene 1 (MTA1) Regulating the Expression of E-cadherin and Promoting the Invasiveness of Prostate Cancer Cells

Human metastasis-associated gene 1 (MTA1) is highly associated with the metastasis of prostate cancer; however, the molecular functions of MTA1 that facilitate metastasis remain unclear. In this study, we demonstrate that the silencing of MTA1 by siRNA treatment results in the upregulation of E-cadherin expression by the phosphorylation of AKT (p-AKT) and decreases the invasiveness of prostate cancer cells. We show that MTA1 is expressed in over 90% of prostate cancer tissues, especially metastatic prostate cancer tissue, comparing to non-expression in normal prostate tissue. RT-PCR analysis and Western blot assay showed that MTA1 expression is significantly higher in highly metastatic prostate cancer PC-3M-1E8 cells (1E8) than in poorly metastatic prostate cancer PC-3M-2B4 cells (2B4). Silencing MTA1 expression by siRNA treatment in 1E8 cells increased the cellular malignant characters, including the cellular adhesive ability, decreased the cellular invasive ability and changed the polarity of cellular cytoskeleton. 1E8 cells over-expressing MTA1 had a reduced expression of E-cadherin, while 1E8 cells treated with MTA1 siRNA had a higher expression of E-cadherin. The expression of phosphorylated AKT (p-AKT) or the inhibition of p-AKT by wortmannin treatment (100 nM) significantly altered the function of MTA1 in the regulation of E-cadherin expression. Alterations in E-cadherin expression changed the role of p-AKT in cellular malignant characters. All of these results demonstrate that MTA1 plays an important role in controlling the malignant transformation of prostate cancer cells through the p-AKT/E-cadherin pathway. This study also provides a new mechanistic role for MTA1 in the regulation of prostate cancer metastasis.     

In vivo knockdown of antisense non‐coding mitochondrial RNAs by a lentiviral‐encoded shRNA inhibits melanoma tumor growth and lung colonization

The family of non‐coding mitochondrial RNAs (ncmtRNA) is differentially expressed according to proliferative status. Normal proliferating cells express sense (SncmtRNA) and antisense ncmtRNAs (ASncmtRNAs), whereas tumor cells express SncmtRNA and downregulate ASncmtRNAs. Knockdown of ASncmtRNAs with oligonucleotides induces apoptotic cell death of tumor cells, leaving normal cells unaffected, suggesting a potential application for developing a novel cancer therapy. In this study, we knocked down the ASncmtRNAs in melanoma cell lines with a lentiviral‐encoded shRNA approach. Transduction with lentiviral constructs targeted to the ASncmtRNAs induced apoptosis in murine B16F10 and human A375 melanoma cells in vitro and significantly retarded B16F10 primary tumor growth in vivo. Moreover, the treatment drastically reduced the number of lung metastatic foci in a tail vein injection assay, compared to controls. These results provide additional proof of concept to the knockdown of ncmtRNAs for cancer therapy and validate lentiviral–shRNA vectors for gene therapy.     

Melanoma-induced suppression of tumor antigen-specific T cell expansion is comparable to suppression of global T cell expansion

We have observed that in vivo interaction between melanoma and resting T cells promotes suppression of antigen-driven proliferative T cell expansion. We hypothesized that this suppression would affect tumor antigen-specific T cell populations more potently than tumor-unrelated T cell populations. A B16F10 cell line was stably transfected to express low levels of the lymphocytic choriomeningitis virus (LCMV) glycoprotein GP33 (B16GP33). Mice bearing B16F10 or B16GP33 tumors were infected with LCMV, and proliferative expansion of LCMV epitope-specific T cell populations was quantified. In vitro and in vivo assays confirmed low levels of antigenic GP33 expression by B16GP33 tumors. Suppressed expansion of GP33-specific T cells was equivalent between mice bearing B16F10 and B16GP33 tumors. These observations suggest that the ability of growing melanoma tumors to impair antigen-driven proliferative expansion of activated T cells is global and not antigen-specific, and provide further insight into the influence of cancer on activated T cell homeostasis.     

An integrated analysis to predict micro‐RNAs targeting both stemness and metastasis in breast cancer stem cells

Several evidences support the idea that a small population of tumour cells representing self‐renewal potential are involved in initiation, maintenance, metastasis, and outcomes of cancer therapy. Elucidation of microRNAs/genes regulatory networks activated in cancer stem cells (CSCs) is necessary for the identification of new targets for cancer therapy. The aim of the present study was to predict the miRNAs pattern, which can target both metastasis and self‐renewal pathways using integration of literature and data mining. For this purpose, mammospheres derived from MCF‐7, MDA‐MB231, and MDA‐MB468 were used as breast CSCs model. They had higher migration, invasion, and colony formation potential, with increasing in stemness‐ and EMT‐related genes expression. Our results determined that miR‐204, ‐200c, ‐34a, and ‐10b contemporarily could target both self‐renewal and EMT pathways. This core regulatory of miRNAs could increase the survival rate of breast invasive carcinoma via up‐regulation of OCT4, SOX2, KLF4, c‐MYC, NOTCH1, SNAI1, ZEB1, and CDH2 and down‐regulation of CDH1. The majority of those target genes were involved in the regulation of pluripotency, MAPK, WNT, Hedgehog, p53, and transforming growth factor β pathways. Hence, this study provides novel insights for targeting core regulatory of miRNAs in breast CSCs to target both self‐renewal and metastasis potential and eradication of breast cancer.     

The Cardioprotective Protein Apolipoprotein A1 Promotes Potent Anti-tumorigenic Effects

Here, we show that apolipoprotein A1 (apoA1), the major protein component of high density lipoprotein (HDL), through both innate and adaptive immune processes, potently suppresses tumor growth and metastasis in multiple animal tumor models, including the aggressive B16F10L murine malignant melanoma model. Mice expressing the human apoA1 transgene (A1Tg) exhibited increased infiltration of CD11b⁺ F4/80⁺ macrophages with M1, anti-tumor phenotype, reduced tumor burden and metastasis, and enhanced survival. In contrast, apoA1-deficient (A1KO) mice showed markedly heightened tumor growth and reduced survival. Injection of human apoA1 into A1KO mice inoculated with tumor cells remarkably reduced both tumor growth and metastasis, enhanced survival, and promoted regression of both tumor and metastasis burden when administered following palpable tumor formation and metastasis development. Studies with apolipoprotein A2 revealed the anti-cancer therapeutic effect was specific to apoA1. In vitro studies ruled out substantial direct suppressive effects by apoA1 or HDL on tumor cells. Animal models defective in different aspects of immunity revealed both innate and adaptive arms of immunity contribute to complete apoA1 anti-tumor activity. This study reveals a potent immunomodulatory role for apoA1 in the tumor microenvironment, altering tumor-associated macrophages from a pro-tumor M2 to an anti-tumor M1 phenotype. Use of apoA1 to redirect in vivo elicited tumor-infiltrating macrophages toward tumor rejection may hold benefit as a potential cancer therapeutic.     

AMPK promotes survival of c‐Myc‐positive melanoma cells by suppressing oxidative stress

Although c‐Myc is essential for melanocyte development, its role in cutaneous melanoma, the most aggressive skin cancer, is only partly understood. Here we used the Nras^Q61KINK4a^?/? mouse melanoma model to show that c‐Myc is essential for tumor initiation, maintenance, and metastasis. c‐Myc‐expressing melanoma cells were preferentially found at metastatic sites, correlated with increased tumor aggressiveness and high tumor initiation potential. Abrogation of c‐Myc caused apoptosis in primary murine and human melanoma cells. Mechanistically, c‐Myc‐positive melanoma cells activated and became dependent on the metabolic energy sensor AMP‐activated protein kinase (AMPK), a metabolic checkpoint kinase that plays an important role in energy and redox homeostasis under stress conditions. AMPK pathway inhibition caused apoptosis of c‐Myc‐expressing melanoma cells, while AMPK activation protected against cell death of c‐Myc‐depleted melanoma cells through suppression of oxidative stress. Furthermore, TCGA database analysis of early‐stage human melanoma samples revealed an inverse correlation between C‐MYC and patient survival, suggesting that C‐MYC expression levels could serve as a prognostic marker for early‐stage disease.     

Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells

Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H₂O₂ treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H₂O₂-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47^phox. Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol.     

Human ribosomal protein S13 promotes gastric cancer growth through down‐regulating p27Kip1

Our previous works revealed that human ribosomal protein S13 (RPS13) was up‐regulated in multidrug‐resistant gastric cancer cells and overexpression of RPS13 could protect gastric cancer cells from drug‐induced apoptosis. The present study was designed to explore the role of RPS13 in tumorigenesis and development of gastric cancer. The expression of RPS13 in gastric cancer tissues and normal gastric mucosa was evaluated by immunohistochemical staining and Western blot analysis. It was found RPS13 was expressed at a higher level in gastric cancer tissues than that in normal gastric mucosa. RPS13 was then genetically overexpressed in gastric cancer cells or knocked down by RNA interference. It was demonstrated that up‐regulation of RPS13 accelerated the growth, enhanced in vitro colony forming and soft agar cologenic ability and promoted in vivo tumour formation potential of gastric cancer cells. Meanwhile, down‐regulation of RPS13 in gastric cancer cells resulted in complete opposite effects. Moreover, overexpression of RPS13 could promote G1 to S phase transition whereas knocking down of RPS13 led to G1 arrest of gastric cancer cells. It was further demonstrated that RPS13 down‐regulated p27^kip1 expression and CDK2 kinase activity but did not change the expression of cyclin D, cyclin E, CDK2, CDK4 and p16^INK4A. Taken together, these data indicate that RPS13 could promote the growth and cell cycle progression of gastric cancer cells at least through inhibiting p27^kip1 expression.     

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.