Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKCα and PKCδ phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.     

Inhibitory effect of luteolin on hepatocyte growth factor/scatter factor-induced HepG2 cell invasion involving both MAPK/ERKs and PI3K-Akt pathways

Hepatocyte growth factor (HGF), also known as scatter factor (SF), and its receptor, the c-Met tyrosine kinase, play roles in cancer invasion and metastasis in a wide variety of tumor cells. Clinical observations suggest that HGF can promote metastasis of hepatoma cells while stimulating tumor invasiveness. We use HGF as an invasive inducer of human hepatoma HepG2 cells to investigate the effect of flavonoids on anti-invasion. In our preliminary study, we investigated the effect of flavonoids including luteolin, quercetin, baicalein, genistein, taxifolin and catechin on HGF-mediated migration and invasion of HepG2 cells. We found that luteolin presented the most potent potential on anti-migration and anti-invasion by Boyden chamber assay. Furthermore, luteolin inhibited HGF-induced cell scattering and cytoskeleton change such as filopodia and lamellipodia was determined by both phase-contrast and fluorescence microscopy studies. In addition, Western blotting and immunoprecipitation were performed to confirm luteolin suppressed the phosphorylation of c-Met, the membrane receptor of HGF, as well as ERK1/2 and Akt, but not JNK1/2, which is activated by HGF. Our investigation demonstrated that luteolin similar to PD98059, which acts as a specific inhibitor of MEK, an up stream kinase regulating ERK1/2, and wortmannin, a PI3K inhibitor, inhibited the invasiveness induced by HGF. In conclusion, the luteolin inhibited HGF-induced HepG2 cell invasion involving both MAPK/ERKs and PI3K-Akt pathways.     

Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation

Pancreatic cancer is characterized by aggressive local invasion and early metastasis formation. Active migration of the pancreatic cancer cells is essential for these processes. We have shown previously that the pancreatic cancer cells lines CFPAC1 and IMIM-PC2 show high migratory activity, and we have investigated herein the reason for this observation. Cell migration was assessed using a three-dimensional, collagen-based assay and computer-assisted cell tracking. The expression of receptor tyrosine kinases was determined by flow-cytometry and cytokine release was measured by an enzyme-linked immunoassay. Receptor function was blocked by antibodies or pharmacological enzyme inhibitors. Both cells lines express the epidermal growth factor receptor (EGFR) as well as its family-member ErbB2 and the platelet-derived growth factor receptor (PDGFR)α, whereas only weak expression was detected for ErbB3 and no expression of PDGFRβ. Pharmacological inhibition of the EGFR or ErbB2 significantly reduced the migratory activity in both cell lines, as did an anti-EGFR antibody. Interestingly, combination of the latter with an anti-PDGFR antibody led to an even more pronounced reduction. Both cell lines release detectable amounts of EGF. Thus, the high migratory activity of the investigated pancreatic cancer cell lines is due to autocrine EGFR activation and possibly of other receptor tyrosine kinases.     

Crosstalk of tight junction components with signaling pathways

Tight junctions (TJs) regulate the passage of ions and molecules through the paracellular pathway in epithelial and endothelial cells. TJs are highly dynamic structures whose degree of sealing varies according to external stimuli, physiological and pathological conditions. In this review we analyze how the crosstalk of protein kinase C, protein kinase A, myosin light chain kinase, mitogen-activated protein kinases, phosphoinositide 3-kinase and Rho signaling pathways is involved in TJ regulation triggered by diverse stimuli. We also report how the phosphorylation of the main TJ components, claudins, occludin and ZO proteins, impacts epithelial and endothelial cell function.     

MicroRNA-181a suppresses salivary adenoid cystic carcinoma metastasis by targeting MAPK–Snai2 pathway

Background

To date microRNAs and their contribution to the onset and propagation of salivary adenoid cystic carcinoma (SACC) are limited. The objective of this study was to identify miR-181a and its mechanism in the metastasis of SACC.

Methods

At first microarray and quantitative RT-PCR were used to investigate microRNA profiles and miR-181a in paired SACC cell lines with different metastatic potential. Then the effect of miR-181a on metastatic potential of SACC was investigated. MiR-181a target genes and Snai2 promoter activity were investigated using luciferase reporter gene assays. Western blot was used to detect MAPK–Snai2 pathway-related protein level.

Results

A panel of deregulated microRNAs (including miR-181a) was identified in paired of SACC cell lines. Functional analysis indicated that miR-181a inhibited SACC cell migration, invasion and proliferation in vitro, and it suppressed tumor growth and lung metastasis in vivo. Direct targeting of miR-181a to MAP2K1, MAPK1 and Snai2 was confirmed by luciferase reporter gene assays. MiR-181a mimic inhibited the expression of MAP2K1, MAPK1 and Snai2 in SACC cells. MAP2K1 or MAPK1 siRNA suppressed Snai2 gene promoter activity and reduced Snai2 expression and the metastatic potential of SACC cells.

Conclusions

Our results indicate that miR-181a plays an important role in the metastasis of SACC, and may serve as a novel therapeutic target for SACC. MiR-181a regulates the MAPK–Snai2 pathway both through direct cis-regulatory mechanism and through indirect trans-regulatory mechanism.

General significance

To our knowledge, this is the first study revealing that miR-181a deregulation mediated the metastasis of SACC by regulating MAPK–Snai2 pathway.     

CUB-domain containing protein 1 represses the epithelial phenotype of pancreatic cancer cells

The prognosis of pancreatic cancer is dismal due to the frequent metastasis and invasion to surrounding organs. Numerous molecules are involved in the malignant behavior of pancreatic cancer cells, but the entire process remains unclear. Several reports have suggested that CUB-domain containing protein-1 (CDCP1) is highly expressed in pancreatic cancer, but its impact on the invasive growth and the upstream regulator remain elusive. To clarify the role of CDCP1 in pancreatic cancer, we here examined the effects of CDCP1 knockdown on the cell behaviors of pancreatic cancer cells. Knockdown of CDCP1 expression in Panc-1 resulted in reduced cellular migration accompanied by the increased expression of E-cadherin and decreased expression of N-cadherin. Knockdown of CDCP1 attenuated the spheroid formation and resistance against gemcitabine, which are some of the cancer stem cell-related phenotypes. Bone morphogenetic protein 4 (BMP4) was found to induce CDCP1 expression via the extracellular signal regulated kinase pathway, suggesting that CDCP1 has a substantial role in the BMP4-induced epithelial-mesenchymal transition. These results indicate that CDCP1 represses the epithelial phenotype of pancreatic cancer cells.     

KITENIN increases invasion and migration of mouse squamous cancer cells and promotes pulmonary metastasis in a mouse squamous tumor model

KAI1 C-terminal interacting tetraspanin (KITENIN) is reported to promote metastasis in mouse colon cancer models. We investigated the role of KITENIN on the progression of squamous cell carcinoma (SCC). In a preliminary clinical study using resected tissues from head and neck SCC patients, KITENIN was highly expressed in tumors and metastatic lymph nodes, while KAI1 was more increased in adjacent mucosa than in tumor. KITENIN-transfected mouse squamous cancer (SCC VII/KITENIN) cells showed significantly higher invasion, migration, and proliferation than empty vector-transfected cells. In syngeneic mouse squamous tumor models, more increased tumor volume and enhanced lung metastasis were found in SCC VII/KITENIN cells-injected mice. Thus, KITENIN increases invasion and migration of squamous cancer cells and thereby promotes distant metastasis in mouse squamous tumor models.     

TGF-β1 inhibits the growth and metastasis of tongue squamous carcinoma cells through Smad4

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that regulates cell growth, differentiation, migration, apoptosis and extracellular matrix remodeling. TGF-β1 transduces signals from the cell membrane to the cell nucleus through serine/threonine kinase receptors and their downstream effectors, Smad molecules. Although many studies have been focused on TGF-β1-Smad signaling pathway, the role of TGF-β1/Smad in tongue squamous cell carcinoma is not fully understood. In the present study, we used a series of cell function assays to examine the role of TGF-β-Smad4 signaling in tongue squamous cell carcinoma. We observed the effects of TGF-β1 on the growth and metastatic potential of the tongue squamous cell carcinoma cell line Ts, which expresses lower level of Smad4 protein. We found that Smad4 could decrease TGF-β1-induced cell proliferation, and that Smad4 overexpression promoted Ts cell apoptosis. In Ts vector control cells, TGF-β1 increased the expression of TβRII, as well as MMP-2, and enhanced cell invasion through the basement membrane, and then induced cell metastasis. However in Ts cells stably expressing Smad4, Smad4 mediated TGF-β1-induced p21 expression promoted cell apoptosis and inhibited cell proliferation, delayed MMP-2 expression, and decreased cell metastasis. Therefore, TGF-β1 plays distinct roles in the Smad4-dependent and -independent signaling pathways.     

乳腺导管原位癌和浸润性导管癌中MMP-7、VEGF及E-cad的表达及临床意义

目的:探讨乳腺导管原位癌(DCIS)和浸润性导管癌(IDC)中基质金属蛋白酶-7(MMP-7)、血管内皮生长因子(VEGF)及钙黏附素E(E-cad)的表达及临床意义。方法:选取2012年1月-2017年8月期间鄂东医疗集团黄石市中心医院乳甲外科的DCIS石蜡包埋标本(DCIS组)59例,IDC石蜡包埋标本(IDC组)32例,另选取同时期正常乳腺组织标本20例为对照组,检测各组MMP-7、VEGF及E-cad的表达情况,并分析MMP-7、VEGF及E-cad的阳性表达率与DCIS、IDC患者临床病理特征的关系,采用Pearson相关性分析MMP-7、VEGF与E-cad之间的相关性。结果:DCIS组、IDC组的MMP-7、VEGF阳性表达率高于对照组,E-cad的强阳性表达率低于对照组(P0.05),DCIS组与IDC组之间的MMP-7、VEGF、E-cad阳性表达率比较差异无统计学意义(P0.05)。MMP-7、VEGF及E-cad的阳性表达率均与患者的年龄、肿瘤大小无关(P0.05),临床分期为Ⅱ-Ⅲ期、中/低分化程度、有淋巴结转移患者的MMP-7、VEGF的阳性表达率高于临床分期为Ⅰ期、高分化程度、无淋巴结转移患者(P0.05),中/低分化程度、有淋巴结转移患者的E-cad的阳性表达率低于高分化程度、无淋巴结转移患者(P0.05)。经Pearson相关性分析显示,MMP-7与VEGF存在正相关关系(r=0.362,P=0.038),MMP-7、VEGF均与E-cad无显著相关性(r=0.071、0.024,P=0.057、0.089)。结论:DCIS和IDC中MMP-7、VEGF表达较高,E-cad表达较低,且与患者临床分期、分化程度、淋巴结转移有关,临床上可以通过检查MMP-7、VEGF、E-cad的表达来评估乳腺癌的发生及发展。     

Association Between the Rat Homologue of CO-029, a Metastasis-associated Tetraspanin Molecule and Consumption Coagulopathy

Recently, we have described a panel of metastasis-associated antigens in the rat, i.e., of molecules expressed on metastasizing, but not on nonmetastasizing tumor lines. One of these molecules, recognized by the monoclonal antibody D6.1 and named accordingly D6.1A, was found to be abundantly expressed predominantly on mesenchyme-derived cells. The DNA of the antigen has been isolated and cloned. Surprisingly, the gene product proved to interfere strongly with coagulation.

The 1.182-kb cDNA codes for a 235–amino acid long molecule with a 74.2% homology in the nucleotide and a 70% homology in the amino acid sequence to CO-029, a human tumor-associated molecule. According to the distribution of hydrophobic and hydrophilic amino acids, D6.1A belongs to the tetraspanin superfamily. Western blotting of D6.1A-positive metastasizing tumor lines revealed that the D6.1A, like many tetraspanin molecules, is linked to further membrane molecules, one of which could be identified as α6β1 integrin. Transfection of a low-metastasizing tumor cell line with D6.1A cDNA resulted in increased metastatic potential and provided a clue as to the functional role of D6.1A. We noted massive bleeding around the metastases and, possibly as a consequence, local infarctions predominantly in the mesenteric region and all signs of a consumption coagulopathy. By application of the D6.1 antibody the coagulopathy was counterregulated, though not prevented.

It has been known for many years that tumor growth and progression is frequently accompanied by thrombotic disorders. Our data suggest that the phenomenon could well be associated with the expression of tetraspanin molecules.

     

Serum midkine depends on lymph node involvement and correlates with circulating VEGF-C in oesophageal squamous cell carcinoma

Lymph node metastasis (LNM) is a key factor for selection of treatment method and patients’ prognosis in oesophageal squamous cell carcinoma (ESCC). However, no biomarkers able to support the clinical detection of LNM have been reported. Recently, vascular endothelial growth factor C (VEGF-C) was found to be a more accurate marker of LNM in lung cancer than computed tomography. Midkine is a multifunctional cytokine involved in cancer development. We investigated circulating midkine levels in ESCC patients (n=73) compared with those in healthy subjects (n=42) with double-antibody-sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA). We found that midkine was elevated in ESCC and involved in metastatic disease. Serum midkine (sMK) was a good marker of LNM, evaluated both clinically and pathologically, as revealed by ROC analysis. It also correlated with serum levels of VEGF-C. The increase of sMK was related to cancer cells, although a weak correlation was observed between sMK and platelet and leucocyte counts.     

Serum midkine depends on lymph node involvement and correlates with circulating VEGF-C in oesophageal squamous cell carcinoma

Lymph node metastasis (LNM) is a key factor for selection of treatment method and patients' prognosis in oesophageal squamous cell carcinoma (ESCC). However, no biomarkers able to support the clinical detection of LNM have been reported. Recently, vascular endothelial growth factor C (VEGF-C) was found to be a more accurate marker of LNM in lung cancer than computed tomography. Midkine is a multifunctional cytokine involved in cancer development. We investigated circulating midkine levels in ESCC patients (n=73) compared with those in healthy subjects (n=42) with double-antibody-sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA). We found that midkine was elevated in ESCC and involved in metastatic disease. Serum midkine (sMK) was a good marker of LNM, evaluated both clinically and pathologically, as revealed by ROC analysis. It also correlated with serum levels of VEGF-C. The increase of sMK was related to cancer cells, although a weak correlation was observed between sMK and platelet and leucocyte counts.     

The Molecular Basis of the Caskin1 and Mint1 Interaction with CASK

Calcium/calmodulin-dependent serine protein kinase (CASK) is a conserved multi-domain scaffolding protein involved in brain development, synapse formation, and establishment of cell polarity. To accomplish these diverse functions, CASK participates in numerous protein-protein interactions. In particular, CASK forms competing CASK/Mint1/Velis and CASK/Caskin1/Velis tripartite complexes that physically associate with the cytoplasmic tail of neurexin, a transmembrane protein enriched at presynaptic sites. This study shows that a short linear EEIWVLRK peptide motif from Caskin1 is necessary and sufficient for binding CASK. We also identified the conserved binding site for the peptide on the CASK calmodulin kinase domain. A related EPIWVMRQ peptide from Mint1 was also discovered to be sufficient for binding. Searching all human proteins for the Mint1/Caskin1 consensus peptide ExIWVxR revealed that T-cell lymphoma invasion and metastasis 1 (TIAM1) contains a conserved EEVIWVRRE peptide that was also found to be sufficient for CASK binding in vitro. TIAM1 is well known for its role in tumor metastasis, but it also possesses overlapping cellular and neurological functions with CASK, suggesting a previously unknown cooperation between the two proteins. This new peptide interaction motif also explains how Caskin1 and Mint1 form competing complexes and suggests a new role for the cellular hub protein CASK.     

MicroRNA-143 functions as a tumor suppressor in human esophageal squamous cell carcinoma

Esophageal cancer is one of the most common cancers worldwide with a poor prognosis. MicroRNAs(miRNAs) are a class of naturally occurring small noncoding RNAs and play an important role in cancer initiation and development. In this study, we demonstrate that the expression levels of miR-143 and miR-145 were significantly decreased in ESCC tissues in comparison with adjacent normal esophageal squamous tissues(NESTs). Furthermore, an inverse correlation between miR-143 and tumor invasion depth and lymph node metastasis was observed. The enforced expression of miR-143 induced growth suppression and apoptosis of ESCC cells. Rescue of miR-143 significantly suppressed the ESCC cells migration and invasion capabilities. Moreover, we show that functions of miR-143 in ESCC are mediated at least in part by the inhibition of extracellular signal regulated kinase-5(ERK-5) activity. These results prove that miR-143 may act as a tumor suppressor in ESCC.     

Mechanisms of resistance to HER family targeting antibodies

The epidermal growth factor (EGF) family of receptor tyrosine kinases consists of four members: EGFR (HER1/ErbB1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Receptor activation via ligand binding leads to downstream signaling that influence cell proliferation, angiogenesis, invasion and metastasis. Aberrant expression or activity of EGFR and HER2 have been strongly linked to the etiology of several human epithelial cancers including but not limited to head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and breast cancer. With this, intense efforts have been made to inhibit the activity of the EGFR and HER2 by designing antibodies against the ligand binding domains (cetuximab, panitumumab and trastuzumab) or small molecules against the tyrosine kinase domains (erlotinib, gefitinib, and lapatinib). Both approaches have shown considerable clinical promise. However, increasing evidence suggests that the majority of patients do not respond to these therapies, and those who show initial response ultimately become refractory to treatment. While mechanisms of resistance to tyrosine kinase inhibitors have been extensively studied, resistance to monoclonal antibodies is less well understood, both in the laboratory and in the clinical setting. In this review, we discuss resistance to antibody-based therapies against the EGFR and HER2, similarities between these resistance profiles, and strategies to overcome resistance to HER family targeting monoclonal antibody therapy.     

Cell cycle arrest or survival signaling through αv integrins,activation of PKC and ERK1/2 lead to anoikis resistance of ovarian cancer spheroids

Ovarian cancer is the most lethal gynecologic cancer mainly due to spheroids organization of cancer cells that disseminate within the peritoneal cavity. We have investigated the molecular mechanisms by which ovarian cancer spheroids resist anoikis, choosing as models the 2 well-characterized human ovarian cancer cell lines IGROV1 and SKOV3. These cell lines have the propensity to float as clusters, and were isolated from tumor tissue and ascites, respectively. To form spheroids, IGROV1 and SKOV3 ovarian adenocarcinoma cells were maintained under anchorage-independent culture conditions, in which both lines survive at least a week. A short apoptotic period prior to a survival signaling commitment was observed for IGROV1 cells whereas SKOV3 cells entered G0/G1 phase of the cell cycle. This difference in behavior was due to different signals. With regard to SKOV3 cells, activation of p38 and an increase in p130/Rb occurred once anchorage-independent culture was established. Analyses of the survival signaling pathway switched on by IGROV1 cells showed that activation of ERK1/2 was required to evade apoptosis, an effect partly dependent on PKC activation and αv integrins. αv-integrin expression is essential for survival through activation of ERK1/2 phosphorylation.     

Autophagy triggered by magnolol derivative negatively regulates angiogenesis

Angiogenesis has a key role in the tumor progression and metastasis; targeting endothelial cell proliferation has emerged as a promising therapeutic strategy for the prevention of cancer. Previous studies have revealed a complex association between the process of angiogenesis and autophagy and its outcome on tumorigenesis. Autophagy, also known as type-II cell death, has been identified as an alternative way of cell killing in apoptotic-resistant cancer cells. However, its involvement in chemoresistance and tumor promotion is also well known. In this study, we used a derivate of natural product magnolol (Ery5), a potent autophagy inducer, to study the association between the autophagy and angiogenesis in both in vitro and in vivo model system. We found that the robust autophagy triggered by Ery5, inhibited angiogenesis and caused cell death independent of the apoptosis in human umbilical cord vein endothelial cells and PC-3 cells. Ery5 induced autophagy effectively inhibited cell proliferation, migration, invasion and tube formation. We further demonstrated that Ery5-mediated autophagy and subsequent inhibition of angiogenesis was reversed when autophagy was inhibited through 3-methyl adenine and knocking down of key autophagy proteins ATG7 and microtubule-associated protein light chain 3. While evaluating the negative regulation of autophagy on angiogenesis, it was interesting to find that angiogenic environment produced by the treatment of VEGF and CoCl2 remarkably downregulated the autophagy and autophagic cell death induced by Ery5. These studies, while disclosing the vital role of autophagy in the regulation of angiogenesis, also suggest that the potent modulators of autophagy can lead to the development of effective therapeutics in apoptosis-resistant cancer.     

Dihydroartemisinin induces apoptosis in human gastric cancer cell line BGC-823 through activation of JNK1/2 and p38 MAPK signaling pathways

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, is associated with a broad range of biological properties including antitumor activity. However, the effect of DHA on gastric cancer has not been clearly clarified. The aim of this study was to investigate the role and mechanism of DHA in human gastric cancer cell line BGC-823. Cell viability was assessed by MTT assay. Cell apoptosis was analyzed with flow cytometry. The expressions of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and their phosphorylated forms as well as apoptosis related proteins were examined by western blot analysis. The results demonstrated that DHA inhibited cell viability of BGC-823 cells in a dose- and time-dependent manner. DHA treatment upregulated the expression of Bax, cleaved caspase-3 and -9, and degraded form of PARP, and downregulated the Bcl-2 expression and Bcl-2/Bax ratio. Meanwhile, DHA increased the phosphorylation of ERK1/2, JNK1/2 and p38 MAPK. Synthetic inhibitors of JNK1/2 or p38 MAPK kinase activity, but not inhibitor of ERK1/2, significantly abolished the DHA-induced activation of caspase-3 and -9. In vivo tumor-suppression assay further indicated that DHA displayed significant inhibitory effect on BGC-823 xenografts in tumor growth. These results indicate that DHA induces apoptosis of BGC-823 cells through JNK1/2 and p38 MAPK signaling pathways and DHA could serve as a potential additional chemotherapeutic agent for treatment of gastric cancer.