Syntaxins 3 and 4 mediate vesicular trafficking of α5β1 and α3β1 integrins and cancer cell migration

Integrins, a family of heterodimeric receptors for cell adhesion to the extracellular matrix (ECM), play key roles in cell migration, cancer progression and metastasis. As transmembrane proteins, integrins are transported in vesicles and delivered to the cell surface by vesicular trafficking. The final step for integrin delivery, i.e., fusion of integrin-containing vesicles with the plasma membrane, is poorly understood at the molecular level. The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins syntaxins 1, 2, 3 and 4 are present at the plasma membrane to drive vesicle fusion. In this study, we examined the roles of syntaxins?1, 2, 3 and 4 in vesicular trafficking of α5β1 and α3β1 integrins. We showed that syntaxins?2, 3 and 4 were expressed in HeLa cervical adenocarcinoma cells and PANC-1 pancreatic adenocarcinoma cells. In migrating HeLa and PANC-1 cells, syntaxins?2, 3 and 4 co-localized with the lipid raft constituent GM1 ganglioside at the leading edge. siRNA knockdown (KD) of syntaxins?3 and 4, but not of syntaxin?2, in HeLa cells reduced cell surface expression of α5β1 and α3β1 integrins and accumulated the integrins in cytoplasmic vesicles, indicating that syntaxins?3 and 4 mediate vesicular trafficking of α5β1 and α3β1 integrins to the cell surface. In addition, KD of syntaxins?3 and 4 inhibited cell adhesion to fibronectin, suppressed chemotactic cell migration and triggered apoptosis. Collectively, these data suggest that syntaxins?3- and 4-dependent integrin trafficking is important in cancer cell migration and survival, and may be a valuable target for cancer therapy.     

β-adrenoceptor signaling and its control of cell replication in MDA-MB-231 human breast cancer cells

MDA-MB-231 human breast cancer cells express high -adrenoceptor levels, predominantly the ₂ subtype. Receptor stimulation by isoproterenol evoked immediate reductions in DNA synthesis which were blocked completely by propranolol and were of the same magnitude as effects elicited by high concentrations of 8-Br-cAMP. Isoproterenol-induced inhibition of DNA synthesis was maintained throughout several days of exposure, resulting in a decrement in total cell number, and the effects were augmented by cotreatment with dexamethason; an even greater effect was seen when cAMP breakdown was inhibited by theophylline, with or without addition of isoproterenol. Despite the persistent effect of isoproterenol, receptor downregulation was evident with as little as 1 h of treatment, and over 90% of the receptors were lost within 24 h. Receptor downregulation was paralleled by homologous desensitization of the adenylyl cyclase response to -adrenoceptor stimulation. Dexamethasone augmented the effects of isoproterenol on DNA synthesis but did not prevent receptor downregulation or desensitization. These results indicate that -adrenoceptors are effectively linked, through cAMP, to the termination of cell replication in MDA-MB-231 human breast cancer cells, and that activation of only a small number of receptors is sufficient for a maximal effect. Novel pharmacologic strategies that focus on cell surface receptors operating through adenylyl cyclase may offer opportunities to combat cancers that are unresponsive to hormonal agents, or that have developed multidrug resistance.     

The α6β1 and α6β4 integrins in human prostate cancer progression

Summary Prostatic secretions are formed by glands composed of basal and luminal cells and surrounded by a basal lamina. The normal basal cells express several integrins (extracellular matrix receptors) including alpha 2, 3, 4, 5, 6, v, beta 1 and beta 4. These integrin units are polarized at the base of the cells adjacent to the basal lamina. The integrin alpha 6 beta 4 is associated with hemidesmosomal-like structures.The natural history of prostate cancer involves the presence of prostatic intraepithelial neoplasia (PIN) lesions (considered precursor lesions), carcinomain situ and invasive carcinoma. Hemidesmosomal proteins and the 31 and 61 integrins (laminin receptors) are retained in the early PIN lesions. Expression of the integrins 2, 4, 5, v and 4 is lost in carcinoma. The 31 and 61 integrins remain associated with invasive carcinoma, the latter being predominant. Integrin expression in carcinoma is diffuse in the plasma membrane and not restricted to the basal aspects of the cell. The 61 integrin is fully functional as judged by an ability to adhere to laminin and contains the wild type 6A cytoplasmic signaling domain. The 61 integrin is a leading candidate for conferring the invasive phenotype in prostatic carcinoma.Tumor cells with high expression of 6 integrin are more invasive when tested in a SCID mouse model system. Following intraperitoneal injection, the human tumor cells invade the mouse diaphragm and move through the muscle on the surface of the laminin coated muscle cells. Our current working hypothesis is that the production of 61 and laminin in human tumor cells contributes to the invasive phenotype. Invasion could occur on the surfaces of laminin coated structures such as the nerves, blood vessels or muscle and account for the known patterns of human prostate tumor progression. Blockage of the expression or function of 61 or laminin or preventing the loss of 4 would be essential steps in confining the carcinoma to the prostate gland where conventional treatment has already proven effective.     

Quantification of thrombospondin-1 secretion and expression of αvβ3 and α3β1 integrins and syndecan-1 as cell-surface receptors for thrombospondin-1 in malignant glioma cells

Malignant glioma cells secrete thrombospondin-1 (TSP-1) which participates in the motility of glioma cells, and binds to cell surface v3 and 31 integrins, and syndecan-1. This study evaluated the amount of TSP-1 secretion from malignant glioma cells, and the expression of v3 and 31 integrins, and syndecan-1. The amounts of TSP-1 in the supernatants from 10 malignant glioma cell lines and eight non-glioma malignant tumor cell lines were measured by enzyme-linked immunosorbent assay. Expression of v3 and 31 integrins, and syndecan-1 were examined by flow cytometry. The amounts of TSP-1 secreted by malignant glioma cells were 43 to 2431 ng/1 × 10⁶ cells/24 h (mean ± SD=626 ± 792). Seven of 10 glioma cell lines secreted more than 100 ng of TSP-1 and three of these cell lines secreted more than 1 g. Seven of eight non-glioma cell lines secreted less than 100 0ng of TSP-1. All glioma cell lines expressed 31 integrin and syndecan-1, and seven of 10 glioma cell lines expressed v3 integrin. Treatment of the glioma cell lines with TGF-2 did not change the expression of v3 integrin. These results suggest that malignant glioma cells secrete high levels of TSP-1, which may be important in the migration of glioma cells via interactions with v3 and 31 integrins, and syndecan-1.     

S100P interacts with integrin α7 and increases cancer cell migration and invasion in lung cancer

S100P, a Ca2⁺ binding protein, has been shown to be overexpressed in various cancers. However, its functional character in lung cancer remains largely unknown. In this study, we show that S100P increases cancer migration, invasion and metastasis in lung cancer cells. Ectopic expression of S100P increases migration, invasion and EMT in less invasive CL1-0 lung cancer cells. Conversely, knockdown of S100P suppressed migration and invasion, and caused a reversion of EMT in highly invasive lung cancer cells. These effects were transduced by increasing the interaction of S100P with integrin α7, which activated focal adhesion kinase (FAK) and AKT. Blocking FAK significantly decreased S100P-induced migration by decreasing Src and AKT activation, whereas inhibiting AKT reduced S100P upregulation on ZEB1 expression. Further study has indicated that S100P knockdown prevents the spread of highly metastatic human lung cancer in animal models. This study therefore suggests that S100P represents a critical activator of lung cancer metastasis. Detection and targeted treatment of S100P-expressing cancer is an attractive therapeutic strategy in treating lung cancer.     

Fibroblast surface-associated FGF-2 promotes contact-dependent colorectal cancer cell migration and invasion through FGFR-SRC signaling and integrin αvβ5-mediated adhesion

Carcinoma-associated fibroblasts were reported to promote colorectal cancer (CRC) invasion by secreting motility factors and extracellular matrix processing enzymes. Less is known whether fibroblasts may induce CRC cancer cell motility by contact-dependent mechanisms. To address this question we characterized the interaction between fibroblasts and SW620 and HT29 colorectal cancer cells in 2D and 3D co-culture models in vitro. Here we show that fibroblasts induce contact-dependent cancer cell elongation, motility and invasiveness independently of deposited matrix or secreted factors. These effects depend on fibroblast cell surface-associated fibroblast growth factor (FGF) -2. Inhibition of FGF-2 or FGF receptors (FGFRs) signaling abolishes these effects. FGFRs activate SRC in cancer cells and inhibition or silencing of SRC in cancer cells, but not in fibroblasts, prevents fibroblasts-mediated effects. Using an RGD-based integrin antagonist and function-blocking antibodies we demonstrate that cancer cell adhesion to fibroblasts requires integrin α_vβ₅. Taken together, these results demonstrate that fibroblasts induce cell-contact-dependent colorectal cancer cell migration and invasion under 2D and 3D conditions in vitro through fibroblast cell surface-associated FGF-2, FGF receptor-mediated SRC activation and α_vβ₅ integrin-dependent cancer cell adhesion to fibroblasts. The FGF-2-FGFRs-SRC-α_vβ₅ integrin loop might be explored as candidate therapeutic target to block colorectal cancer invasion.     

Interleukin-8 promotes cell migration through integrin αvβ6 upregulation in colorectal cancer

Colorectal cancer (CRC), which is notorious for high morbidity and mortality around the world, shows a predilection for metastasis to liver. Interleukin-8 (IL-8), a chemokine with a defining CXC amino acid motif, has been reported to promote CRC cell migration and is associated with poor prognosis of CRC. However, the underlying molecular mechanism of IL-8-mediated migration remains obscure. In this study, we first demonstrated the cross talk between IL-8 and integrin αvβ6. We analyzed 139 human CRC samples, and found that the immunohistochemical expression of αvβ6 was significantly correlated with expression of IL-8. Furthermore, IL-8 increased the migration through integrin αvβ6 in human CRC cells, and both CXCR1 and CXCR2 were primarily involved during the process. IL-8 upregulated αvβ6 expression in a dose-dependent manner through activation of ERK and Ets-1 signaling pathway. Taken together, our results indicated that IL-8 enhances the migration of CRC cells by increasing αvβ6 integrin expression through the ERK/Ets-1 pathway. Targeting integrin αvβ6 in IL-8 expressing tumors might be a potential therapeutic strategy for CRC patients.     

CD151 promotes α3β1 integrin-dependent organization of carcinoma cell junctions and restrains collective cell invasion

Integrins function in collective migration both as major receptors for extracellular matrix and by crosstalk to adherens junctions. Despite extensive research, important questions remain about how integrin signaling mechanisms are integrated into collective migration programs. Tetraspanins form cell surface complexes with a subset of integrins and thus are good candidates for regulating the balance of integrin functional inputs into cell-matrix and cell-cell interactions. For example, tetraspanin CD151 directly associates with α3β1 integrin in carcinoma cells and promotes rapid α3β1-dependent single cell motility, but CD151 also promotes organized adherens junctions and restrains collective carcinoma cell migration on 2D substrates. However, the individual roles of CD151s integrin partners in CD151s pro-junction activity in carcinoma cells were not well understood. Here we find that CD151 promotes organized carcinoma cell junctions via α3β1 integrin, by a mechanism that requires the a3b1 ligand, laminin-332. Loss of CD151 promotes collective 3D invasion and growth in vitro and in vivo, and the enhanced invasion of CD151-silenced cells is α3 integrin dependent, suggesting that CD151 can regulate the balance between α3β1s pro-junction and pro-migratory activities in collective invasion. An analysis of human cancer cases revealed that changes in CD151 expression can be linked to either better or worse clinical outcomes depending on context, including potentially divergent roles for CD151 in different subsets of breast cancer cases. Thus, the role of the CD151-α3β1 complex in carcinoma progression is context dependent, and may depend on the mode of tumor cell invasion.     

TNF-��/NF-��B/Snail pathway in cancer cell migration and invasion

Tumour necrosis factor-alpha (TNF-α) is an important inflammatory factor that acts as a master switch in establishing an intricate link between inflammation and cancer. A wide variety of evidence has pointed to a critical role of TNF-α in tumour proliferation, migration, invasion and angiogenesis. The function of TNF-α as a key regulator of the tumour microenvironment is well recognised. We will emphasise the contribution of TNF-α and the nuclear factor-κB pathway on tumour cell invasion and metastasis. Understanding the mechanisms underlying inflammation-mediated metastasis will reveal new therapeutic targets for cancer prevention and treatment.     

Regulation of p53 and cell proliferation by resveratrol and its derivatives in breast cancer cells: an in silico and biochemical approach targeting integrin αvβ3

Resveratrol is a grape polyphenol with cancer preventative activities in tissue culture and animal model studies. Potential of resveratrol as a broad-based chemopreventive agent have been questioned by its limited bioavailability. The bioefficacy of resveratrol was compared with its derivatives, triacetyl-resveratrol (trans-3,5,4'-triacetylstilbene) and trimethoxy-resveratrol (trans-3,5,4'-trimethoxystilbene) in both estrogen receptor-α (ERα)-positive MCF-7 and ERα-negative MDA-MB-231 breast cancer cells. Binding to integrin αvβ3 and control of cell proliferation and p53 were chosen as targets for comparative analysis using an in silico and biochemical approach. Resveratrol and triacetyl-resveratrol interacted avidly and specifically with integrin αvβ3 through binding at the site targeted by the high affinity cyclic Arg-Gly-Asp (RGD) peptide. In contrast, binding of trimethoxy-resveratrol to this site was substantially less robust. Moreover, the different stilbenes also elicited diverse cellular and signaling responses in MCF-7 and MDA-MB-231 cells, as evidenced by analysis of colony formation, cell proliferation, cell cycle phase transition, the extent of phosphorylation of p53 at Ser15 and p53-inducible proteins, p21 and p53R2, respectively. Further, stilbene-elicited signaling cascade leading to p53 activation was examined in MCF-7 cells and results showed that resveratrol and triacetyl-resveratrol induced both ERK and p38 phosphorylation, whereas only marginal changes in state of phosphorylation in these two kinases were observed in trimethoxy-resveratrol-treated cells. Taken together, these results support that resveratrol and triacetyl-resveratrol regulate proliferation and gene expression in breast cancer cells by utilizing largely similar signaling molecules and pathways and cellular events, which appear quite distinct from those targeted by trimethoxy-resveratrol.     

PTHrP promotes colon cancer cell migration and invasion in an integrin α6β4-dependent manner through activation of Rac1

Parathyroid hormone-related protein (PTHrP) is expressed by human colon cancer tissue and cell lines. Rac1 GTPase enhances colon cancer cell migration and invasion. Here we report a positive correlation between PTHrP expression and Rac1 activity in LoVo (human colon cancer) cells. The positive effects of PTHrP on Rac1 activity and on cell migration and invasion are mediated via the guanine nucleotide exchange factor Tiam1. Knockdown of integrin α6β4, which is upregulated by PTHrP, negates the PTHrP-mediated increase in Rac1 activation. Integrin α6β4 signals synergistically with growth factor receptors to activate the phosphatidylinositol 3-kinase (PI3-K) pathway. Chemical inhibition of PI3-K negates the PTHrP-mediated effects on Tiam1 and Rac1 activity. Tumors from PTHrP-overexpressing LoVo cells also show increased expression of Tiam1. Taken together, these observations provide evidence of a link between PTHrP and Rac1 activity through integrin α6β4, resulting in enhanced cell migration and invasion. Targeting PTHrP production in colon cancer may thus prove therapeutically beneficial.     

Psoriasin (S100A7) associates with integrin β6 subunit and is required for αvβ6-dependent carcinoma cell invasion

Expression of the integrin αvβ6 is upregulated in a variety of carcinomas where it appears to be involved in malignant progression, although the biology of this integrin is not fully explored. We have generated oral carcinoma cells that express αvβ6 composed of wild-type αv and a mutant β6 that lacks the unique C-terminal 11 amino acids (aa). We found that these residues, although not required for αvβ6-dependent adhesion or migration, are essential for αvβ6-dependent invasive activity. We have used a proteomic approach to identify novel binding partners for the β6 subunit cytoplasmic tail and report that psoriasin (Psor) (S100A7) bound preferentially to the recombinant β6 cytoplasmic domain, though not in the absence of the unique C-terminal 11aa. Endogenous cellular Psor co-precipitated with endogenous β6 and colocalised with αvβ6 at the cell membrane and intracellular vesicles. Knockdown of Psor, with small interfering RNA, had no effect on αvβ6-dependent adhesion or migration but abrogated αvβ6-mediated oral carcinoma cell invasion both in Transwell and, the more physiologically relevant, organotypic invasion assays, recapitulating the behaviour of the β6-mutant cell line. Membrane-permeant Tat-peptides encoding the unique C-terminal residues of β6, bound directly to recombinant Psor and inhibited cellular Psor binding to β6; this blocked αvβ6-dependent, but not αvβ6-independent, invasion. These data identify a novel interaction between Psor and β6 and demonstrate that it is required for αvβ6-dependent invasion by carcinoma cells. Inhibition of this interaction may represent a novel therapeutic strategy to target carcinoma invasion.     

The TGF-β/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system

Transforming growth factor-β (TGF-β) has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stages. In contrast to the mechanisms by which TGF-β induces growth arrest, the pathways that mediate tumor invasion are not well understood. Here, we describe a TGF-β-dependent invasion assay system consisting of spheroids of MCF10A1 normal breast epithelial cells (M1) and RAS-transformed (pre-)malignant derivatives (M2 and M4) embedded in collagen gels. Both basal and TGF-β-induced invasion of these cell lines was found to correlate with their tumorigenic potential; M4 showing the most aggressive behavior and M1 showing the least. Basal invasion was strongly inhibited by the TGF-β receptor kinase inhibitor SB-431542, indicating the involvement of autocrine TGF-β or TGF-β-like activity. TGF-β-induced invasion in premalignant M2 and highly malignant M4 cells was also inhibited upon specific knockdown of Smad3 or Smad4. Interestingly, both a broad spectrum matrix metalloproteinase (MMP) inhibitor and a selective MMP2 and MMP9 inhibitor mitigated TGF-β-induced invasion of M4 cells, while leaving basal invasion intact. In line with this, TGF-β was found to strongly induce MMP2 and MMP9 expression in a Smad3- and Smad4-dependent manner. This collagen-embedded spheroid system therefore offers a valuable screening model for TGF-β/Smad- and MMP2- and MMP9-dependent breast cancer invasion.     

Tumor-associated macrophages regulate gastric cancer cell invasion and metastasis through TGFβ2/NF-κB/Kindlin-2 axis

Objective: Recent studies have shown that tumor-associated macrophages(TAMs) play an important role in cancer invasion and metastasis. Our previous studies have reported that TAMs promote the invasion and metastasis of gastric cancer(GC) cells through the Kindlin-2 pathway. However, the mechanism needs to be clarified.Methods: THP-1 monocytes were induced by PMA/interleukin(IL)-4/IL-13 to establish an efficient TAM model in vitro and M2 macrophages were isolated via flow cytometry. A dual lucife...     

The TGF-β/Smad2/3 signaling pathway is involved in Musashi2-induced invasion and metastasis of colorectal cancer

To identify Musashi2 as an effective biomarker regulated by the TGF-β/Smad_2/3 signaling pathway for the precise diagnosis and treatment of colorectal cancer (CRC) through bioinformatic tools and experimental verification. The Cancer Genome Atlas, Timer, and Kaplan−Meier analyses were performed to clarify the expression of Musashi2 and its influence on the prognosis of CRC. Transforming growth factor beta 1 (TGF-β1) was used to activate the TGF-β/Smad_2/3 signaling pathway to identify whether it could regulate the expression and function of Musashi2. Western blot analysis and quantitative PCR analyses were conducted to verify the expression of Musashi2. Cell counting kit-8 (CCK8), EdU, wound healing, and Transwell assays were conducted to reveal the role of Musashi2 in the proliferation, migration, and invasion of CRC. Musashi2 was upregulated in CRC and promoted proliferation and metastasis. TGF-β1 increased the expression of Musashi2, while the antagonist inducer of type II TGF-β receptor degradation-1 (ITD-1) decreased the expression. CCK8 and EdU assays demonstrated that inhibition of Musashi2 or use of ITD-1 lowered proliferation ability. The Transwell and wound healing assays showed that the migration and invasion abilities of CRC cells could be regulated by Musashi2. The above functions could be enhanced by TGF-β1 by activating the TGF-β/Smad_2/3 signaling pathway and reversed by ITD-1. A positive correlation was found between Musashi2 and the TGF-β/Smad_2/3 signaling pathway. TGF-β1 activates the TGF-β/Smad_2/3 pathway to stimulate the expression of Musashi2, which promotes the progression of CRC. Musashi2 might become a target gene for the development of new antitumor drugs.