Inhibition Of Tumor Cell Motility Research Articles

The role of ICAM-2 in neuroblastoma

The role of immunoglobulin superfamily cell adhesion molecules (CAMs) in facilitating immune responses in normal and tumor cells is well established. Cell adhesion molecule-1 (CADM1), for example, suppresses development of mouse mammary tumor cell metastasis by interacting with CD8+ T cells in immune-competent hosts [1]. Similarly, co-expression of Intercellular Adhesion Molecule-2 (ICAM-2) and chemokine C-X-C motif ligand 17 (CXCL17) elicits anti-tumor immune responses and suppresses tumor growth [2]. Although controversial, current literature suggests that proteins of the immunoglobulin superfamily CAMs also have functions that may well be distinct from their roles in mediating immune responses. Junctional adhesion molecule-A (JAM-A), for example, negatively regulates breast cancer cell invasiveness by disrupting tight junctions [3]; and a member of the B7 family of the immunoglobulin superfamily proteins, B7-H3, impairs osteogenic differentiation in vitro and in vivo [4]. Our lab demonstrated that ICAM-2 inhibited the development of disseminated neuroblastoma tumors in a preclinical model of metastatic neuroblastoma [5-7]. This inhibition depended on the interaction of ICAM-2 with the actin cytoskeletal linker protein α-actinin, an interaction that inhibited cell motility [7]. Ectopic expression of ICAM-2 did not affect the tumorigenic potential of neuroblastoma cells [7]. Importantly, immunohistochemical analyses of primary neuroblastoma tumor specimens demonstrated that neuroblastoma cells expressing ICAM-2 are phenotypically and histologically those recognized clinically to have limited metastatic potential [5]. Since metastatic disease is responsible for >90% of cancer-related deaths for multiple types of solid tumors, we suggest that elucidation of the molecular mechanism by which ICAM-2 suppresses the metastatic potential of neuroblastoma cells would identify proteins or pathways that might be exploited therapeutically to prevent metastatic disease progression. In normal tissues ICAM-2 is expressed predominantly by neovascular endothelial cells, and at lower levels by established vasculature and some leukocytes. The 202 amino acids comprising its extracellular domain mediate binding of ICAM-2 on endothelial cells to β2-integrins on the surface of leukocytes, to facilitate migration of neutrophils through the vascular endothelium as a component of immune reactions [8]. In neuroblastoma cells ICAM-2 inhibits cell motility independent of immune response, as we observed this inhibition in wound healing and modified Boyden chamber assays in vitro, assays that clearly lack an immune component [5,7]. In silico modeling indicated that ICAM-2 with mutations in the proposed α-actinin binding domain had a more ‘closed’ configuration than the wild type protein, and predicted that these ICAM-2 mutants would not interact with α-actinin [7]. Co-immunoprecipitation experiments confirmed in silico predictions [7]. In support of these findings, the interaction of ICAM-2 with α-actinin was essential to inhibit development of disseminated tumors in vivo [7]. As mentioned, ICAM-2 did not affect tumorigenic potential, and unpublished data showed no effect of ICAM-2 on expression of epithelial-mesenchymal transition (EMT) or stemness markers. Although we regard the inhibition of tumor cell motility by ICAM-2 in vitro to be compelling evidence that ICAM-2 participates in cell functions distinct from immune responses, we acknowledge the complex nature of metastatic tumor progression and propose that our data suggest multiple hypotheses with respect to the mechanism by which ICAM-2-inhibits neuroblastoma cell motility. The first hypothesis is that the intracellular interaction of ICAM-2 with α-actinin initiates ‘inside out’ signaling, and causes conformational changes in the extracellular domain of ICAM-2 that facilitate (or inhibit) interactions with specific extracellular matrix proteins that play an integral role in cell motility. The second is that the interaction of ICAM-2 with α-actinin alters the conformation of α-actinin to facilitate (or inhibit) the association of this actin cytoskeletal protein with alternative binding partners, with ICAM-2 acting as an ‘activator’ (or inhibitor) of α-actinin rather than as a membrane anchor protein. The third hypothesis, based on unpublished microarray data, is that ICAM-2 expression indirectly up-regulates protein tyrosine phosphatases (PTPs) involved in the formation and maintenance of focal adhesions such as focal adhesion kinase (FAK), Src, or Rac and Rho GTPases, each of which has crucial role in tumor cell motility. Our data show that ICAM-2 inhibits tumor cell motility and suppresses the metastatic potential of neuroblastoma cells. We propose that elucidating molecular events associated with ICAM-2 expression will identify key protein interactions that regulate the metastatic process in this cell type.

Integrin-free tetraspanin CD151 can inhibit tumor cell motility upon clustering and is a clinical indicator of prostate cancer progression.

Normal physiology relies on the organization of transmembrane proteins by molecular scaffolds, such as tetraspanins. Oncogenesis frequently involves changes in their organization or expression. The tetraspanin CD151 is thought to contribute to cancer progression through direct interaction with the laminin-binding integrins α3β1 and α6β1. However, this interaction cannot explain the ability of CD151 to control migration in the absence of these integrins or on non-laminin substrates. We demonstrate that CD151 can regulate tumor cell migration without direct integrin binding and that integrin-free CD151 (CD151(free)) correlates clinically with tumor progression and metastasis. Clustering CD151(free) through its integrin-binding domain promotes accumulation in areas of cell-cell contact, leading to enhanced adhesion and inhibition of tumor cell motility in vitro and in vivo. CD151(free) clustering is a strong regulator of motility even in the absence of α3 expression but requires PKCα, suggesting that CD151 can control migration independent of its integrin associations. The histologic detection of CD151(free) in prostate cancer correlates with poor patient outcome. When CD151(free) is present, patients are more likely to recur after radical prostatectomy and progression to metastatic disease is accelerated. Multivariable analysis identifies CD151(free) as an independent predictor of survival. Moreover, the detection of CD151(free) can stratify survival among patients with elevated prostate-specific antigen levels. Cumulatively, these studies demonstrate that a subpopulation of CD151 exists on the surface of tumor cells that can regulate migration independent of its integrin partner. The clinical correlation of CD151(free) with prostate cancer progression suggests that it may contribute to the disease and predict cancer progression.

STAT5b as Molecular Target in Pancreatic Cancer—Inhibition of Tumor Growth, Angiogenesis, and Metastases

The prognosis of patients suffering from pancreatic cancer is still poor and novel therapeutic options are urgently needed. Recently, the transcription factor signal transducer and activator of transcription 5b (STAT5b) was associated with tumor progression in human solid cancer. Hence, we assessed whether STAT5b might serve as an anticancer target in ductal pancreatic adenocarcinoma (DPAC). We found that nuclear expression of STAT5b can be detected in approximately 50% of DPAC. Blockade of STAT5b by stable shRNA-mediated knockdown showed no effects on tumor cell growth in vitro. However, inhibition of tumor cell motility was found even in response to stimulation with epidermal growth factor or interleukin-6. These findings were paralleled by a reduction of prometastatic and proangiogenic factors in vitro. Subsequent in vivo experiments revealed a strong growth inhibition on STAT5b blockade in subcutaneous and orthotopic models. These findings were paralleled by impaired tumor angiogenesis in vivo. In contrast to the subcutaneous model, the orthotopic model revealed a strong reduction of tumor cell proliferation that emphasizes the meaning of assessing targets in an appropriate microenvironment. Taken together, our results suggest that STAT5b might be a potential novel target for human DPAC.

Blocking αvβ3 integrin by a recombinant RGD disintegrin impairs VEGF signaling in endothelial cells

Vascular endothelial growth factor (VEGF) and αvβ3 integrin are key molecules that actively participate in tumor angiogenesis and metastasis. Some integrin-blocking molecules are currently under clinical trials for cancer and metastasis treatment. However, the mechanism of action of such inhibitors is not completely understood. We have previously demonstrated the anti-angiogenic and anti-metastatic properties of DisBa-01, a recombinant His-tag RGD-disintegrin from Bothrops alternatus snake venom in some experimental models. DisBa-01 blocks αvβ3 integrin binding to vitronectin and inhibits integrin-mediated downstream signaling cascades and cell migration. Here we add some new information on the mechanism of action of DisBa-01 in the tumor microenvironment. DisBa-01 supports the adhesion of fibroblasts and MDA-MB-231 breast cancer cells but it inhibits the adhesion of these cells to type I collagen under flow in high shear conditions, as a simulation of the blood stream. DisBa-01 does not affect the release of VEGF by fibroblasts or breast cancer cells but it strongly decreases the expression of VEGF mRNA and of its receptors, vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2) in endothelial cells. DisBa-01 at nanomolar concentrations also modulates metalloprotease 2 (MMP-2) and 9 (MMP-9) activity, the latter being decreased in fibroblasts and increased in MDA-MB-231 cells. In conclusion, these results demonstrate that αvβ3 integrin inhibitors may induce distinct effects in the cells of the tumor microenvironment, resulting in blockade of angiogenesis by impairing of VEGF signaling and in inhibition of tumor cell motility.

Abstract 3421: Nm23, a metastasis suppressor gene, binds the cytoskeletal linker Ezrin to regulate motility

Abstract Nm23 was the first metastasis suppressor gene characterized. One of the best correlates of the metastasis suppressive effect of Nm23 is its inhibition of tumor cell motility, but the mechanistic basis for this phenotype have not been elucidated. Our data in this abstract describe a novel interaction of Nm23-H1 with the FERM domain of Ezrin, which competitively inhibits the ability of FAK to bind Ezrin and induce motility. We hypothesized that Nm23-H1 may suppress motility and metastasis through the binding and inactivation of proteins that promote aggressive behavior. An unbiased survey of Nm23-H1 binding proteins was conducted in a 4T1 murine mammary carcinoma system. Flag tagged Nm23-H1 and its mouse variant, M1, were transfected into 4T1 cancer cells. Primary tumor size was unaffected by over-expression of Nm23-H1 or M1. Histological examination revealed a 75% decrease in liver metastasis by Nm23-H1 or M1 over-expression (P < 0.001). To characterize Nm23-H1/M1 binding proteins, Flag tagged Nm23 from both H1 and M1 clones were immunoprecipitated (IP) from cell lines in vitro as well as isolated primary tumors and metastases. Mass spectrometry analysis of the IP samples identified Ezrin as a potential binding partner. Ezrin is a member of the ERM family of proteins, which have been described to link the actin cytoskeleton to membrane associated proteins. Using co-IP, Ezrin was validated as a binding partner of Nm23-H1 and M1 in both the 4T1 and human MDA-MB-231 cell lines. Nm23-H1 and M1 were found to bind to the N terminal of Ezrin, which contains the highly conserved FERM domain. Deletion mutants established the interaction of Nm23 in both cell lines with the FERM-C portion, corresponding to Ezrin amino acids 205 to 300. Ezrin exists as a folded circular protein that, when activated by phosphorylation at amino acid 567 unfurls, exposing the FERM domain. Nm23-H1 co-immunoprecipitated constitutively active (T567D) but not inactive (T567A) mutants of Ezrin, suggesting that the active “open” structure permitted access to the FERM domain. As focal adhesion kinase (FAK) binds to Ezrin also through the FERM domain, it was hypothesized that Nm23-H1 would competitively prevent this association, confirmed in triple co-IP experiments. Expression of the T567D mutant of Ezrin increased cell motility of the 4T1 cells by an average of 148% (P<0.01). The expression of the T567D mutant did not overcome the motility inhibiting phenotype of Nm23 when co-expressed in 4T1 cells. We therefore hypothesize that Nm23 functions as a secondary regulator for Ezrin, binding to the N terminus and preventing the FERM domain from interacting with cell membrane associated proteins such as FAK that control tumor cell motility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3421. doi:1538-7445.AM2012-3421

Inhibition of Tumor Cell Motility by the Interferon-inducible GTPase MxA

To identify pathways controlling prostate cancer metastasis we performed differential display analysis of the human prostate carcinoma cell line PC-3 and its highly metastatic derivative PC-3M. This revealed that a 78-kDa interferon-inducible GTPase, MxA, was expressed in PC-3 but not in PC-3M cells. The gene encoding MxA, MX1, is located in the region of chromosome 21 deleted as a consequence of fusion of TMPRSS2 and ERG, which has been associated with aggressive, invasive prostate cancer. Stable exogenous MxA expression inhibited in vitro motility and invasiveness of PC-3M cells. In vivo exogenous MxA expression decreased the number of hepatic metastases following intrasplenic injection. Exogenous MxA also reduced motility and invasiveness of highly metastatic LOX melanoma cells. A mutation in MxA that inactivated its GTPase reversed inhibition of motility and invasion in both tumor cell lines. Co-immunoprecipitation studies demonstrated that MxA associated with tubulin, but the GTPase-inactivating mutation blocked this association. Because MxA is a highly inducible gene, an MxA-targeted drug discovery screen was initiated by placing the MxA promoter upstream of a luciferase reporter. Examination of the NCI diversity set of small molecules revealed three hits that activated the promoter. In PC-3M cells, these drugs induced MxA protein and inhibited motility. These data demonstrate that MxA inhibits tumor cell motility and invasion, and that MxA expression can be induced by small molecules, potentially offering a new approach to the prevention and treatment of metastasis.

A phase III randomized placebo controlled NCCTG trial of carboxyaminoimidazole (CAI) in patients with advanced non-small cell lung cancer (NSCLC)

7054 Background: Benefit from chemotherapy (ctx) for NSCLC has plateaued. Agents with novel mechanisms may provide further gain. CAI has in-vitro effects including inhibition of tumor cell motility, adhesion, and growth. Methods: CAI/placebo at 250 mg/day was given orally as maintenance therapy in stage 3 or 4 NSCLC patients within 6 weeks of completion of first line ctx (Stage 3 patients may have received thoracic radiotherapy). Eligible patients had stable or responding disease to one ctx regimen of 3–6 months (mo) duration; ECOG performance status of 0,1,2; absence of untreated brain metastases and adequate hematologic, hepatic and renal function. The study closed early due to slow accrual (targeted 360, observed 186). 360 patients followed for a minimum of 1 year would have provide 80% power to detect a 40% increase in the primary endpoint of median survival with a two-sided 5% type I error rate. The 154 observed deaths in the 186 patients accrued provide 80% power to detect a 60% increase in median survival. Results: Between 4/99 and 8/04, 186 patients (94 CAI, 92 placebo) were randomized with balance in median age, performance status, gender, histology, stage and response status to initial ctx. Incidence of severe (grade 3+) adverse events (AE) was not significantly different for CAI compared to placebo (42% vs. 34%, p=0.24). AE (primarily grade 1,2) observed more often in the CAI group included fatigue (55.6% vs 29.4%, p=0.0003), anorexia (31.1% vs 13.0%, p=0.003), nausea (61.1% vs 30.4%, p<0.0001), and vomiting (32.2% vs 14.1%, p=0.004); and grade 3+ AE included neurosensory (8.5% vs. 0.0%, p=0.004) and ataxia (11.1% vs. 3.3%, p=0.05). Patients discontinued treatment for AE/refusal more often in the CAI group (34.5% vs 6.8%, p<0.0001). Median follow-up is 20.0 mo. Both median survival and time to progression are not significantly different between the two groups (11.8 mo vs 10.5 mo, p= 0.93; 3.2 mo vs 2.5 mo, p=0.88). Conclusion: The addition of CAI following chemotherapy does not provide clinical benefit over placebo in advanced NSCLC. No significant financial relationships to disclose.

Repressions of actin assembly and RhoA localization are involved in inhibition of tumor cell motility by lipophilic ascorbyl phosphate

Our previous study showed that tumor invasion of human fibrosarcoma cells HT-1080 is hardly inhibited by ascorbic acid itself (Asc), but inhibited by 2-O-phosphorylated Asc-6-O-palmitylester (Asc2P6Plm) more markedly than 2-O-phosphorylated Asc or Asc-6-O-palmitylester, and that the inhibitory effect may be attributed to an increase in intracellular Asc derived from Asc2P6Plm. In the present study, the mechanism underlying the inhibitory effect of Asc2P6Plm on tumor invasion was analyzed. Migratory ability of the tumor cells was shown to be inhibited in a dose-dependent manner by either treatment with Asc2P6Plm at 50-300 micro M for 1 h or at 10-50 micro M for 18 h as assessed by cell sheet scratching assay. Hydroxyl radicals in homogenates of Asc2P6Plm-treated HT-1080 cells were markedly diminished relative to those of non-treated cells as evaluated by electron spin resonance method using the spin trapping agent DMPO. This may be closely related to attenuation of intracellular gross reactive oxygen species by Asc2P6Plm as was shown with the redox indicator CDCFH-DA. Actin was localized in the vicinity of the cell membrane abundantly in non-treated cells, but was diminished in a time-dependent manner in Asc2P6Plm-treated cells together with disappearance of pseudopods as shown with the actin-directed agent NBD-phallacidin and by immunocytochemical stain. The cell adhesion-controling molecule RhoA was increased time-dependently in the cytoplasm of Asc2P6Plm-treated cells as shown by Western blots. Thus the inhibition of tumor invasion by Asc2P6Plm was shown to be attributed to decrease in both the cell migratory ability and the actin localization near the cell membrane, which may result from an increase in cytoplasmic RhoA and reduction of intracellular ROS that is achieved by enrichment of intracellular Asc derived from Asc2P6Plm.

Tumor Cell Motility as a Novel Target in Cancer – Experimental and Clinical Results

Background: Surgery, chemotherapy and radiotherapy are the mainstay of tumor management. However, in systemic disease cure can be achieved in yet a few tumor entities. Based on cellbiological research we have characterized the process of tumor progression and metastasis and disclosed that the loss of cell-cell adhesion in association with an increased tumor cell motility is an essential feature of the malignant potential of a tumor. Methods: According to this principle we derived therapeutical methods differing from hitherto existing treatments by being exclusively focused on tumor cell motility. Characterization of so-called anti-motility factors was performed biochemically as well as with motility assays by in vitro studies in established bladder carcinoma cell lines. Results: We evaluated the potential therapeutic benefit in a model of chemically induced bladder carcinoma followed by a phase I/II trial applying antimotility factors in patients with advanced bladder cancer. Conclusion: Both basic research as well as the results of first clinical trials confirm, that advanced carcinomas can be influenced by inhibition of tumor cell motility.

Sphingosine-1-Phosphate: A Platelet-Activating Sphingolipid Released From Agonist-Stimulated Human Platelets

Sphingosine-1-phosphate (Sph-1-P) is the initial product of catabolism of sphingosine by sphingosine kinase and is cleaved by Sph-1-P lyase to a fatty aldehyde and ethanolamine phosphate. This phosphorylated sphingoid base is not only an intermediary catabolite, but also a bioactive lipid with important functions, including stimulation of cell proliferation in Swiss 3T3 fibroblasts and inhibition of tumor cell motility. In the present study, we examined functional roles of Sph-1-P in human platelets. Sph-1-P induced platelet shape change and aggregation reactions, although it failed to elicit secretion. Sphingosine, ceramide, sphingomyelin, and N,N-dimethylsphingosine did not mimic the positive effects of Sph-1-P on platelets. Subthreshold concentrations of Sph-1-P and weak platelet agonists such as adenosine diphosphate (ADP) and epinephrine synergistically elicited aggregation, which may be important for efficient amplification of platelet activation. Sph-1-P induced intracellular Ca2+ mobilization and the dose-response for Ca2+ release correlated closely with the concentration required for induction of shape change. On addition of [3H]sphingosine to intact platelets, the label was rapidly converted to Sph-1-P, and subsequently to ceramide and sphingomyelin. Interestingly, the Sph-1-P formed was specifically released into medium on stimulation of platelets with physiologic agonists. The amount of Sph-1-P in platelets, as measured by its conversion into radiolabeled N-acetyl-Sph-1-P, was 1.4 nmol/10(9) cells and was about four times higher than the mass of Sph present. When compared by mole percent Sph-1-P/phospholipid, the value for platelets is over 10 times higher than that for neutrophils. Our results suggest that Sph-1-P, rapidly converted from sphingosine, abundantly stored in platelets, and released on the cell activation, may play a physiologic role in thrombosis, hemostasis, and the natural wound-healing processes.