Migration Of Metastatic Cancer Cells Research Articles

Integrins and the Metastasis-like Dissemination of Acute Lymphoblastic Leukemia to the Central Nervous System.

Acute lymphoblastic leukemia (ALL) disseminates with high prevalence to the central nervous system (CNS) in a process resembling aspects of the CNS surveillance of normal immune cells as well as aspects of brain metastasis from solid cancers. Importantly, inside the CNS, the ALL blasts are typically confined within the cerebrospinal fluid (CSF)-filled cavities of the subarachnoid space, which they use as a sanctuary protected from both chemotherapy and immune cells. At present, high cumulative doses of intrathecal chemotherapy are administered to patients, but this is associated with neurotoxicity and CNS relapse still occurs. Thus, it is imperative to identify markers and novel therapy targets specific to CNS ALL. Integrins represent a family of adhesion molecules involved in cell-cell and cell-matrix interactions, implicated in the adhesion and migration of metastatic cancer cells, normal immune cells, and leukemic blasts. The ability of integrins to also facilitate cell-adhesion mediated drug resistance, combined with recent discoveries of integrin-dependent routes of leukemic cells into the CNS, have sparked a renewed interest in integrins as markers and therapeutic targets in CNS leukemia. Here, we review the roles of integrins in CNS surveillance by normal lymphocytes, dissemination to the CNS by ALL cells, and brain metastasis from solid cancers. Furthermore, we discuss whether ALL dissemination to the CNS abides by known hallmarks of metastasis, and the potential roles of integrins in this context.

Abstract 2326: Differential lipid metabolism in mammary cancer cell progression

Abstract There is an association between increased lipid accumulation and aggressive breast cancer phenotypes, and therefore poorer clinical outcomes. The mechanism by which dysregulated lipid metabolism may promote metastasis remains incompletely understood. Several potential mechanisms by which the neutral lipid storage organelles, cytoplasmic lipid droplets, may promote metastasis include utilization of fatty acids (FAs) for oxidation, protection against lipotoxcity, and production of lipid signaling mediators. We have previously demonstrated that metastatic MCF10CA1a mammary cancer cells rely on fatty acid oxidation (FAO) compared to non-metastatic MCF10A-ras cells, using etomoxir to inhibit the rate-limiting enzyme of FAO, carnitine palmitoyltransferase 1 (CPT1). Previous literature indicates that there is an increase in various lipid metabolism pathways, including FA uptake, de novo lipogenesis, and FA storage in neutral lipid. We found that there is no significant difference between palmitic acid uptake between the non-metastatic and metastatic cells. In the current study, we show that MCF10CA1a cells have significantly higher levels of carbon incorporation into palmitic and oleic FAs from 13C-acetate (25% and 20% greater), 13C-glucose (21% and 11% greater), and 13C-lactate (29% and 22% greater) respectively, compared to the MCF10A-ras cells, as detected by LC-MS/MS. In order to determine the source of FAs that contributes to the migration-dependent FAO, we assessed MCF10CA1a cell migration following inhibition of lipolysis (adipose triglyceride lipase—ATGL; ATGListatin) alone or in combination with the FAO inhibitor, etomoxir. We determined that inhibition of ATGL, the initial enzyme of triglyceride lipolysis pathway, significantly reduces MCF10CA1a cell migration (20% decrease compared to vehicle). Inhibition of FAO and lipolysis did not induce additional reduction in cell migration. We also showed that MCF10CA1a cells are more dependent on FA metabolism than the non-metastatic MCF10A-ras cells, demonstrated by MCF10CA1a cells having a greater decrease in cell viability compared to the MCF10A-ras cells given each FA metabolism inhibition (p<0.05: 22% greater decrease for FAO inhibition and 16% greater decrease for dual FAO and lipolysis inhibition, p=0.05: 17% greater decrease for FA synthesis inhibition). These data suggest that metastatic MCF10CA1a cells rely on their increased FA metabolism to survive and migrate, and specifically that the catabolism of FAs from triglyceride supports the FAO-driven migration of metastatic cancer cells. Citation Format: Chaylen Andoline, Parik Pawar, Dorothy Teegarden. Differential lipid metabolism in mammary cancer cell progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2326.

Abstract 1477: Co-targeting c-Met and c-Src pathways to inhibit metastatic cancer cell migration and invasion

Abstract Overexpression of tyrosine kinases c-Src and c-Met has been shown to be highly associated with disease progression and poor clinical outcomes in many types of cancers. Although small molecule Src or Met inhibitors are being individually evaluated in clinical trials, patient data suggest that single treatment with any of these inhibitors only yield limited effects. One possibility is that redundant signaling pathways in tumor cells may allow tumor cell functions less interrupted upon inhibition of only one pathway. We therefore propose that c-Src and c-Met are two compensatory pathways that may cooperate to promote cancer metastasis. In order to delineate this hypothesis, we studied treatment effects of combining inhibitors targeting c-Src (dasatinib) and c-Met (ASLAN002) on multiple functional behaviors associated with tumor metastasis in c-Src and c-Met expressing metastatic cancer cells. Combination of Src- and Met-inhibitors was found to strongly inhibit cell colony formation by clonogenic survival assay. In a wound-healing assay, compared to either treatment alone, the combinatorial treatment exhibited greater suppression on cell motility. In addition, using a Matrigel-based Transwell assay, dasatinib plus ASLAN002 resulted in significant reduction of cell invasion in the presence or absence of HGF. Western blot analysis demonstrated that co-targeting Met and Src kinases lead to not only inhibition on c-Met and c-Src autophosphorylation but also multiple downstream kinases such as focal adhesion kinase (FAK), Akt and extracellular signal-regulated kinase (ERK). These data indicate that combined application of small molecule Src and Met inhibitors cause significant impairment on functional behaviors associated with cancer metastasis in vitro including survival, migration and invasion, suggesting that this combination intervention modality may serve as a potential anti-metastatic strategy that could be further tested in metastatic models in vivo. Citation Format: Yao Dai, Dietmar W. Siemann. Co-targeting c-Met and c-Src pathways to inhibit metastatic cancer cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1477.

Chemo-Mechanical Modulation of Cell Motions Using DNA Nanosprings.

Cell motions such as migration and change in cellular morphology are essential activities for multicellular organism in response to environmental stimuli. These activities are a result of coordinated clustering/declustering of integrin molecules at the cell membrane. Here, we prepared DNA origami nanosprings to modulate cell motions by targeting the clustering of integrin molecules. Each nanospring was modified with arginyl-glycyl-aspartic acid (RGD) domains with a spacing such that when the nanospring is coiled, the RGD ligands trigger the clustering of integrin molecules, which changes cell motions. The coiling or uncoiling of the nanospring is controlled, respectively, by the formation or dissolution of an i-motif structure between neighboring piers in the DNA origami nanodevice. At slightly acidic pH (<6.5), the folding of the i-motif leads to the coiling of the nanospring, which inhibits the motion of HeLa cells. At neutrality (pH 7.4), the unfolding of the i-motif allows cells to resume mechanical movement as the nanospring becomes uncoiled. We anticipate that this pH-responsive DNA nanoassembly is valuable to inhibit the migration of metastatic cancer cells in acidic extracellular environment. Such a chemo-mechanical modulation provides a new mechanism for cells to mechanically respond to endogenous chemical cues.

Thy-1 (CD90)-Induced Metastatic Cancer Cell Migration and Invasion Are β3 Integrin-Dependent and Involve a Ca2+/P2X7 Receptor Signaling Axis.

Cancer cell adhesion to the vascular endothelium is an important step in tumor metastasis. Thy-1 (CD90), a cell adhesion molecule expressed in activated endothelial cells, has been implicated in melanoma metastasis by binding to integrins present in cancer cells. However, the signaling pathway(s) triggered by this Thy-1-Integrin interaction in cancer cells remains to be defined. Our previously reported data indicate that Ca2+-dependent hemichannel opening, as well as the P2X7 receptor, are key players in Thy-1-αVβ3 Integrin-induced migration of reactive astrocytes. Thus, we investigated whether this signaling pathway is activated in MDA-MB-231 breast cancer cells and in B16F10 melanoma cells when stimulated with Thy-1. In both cancer cell types, Thy-1 induced a rapid increase in intracellular Ca2+, ATP release, as well as cell migration and invasion. Connexin and Pannexin inhibitors decreased cell migration, implicating a requirement for hemichannel opening in Thy-1-induced cell migration. In addition, cell migration and invasion were precluded when the P2X7 receptor was pharmacologically blocked. Moreover, the ability of breast cancer and melanoma cells to transmigrate through an activated endothelial monolayer was significantly decreased when the β3 Integrin was silenced in these cancer cells. Importantly, melanoma cells with silenced β3 Integrin were unable to metastasize to the lung in a preclinical mouse model. Thus, our results suggest that the Ca2+/hemichannel/ATP/P2X7 receptor-signaling axis triggered by the Thy-1-αVβ3 Integrin interaction is important for cancer cell migration, invasion and transvasation. These findings open up the possibility of therapeutically targeting the Thy-1-Integrin signaling pathway to prevent metastasis.

Abstract 4018: Interaction site elucidation of the Rac1/Cdc42 inhibitor, MBQ-167

Abstract Metastasis continues to be the primary cause of cancer-related death. Therefore, we focus on designing small molecules inhibitors to block metastasis. The Rho GTPase family members Rac1, and Cdc42 are critical regulators of cancer cell migration and invasion, and thus, metastasis. Rac1 is activated by Guanine Nucleotide Exchange Factors (GEFs), which promote the exchange of GDP to GTP. In most cancers, the homologous Rac1 and Cdc42 are hyperactivated due to the overexpression of GEFs. Therefore, we designed inhibitors to block the activation and interaction of Rac1 and Cdc42 with their GEFs. Our lead compound MBQ-167, is a dual inhibitor of Rac1 and Cdc42 with IC50s of 103 nM and 78 nM, respectively. MBQ-167 inhibited the viability and migration of metastatic cancer cells without affecting non-metastatic or non-cancer cells. Moreover, MBQ-167 reduced mammary tumor growth and metastasis of HER2 positive and triple-negative breast cancer cells in immunocompromised mice by ~ 90%. To further elucidate the mechanism of action of MBQ-167, we tested the hypothesis that MBQ-167 blocks the interaction of specific Rac1/Cdc42 GEFs by interaction with particular amino acid residues in the switch I and II homologous regions of Rac1 and Cdc42 proteins. Therefore, to achieve the objective of elucidating the interaction site of MBQ-167 on Rac1 and Cdc42, we used in silico analysis and Nuclear Magnetic Resonance (NMR) of MBQ-167 with Rac1 in a GDP bound state. These results were biochemically validated by pulldown assays using a G15A nucleotide-free mutant predicted to interact tightly with Rac1/Cdc42 GEFs. In silico, using Autodock Tools, MBQ-167 was docked to the GEF binding domain of Rac1 and Cdc42 with low binding energy, indicating a tight interaction. For the NMR structural analyses, Rac1 was isotopically labeled with 15N and titrated with several concentrations of MBQ-167, at ratios of 1:1, 1:2, 1:3, and 1:4. We were able to detect chemical shift perturbations in key residues in the switch I and II regions that interact with Rac1/Cdc42 GEFs and regulate the activity. Moreover, these perturbations were more intense with increasing amounts of MBQ-167, confirming the specificity of the interaction. Therefore, our data signify the direct interaction of MBQ-167 with Rac1, thus validating the role of MBQ-167 as a specific Rac1/Cdc42 inhibitor. Citation Format: Julia I. Medina, Marvin Bayro, Eliud Hernandez, Cornelis Vlaar, Ricardo Gonzalez, Suranganie Dharmawardhane. Interaction site elucidation of the Rac1/Cdc42 inhibitor, MBQ-167 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4018.

Elevated Extracellular Fluid Viscosity Stimulates Migration of Metastatic Cancer Cells

Elevated Extracellular Fluid Viscosity Stimulates Migration of Metastatic Cancer Cells

A hybrid discrete–continuous model of metastatic cancer cell migration through a remodeling extracellular matrix

AbstractA major cause of cancer mortality is metastasis of cancer cells from a localized tumor to other parts of the body. The interactions between cancer cells and the surrounding tumor microenvironment, particularly the extracellular matrix (ECM), are central to metastatic migration. During metastasis, enzymes are secreted to degrade and crosslink collagen fibers in the ECM, inducing structural alterations and changes in the properties of the ECM. The mechanism of how these changes facilitate the directional motility of cancer cells along the collagen fibers remains unclear. A hybrid discrete–continuous computational model is developed via the software CompuCell3D to simulate the migration of metastatic cancer cells through the ECM during chemical and physical remodeling. Cancer cells are treated as discrete agents, and ECM components (collagen fibers and remodeling enzymes) are modeled by partial differential equations. The computational model of cancer migration provides understanding of how ECM remodeling affects migration efficiency.

Matrix metalloproteinase targeted peptide vesicles for delivering anticancer drugs.

A matrix metalloproteinase (MMP) targeted tetrapeptide vesicle has been designed and developed, which strongly binds at a MMP9 enzymatic site. Interestingly, it has a propensity to encapsulate and deliver the doxorubicin drug specifically to the cancer cell, induces superior apoptotic death, and inhibits the metastatic cancer cell migration and growth of multicellular 3D spheroids.

Abstract B094: The dual Rac/Cdc42 inhibitor MBQ-167 and derivatives as anticancer compounds

Abstract Metastatic disease lacks effective treatments, and remains the primary cause of mortality from epithelial cancers. The Rho family GTPases Rac and Cdc42 and their downstream effector p21-activated kinase (PAK) are pivotal regulators of metastatic cancer cell migration and invasion; thus, overexpression of Rac/Cdc42/PAK has been correlated with reduced patient survival in breast, gastric, and lung cancer. Our studies on the efficacy and pharmacokinetics of the Rac inhibitor EHop-016 (US patents # 8,884,006 B2; 9,278,956B1) validated the development of Rac and Cdc42 inhibitors as anti-metastatic cancer therapeutics. To improve the pharmacologic utility of EHop-016, structural derivatives were screened for enhanced efficacy and bioavailability. We recently identified a dual Rac and Cdc42 inhibitor, MetaBloq(MBQ)-167, which inhibits Rac and Cdc42 activation with IC50s of 103 nM for Rac inhibition and 78 nM for Cdc42 inhibition. Moreover, MBQ-167 is specific to cancer cells that have undergone epithelial-to-mesenchymal transition (EMT), but not epithelial cancer cells or noncancer cells. In metastatic cancer cells, MBQ-167 induces a unique phenotype of loss in cell polarity, cell surface actin extensions, and cell-substrate attachments to undergo anoikis (apoptosis due to inadequate cell-matrix interactions). In vivo, MBQ-167 inhibits HER2 type mammary tumor growth and metastasis in immunocompromised mice by ~90-100% (Humphries-Bickley et al., Mol Cancer Ther 2017; patent applications US 15/499532, PCT/US17/29921). In this study, we have further tested the pharmacokinetics and utility of MBQ-167 in additional models of cancer and screened MBQ-167 derivatives for their utility as Rac/Cdc42 inhibitors. Similar to the results with HER2 type breast cancer, in severe combined immunodeficiency (SCID) mice with mammary fat pad tumors from the triple-negative MDA-MB-231 human breast cancer cell line, the effect of MBQ-167 saturated at 1 mg/kg BW with a 80% reduction in tumor growth and a 100% reduction in metastasis. In gastric cancer, MBQ-167 reduced the viability of metastatic NCI-N87 gastric cancer cells, induced anoikis without affecting the AGS nonmetastatic gastric cancer cells, and inhibited tumor growth in SCID mice. We have screened a range of MBQ-167 derivatives for cell viability and the “anoikis” phenotype and isolated 4 compounds that reduced breast cancer cell viability with GI50s in the nM range. From this screen, MBQ-14 acts similar to MBQ-167, and thus is considered to be a viable inhibitor for further investigation. We have developed a quantitative method for identifying MBQ-167 and MBQ-14 from mouse plasma using ultra-performance convergence chromatography (UPC2)/MS/MS, and find that MBQ-167 is bioavailable in mouse plasma with a half-life of ~4h following oral or intraperitoneal administration. Overall our data demonstrate the utility of further developing MBQ-167 and derivatives as anti-metastatic cancer therapeutics. Citation Format: Linette Castillo-PIchardo, Maria Del Mar Maldonado, Jean Ruiz-Calderon, Jose F. Rodriguez-Orengo, Eliud Hernandez, Cornelis Vlaar, Surangani F. Dharmawardhane Flanagan. The dual Rac/Cdc42 inhibitor MBQ-167 and derivatives as anticancer compounds [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B094.

The anti-invasive role of novel synthesized pyridazine hydrazide appended phenoxy acetic acid against neoplastic development targeting matrix metallo proteases

Neoplastic metastasis is a major process where tumor cells migrate from the primary tumor and colonize at other parts of our body to form secondary tumor. Cancer incidences are rising and novel anti-neoplastic compounds with new mechanism of actions are essential for preventing cancer related deaths. In the current examination, a novel series of pyridazine analogues 6a-l was synthesized and evaluated against metastatic neoplastic cells. Experimental data postulated compound 6j has potential cytotoxic efficacy with prolonged activity against various cancer cells, including A549, HepG2, A498, CaSki and SiHa cells. Moreover, compound 6j arrests the A549 migration and invasions markedly by counteracting matrix metalloproteinase (MMP)-2 and MMP-9 expressions. Also, compound 6j proved its potentiality against Dalton’s solid lymphoma progression in-vivo by abridging MVD and MMP expressions. Compound 6j interacts with MMP-2 and MMP-9 by H- bond in-silico, thereby down regulates the MMPs action in tumourigenesis. Altogether, we concluded that compound 6j down regulates MMP-2 and MMP-9 and thereby impairs metastatic cancer cell migration and invasions which can be translated into a potent anti-neoplastic agent.

The metastatic microenvironment of human liver characterised by increased levels of inflammatory cytokines promotes cancer cell chemotaxis RN

Objective: Healthy human liver is immunologically equipped for effective tumour surveillance, with repertoires of potent cytotoxic lymphoid populations. Despite this, hepatic metastases are common with little known of the immune landscape that remains following hepatectomy. How the immune microenvironment of the liver responds in the presence of metastatic disease, and whether this response dictates hepatic metastatic recurrence has yet to be elucidated. We hypothesise the cytokine milieu across metastatic liver can modify the local anti-tumour response and thus influence further tumour growth. Methods: Biopsies of freshly resected tumour, tumour adjacent liver and liver at the distal resection margin (n=25) were cultured in vitro to obtain metastatic conditioned media (CM). Using an antibody array, this CM was screened for 102 cytokines, compared to CM from healthy control liver. Differentially expressed cytokines (n=12) were quantified using Luminex-based multiplex analysis. Results: IL6, CXCL1(GRO ), CXCL5(ENA 78), GCSF, GMCSF, VEGF, LIF, CCL 3(MIP-1) demonstrated significant variation in distal metastatic liver when compared to healthy donor. When analyzed by chemotaxis assay, all areas of metastatic liver induced migration of metastatic cancer cells. Furthermore, depletion of neutrophils at the distal resection margin significantly correlated with recurrent hepatic metastases. Conclusion: Healthy human liver has a highly evolved anti-tumour immune repertoire which is maintained by a complex cytokine environment. This immune landscape is perturbed in the presence of metastases, with increased expression of chemotactic cytokines and altered immune cell populations, inducing metastatic cell migration and tumour recurrence.

Two-Pore Channel Function Is Crucial for the Migration of Invasive Cancer Cells.

Metastatic invasion is the major cause of cancer-related deaths. In this study, we introduce two-pore channels (TPC), a recently described class of NAADP- and PI(3,5)P2-sensitive Ca2+-permeable cation channels in the endolysosomal system of cells, as candidate targets for the treatment of invasive cancers. Inhibition of the channel abrogated migration of metastatic cancer cells in vitro Silencing or pharmacologic inhibition of the two-pore channel TPC2 reduced lung metastasis of mammary mouse cancer cells. Disrupting TPC function halted trafficking of β1-integrin, leading to its accumulation in EEA1-positive early endosomes. As a consequence, invasive cancer cells were no longer able to form leading edges, which are required for adequate migration. Our findings link TPC to cancer cell migration and provide a preclinical proof of concept for their candidacy as targets to treat metastatic cancers. Cancer Res; 77(6); 1427-38. ©2017 AACR.

General Introduction to Drebrin.

Drebrin was first discovered by our group as "developmentally regulated brain protein" from the chicken optic tectum. Drebrin is an actin-binding protein, which is classified into two major isoforms produced by alternative splicing from a single DBN1 gene. The isoform predominantly expressed in the adult brain (drebrin A) is neuron specific, containing a neuron-specific sequence (Ins2) in the middle of the molecule. Drebrin A is highly concentrated in dendritic spines, and its accumulation level is regulated by synaptic activity. In contrast, drebrin E, which lacks Ins2, is found in widespread but not ubiquitous cell types in various tissues. The isoform conversion from drebrin E to drebrin A occurs in parallel with synaptogenesis. Drebrin decorating F-actin is found at the recipient side of cell-cell communication systems, such as gap junctions, adherens junctions, immunological synapses, and neuronal synapses. In addition, it is involved in the cellular mechanisms of cell migration, cell process formation, cancer metastasis, and spermatogenesis. Lack of drebrin leads to the dysfunction of cell-cell communication, resulting in aberrant migration of metastatic cancer cells, aberrant synaptic function in dementia, and rupture of endothelial integrity. Because drebrin forms a unique F-actin with a longer helical crossover, drebrin may create an F-actin platform for molecular assembly and play a pivotal role in intercellular communication.

Direct mapping of melanoma cell - endothelial cell interactions.

The most life-threatening aspect of cancer is metastasis; cancer patient mortality is mainly due to metastasis. Among all metastases, presence of brain metastasis is one with the poorest prognosis; the median survival time can be counted in months. Therefore, prevention or decreasing their incidence would be highly desired both by patients and physicians. Metastatic cells invading the brain must breach the cerebral vasculature, primarily the blood-brain barrier. The key step in this process is the establishment of firm adhesion between the cancer cell and the cerebral endothelial layer. Using the atomic force microscope, a high-resolution force spectrograph, our aim was to explore the connections among the cell morphology, cellular mechanics, and biological function in the process of transendothelial migration of metastatic cancer cells. By immobilization of a melanoma cell to an atomic force microscope's cantilever, intercellular adhesion was directly measured at quasi-physiological conditions. Hereby, we present our latest results by using this melanoma-decorated probe. Binding characteristics to a confluent layer of brain endothelial cells was directly measured by means of single-cell force spectroscopy. Adhesion dynamics and strength were characterized, and we present data about spatial distribution of elasticity and detachment strength. These results highlight the importance of cellular mechanics in brain metastasis formation and emphasize the enormous potential toward exploration of intercellular dynamic-related processes.

Synergistic Anticancer Effect of Peptide-Docetaxel Nanoassembly Targeted to Tubulin: Toward Development of Dual Warhead Containing Nanomedicine.

Microtubule dynamics play a crucial role in cancer cell division. Various drugs are developed to target microtubule. Although a few of them show potential in treatment of cancer, but success rate is limited due to their poor bioavailability and lack of specificity. Thus, development of highly bioavailable and target specific anticancer drug is extremely necessary. To address these key issues, here, a combination of approaches such as development of a dodecapeptide-docetaxel nanoassembly targeted to tubulin and MUC1 (mucin 1, cell surface associated glycoprotein) targeting oligonucleotide aptamer conjugated liposome for delivering peptide-docetaxel nanoassembly into the breast cancer cell have been demonstrated. These studies reveal that the peptide forms nanoassembly and entraps docetaxel drug. Further, the liposomal formulation of peptide-docetaxel exerts synergistic anticancer effect, activates key mitotic check point proteins, and inhibits bipolar spindle formation, metastatic cancer cell migration, and growth of tumor mimicking 3D multicellular spheroid.

Science Signaling Podcast for 9 February 2016: Transendothelial migration

Phosphoproteomics analysis identifies signaling events required for transendothelial migration of metastatic cancer cells.

Metastatic bladder cancer cells distinctively sense and respond to physical cues of collagen fibril-mimetic nanotopography.

Tumor metastasis is characterized by enhanced invasiveness and migration of tumor cells through the extracellular matrix (ECM), resulting in extravasation into the blood and lymph and colonization at secondary sites. The ECM provides a physical scaffold consisting of components such as collagen fibrils, which have distinct dimensions at the nanoscale. In addition to the interaction of peptide moieties with tumor cell integrin clusters, the ECM provides a physical guide for tumor cell migration. Using nanolithography we set out to mimic the physical dimensions of collagen fibrils using lined nanotopographical silicon surfaces and to explore whether metastatic tumor cells are uniquely able to respond to these physical dimensions. Etched silicon surfaces containing nanoscale lined patterns with varying trench and ridge sizes (65-500 nm) were evaluated for their ability to distinguish between a non-metastatic (253 J) and a highly metastatic (253 J-BV) derivative bladder cancer cell line. Enhanced alignment was distinctively observed for the metastatic cell lines on feature sizes that mimic the dimensions of collagen fibrils (65-100 nm lines, 1:1-1:1.5 pitch). Further, these sub-100 nm lines acted as guides for migration of metastatic cancer cells. Interestingly, even at this subcellular scale, metastatic cell migration was abrogated when cells were forced to move perpendicular to these lines. Compared to flat surfaces, 65 nm lines enhanced the formation of actin stress fibers and filopodia of metastatic cells. This was accompanied by increased formation of focal contacts, visualized by immunofluorescent staining of phospho-focal adhesion kinase along the protruding lamellipodia. Simple lined nanotopography appears to be an informative platform for studying the physical cues of the ECM in a pseudo-3D format and likely mimics physical aspects of collagen fibrils. Metastatic cancer cells appear distinctively well adapted to sense these features using filopodia protrusions to enhance their alignment and migration.

Combined inhibition of AXL, Lyn and p130Cas kinases block migration of triple negative breast cancer cells

Blocking the migration of metastatic cancer cells is a major goal in the therapy of cancer. The receptor tyrosine kinase AXL is one of the main triggers for cancer cell migration in neoplasia of breast, colon, skin, thyroid and prostate. In our study we analyzed the effect of AXL inhibition on cell motility and viability in triple negative breast cancer cell lines overexpressing AXL. Thereby we reveal that the compound BMS777607, exhibiting the lowest IC50 values for inhibition of AXL kinase activity in the studied cell lines, attenuates cell motility to a lower extent than the kinase inhibitors MPCD84111 and SKI606. By analyzing the target kinases of MPCD84111 and SKI606 with kinase profiling assays we identified Lyn, a Src family kinase, as a target of both compounds. Knockdown of Lyn and the migration-related CRK-associated substrate (p130Cas), had a significant inhibitory effect on cell migration. Taken together, our findings highlight the importance of combinatorial or multikinase inhibition of non-receptor tyrosine kinases and AXL receptor tyrosine kinase in the therapy of triple negative breast cancer.

Modification and biological evaluation of thiazole derivatives as novel inhibitors of metastatic cancer cell migration and invasion.

Fascin has recently emerged as a potential therapeutic target, as its expression in cancer cells is closely associated with tumor progression and metastasis. Following the initial discovery of a series of thiazole derivatives that demonstrated potent antimigration and antiinvasion activities via possible inhibition of fascin function, we report here the design and synthesis of 63 new thiazole derivatives by further structural modifications in search of more potent fascin inhibitors. The 5 series of analogues with longer alkyl chain substitutions on the thiazole nitrogen exhibited greater antimigration activities than those with other structural motifs. The most potent analogue, 5p, inhibited 50% of cell migration at 24 nM. Moreover, the thiazole analogues showed strong antiangiogenesis activity, blocking new blood vessel formation in a chicken embryo membrane assay. Finally, a functional study was conducted to investigate the mechanism of action via interaction with the F-actin bundling protein fascin.