Metastasis is the main cause of prostate cancer-associated deaths. While significant progerss has been made in the treatment of primary tumors, efficent therapies that target the metastatic spread of prostate cancer are far from clinical reality. To efficiently treat cancer we need be able to impede its spread. Unfortunately, the majority of current therapeutics approved to treat metastatic cancer were originally selected based on their ability to inhibit primary tumor growth. This inherent flaw precluded these therapies from efficiently targeting the development of secondary metastatic lesions, a process that is distinct from that of primary tumor progression. In this review we will summarize the conceptual, cellular and molecular targets that should be considered to design effective anti-metastatic therapies.
Tumor cell extravasation is a key step during cancer metastasis, yet the precise mechanisms that regulate this dynamic process are unclear. We utilized a high-resolution time-lapse intravital imaging approach to visualize the dynamics of cancer cell extravasation in vivo. During intravascular migration, cancer cells form protrusive structures identified as invadopodia by their enrichment of MT1-MMP, cortactin, Tks4, and importantly Tks5, which localizes exclusively to invadopodia. Cancer cells extend invadopodia through the endothelium into the extravascular stroma prior to their extravasation at endothelial junctions. Genetic or pharmacological inhibition of invadopodia initiation (cortactin), maturation (Tks5), or function (Tks4) resulted in an abrogation of cancer cell extravasation and metastatic colony formation in an experimental mouse lung metastasis model. This provides direct evidence of a functional role for invadopodia during cancer cell extravasation and distant metastasis and reveals an opportunity for therapeutic intervention in this clinically important process.
It is well established that widely expressed PTK7 is essential for vertebrate tissue morphogenesis. In cancer, the functionality of PTK7 is selectively regulated by membrane type-1 matrix metalloproteinase (MT1-MMP), ADAMs (a disintegrin domain and metalloproteinases), and ?-secretase proteolysis. Here, we established that the full-length membrane PTK7, its Chuzhoi mutant with the two functional MT1-MMP cleavage sites, and its L622D mutant with the single inactivated MT1-MMP cleavage site differentially regulate cell motility in a two-dimensional versus three-dimensional environment. We also demonstrated that in polarized cancer cells, the levels of PTK7 expression and proteolysis were directly linked to the structure and kinetics of cell protrusions, including lamellipodia and invadopodia. In the functionally relevant and widely accepted animal models of metastasis, mouse and chick embryo models, both the overexpression and knock-out of PTK7 in HT1080 cells abrogated metastatic dissemination. Our analysis of human tissue specimens confirmed intensive proteolysis of PTK7 in colorectal cancer tumors, but not in matching normal tissue. Our results provide convincing evidence that both PTK7 expression and proteolysis, rather than the level of the cellular full-length PTK7 alone, contribute to efficient directional cell motility and metastasis in cancer.
Breast cancer and melanoma cells commonly metastasize to the brain using homing mechanisms that are poorly understood. Cancer patients with brain metastases display poor prognosis and survival due to the lack of effective therapeutics and treatment strategies. Recent work using intravital microscopy and preclinical animal models indicates that metastatic cells colonize the brain, specifically in close contact with the existing brain vasculature. However, it is not known how contact with the vascular niche promotes microtumor formation. Here, we investigate the role of connexins in mediating early events in brain colonization using transparent zebrafish and chicken embryo models of brain metastasis. We provide evidence that breast cancer and melanoma cells utilize connexin gap junction proteins (Cx43, Cx26) to initiate brain metastatic lesion formation in association with the vasculature. RNAi depletion of connexins or pharmacological blocking of connexin-mediated cell-cell communication with carbenoxolone inhibited brain colonization by blocking tumor cell extravasation and blood vessel co-option. Activation of the metastatic gene twist in breast cancer cells increased Cx43 protein expression and gap junction communication, leading to increased extravasation, blood vessel co-option and brain colonization. Conversely, inhibiting twist activity reduced Cx43-mediated gap junction coupling and brain colonization. Database analyses of patient histories revealed increased expression of Cx26 and Cx43 in primary melanoma and breast cancer tumors, respectively, which correlated with increased cancer recurrence and metastasis. Together, our data indicate that Cx43 and Cx26 mediate cancer cell metastasis to the brain and suggest that connexins might be exploited therapeutically to benefit cancer patients with metastatic disease.
Signaling by urokinase-type plasminogen activator receptor (uPAR) can cause epithelial-mesenchymal transition (EMT) in cultured breast cancer cells. In this report, we show that uPAR signaling can also induce cancer stem cell (CSC)-like properties. Ectopic overexpression of uPAR in human MDA-MB-468 breast cancer cells promoted the emergence of a CD24(-)/CD44(+) phenotype, characteristic of CSCs, while increasing the cell surface abundance of integrin subunits ?1/CD29 and ?6/CD49f that represent putative mammary gland stem cell biomarkers. uPAR overexpression increased mammosphere formation in vitro and tumor formation in an immunocompromized severe combined immunodeficient (SCID) mouse model of orthotopic breast cancer. Hypoxic conditions that are known to induce EMT in MDA-MB-468 cells also increased cell surface ?1/CD29, mimicking the effects of uPAR overexpression. Antagonizing uPAR effector signaling pathways reversed the increase in cell surface integrin expression. Whereas uPAR overexpression did not induce EMT in MCF-7 breast cancer cells, CSC-like properties were nevertheless still induced along with an increase in tumor initiation and growth in the orthotopic setting in SCID mice. Notably, in MCF-7 cell mammospheres, which display a well-defined acinus-like structure with polarized expression of E-cadherin and ?1-integrin, cell collapse into the central cavity was decreased by uPAR overexpression, suggesting that uPAR signaling may stabilize epithelial morphology. In summary, our findings show that uPAR signaling can induce CSC-like properties in breast cancer cells, either concomitantly with or separately from EMT.
Angiogenesis is controlled by signals that stimulate motility in endothelial cells at the tips of vascular sprouts while maintaining cell-cell adhesion in the stalks of angiogenic sprouts. We show here that Gs-linked G protein-coupled receptor activation of cAMP-dependent protein kinase (PKA) plays an important role in regulating the switch between endothelial cell adhesion and migration by activating C-terminal Src kinase, leading to inhibition of pp60Src. Activated PKA blocks pp60Src-dependent vascular endot helial-cadherin phosphorylation, thereby stimulating cell-cell adhesion while suppressing endothelial cell polarization, motility, angiogenesis, and vascular permeability. Similar to the actions of Notch and Dll4, PKA activation blocks sprouting in newly forming embryonic blood vessels, while PKA inhibition promotes excessive sprouting in these vessels. These findings demonstrate that G protein-coupled receptors and PKA regulate vascular sprouting during angiogenesis by controlling endothelial cell migration and cell-cell adhesion through their actions on pp60Src.
Little is known about how metastatic cancer cells arrest in small capillaries and traverse the vascular wall during extravasation in vivo. Using real-time intravital imaging of human tumor cells transplanted into transparent zebrafish, we show here that extravasation of cancer cells is a highly dynamic process that involves the modulation of tumor cell adhesion to the endothelium and intravascular cell migration along the luminal surface of the vascular wall. Tumor cells do not damage or induce vascular leak at the site of extravasation, but rather induce local vessel remodeling characterized by clustering of endothelial cells and cell-cell junctions. Intravascular locomotion of tumor cells is independent of the direction of blood flow and requires beta1-integrin-mediated adhesion to the blood-vessel wall. Interestingly, the expression of the pro-metastatic gene Twist in tumor cells increases their intravascular migration and extravasation through the vessel wall. However, in this case, Twist expression causes the tumor cells to switch to a beta1-integrin-independent mode of extravasation that is associated with the formation of large dynamic rounded membrane protrusions. Our results demonstrate that extravasation of tumor cells is a highly dynamic process influenced by metastatic genes that target adhesion and intravascular migration of tumor cells, and induce endothelial remodeling.
Kinases are known to regulate fundamental processes in cancer including tumor proliferation, metastasis, neovascularization, and chemoresistance. Accordingly, kinase inhibitors have been a major focus of drug development, and several kinase inhibitors are now approved for various cancer indications. Typically, kinase inhibitors are selected via high-throughput screening using catalytic kinase domains at low ATP concentration, and this process often yields ATP mimetics that lack specificity and/or function poorly in cells where ATP levels are high. Molecules targeting the allosteric site in the inactive kinase conformation (type II inhibitors) provide an alternative for developing selective inhibitors that are physiologically active. By applying a rational design approach using a constrained amino-triazole scaffold predicted to stabilize kinases in the inactive state, we generated a series of selective type II inhibitors of PDGFRbeta and B-RAF, important targets for pericyte recruitment and endothelial cell survival, respectively. These molecules were designed in silico and screened for antivascular activity in both cell-based models and a Tg(fli1-EGFP) zebrafish embryogenesis model. Dual inhibition of PDGFRbeta and B-RAF cellular signaling demonstrated synergistic antiangiogenic activity in both zebrafish and murine models of angiogenesis, and a combination of previously characterized PDGFRbeta and RAF inhibitors validated the synergy. Our lead compound was selected as an orally active molecule with favorable pharmacokinetic properties which demonstrated target inhibition in vivo leading to suppression of murine orthotopic tumors in both the kidney and pancreas.
Lipid accumulation in arteries induces vascular inflammation and atherosclerosis, the major cause of heart attack and stroke in humans. Extreme hyperlipidemia induced in mice and rabbits enables modeling many aspects of human atherosclerosis, but microscopic examination of plaques is possible only postmortem. Here we report that feeding adult zebrafish (Danio rerio) a high-cholesterol diet (HCD) resulted in hypercholesterolemia, remarkable lipoprotein oxidation, and fatty streak formation in the arteries. Feeding an HCD supplemented with a fluorescent cholesteryl ester to optically transparent fli1:EGFP zebrafish larvae in which endothelial cells express green fluorescent protein (GFP), and using confocal microscopy enabled monitoring vascular lipid accumulation and the endothelial cell layer disorganization and thickening in a live animal. The HCD feeding also increased leakage of a fluorescent dextran from the blood vessels. Administering ezetimibe significantly diminished the HCD-induced endothelial cell layer thickening and improved its barrier function. Feeding HCD to lyz:DsRed2 larvae in which macrophages and granulocytes express DsRed resulted in the accumulation of fluorescent myeloid cells in the vascular wall. Using a fluorogenic substrate for phospholipase A(2) (PLA(2)), we observed an increased vascular PLA(2) activity in live HCD-fed larvae compared to control larvae. Furthermore, by transplanting genetically modified murine cells into HCD-fed larvae, we demonstrated that toll-like receptor-4 was required for efficient in vivo lipid uptake by macrophages. These results suggest that the novel zebrafish model is suitable for studying temporal characteristics of certain inflammatory processes of early atherogenesis and the in vivo function of vascular cells.
Early biomarkers and effective therapeutic strategies are desperately needed to treat pancreatic ductal adenocarcinoma (PDAC), which has a dismal 5-year patient survival rate. Here, we report that the novel tyrosine kinase PEAK1 is upregulated in human malignancies, including human PDACs and pancreatic intraepithelial neoplasia (PanIN). Oncogenic KRas induced a PEAK1-dependent kinase amplification loop between Src, PEAK1, and ErbB2 to drive PDAC tumor growth and metastasis in vivo. Surprisingly, blockade of ErbB2 expression increased Src-dependent PEAK1 expression, PEAK1-dependent Src activation, and tumor growth in vivo, suggesting a mechanism for the observed resistance of patients with PDACs to therapeutic intervention. Importantly, PEAK1 inactivation sensitized PDAC cells to trastuzumab and gemcitabine therapy. Our findings, therefore, suggest that PEAK1 is a novel biomarker, critical signaling hub, and new therapeutic target in PDACs.
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