During tumor progression, malignant cells acquire the ability to overcome cell-cell adhesion and invade surrounding tissues. Almost without exception, what kills cancer patients is metastasis, not the primary tumor. Therefore, understanding tumor metastasis and developing treatments that target metastatic cells present the best hope for long-term tumor regression.
The primary mission of our laboratory is to develop novel treatments that prevent metastasis, or selectively target metastatic cancer cells.
Metastasis is not well understood at the molecular level, but is strongly associated with loss of E-cadherin function in epithelial cells (see Figure 1). E-cadherin is the main epithelial cell-cell adhesion molecule and is either functionally disrupted, or its expression is lost altogether during tumor progression. The effect is likely to be direct, because re-establishing E-cadherin function in cadherin-deficient cell lines can reverse the invasive phenotype. Moreover, experiments in transgenic mice strongly suggest that loss of E-cadherin directly promotes the transition of a benign adenoma into a carcinoma. The E-cadherin gene is frequently affected by loss of heterozygosity in gastric and breast cancer. Inactivating mutations of the wild type E-cadherin allele are typically seen in highly invasive diffuse gastric carcinomas, as well as in invasive lobular breast carcinomas. In most other tumors there is either a heterogeneous loss of E-cadherin expression, or a loss of E-cadherin function, both of which are thought to occur epigenetically. Several mechanisms may account for the epigenetic downregulation of E-cadherin expression, including promoter hypermethylation, and/or increased expression and binding of transcriptional repressors (like Snail, Slug, SIP1, or Twist). Loss of cadherin function is associated with increased tyrosine phosphorylation of the cadherin-catenin complex, which is promoted by sustained activation of several RTKs, or soluble tyrosine kinases (e.g. Src, Fyn, etc.).
The mechanism by which E-cadherin promotes suppression of invasiveness is still unclear. However, recent data argue that the adhesive function, which is mediated by its extracellular domain, is not involved in this effect. The intracellular domain of E-cadherin interacts directly with β-catenin and p120, via separate conserved interaction domains. β-catenin interacts further with α-catenin, which is required for cell-cell adhesion. α-catenin is also implicated in the association of the cadherin complex to the actin cytoskeleton (although recent data have contradicted this notion), and with receptor tyrosine kinases (RTKs; c-erbB1, c-erbB2 [Her2], and c-Met), which modulate intercellular adhesion by inducing tyrosine phosphorylation of the cadherin-catenin complex, and are known to promote cell motility. On the other hand, p120 binding stabilizes the cadherin complex thereby promoting cell adhesion (Ireton et al., JCB 2002). Loss of p120 expression is prevalent in certain human cancers and is associated with the subsequent loss of cadherin expression, suggesting that p120 can act as a tumor suppressor. However, the phenotype mostly encountered in human carcinomas is p120 mislocalization and accumulation in the cytoplasm and the nucleus, as a result of cadherin loss. p120 overexpression induces dramatic changes in cell morphology and increases cell motility. These effects are apparently mediated by modulation of Rho GTPases (such as RhoA, Rac1 and Cdc42), a family of molecular switches that play instrumental roles in the organization of the cytoskeleton and cell motility. These data led to the hypothesis that endogenous p120 may regulate the balance between adhesive and motile cellular phenotypes. Therefore, both β-catenin and p120 are good candidates for mediating the invasion suppressive effects of E-cadherin. Several ongoing projects are focused on elucidating the role of cadherins and catenins in promoting invasion and metastasis.
A variety of techniques are currently in use in the lab. Protein-protein interactions are studied by specific pulldown assays followed by mass-spec analysis of co-eluting proteins, immunoprecipitation, ELISA, or FRET. Structure-function analysis of interacting proteins is performed by using panels of deletion or point mutants. We study cell migration by scratch-wound assays, by measuring the velocity and directionality of cell movement using a live-cell imaging workstation, or through the use of a transwell assay with the aid of specific chemoattractants. Invasion in vitro, is tested again by the use of a transwell assay, with the difference that cells have to invade through a layer of matrigel prior to migrating to the underside of the transwell filter. Other functional assays routinely used in the lab, include cell aggregation and adhesion strength assays, anchorage-independent growth assays, immunofluorescence, as well as live imaging of microinjected cells, or cells expressing GFP, CFP, or YFP-tagged proteins. Ectopic protein expression (or expression of specific siRNAs) is routinely accomplished by retroviral infection, Amaxa electroporation, or transient transfection. The activation state of Rho family GTPases is determined by specific pulldown assays in cell lysates, or by FRET in live cells using Raichu-constructs. Tumor growth and invasiveness are also studied following the injection of human tumor cells in nude mice. Whole animal fluorescence is used to determine the presence and extent of metastasis. Finally, the expression of cadherin/catenin genes in human cancer is tested by real-time PCR, western blotting, or immunohistochemistry of matched tumor and control samples individually, or after constructing tissue arrays.
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