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Understanding how RhoC expression and activation are regulated is essential for deciphering its contribution to tumorigenesis. Here, we report that RhoC expression and activation are induced by the epithelial to mesenchymal transition (EMT) of colon carcinoma. Using LIM 1863 colon cancer cells, RhoC protein expression and subsequent activation were detected coincident with the loss of E-cadherin and acquisition of mesenchymal characteristics. Several Ets-1 binding sites were identified in the RhoC promoter, and evidence was obtained using chromatin immunoprecipitation that Ets-1 can regulate RhoC expression during the EMT. Interestingly, a marked decrease in RhoA activation associated with the EMT was observed that corresponds to the increase in RhoC expression. Use of shRNA established that RhoA inhibits and RhoC promotes post-EMT cell migration, demonstrating functional significance for their coordinate regulation. To assess the importance of RhoC expression in colon cancer, immunohistochemistry was performed on 566 colorectal tumors with known clinical outcome. The level of RhoC ranged from no expression to high expression, and statistical analysis revealed that elevated RhoC expression correlates with poor outcome as well as aberrant expression and localization of E-cadherin. These data provide one mechanism for how RhoC expression is regulated in colon carcinoma and substantiate its utility as a prognostic marker.

Superficial injury involving the mucosa of the gastrointestinal tract heals by a process termed restitution that involves epithelial sheet movement into the damaged area. The forces that drive epithelial sheet movement are only partially understood, although it is known to involve changes in the morphology of cells bordering the damage, such as the formation of large, flat, cytoplasmic extensions termed lamellae. We investigated the mechanism of epithelial sheet movement by following the response of the actin cytoskeleton and specific integrins (α6β4, α6β1, and α3β1) to wounding. To model this event in vitro, monolayers of T84 cells, well-differentiated colon carcinoma cells, were damaged by aspiration and the ensuing response was analyzed by a combination of time-lapse video microscopy, fluorescence confocal microscopy and antibody inhibition assays. We show that wound healing begins with retraction of the monolayer. α6β4 integrin is localized on the basal surface in structures referred to as type II hemidesmosomes that persist throughout this early stage. We hypothesize that these structures adhere to the substrate and function to retard retraction. Once retraction ceases, the wound is contracted initially by actin purse strings and then lamellae. Purse strings and lamellae produce a pulling force on surrounding cells, inducing them to flatten into the wound. In the case of lamellae, we detected actin suspension cables that appear to transduce this pulling force. As marginal cells produce lamellae, their basal type II hemidesmosomes disappear and the α6 integrins appear evenly distributed over lamellae surfaces. Antibodies directed against the α6 subunit inhibit lamellae formation, indicating that redistribution of the α6 integrins may contribute to the protrusion of these structures. Antibodies directed against the α3β1 integrin also reduce the size and number of lamellae. This integrin's contribution to lamellae extension is most likely related to its localization at the leading edge of emerging protrusions. In summary, wounds in epithelial sheets initially retract, and then are contracted by first an actin purse string and then lamellae, both of which serve to pull the surrounding cells into the denuded area. The α6 integrins, particularly α6β4, help contain retraction and both the α6 integrins and α3β1 integrin contribute to lamellae formation.

Insulin-like growth factor II (IGF-II) mRNA-binding protein 3 (IMP3) is emerging as a useful indicator of the progression and outcome of several cancers. IMP3 expression is associated with triple-negative breast carcinomas (TNBCs), which are aggressive tumors associated with poor outcome. In this study, we addressed the hypothesis that signaling pathways, which are characteristic of TNBCs, impact the expression of IMP3 and that IMP3 contributes to the function of TNBCs. The data obtained reveal that IMP3 expression is repressed specifically by estrogen receptor β (ERβ) and its ligand 3βA-diol but not by ERα. Epidermal growth factor receptor (EGFR) signaling and consequent activation of the mitogen-activated protein kinase pathway induce IMP3 transcription and expression. Interestingly, we discovered that the EGFR promoter contains an imperfect estrogen response element and that ERβ represses EGFR transcription. These data support a mechanism in which ERβ inhibits IMP3 expression indirectly by repressing the EGFR. This mechanism relates to the biology of TNBC, which is characterized by diminished ERβ and increased EGFR expression. We also demonstrate that IMP3 contributes to the migration and invasion of breast carcinoma cells. Given that IMP3 is an mRNA-binding protein, we determined that it binds several key mRNAs that could contribute to migration and invasion, including CD164 (endolyn) and MMP9. Moreover, expression of these mRNAs is repressed by ERβ and enhanced by EGFR signaling, consistent with our proposed mechanism for the regulation of IMP3 expression in breast cancer cells. Our findings show that IMP3 is an effector of EGFR-mediated migration and invasion and they provide the first indication of how this important mRNA-binding protein is regulated in cancer.

The Mac-2 lectin (carbohydrate binding protein 35) is a soluble, 32- to 35-kDa phosphoprotein that binds galactose-containing glycoconjugates. We report here that the colonic epithelium is a major site of Mac-2 expression in vivo based on immunohistochemistry of human tissue specimens. In this epithelium, proliferating cells at the base of the crypts do not express Mac-2 but its expression increases with differentiation along the crypt-to-surface axis. Mac-2 expression is concentrated in the nuclei of these differentiated epithelial cells. The progression from normal mucosa to adenoma to carcinoma is associated with significant changes in Mac-2 nuclear localization and expression. In all adenomas (9/9) and carcinomas (13/13) examined, Mac-2 was not present in the nucleus but was localized in the cytoplasm. Sequencing of Mac-2 cDNAs from normal mucosa and carcinoma revealed no specific mutations that could account for this loss of nuclear localization. We also observed a 5- to 10-fold decrease in Mac-2 mRNA levels in cancer compared to normal mucosa as well as a significant reduction in the amount of Mac-2 protein expressed. These observations suggest that Mac-2 exclusion from the nucleus and its decreased expression may be related to the neoplastic progression of colon cancer.

Disruptions in the mucosal lining of the gastrointestinal tract reseal by epithelial cell migration, a process termed restitution. We examined the involvement of laminin isoforms and their integrin receptors in restitution using the intestinal epithelial cell line T84. T84 cells express primarily laminins 5, 6, and 7 as indicated by immunostaining using laminin subunit-specific monoclonal antibodies (MAbs). A MAb (BM2) specific for the laminin alpha 3 subunit, a component of laminins 5, 6, and 7, completely inhibited the closure of mechanical wounds in T84 monolayers. Confocal microscopy using MAbs BM2 (laminin alpha 3 subunit) and 6F12 (laminin beta 3 subunit) revealed that laminin-5 is deposited in a basal matrix that extends into the wound. The MAbs 4E10 (laminin beta 1 subunit) and C4 (laminin beta 2 subunit) stained the lateral membranes between T84 cells. This staining was enhanced in cells adjoining wounds. Because T84 cells stained faintly with MAbs 4C7 (laminin alpha 1 subunit) and with MAbs 4F11 and 1B4 (laminin alpha 2 subunit), we suggest that expression of laminins 6 and 7 is enhanced in response to wounding. The alpha 3 beta 1 integrin and the alpha 6-containing integrins function in wound closure because MAbs specific for the beta 1 integrin subunit (MAb13), the alpha 3 subunit (IVA5), and the alpha 6 subunit (2B7) potently inhibited T84 migration into wounds. Immunofluorescence using UMA9, a beta 4-integrin-specific MAb, revealed that alpha 6 beta 4 integrin exists in a Triton-X-100-insoluble structure at the basal surface and that the staining of this structure is enhanced in cells adjoining wounds. In addition, a Triton-X-100-soluble pool of alpha 6 beta 4, as well as alpha 3 beta 1 and presumably alpha 6 beta 1, was found along lateral surfaces of T84 cells. On flattened cells adjoining wounds, staining for these integrins was distributed diffusely, suggesting a redistribution that accompanies cell migration. Taken together, these data suggest that wound-induced epithelial cell migration is a finely tuned process that is dependent upon the regulated function and localization of specific laminins and their integrin receptors.

Key Points Tumour cells express vascular endothelial growth factor (VEGF) receptors and respond to autocrine and paracrine VEGF signals. VEGF signalling in tumour cells affects tumour functions independently of angiogenesis. VEGF signalling in tumour cells is mediated by VEGF receptor tyrosine kinases (RTKs) and neuropilins (NRPs). NRPs may be at the centre of VEGF signalling because they regulate the function of RTKs and integrins that are crucial for tumour cell function. Autocrine VEGF signalling may be essential for tumour initiation because it regulates the size of the cancer stem cell pool and the self-renewal of cancer stem cells. Therapeutic approaches that aim to target NRPs and VEGF RTKs on tumour cells could be useful to promote tumour regression and to diminish the probability of tumour recurrence, especially when used in combination with VEGF-specific antibodies and other modes of therapy. The roles of vascular endothelial growth factor (VEGF) in cancer go beyond effects on the vasculature. VEGF signalling in tumour cells, which is mediated by VEGF receptor tyrosine kinases and the neuropilins, contributes to many aspects of tumorigenesis, as highlighted in this Review. The function of vascular endothelial growth factor (VEGF) in cancer is not limited to angiogenesis and vascular permeability. VEGF-mediated signalling occurs in tumour cells, and this signalling contributes to key aspects of tumorigenesis, including the function of cancer stem cells and tumour initiation. In addition to VEGF receptor tyrosine kinases, the neuropilins are crucial for mediating the effects of VEGF on tumour cells, primarily because of their ability to regulate the function and the trafficking of growth factor receptors and integrins. This has important implications for our understanding of tumour biology and for the development of more effective therapeutic approaches.

This review explores the mechanistic basis of breast carcinoma progression by focusing on the contribution of integrins. Integrins are essential for progression not only for their ability to mediate physical interactions with extracellular matrices but also for their ability to regulate signaling pathways that control actin dynamics and cell movement, as well as for growth and survival. Our comments center on the alpha6 integrins (alpha6beta1 and alpha6beta4), which are receptors for the laminin family of basement membrane components. Numerous studies have implicated these integrins in breast cancer progression and have provided a rationale for studying the mechanistic basis of their contribution to aggressive disease. Recent work by our group and others on mechanisms of breast carcinoma invasion and survival that are influenced by the alpha6 integrins are discussed.

The role of the integrin alpha 6 beta 1 in breast carcinoma progression was studied by targeted elimination of this integrin in MDA-MB-435 cells, a human breast carcinoma cell line that is highly metastatic in athymic mice. The strategy used is based on the finding that expression of a cytoplasmic domain deletion mutant of the beta 4-integrin subunit (beta 4-delta CYT) in MDA-MB-435 cells eliminates formation of the alpha 6 beta 1 heterodimer. MDA-MB-435 cells that lacked alpha 6 beta 1 expression (beta 4-delta CYT transfectants) formed tumors in athymic mice that were suppressed in their growth and that exhibited a significant increase in apoptosis in comparison to the control tumors. Unlike the control MDA-MB-435 cells, the beta 4-delta CYT transfectants were unable to establish metastatic foci in the lungs. Also, the control transfectants grew substantially better than the beta 4-delta CYT transfectants in the liver after intrahepatic injection because of extensive apoptosis in the beta 4-delta CYT transfectants. These data suggest that a major function of the alpha 6 beta 1 integrin in breast carcinoma is to facilitate tumorigenesis and promote tumor cell survival in distant organs.

The components of tumor cell surfaces that participate in the recognition and lysis of these cells by activated macrophages have not been identified. One plausible hypothesis is that these components are specific carbohydrate structures. As an initial test of this hypothesis, I have made use of the oligosaccharide processing inhibitors 1-deoxynojirimycin (dNM) and 1-deoxymannojirimycin (dMM). dNM is an inhibitor of the glucosidases involved in the initial steps of oligosaccharide processing. dMM inhibits mannosidase I. P815 cells incubated in the presence of 1-2 mM dNM for 24 hr synthesized mature glycoproteins that contained glucosylated high-mannose asparagine-linked oligosaccharides instead of complex forms. The glucosylated oligosaccharides were present in trypsin digests of the cell surface. The dNM treatment resulted in a diminution in the amount of surface galactose residues as evidenced by neuraminidase/galactose oxidase/ NaB3H4 labeling of surface glycopeptides. It did not, however, inhibit protein synthesis or alter the surface polypeptide profile of the tumor cells. P815 and R1- cells incubated in the presence of 1-3 mM dNM for 24 hr were considerably less sensitive to lysis by interferon-γ -activated macrophages than were cells incubated in control medium. At a dNM concentration of 3 mM, a 71% inhibition of P815 cell lysis was observed. Similarly, P815 and R1- cells incubated in the presence of 2 mM dMM were also less sensitive to macrophage-mediated lysis than were control cells. The inhibitors did not affect cell viability, growth, or gross morphology. These observations suggest that complex asparagine-linked oligosaccharides on tumor cell surfaces may participate in recognition and lysis by activated macrophages.

The intracellular levels of many proteins are regulated by ubiquitin-dependent proteolysis. One of the best-characterized enzymes that catalyzes the attachment of ubiquitin to proteins is a ubiquitin ligase complex, Skp1-Cullin-F box complex containing Hrt1 (SCF). We sought to artificially target a protein to the SCF complex for ubiquitination and degradation. To this end, we tested methionine aminopeptidase-2 (MetAP-2), which covalently binds the angiogenesis inhibitor ovalicin. A chimeric compound, proteintargeting chimeric molecule 1 (Protac-1), was synthesized to recruit MetAP-2 to SCF. One domain of Protac-1 contains the IκBα phosphopeptide that is recognized by the F-box protein β-TRCP, whereas the other domain is composed of ovalicin. We show that MetAP-2 can be tethered to SCFβ - TRCP, ubiquitinated, and degraded in a Protac-1-dependent manner. In the future, this approach may be useful for conditional inactivation of proteins, and for targeting disease-causing proteins for destruction.