Recent studies have demonstrated the importance of E-cadherin, a homophilic cell–cell adhesion molecule, in contact inhibition of growth of normal epithelial cells. Many tumor cells also maintain strong intercellular adhesion, and are growth-inhibited by cell– cell contact, especially when grown in three-dimensional culture. To determine if E-cadherin could mediate contact-dependent growth inhibition of nonadherent EMT/6 mouse mammary carcinoma cells that lack E-cadherin, we transfected these cells with an exogenous E-cadherin expression vector. E-cadherin expression in EMT/6 cells resulted in tighter adhesion of multicellular spheroids and a reduced proliferative fraction in three-dimensional culture. In addition to increased cell–cell adhesion, E-cadherin expression also resulted in dephosphorylation of the retinoblastoma protein, an increase in the level of the cyclin-dependent kinase inhibitor p27kip1 and a late reduction in cyclin D1 protein. Tightly adherent spheroids also showed increased levels of p27 bound to the cyclin E-cdk2 complex, and a reduction in cyclin E-cdk2 activity. Exposure to E-cadherin–neutralizing antibodies in three-dimensional culture simultaneously prevented adhesion and stimulated proliferation of E-cadherin transfectants as well as a panel of human colon, breast, and lung carcinoma cell lines that express functional E-cadherin. To test the importance of p27 in E-cadherin–dependent growth inhibition, we engineered E-cadherin–positive cells to express inducible p27. By forcing expression of p27 levels similar to those observed in aggregated cells, the stimulatory effect of E-cadherin–neutralizing antibodies on proliferation could be inhibited. This study demonstrates that E-cadherin, classically described as an invasion suppressor, is also a major growth suppressor, and its ability to inhibit proliferation involves upregulation of the cyclin-dependent kinase inhibitor p27.

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Previous gene targeting studies have implicated an indispensable role of vascular endothelial growth factor (VEGF) in tumor angiogenesis, particularly in tumors of embryonal or endocrine origin. In contrast, we report here that transformation of VEGF-deficient adult fibroblasts (MDF528) with ras or neu oncogenes gives rise to highly tumorigenic and angiogenic fibrosarcomas. These aggressive VEGF-null tumors (528ras, 528neu) originated from VEGF(-/-) embryonic stem cells, which themselves were tumorigenically deficient. We also report that VEGF production by tumor stroma has a modest role in oncogene-driven tumor angiogenesis. Both ras and neu oncogenes down-regulated at least two endogenous inhibitors of angiogenesis [pigment epithelium derived factor (PEDF) and thrombospondin 1 (TSP-1)]. This is functionally important as administration of an antiangiogenic TSP-1 peptide (ABT-526) markedly inhibited growth of VEGF(-/-) tumors, with some ingress of pericytes. These results provide the first definitive genetic demonstration of the dispensability of tumor cell-derived VEGF in certain cases of 'adult' tumor angiogenesis, and thus highlight the importance of considering VEGF-independent as well as VEGF-dependent pathways when attempting to block this process pharmacologically.

Other tumor-specific "anti-adhesives" should be explored that can be effective chemosensitizers when used in combination with cell cycle-specific drugs for the treatment of small, solid tumors.

Although in vitro anchorage-independent growth is widely used as a marker of cell transformation, the biological implications of this trait are poorly understood. We previously demonstrated that enforced anchorage-independent growth of a nontumorigenic, immortalized epithelial cell line (IEC-18) in multicellular spheroid culture results in massive apoptotic cell death. This death process, termed anoikis, is prevented by expression of transforming oncogenes, which also confer tumorigenic competence. This study examines whether acquisition of an anoikis-resistant phenotype is causally related to the tumorigenic capacity of transformed epithelial cells. Parental IEC-18 cells were subjected to 10 cycles of selection for survival in speroid culture. Unlike parental cells, the resulting anoikis-resistant variants (AR1.10 and AR2.10) formed relatively large tumors in nude mice. Both anoikis-resistant sublines displayed upregulated expression of vascular endothelial growth factor (VEGF), a potent angiogenesis stimulator. VEGF121 overexpression alone did not induce tumorigenic conversion of parental IEC-18 cells, which remained highly susceptible to anoikis. We postulate that both anoikis-resistance and angiogenic-competence contribute to tumor formation. Development of anoikis-resistance can be then viewed as a precondition for expression of the tumorigenic phenotype. Our results suggest that even when angiogenesis is not a rate limiting factor (e.g. in vitro) the selective pressures of solid tumor-like, 3-dimensional growth conditions favoring anoikis resistance result in collateral induction of a proangiogenic phenotype.

We have previously shown, using tumor cell populations genetically tagged by random integrations of plasmid DNA, that metastatically-competent clonal cell variants have a strong growth advantage within primary tumors over their non-metastatic counterparts. As a result, primary tumors can become overgrown by the progeny of such cells, a process referred to as "clonal dominance" of primary tumors by metastatically-competent cells. Because of the well-known "metastatic inefficiency" of the multi-step cascade process of spread and growth, clonal dominance within primary tumors may be necessary for distant metastatic spread or increase the probability of its occurrence. To examine this hypothesis mice were inoculated s.c. with mixture of non-metastatic and genetically tagged, metastatically-competent mouse mammary carcinoma cells in defined ratios, but always containing an excess of the unmarked non-metastatic population. Progressive overgrowth of the metastatic subpopulation was monitored as a function of time by Southern analysis of DNA obtained from mixed primary tumors. This allowed us to evaluate the effects that surgical removal of the primary tumor had before, during and after effective clonal dominance, and what influence this had on the subsequent formation of distant metastases. Surgical removal of primary tumors before metastatic clonal dominance resulted in a low (0.25%) frequency of lung metastases, whereas removal just 1 or 2 weeks later during or after clonal dominance was achieved resulted in a high (75-100%) frequency of such metastases. Our results support the hypothesis that dominance of primary tumors by metastatically competent cells may be necessary for distant metastatic spread, and also suggest that clonal interactions play a significant role in modulating the metastatic ability of tumor cells in vivo.

It has been suggested that transforming growth factor beta (TGF-beta) may be a potential negative autocrine growth regulator of carcinomas including mammary carcinomas. To directly test this hypothesis we have cloned and expressed human TGF-beta 1 cDNA in a murine mammary adenocarcinoma which is normally growth-inhibited by addition of exogenous TGF-beta in vitro. A number of transfectants over-expressing the foreign TGF-beta 1 mRNA were selected and compared to transfectants which did not overexpress the exogenous TGF-beta 1 cDNAS. Cell lines overexpressing the transfected TGF-beta 1 mRNA were found to produce total levels of TGF-beta 7 to 10 fold greater than the parental cells or control transfected clones. However, when levels of active fractions of TGF-beta were compared in cell lines overexpressing TGF-beta 1 to those which did not, no differences were found. This suggests that the activation mechanism is not necessarily induced or altered by increasing levels of latent TGF-beta 1 production in a given tumor cell line. The basal in vitro doubling time of TGF-beta 1 overexpressing clones was identical to the control populations. Similarly, in vivo tumor growth rates after s.c. injection were similar to that of the parental line. Thus the precise role of TGF-beta in mediating either the in vitro or in vivo growth control of a sensitive mammary adenocarcinoma cell line remains unclear. It may be that cellular over-secretion of latent TGF-beta must be coupled with enhanced cellular TGF-beta activation prior to any observed effect on growth rate in vitro or in vivo; this latter event may constitute the "rate-limiting" step of TGF-beta activity on tumor behavior.