Angiogenesis requires endothelial cell invasion and is crucial for wound healing and for tumor growth and metastasis. Invasion of native collagen is mediated by the alpha(5)beta(1) integrin fibronectin receptor. Thus, alpha(5)beta(1) up-regulation on the surfaces of endothelial cells may induce endothelial cell invasion to stimulate angiogenesis. We report that the interaction of alpha(5)beta(1) with its PHSRN peptide ligand induces human microvascular endothelial cell invasion and that PHSRN-induced endothelial cell invasion is regulated by alpha(4)beta(1) integrin and requires matrix metalloproteinase 1 (MMP-1). Moreover, our results show that exposure to PHSRN causes rapid, specific up-regulation of surface levels of alpha(5)beta(1) integrin and significantly increases alpha(5) integrin mRNA in microvascular endothelial cells. Consistent with these results, alpha(5) small interfering RNA abrogates PHSRN-induced surface alpha(5) and MMP-1 up-regulation, as well as blocking invasion induction. We also observed dose-dependent, PHSRN-induced alpha(5)beta(1) integrin up-regulation on endothelial cells in vivo in Matrigel plugs. We further report that the PHSCN peptide, an alpha(5)beta(1)-targeted invasion inhibitor, blocks PHSRN-induced invasion, alpha(5)beta(1) up-regulation, alpha(5) mRNA induction, and MMP-1 secretion in microvascular endothelial cells and that systemic PHSCN administration prevents PHSRN-induced alpha(5)beta(1) up-regulation and angiogenesis in Matrigel plugs. These results demonstrate a critical role for alpha(5)beta(1) integrin and MMP-1 in mediating the endothelial cell invasion and angiogenesis and suggest that PHSRN-induced alpha(5) transcription and alpha(5)beta(1) up-regulation may form an important feed-forward mechanism for stimulating angiogenesis.

In the present study, we demonstrate that Ca 2 þ -induced growth inhibition and induction of differentiation in a line of human colon carcinoma cells (CBS) is dependent on mitogen-activated protein (MAP) kinase signaling and is associated with upregulation of extracellular calcium-sensing receptor (CaSR) expression. When CBS cells were grown in Ca 2 þ -free medium and then switched to medium supplemented with 1.4 mM Ca 2 þ , proliferation was reduced and morphologic features of differentiation were expressed. E-cadherin, which was minimally expressed in nonsupplemented medium, was rapidly induced in response to Ca 2 þ stimulation. Sustained activation of the extracellular signal-regulated kinase (ERK) occured in Ca 2 þ -supplemented medium. When an inhibitor of ERK activation (10 mM U0126) was included in the Ca 2 þ -supplemented culture medium, ERK-activation did not occur. Concomitantly, E-cadherin was not induced, cell proliferation remained high and differentiation was not observed. The same level of Ca 2 þ supplementation that induced MAP kinase activation also stimulated CaSR upregulation in CBS cells. A clonal isolate of the CBS line that did not upregulate CaSR expression in response to extracellular Ca 2 þ was isolated from the parent cells. This isolate failed to produce E-cadherin or undergo growth inhibition/induction of differentiation when exposed to Ca 2 þ in the culture medium. However, ERK-activation occurred as efficiently in this isolate as in parent CBS cells or in a cloned isolate that underwent growth reduction and differentiation in response to Ca 2 þ stimulation. Together, these data indicate that CaSR upregulation and MAP kinase signalling are both intermediates in the control of colon carcinoma cell growth and differentiation. They appear to function, at least in part, independently of one another.

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Previous studies have demonstrated that all-trans retinoic acid (RA) increases collagen production and decreases matrix metalloproteinase (MMP) activity in organ-cultured human skin. Decreased MMP activity is associated with up-regulation of tissue inhibitor of metalloproteinase-1 (TIMP-1). These changes are accompanied by a hyperplastic response in the epidermis. Here we show that a synthetic picolinic ester-substituted retinoid (designated as MDI 301) has comparable effects to those of RA in regard to these activities. What makes these findings of interest is that RA also stimulates elaboration of several pro-inflammatory cytokines and up-regulates leukocyte adhesion molecules in organ-cultured skin. MDI 301 does not induce such changes or is much less active. In a past study we showed that while RA was irritating to the skin of topically treated hairless mice, MDI 301 was essentially non-irritating under the same conditions [Varani et al. (2003) Arch. Dermatol Res 295:255-262]. Taken in conjunction with the findings from the past study, the present data suggest that MDI 301 will be similar to RA in capacity to repair damaged skin, but will be effective under conditions that are not irritating. These findings, thus, suggest that retinoid efficacy and clinically relevant irritancy are not inextricably linked. Potential for efficacy under conditions in which irritation is not observed is a strong rationale for further development of MDI 301 as a skin-repair agent.

The α5β1 integrin fibronectin receptor is an attractive therapeutic target in breast cancer because it plays key roles in invasion and metastasis. While its inactive form is widely expressed, activated α5β1 occurs only on tumor cells and their associated vasculature. The PHSCN peptide has been shown to bind activated α5β1 preferentially, thereby blocking invasion in vitro, and inhibiting growth, metastasis and tumor recurrence in preclinical models. Moreover in a recent Phase I clinical trial, systemic PHSCN monotherapy was well tolerated, and metastatic disease failed to progress for 4-14 months in 38% of patients receiving it. A significantly more potent PHSCN derivative, the PHSCN-polylysine dendrimer (Ac-PHSCNGGK-MAP) has recently been developed. We report that it is 1280- to 6700-fold more potent than the PHSCN peptide at blocking α5β1 mediated SUM-149 PT and MDA-MB-231 human breast cancer cell invasion of naturally occurring basement membranes in vitro. Chou-Talalay analysis of these data suggested that invasion inhibition by the PHSCN dendrimer was highly synergistic. We also report that, consistent with its enhanced invasion-inhibitory potency, the PHSCN dendrimer is 700- to 1100-fold more effective than the PHSCN peptide at preventing SUM-149 PT and MDA-MB-231 extravasation in the lungs of athymic, nude mice. Our results also show that many extravasated SUM-149 PT and MDA-MB-231 cells go on to develop into metastatic colonies, and that pretreatment with the PHSCN dendrimer is more than 100-fold more effective at reducing lung colony formation. Since many patients newly diagnosed with breast cancer already have locally advanced or metastatic disease, the availability of a well-tolerated, nontoxic systemic therapy that can prevent metastatic progression by blocking invasion could be very beneficial.