Integrins contribute to progression in many cancers, including breast cancer. For example, the interaction of ␣ 5 ␤ 1 with plasma fibronectin causes the constitutive invasiveness of human prostate cancer cells. Inhibition of this process reduces tumorigenesis and prevents metastasis and recurrence. In this study, naturally serum-free basement membranes were used as invasion substrates. Immunoassays were used to compare the roles of

IntroductionRapid induction of keratinocyte and fibroblast migration into wounds is necessary for tissue repair. Reepithelialization begins soon after injury as epidermal cells become migratory (1, 2). Because plasma fibronectin (pFn) circulates in the blood at 0.3-0.5 mg/mL (reviewed in ref.3) and binds to fibrin, it is a significant component of clot provisional matrix. If the basement membrane is destroyed, epidermal cells migrate over the pFnrich provisional matrix (4-6). The fibronectin of the provisional matrix is also known to promote the movement of fibroblasts, macrophages, and blood vessels into the wound space (1, 7). Keratinocytes, other epithelial cell types such as mammary and prostate epithelial cells, and fibroblasts express the α5β1 integrin fibronectin receptor (8-11). Although keratinocytes express less α5β1 than α3β1, α5β1 is more important for migration (8). Also, expression of α5β1 (12, 13) and specific metalloproteinases (14) are increased in motile keratinocytes at the leading edge of the new epithelium. Keratinocyte and fibroblast cell movement is supported by pFn (15,16). Fragments of pFn containing the cell-binding domain, which are found in wounds due to localized proteinase activity (17, 18), stimulate monocyte (7) and fibroblast chemotaxis through the dermis (19-21), as well as matrix metalloproteinase expression by fibroblasts (11). This interaction also stimulates cellular invasion of the provisional matrix and wound stroma (16; reviewed in ref. 22). The pFn cell-binding domain also induces monocytes to extravasate from proximal vessels, enabling them to enter the wound and differentiate into inflammatory macrophages (23), and can stimulate normal human endothelial cell motility in vitro (24). Thus, the invasive/migratory phenotype expressed by epithelial cells and fibroblasts after wounding may result from pFn fragmentation and α5β1 integrin-mediated signaling. Furthermore, this interaction promotes the immigration or motility of a number of cell types involved in healing through a variety of extracellular matrices, including the stroma as well as vascular basement membranes (23,24).To define the invasion-stimulatory sequences of pFn, the naturally serum-free basement membranes and associated extracellular matrices of sea urchin embryos (SU-ECM) were used as in vitro invasion substrates (25). We report that the PHSRN sequence of pFn stimulates SU-ECM invasion by keratinocytes and fibroblasts. Acetylated and amidated PHSRN peptide (Ac-PHSRN-NH 2 ) is greatly increased in activity, whereas a randomized sequence peptide, Ac-HSPNR-NH 2 , is inactive. In derivatives of the PHSRN peptide, substitution of arginine by alanine or glutamic acid also results in inactivity. The PHSRN sequence of the plasma fibronectin (pFn) cell-binding domain induces human keratinocytes and fibroblasts to invade the naturally serum-free extracellular matricies of sea urchin embryos. The potency of acetylated, amidated PHSRN (Ac-PHSRN-NH 2 ) is significantly increased, making it more active on a molar basis ...

A 5 B 1 Integrin interacts with the PHSRN sequence of plasma fibronectin, causing constitutive invasion by human prostate cancer cells. Inhibition of this process reduces tumorigenesis and prevents metastasis and recurrence. In this study, naturally serum-free basement membranes were used as in vitro invasion substrates. Immunoassays were employed to dissect the roles of focal adhesion kinase (FAK), phosphatidylinositol 3 ¶-kinase (PI3K), and protein kinase CD (PKCD) in A 5 B 1 -mediated, matrix metalloproteinase-1 (MMP-1)-dependent invasion by metastatic human DU 145 prostate cancer cells. We found that a peptide composed of the PHSRN sequence induced rapid FAK phosphorylation at Tyr 397 (Y397), a site whose phosphorylation is associated with kinase activation. The technique of RNA silencing [small interfering RNA (siRNA)] confirmed the role of FAK in PHSRN-induced invasion. PHSRN also induced the association of the p85-regulatory subunit of PI3K with FAK at a time corresponding to FAK phosphorylation and activation, and maximal PI3K activity occurred at this same time. The necessity of PI3K activity in both PHSRN-induced invasion and MMP-1 expression was confirmed by using specific PI3K inhibitors. By employing a specific inhibitor, Rottlerin, and by using siRNA, we also found that PKCD, a PI3K substrate found in focal adhesions, functions in PHSRN-induced invasion. In addition, the induction of MMP-1 in PHSRN-treated DU 145 cells was shown by immunoblotting, and the role of MMP-1 in PHSRNinduced invasion was confirmed by the use of blocking anti-MMP-1 monoclonal antibody. Finally, a close temporal correspondence was observed between PHSRN-induced invasion and PHSRN-induced MMP-1 activity in DU 145 cells.

Amplification and overexpression of ERBB-2 in human breast cancer is thought to play a significant role in the progression of the disease; however, its precise role in the aetiology of altered phenotypes associated with human breast cancer is unknown. We have previously shown that exogenous overexpression of ERBB-2 conferred growth factor independence on human mammary epithelial cells. In this study, we show that ERBB-2 overexpression also causes the cells to acquire other characteristics exhibited by human breast cancer cells, such as anchorage-independent growth and invasion capabilities. ERBB-2-induced invasion is dependent on fibronectin and correlates with the down-regulation of cell surface α4 integrin. In addition ERBB-2 co-immunoprecipitates with focal adhesion kinase (FAK) in these cells. We have also shown, by use of exogenously expressed PTEN and by treatment with the PI3′-kinase inhibitor LY294002, that ERBB-2-induced invasion is dependent on the PI3′-kinase pathway; however, PTEN does not dephosphorylate FAK in these cells. © 2000 Cancer Research Campaign

Purpose: Oral mucositis is a common acute morbidity associated with radiation and/or chemotherapy treatment for cancer. D-Methionine (D-Met), the dextro-isomer of the common amino acid L-methionine, has been documented to protect normal tissues from a diverse array of oxidative insults. Experimental Design: We evaluated if D-Met could selectively prevent radiation-induced oral mucositis using in vitro cell culture models as well as an in vivo model of radiation injury to the oral mucosa in C3H mice. Results: Unlike free-radical scavengers, which protected both normal and transformed tumor cells in vitro from radiation-induced cell death, treatment with D-Met in culture protected nontransformed primary human cells from radiation-induced cell death (protective factor between 1.2 and 1.6; P < 0.05) whereas it did not confer a similar protection on transformed tumor cells. D-Met treatment also provided significant protection to normal human fibroblasts, but not to tumor cell lines, from radiation-induced loss of clonogenicity (protection factor,1.6 F 0.15). D-Met treatment did not alter DNA damage (as measured by histone phosphorylation) following irradiation but seemed to selectively mitigate the loss of mitochondrial membrane potential in nontransformed cells, whereas it did not provide a similar protection to tumor cells.Tumor control of implanted xenografts treated with radiation or concurrent cisplatin and radiation was not altered by D-Met treatment. Pharmacokinetics following administration of a liquid suspension of D-Met in rats showed 68% bioavailability relative to i.v. administration. Finally, in a murine modelof mucositis, a dose-dependent increase inprotection was observed with the protective factor increasing from 1.6 to 2.6 over a range of oral D-Met doses between 200 and 500 mg/kg (P < 0.0003).Conclusions: D-Met protected normal tissues, but not tumor cells, in culture from radiationinduced cell death; it also protected normal cells from radiation-induced mucosal injury in a murine model but did not alter tumor response to therapy. Further studies on the use of D-Met to protect from oral mucositis are warranted.

Idiopathic pulmonary fibrosis is a disease that is characterized by fibroblast accumulation and activation in the distal airspaces of the lung. We hypothesized that fibrotic lung fibroblasts migrate/ invade across basement membranes by integrin-mediated mechanisms as a means of entering alveoli. We demonstrate that in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis, fibronectin signaling is both necessary and sufficient for basement membrane migration/invasion across basement membranes. This effect is mediated through the α 5 β 1 integrin because blockade of fibronectin-α 5 integrin ligation attenuated this response. In contrast, ligation of α 4 β 1 integrin inhibits basement membrane invasion by normal lung fibroblasts but not by fibrotic lung fibroblasts. This phenotypic difference is not related to surface expression of the α 4 β 1 integrin, as demonstrated by flow cytometry. In normal lung fibroblasts but not in fibrotic lung fibroblasts, we show that ligation of α 4 β 1 integrin induces a significant increase in phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activity. Fibrotic lung fibroblasts express constitutively less PTEN mRNA and protein as well as phosphatase activity in comparison to normal lung fibroblasts. Together, these data suggest that a loss of α 4 β 1 signaling via PTEN confers a migratory/invasive phenotype to fibrotic lung fibroblasts. Furthermore, this study implicates a loss of PTEN function in the pathophysiology of idiopathic pulmonary fibrosis. Keywords pulmonary fibrosis; extracellular matrix; fibronectins; cell movement Pulmonary fibrosis is the end result of injury to the lung (1). Antecedent injuries to the lung may be recognized, as with chemotherapy (2), collagen vascular disease (3), or inhalational injury (4). However, in the idiopathic interstitial pneumonias, the inciting insult remains unidentified (5). Of the various idiopathic interstitial pneumonias, usual interstitial pneumonia (UIP) has the worst prognosis and is the histologic pattern that is associated with the clinical entity known as idiopathic pulmonary fibrosis (IPF). UIP/IPF is characterized by a lower lobe predominant, subpleural deposition of scar tissue with a relative paucity of inflammatory cells (6). Although most fibrotic zones are composed of "old," relatively acellular collagen bundles, NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript small aggregates of intraalveolar myofibroblasts and fibroblasts have been identified in "fibroblastic foci" of UIP (7). Myofibroblasts within fibroblastic foci are the predominant source of secreted collagen (8). Intraalveolar fibroblasts are found in proximity to both collagen-and fibronectin-rich areas in pulmonary fibrosis (9), suggesting a possible role for extracellular matrix (ECM) signaling in the pathophysiology of UIP (10).Integrins are the major receptors for ECM proteins (11) and are comprised of two noncovalently linked subunits, termed α and β, that together form heterodimer pairs. T...

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.

Purpose-External beam radiation therapy is often used as in an attempt to cure localized prostate cancer (PCa), but is only palliative against disseminated disease. Raf Kinase Inhibitory Protein (RKIP) is a metastasis suppressor whose expression is reduced in approximately 50% of localized PCa tissues and is absent in metastases. Chemotherapeutic agents have been shown to induce tumor apoptosis through induction of RKIP expression. Our goal was to test if radiation therapy similarly induces apoptosis through induction of RKIP expression.Methods-The C4-2B PCa cell line was engineered to over express or under express RKIP. The engineered cells were tested for apoptosis in cell culture and tumor regression in mice following radiation treatment.Results-Radiation induced both RKIP expression and apoptosis of PCa cells. Over expression of RKIP sensitized PCa cells to radiation-induced apoptosis; whereas, short-hairpin targeting of RKIP, so that radiation could not induce RKIP expression, protected cells from radiation-induced apoptosis. In a murine model, knockdown of RKIP in PCa cells diminished radiation-induced apoptosis. Molecular concept mapping of genes altered upon manipulation of RKIP expression revealed that an inverse correlation with the concept of genes altered by irradiation. Conclusion-The data presented here indicate that the loss of RKIP, as seen in primary PCa tumors and metastases, confers protection against radiation-induced apoptosis. Therefore, it is conceivable that loss of RKIP confers a growth advantage upon PCa cells at distant sites since loss of RKIP would decrease apoptosis, favoring proliferation.