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Integrins regulate cell-cell and cell-matrix adhesion and thereby play critical roles in tumor progression and metastasis. Although work in preclinical models suggests that β1 integrins may stimulate metastasis of a number of cancers, expression of the β1 subunit alone has not been shown to be a useful prognostic indicator in human cancer patients. Here we have demonstrated that the α2β1 integrin suppresses metastasis in a clinically relevant spontaneous mouse model of breast cancer. These data are consistent with previous studies indicating high expression of α2β1 integrin in normal breast epithelium and loss of α2β1 in poorly differentiated breast cancer. They are also consistent with our systematic analysis of microarray databases of human breast and prostate cancer, which revealed that decreased expression of the gene encoding α2 integrin, but not genes encoding α1, α3, or β1 integrin, was predictive of metastatic dissemination and decreased survival. The predictive value of α2 expression persisted within both good-risk and poor-risk cohorts defined by estrogen receptor and lymph node status. Thus, the α2β1 integrin functionally inhibits breast tumor metastasis, and α2 expression may serve as an important biomarker of metastatic potential and patient survival.

Phosphoinositide 3-kinase (PI(3)K) is a unique enzyme characterized by both lipid and protein kinase activities. Here, we demonstrate a requirement for the protein kinase activity of PI(3)K in agonist-dependent beta-adrenergic receptor (betaAR) internalization. Using PI(3)K mutants with either protein or lipid phosphorylation activity, we identify the cytoskeletal protein non-muscle tropomyosin as a substrate of PI(3)K, which is phosphorylated in a wortmannin-sensitive manner on residue Ser 61. A constitutively dephosphorylated (S61A) tropomyosin mutant blocks agonist-dependent betaAR internalization, whereas a tropomyosin mutant that mimics constitutive phosphorylation (S61D) complements the PI(3)K mutant, with only lipid phosphorylation activity reversing the defective betaAR internalization. Notably, knocking down endogenous tropomyosin expression using siRNAs that target different regions if tropomyosin resulted in complete inhibition of betaAR endocytosis, showing that non-muscle tropomyosin is essential for agonist-mediated receptor internalization. These studies demonstrate a previously unknown role for the protein phosphorylation activity of PI(3)K in betaAR internalization and identify non-muscle tropomyosin as a cellular substrate for protein kinase activity of PI(3)K.

The α2β1 integrin plays an important but complex role in angiogenesis and vasculopathies. Published GWAS studies established a correlation between genetic polymorphisms of the α2β1 integrin gene and incidence of diabetic retinopathy. Recent studies indicated that α2-null mice demonstrate superior vascularization in both the wound and diabetic microenvironments. The goal of this study was to determine whether the vasculoprotective effects of α2-integrin deficiency extended to the retina, using the oxygen-induced retinopathy (OIR) model for retinopathy of prematurity (ROP).In the OIR model, wild-type (WT) and α2-null mice were exposed to 75% oxygen for 5 days (postnatal day [P] 7 to P12) and subsequently returned to room air for 6 days (P12-P18). Retinas were collected at postnatal day 7, day 13, and day 18 and examined via hematoxylin and eosin and Lectin staining. Retinas were analyzed for retinal vascular area, neovascularization, VEGF expression, and Müller cell activation. Primary Müller cell cultures from WT and α2-null mice were isolated and analyzed for hypoxia-induced VEGF-A expression.In the retina, the α2β1 integrin was minimally expressed in endothelial cells and strongly expressed in activated Müller cells. Isolated α2-null primary Müller cells demonstrated decreased hypoxia-induced VEGF-A expression. In the OIR model, α2-null mice displayed reduced hyperoxia-induced vaso-attenuation, reduced pathological retinal neovascularization, and decreased VEGF expression as compared to WT counterparts.Our data suggest that the α2β1 integrin contributes to the pathogenesis of retinopathy. We describe a newly identified role for α2β1 integrin in mediating hypoxia-induced Müller cell VEGF-A production.

Airway smooth muscle (ASM) from infant guinea pigs has less spontaneous relaxation during stimulation than ASM from adults. Inhibition of cyclooxygenase (COX), which catalyzes the production of prostanoids, increases this relaxation in infant ASM and abolishes age differences, thus suggesting that prostanoids reduce relaxation in infant ASM. In this study, we investigated whether leukotrienes are also involved in reducing spontaneous relaxation; whether the two COX isoforms, COX-1 and COX-2, differentially regulate spontaneous relaxation; and whether prostanoid release is developmentally regulated in guinea pig ASM. In different age groups, we measured relaxation during and after electrical stimulation in tracheal strips as well as prostanoid release from tracheal segments. Relaxation was studied in the absence and in the presence of a lipoxygenase inhibitor, a cysteinyl leukotriene receptor-1 antagonist, a COX-1 inhibitor, or a COX-2 inhibitor. We found that inhibition of lipoxygenase or cysteinyl leukotriene receptor-1 antagonism did not increase spontaneous relaxation at any age, thus excluding a role for leukotrienes in this phenomenon. Inhibition of COX-2, but not COX-1, promoted spontaneous relaxation. The basal release of prostanoids was more abundant in tissue from infant animals and decreased significantly with age. Thromboxane B2 was the most abundant metabolite released at all ages. Electrical stimulation and epithelium removal did not affect the age difference in prostanoid release. We conclude that increased basal prostanoid release contributes to the reduced spontaneous relaxation in immature guinea pig ASM compared with older animals. By regulating ASM relaxation, prostanoids may play a role in the airway hyperresponsiveness at a young age.

Angiogenesis expands the vascular network during normal development and in response to angiogenic stress. Dysregulation of this dynamic process contributes to tumor progression and to the pathogenesis of many diseases. Evidence suggests that the alpha2beta1 integrin, a collagen and laminin receptor, plays an important role in angiogenesis. In the wound-healing and tumor microenvironment, deletion of the alpha2beta1 integrin has been reported to increase neoangiogenesis. In contrast, small molecule inhibitor (SMI) targeting of the alpha2 integrin blocks sprouting angiogenesis. To reconcile these divergent findings and gain a fuller understanding of alpha2beta1 integrin's role in angiogenesis we turned to the retina. The retinal model is uniquely suited for angiogenesis investigations as the retinal vasculature develops postnatally in a 2-dimensional plane in a well-characterized manner. Evaluation of the alpha2-null retina reveals a constellation of defects and delays in vascular development, including delayed vessel outgrowth, and increased vessel irregularity and decreased plexus density at the vascular front. Additionally we determined that alpha2 integrin-deletion has a protective effect in an oxygen-induced retinopathy model of retinopathy of prematurity (ROP) in mice by inhibiting both hyperoxia-induced vaso-obliteration and hypoxia-induced pathologic neovascularization. Confirming this result, our analysis of human microarray data shows, for the first time, that preterm infants with lower ITGA2 expression are less likely to suffer from ROP. This work clarifies the role of alpha2beta1 integrin in sprouting angiogenesis and raises the intriguing possibility of alpha2 integrin targeted therapies for prevention of ROP. These changes are reminiscent of changes observed in other models with dysregulated notch signaling. Recent studies reported that the alpha2beta1 integrin regulates sprouting angiogenesis by inducing DLL4 in ‘tip cells’. We show, for the first time, notch induced downregulation of alpha2beta1 integrin expression in ‘stalk cells’. Together these results suggest that the alpha2beta1 integrin coordinates endothelial notch signaling by stabilizing tip-stalk status. The apparent discrepancy between the effects of the alpha2 integrin inhibition and integrin-deletion may reflect differences between acute and chronic upregulation of notch signaling. We propose that synergistic use of notch and alpha2 integrin targeted therapies may provide enhanced anti-tumor angiogenesis. Citation Format: Aasakiran Madamanchi, Megan Capozzi, Ling Geng, Zhengzhi Li, Zhonghua Zhang, Richard Friedman, Kent Dickeson, John Penn, Mary Zutter. Alpha2beta1 integrin regulation of endothelial notch signaling in the retina. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3900. doi:10.1158/1538-7445.AM2013-3900