Humphrey Gardner

Cyclin D1 provides a link between development and oncogenesis in the retina and breast

Mice lacking cyclin D1 have been generated by gene targeting in embryonic stem cells. Cyclin D1-deficient animals develop to term but show reduced body size, reduced viability, and symptoms of neurological impairment. Their retinas display a striking reduction in cell number due to proliferative failure during embryonic development. In situ hybridization studies of normal mouse embryos revealed an extremely high level of cyclin D1 in the retina, suggesting a special dependence of this tissue on cyclin D1. In adult mutant females, the breast epithelial compartment fails to undergo the massive proliferative changes associated with pregnancy despite normal levels of ovarian steroid hormones. Thus, steroid-induced proliferation of mammary epithelium during pregnancy may be driven through cyclin D1.

Cyclin E Ablation in the Mouse

E type cyclins (E1 and E2) are believed to drive cell entry into the S phase. It is widely assumed that the two E type cyclins are critically required for proliferation of all cell types. Here, we demonstrate that E type cyclins are largely dispensable for mouse development. However, endoreplication of trophoblast giant cells and megakaryocytes is severely impaired in the absence of cyclin E. Cyclin E-deficient cells proliferate actively under conditions of continuous cell cycling but are unable to reenter the cell cycle from the quiescent G(0) state. Molecular analyses revealed that cells lacking cyclin E fail to normally incorporate MCM proteins into DNA replication origins during G(0)-->S progression. We also found that cyclin E-deficient cells are relatively resistant to oncogenic transformation. These findings define a molecular function for E type cyclins in cell cycle reentry and reveal a differential requirement for cyclin E in normal versus oncogenic proliferation.

Regulation of inflammation by collagen-binding integrins α1β1 and α2β1 in models of hypersensitivity and arthritis

Adhesive interactions play an important role in inflammation by promoting leukocyte attachment and extravasation from the vasculature into the peripheral tissues. However, the importance of adhesion molecules within the extracellular matrix-rich environment of peripheral tissues, in which cells must migrate and be activated, has not been well explored. We investigated the role of the major collagen-binding integrins, alpha1beta1 and alpha2beta1, in several in vivo models of inflammation. mAbs against murine alpha1 and alpha2 were found to significantly inhibit effector phase inflammatory responses in animal models of delayed-type hypersensitivity (DTH), contact hypersensitivity (CHS), and arthritis. Mice that were alpha1-deficient also showed decreased inflammatory responses in the CHS and arthritis models when compared with wild-type mice. Decreased leukocyte infiltration and edema formation accompanied inhibition of antigen-specific models of inflammation, as nonspecific inflammation induced by croton oil was not inhibited. This study demonstrates the importance in vivo of alpha1beta1 and alpha2beta1, the collagen-binding integrins, in inflammatory diseases. The study also extends the role of integrins in inflammation beyond leukocyte attachment and extravasation at the vascular endothelial interface, revealing the extracellular matrix environment of peripheral tissues as a new point of intervention for adhesion-based therapies.

Profiles of Matrix Metalloproteinases, Their Inhibitors, and Laminin in Stroke Patients Influence of Different Therapies

BACKGROUND AND PURPOSE The goal of this study was to determine the temporal profile of several matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), and laminin (an MMP substrate) in human stroke under different treatment paradigms, including thrombolysis and hypothermia. METHODS We serially measured the serum levels of MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, and laminin in 50 patients with acute ischemic stroke using zymography or enzyme-linked immunosorbent assay. Patients were treated with heparin, therapeutic thrombolysis, or hypothermia. Scandinavian Stroke Scale scores were obtained at baseline. Infarct volume was measured with CT scanning on day 4 after stroke onset. Healthy persons were used as control subjects. RESULTS MMP-2 and MMP-9 increased during the course of ischemia, whereas intact laminin and TIMP-2 decreased significantly (P<0.05). MMP-9 and laminin levels varied significantly by infarct size (P=0.001) and therapy (P=0.0005). MMP-9 levels were significantly higher in patients treated with tissue plasminogen activator (tPA) compared with patients treated with hypothermia. The cleaved form of MMP-9 was found solely in 4 patients treated with tPA. Intact laminin levels were significantly lower in the tPA group than in the hypothermia group. CONCLUSIONS Selected MMPs and TIMPs are involved in the pathophysiology of acute stroke. This is also reflected by changes in laminin. Treatment paradigms differentially influence levels of MMP-9 and laminin. Combination therapies explicitly involving MMP inhibition could be of value in future treatment strategies.

|[alpha]|1|[beta]|1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis

Psoriasis is a common T cell–mediated autoimmune inflammatory disease. We show that blocking the interaction of α1β1 integrin (VLA-1) with collagen prevented accumulation of epidermal T cells and immunopathology of psoriasis. α1β1 integrin, a major collagen-binding surface receptor, was exclusively expressed by epidermal but not dermal T cells. α1β1-positive T cells showed characteristic surface markers of effector memory cells and contained high levels of interferon-γ but not interleukin-4. Blockade of α1β1 inhibited migration of T cells into the epidermis in a clinically relevant xenotransplantation model. This was paralleled by a complete inhibition of psoriasis development, comparable to that caused by tumor necrosis factor-α blockers. These results define a crucial role for α1β1 in controlling the accumulation of epidermal type 1 polarized effector memory T cells in a common human immunopathology and provide the basis for new strategies in psoriasis treatment focusing on T cell–extracellular matrix interactions.

Integrin α1β1 and Transforming Growth Factor-β1 Play Distinct Roles in Alport Glomerular Pathogenesis and Serve as Dual Targets for Metabolic Therapy

Alport syndrome is a genetic disorder resulting from mutations in type IV collagen genes. The defect results in pathological changes in kidney glomerular and inner-ear basement membranes. In the kidney, progressive glomerulonephritis culminates in tubulointerstitial fibrosis and death. Using gene knockout-mouse models, we demonstrate that two different pathways, one mediated by transforming growth factor (TGF)-β1 and the other by integrin α1β1, affect Alport glomerular pathogenesis in distinct ways. In Alport mice that are also null for integrin α1 expression, expansion of the mesangial matrix and podocyte foot process effacement are attenuated. The novel observation of nonnative laminin isoforms (laminin-2 and/or laminin-4) accumulating in the glomerular basement membrane of Alport mice is markedly reduced in the double knockouts. The second pathway, mediated by TGF-β1, was blocked using a soluble fusion protein comprising the extracellular domain of the TGF-β1 type II receptor. This inhibitor prevents focal thickening of the glomerular basement membrane, but does not prevent effacement of the podocyte foot processes. If both integrin α1β1 and TGF-β1 pathways are functionally inhibited, glomerular foot process and glomerular basement membrane morphology are primarily restored and renal function is markedly improved. These data suggest that integrin α1β1 and TGF-β1 may provide useful targets for a dual therapy aimed at slowing disease progression in Alport glomerulonephritis.

Treatment of murine lupus with cDNA encoding IFN-γR/Fc

IFN-γ, a pleiotropic cytokine, is a key effector molecule in the pathogenesis of several autoimmune diseases, including lupus. Importantly, deletion of IFN-γ or IFN-γR in several lupus-predisposed mouse strains resulted in significant disease reduction, suggesting the potential for therapeutic intervention. We evaluated whether intramuscular injections of plasmids with cDNA encoding IFN-γR/Fc can retard lupus development and progression in MRL-Faslpr mice. Therapy significantly reduced serum levels of IFN-γ, as well as disease manifestations (autoantibodies, lymphoid hyperplasia, glomerulonephritis, mortality), when treatment was initiated at the predisease stage, particularly when IFN-γR/Fc expression was enhanced by electroporation at the injection site. Remarkably, disease was arrested and even ameliorated when this treatment was initiated at an advanced stage. This therapy represents a rare example of disease reversal and makes application of this nonviral gene therapy in humans with lupus (and perhaps other autoimmune/inflammatory conditions) highly promising.

Low plasma levels of matrix metalloproteinase 9 permit increased tumor angiogenesis.

Angiogenesis is essential for tumor growth and blocking this process might be a valid tool for the control of cancer growth. We showed previously that tumor angiogenesis in integrin α1-null mice is reduced compared to that of wild type animals and that over-expression of matrix metalloproteinase 9 (MMP-9) in the α1-null and consequent generation of angiostatin (an inhibitor of endothelial cell growth) from circulating plasminogen was implicated in the mechanism of tumor inhibition. Our findings suggested that secretion of excess MMPs generates inhibitors of endothelial cell proliferation, including but not necessarily limited to angiostatin, resulting ultimately in auto-inhibition of angiogenesis. Thus MMP inhibitors used as anti-tumor drugs might in fact cause a paradoxical increase in tumor angiogenesis and tumor growth. In order to determine whether MMP-9 expression was directly involved in the regulation of tumor growth, we specifically inhibited or enhanced MMP-9 synthesis in vitro and in vivo, and subsequently analysed primary endothelial cell proliferation and angiostatin synthesis, as well as tumor vascularization and development. We provide evidence that reduction of plasma levels of MMP-9 in either normal or integrin α1-null mice leads to decreased synthesis of angiostatin and consequent increased tumor growth and vascularization. In contrast, specifically enhancing MMP-9 expression in vivo caused a reduction in tumor vascularization. These findings are the opposite to other studies suggesting a pro-tumorigenic role for MMP-9, and may account for some of the recently observed failures of anti MMP therapy in tumor treatment.

PIK3CA Mutations May Be Discordant between Primary and Corresponding Metastatic Disease in Breast Cancer

Purpose:PIK3CA mutations are frequent in breast cancer and activate the PI3K/Akt pathway. Unexpectedly, PIK3CA mutation appears in general to be associated with better outcome. In a cohort of patients where both primary and metastatic lesions were available, the objective was to assess changes in PIK3CA mutations. We wished to discern whether selective pressures occur and the influence of PIK3CA mutation on time to recurrence. Experimental Design: Formalin-fixed paraffin-embedded tumor blocks were obtained from 104 patients with paired samples from primary tumors and corresponding asynchronous metastatic breast tumors. Samples were analyzed for PIK3CA mutations (exons 9 and 20) as well as immunohistochemical evaluation for PTEN, pAKT, Ki67, ER, and HER2. Results:PIK3CA mutation was detected in 45% of the primary tumors. Overall, there was a net gain in mutation in metastatic disease, to 53%; nonetheless, there were instances where metastases were wild type in patients with PIK3CA mutant primary tumors. Laser capture microdissection on a subset of cases revealed microheterogeneity for PIK3CA mutational status in the primary tumor. PIK3CA mutants overall showed a significantly longer time to first recurrence than wild type cases (P = 0.03). Conclusion:PIK3CA mutations occur at high frequency in primary and metastatic breast cancer; these may not necessarily confer increased aggressiveness as mutants had a longer time to recurrence. Because PIK3CA status quite frequently changes between primary and metastatic disease, it emphasizes the necessity of assessing the PIK3CA status in the metastatic lesion for selection of PIK3CA inhibitor therapy. Clin Cancer Res; 17(4); 667–77. ©2010 AACR.

Antibody-mediated blockade of integrin ?v?6 inhibits tumor progression in vivo by a transforming growth factor-?–regulated mechanism

The alpha(v)beta(6) integrin is up-regulated on epithelial malignancies and has been implicated in various aspects of cancer progression. Immunohistochemical analysis of alpha(v)beta(6) expression in 10 human tumor types showed increased expression relative to normal tissues. Squamous carcinomas of the cervix, skin, esophagus, and head and neck exhibited the highest frequency of expression, with positive immunostaining in 92% (n = 46), 84% (n = 49), 68% (n = 56), and 64% (n = 100) of cases, respectively. We studied the role of alpha(v)beta(6) in Detroit 562 human pharyngeal carcinoma cells in vitro and in vivo. Prominent alpha(v)beta(6) expression was detected on tumor xenografts at the tumor-stroma interface resembling the expression on human head and neck carcinomas. Nonetheless, coculturing cells in vitro with matrix proteins did not up-regulate alpha(v)beta(6) expression. Detroit 562 cells showed alpha(v)beta(6)-dependent adhesion and activation of transforming growth factor-beta (TGF-beta) that was inhibited >90% with an alpha(v)beta(6) blocking antibody, 6.3G9. Although both recombinant soluble TGF-beta receptor type-II (rsTGF-beta RII-Fc) and 6.3G9 inhibited TGF-beta-mediated Smad2/3 phosphorylation in vitro, there was no effect on proliferation. Conversely, in vivo, 6.3G9 and rsTGF-beta RII-Fc inhibited xenograft tumor growth by 50% (n = 10, P < 0.05) and >90% (n = 10, P < 0.001), respectively, suggesting a role for the microenvironment in this response. However, stromal collagen and smooth muscle actin content in xenograft sections were unchanged with treatments. Although further studies are required to consolidate in vitro and in vivo results and define the mechanisms of tumor inhibition by alpha(v)beta(6) antibodies, our findings support a role for alpha(v)beta(6) in human cancer and underscore the therapeutic potential of function blocking alpha(v)beta(6) antibodies.