John J. Grzesiak

The integrin-extracellular matrix axis in pancreatic cancer.

Pancreatic cancer is the fifth leading cause of adult cancer death in the United States, with 5-year survival rates of only 1% to 4%. Current therapeutic strategies generally result in only a few months of extended life. Recent evidence from several independent laboratories in vitro and in vivo indicate that integrin-mediated cell attachment to the extracellular matrix (ECM), components of which are highly up-regulated in pancreatic cancer, evokes phenotypes and signaling pathways that regulate tumor cell growth and migration. In this review, we will discuss our current understanding of the role of the ECM in directing pancreatic cancer growth, progression, and metastasis. Topics covered include a survey of the existing literature regarding the in vivo and in vitro expression of the ECM and its cell surface receptors, the integrins, in pancreatic cancer; mechanisms involved in the integrin-ECM-mediated malignant phenotype; and future directions for the study of the integrin-ECM axis and its role in pancreatic cancer progression, including potential therapeutic strategies.

Determination of the ligand-binding specificities of the alpha2beta1 and alpha1beta1 integrins in a novel 3-dimensional in vitro model of pancreatic cancer.

Objectives: Pancreatic cancer cells express 2 known collagen-binding integrins, &agr;2&bgr;1 and &agr;1&bgr;1. The ligand-binding specificity of &agr;1&bgr;1 and the integrin/s responsible for mediating the malignant phenotype on type I collagen in the 3-dimensional (3D) tumor microenvironment have not been determined in pancreatic cancer. The aim of the present study was to determine the ligand-binding specificities of the &agr;2&bgr;1 and &agr;1&bgr;1 integrins using a novel 3D in vitro model of pancreatic cancer. Methods: We used 3D type I collagen-glycosaminoglycan scaffolds in adhesion and proliferation assays with pancreatic cancer cell lines, as well as affinity chromatography and inhibition of adhesion assays. Results: We demonstrate for the first time that CFPAC, BxPC-3, Colo-357, FG, and Panc-1 cells attach to 3D type I collagen scaffolds in an &agr;2&bgr;1-specific manner and that this integrin-specific adhesion is required for subsequent cell proliferation. MiaPaCa-2 cells, which do not express the &agr;2&bgr;1 or &agr;1&bgr;1 integrins, do not attach or proliferate on 3D type I collagen scaffolds. We also demonstrate the novel finding that the &agr;1&bgr;1 integrin is a type IV collagen receptor in pancreatic cancer cells. Conclusions: These data indicate that targeting &agr;2&bgr;1 integrin-specific type I collagen adhesion may have therapeutic value in the treatment of pancreatic cancer.

Alpha-synuclein overexpression in oligodendrocytic cells results in impaired adhesion to fibronectin and cell death

The cardinal pathological features of multiple system atrophy (MSA) are the presence of glial cytoplasmic inclusions (GCIs) in oligodendrocytes and loss of oligodendrocytes. To understand the mechanisms underlying MSA, we examined the effects of overexpression of human alpha-synuclein (halpha-syn) in CG-4 oligodendrocytic progenitor cells. CG-4 cells overexpressing halpha-syn (halpha-syn CG-4) demonstrated severely impaired adhesion and increased cell death when plated on fibronectin compared to laminin. The expression of the alphav integrin subunit in whole cell lysates was also significantly downregulated in halpha-syn CG-4. These results demonstrate a cytotoxic consequence of halpha-syn overexpression in CG-4. This cytotoxicity appears to be the result of alterations in cell-extracellular matrix interactions, where impaired adhesion to fibronectin is associated with downregulation of the alphav integrin subunit and increased cell death. It may, therefore, be one of the mechanisms underlying the loss of oligodendrocytes in MSA.

GSK3 and PKB/Akt are associated with integrin‐mediated regulation of PTHrP, IL‐6 and IL‐8 expression in FG pancreatic cancer cells

We have demonstrated recently that PTHrP is upregulated in pancreatic adenocarcinoma and that the ECM exerts regulatory control, at least in part, over PTHrP expression. In our present study, we examined the potential signaling interactions between these 2 pathways. Our results demonstrate that, under serum‐free conditions, adhesion of FG pancreatic adenocarcinoma cells on Fn is mediated by the α5β1 integrin, whereas adhesion to Type I collagen is mediated by the α2β1 integrin. α5β1 integrin‐mediated adhesion to Fn results in a phenotype that includes a reduction in cell proliferation, increased E‐cadherin localization in cell–cell contacts, increased β‐catenin localization throughout the cell, inhibition of haptokinetic cell migration, and increased expression of PTHrP, IL‐6 and IL‐8 relative to α2β1 integrin‐mediated adhesion on Type I collagen. A phosphoprotein immunoblotting screen of FG pancreatic cancer cells grown on either Fn or Type I collagen indicates that GSK3 and PKB/Akt are differentially phosphorylated on these 2 substrates. These results implicate GSK3 and PKB/Akt in the integrin‐mediated regulation of PTHrP, IL‐6 and IL‐8 in pancreatic cancer.

The extracellular matrix differentially regulates the expression of PTHrP and the PTH/PTHrP receptor in FG pancreatic cancer cells.

Objectives: Previous studies by our laboratory have demonstrated that parathyroid hormone–related protein (PTHrP) and its receptor (PTH/PTHrP receptor) are commonly expressed in pancreatic cancer and suggest their participation in the progression of this devastating disease. It has also been demonstrated that one of the major hallmarks of pancreatic adenocarcinoma is an increased production of the extracellular matrix (ECM), a critical regulator of diverse cellular processes such as differentiation, proliferation, and angiogenesis. The present study focused on the relationship between the PTHrP and ECM axes in the pathobiology of pancreatic cancer. Method and Results: Using the FG pancreatic adenocarcinoma cell line, we demonstrate a significant inverse correlation between FG cell proliferation and PTHrP expression that depended on the ECM protein on which the cells were cultured (P < 0.05). Generally, ECM proteins that promoted the strongest proliferation, including type I collagen, type IV collagen, and laminin, resulted in decreased expression of PTHrP. Conversely, ECM proteins that promoted the weakest proliferation, including fibronectin, vitronectin, and BSA, resulted in increased expression of PTHrP. A similar trend was found between FG cell proliferation and the PTH/PTHrP receptor expression, with Pearson correlation coefficients of 0.480 (mRNA) and -0.591 (protein). Conclusion: These observations demonstrate a unique functional relationship between the ECM and PTHrP axes and have important implications for our understanding of the complex mechanisms responsible for the progression of pancreatic cancer and its metastases.

Type I collagen and divalent cation shifts disrupt cell-cell adhesion, increase migration, and decrease PTHrP, IL-6, and IL-8 expression in pancreatic cancer cells

Background: We have shown in FG pancreatic cancer cells that α2β1 integrin-mediated type I collagen adhesion decreases parathyroid hormone-related protein (PTHrP), inerleukin-6 (IL-6), and interleukin-8 (IL-8) expression, decreases the localization of E-cadherin and β-catenin in cell-cell contacts, increases cell migration, and increases glycogen synthase kinase 3 (GSK3) and protein kinase B (PKB/Akt) phosphorylation states relative to α5β1 integrin-mediated fibronectin (Fn) adhesion.Aim of the Study: To extend our observations in FG cells to other pancreatic cancer cell lines, and to determine whether E-cadherin-mediated cell-cell adhesion and its downstream effectors were functionally involved in the ECM-mediated regulation of PTHrP, IL-6, and IL-8.Methods: We used standard biochemical techniques to determine ECM-specific differences in E-cadherin and β-catenin localization, GSK3 and PKB/Akt phosphorylation, haptokinetic cell migration, and cytokine expression in pancreatic cancer cells. We also conducted functional studies using pharmacological inhibitors for GSK3 and PKB/Akt, as well as elevated Mg2+/Ca2+ ratios similar to pancreatic juice, and examined their effects on cytokine expression.Results: Differences in E-cadherin and β-catenin localization along with GSK3 and PKB/Akt phosphorylation occur in multiple pancreatic cancer cell lines, resulting in differences in ECM-mediated haptokinesis and cytokine expression that are generally consistent with previous observations in FG cells. Our functional studies also suggest that E-cadherin-mediated cell-cell adhesion and downstream effectors are involved in PTHrP, IL-6, and IL-8 expression.Conclusions: These data indicate that α2β1 integrin-mediated type I collagen adhesion disrupts cell-cell adhesion architecture, resulting in increased migration and decreased PTHrP, IL-6, and IL-8 expression in pancreatic cancer cells.

Activation of the α2β1 integrin‐mediated malignant phenotype on type I collagen in pancreatic cancer cells by shifts in the concentrations of extracellular Mg2+ and Ca2+

The authors have previously demonstrated that α2β1 integrin‐mediated pancreatic cancer cell adhesion to Type I collagen is Mg2+‐dependent, inhibited by Ca2+, and that this integrin, purified from cell lysates using Type I‐collagen‐sepharose in Mg2+, can be eluted with Ca2+. In the present study, the authors examined the divalent cation‐dependency of α2β1 integrin‐mediated pancreatic cancer cell adhesion, migration and proliferation on Type I collagen, an extracellular matrix protein shown to be highly up‐regulated, and to promote the malignant phenotype in vitro and in vivo. The results indicate that cells attach to Type I collagen maximally when Mg2+ is greater than 1 mM, and that addition of increasing concentrations of Ca2+ reduces this adhesion. These effects are reversible, in that previous cell attachment in Mg2+ can be reversed by adding Ca2+, and vice versa. They also demonstrate that pancreatic cancer cells migrate and proliferate on Type I collagen in Mg2+ alone, but maximally when Mg2+ is present at concentrations that promote maximal cell adhesion and Ca2+ is present at concentrations less than Mg2+. Cell adhesion and proliferation assays, as well as affinity chromatography on Type I collagen using anti‐integrin function‐blocking monoclonal antibodies indicate that the effects of these divalent cation shifts are mediated specifically by the α2β1 integrin. As pancreatic juice contains over 1,200‐fold more Mg2+ than Ca2+ and solid tumors are characterized by increased magnesium load, these data indicate that such pathophysiological divalent cation shifts could be involved in the activation of the α2β1 integrin‐mediated malignant phenotype on Type I collagen in the pancreatic cancer.

Divalent cations modulate the integrin‐mediated malignant phenotype in pancreatic cancer cells

We have previously demonstrated that pathophysiological shifts in the concentrations of extracellular Mg2+ and Ca2+ activate the α2β1 integrin‐mediated malignant phenotype on type I collagen in pancreatic cancer cells, as evidenced by increased adhesion, migration and proliferation. In the present study, we examined the integrin and divalent cation specificity of pancreatic cancer cell interactions with other physiologically relevant extracellular matrix proteins, including fibronectin, type IV collagen, laminin and vitronectin. Our results indicate that, like α2β1 integrin‐mediated interactions with type I collagen, β1 integrin‐mediated adhesion to fibronectin, type IV collagen and laminin are promoted by Mg2+ but not by Ca2+. On vitronectin, cells attach via αvβ5 and β1 integrins, and in the presence of either divalent cation. We also demonstrate that, like type I collagen, pancreatic cancer cell migration and proliferation on fibronectin, laminin and type IV collagen is maximal when Mg2+ is present at concentrations that promote optimal adhesion and Ca2+ is present at concentrations less than Mg2+. On vitronectin, Panc‐1 cell migration is maximal with decreased Mg2+ and increased Ca2+, but the reverse is true for BxPC‐3 cells. Both cell lines exhibited maximal proliferation with increased Mg2+ and decreased Ca2+, however. Together with evidence indicating that the in vivo local tumor microenvironment contains increased Mg2+ and decreased Ca2+, our studies demonstrate that such divalent cation shifts could activate the integrin‐mediated malignant phenotype in pancreatic cancer. (Cancer Sci 2008; 99: 1553–1563)

Divalent cations modulate alpha2beta1 integrin-mediated malignancy in a novel 3-dimensional in vitro model of pancreatic cancer.

Objectives: We previously showed that divalent cations regulate &agr;2&bgr;1 integrin-mediated pancreatic cancer cell interactions with type I collagen in 2 dimensions (2D), including cell adhesion, migration, and proliferation. Presently, we examined divalent cation-dependent &agr;2&bgr;1 integrin-mediated pancreatic cancer cell adhesion and proliferation on type I collagen in a novel 3D in vitro model. Methods: Cell attachment, proliferation, and antibody inhibition assays on type I collagen in both 2D and 3D, and microscopy and immunoblotting were used for these studies. Results: As in 2D, cell attachment on type I collagen in 3D is Mg2+-dependent and inhibited by Ca2+. Proliferation in 3D is also Mg2+-dependent, but maximal when Mg2+ is present at concentrations that promote maximal cell adhesion and Ca2+ is present at concentrations less than Mg2+. Immunoblotting studies demonstrate that the divalent cation-dependent changes in cell-cell adhesion observed on type I collagen in both 2D and 3D are associated with the changes in E-cadherin and &bgr;-catenin expression. Antibody inhibition assays indicate further that the &agr;2&bgr;1 integrin specifically mediates proliferation on type I collagen in 3D under altered divalent cation conditions. Conclusions: Divalent cation shifts could activate &agr;2&bgr;1 integrin-mediated malignancy in the type I collagen-rich 3D tumor microenvironment of pancreatic cancer.

Identification of DU 145 prostate cancer cell proteins that bind to the carboxy-terminal peptide of human PTHrP in vitro

Peptides spanning the range of human parathyroid hormone-related protein (PTHrP) have been shown to bind heat shock protein-70 expressed on the surface of cancer cells with cytoprotective consequences in vitro. The present study focused on identification of intracellular proteins that interact with the carboxy-terminal peptide of human PTHrP. Using affinity chromatography, we applied extracts of DU 145 prostate cancer cells over PTHrP (140-173)-Sepharose and eluted with 8 M urea. After concentration and electrophoresis, protein bands were excised and subjected to mass spectroscopy analyses. Proteins identified included those associated with protection from oxidative stress, DNA repair, protection from apoptosis, and proteins involved in membrane trafficking and cytoskeletal rearrangement. These novel protein-protein interactions further support the hypothesis that the carboxy-terminus of PTHrP plays a role in cell survival.