Jacqueline Banyard

αVβ3 Integrin-Targeted PLGA-PEG Nanoparticles for Enhanced Anti-tumor Efficacy of a Pt(IV) Prodrug

Targeted delivery of therapeutics to tumor neovasculature is potentially a powerful approach for selective cancer treatment. Integrins are heterodimeric transmembrane proteins involved in cell adhesion and cell signaling, and their expression is commonly upregulated in cancers and inflammatory diseases. The α(v)β(3) integrin is differentially upregulated on angiogenic endothelial cells as well as on many cancer cells. Here we demonstrate the differential targeting of cisplatin prodrug-encapsulated poly(d,l-lactic-co-glycolic acid)-block-polyethylene glycol (PLGA-PEG) nanoparticles (NPs) to the α(v)β(3) integrin on cancer cells using the cyclic pentapeptide c(RGDfK). Cisplatin is one of the most widely used anticancer drugs, and approaches that can improve its therapeutic index are of broad importance. The RGD-targeted Pt(IV)-encapsulated NPs displayed enhanced cytotoxicity as compared to cisplatin administered in its conventional dosage form in model prostate and breast cancer epithelial cells in vitro. Cytotoxicities were also elevated in comparison to those of previously reported systems, a small molecule Pt(IV)-RGD conjugate and a Pt(IV) nanoscale coordination polymer carrying RGD moieties. This result encouraged us also to evaluate the anticancer effect of the new construct in an animal model. The RGD-targeted PLGA-PEG NPs were more efficacious and better tolerated by comparison to cisplatin in an orthotopic human breast cancer xenograft model in vivo.

Motility and invasion are differentially modulated by Rho family GTPases

Cell migration in vivo often requires invasion through tissue matrices. Currently little is known regarding the signaling pathways that regulate cell invasion through three-dimensional matrices. The small GTPases Cdc42, Rac and Rho are key regulators of actin cytoskeletal and adhesive structures. We now show that expression of dominant negative forms of either Cdc42, Rac or Rho inhibited PDGF-BB-stimulated Rat1 fibroblast invasion into 3D collagen matrices, indicating that the activity of each of these GTPases is necessary for cell invasion. In contrast, only Rac activation was required for PDGF-BB-stimulated locomotion across a planar substrate in the Boyden chamber. Interestingly, PDGF-induced invasion was also strongly inhibited by expression of constitutively active forms of Cdc42 or Rho, and to a lesser extent by constitutively active Rac. We also show that constitutively active V12-Rac significantly stimulated basal Rat1 fibroblast invasion, independent of PI-3-kinase activity, and that this effect was suppressed by the effector mutant V12/H40-Rac. These results suggest that cellular invasion may require an optimal level of activation of Cdc42, Rho and Rac, and that migration and invasion are differentially modulated by Rho family GTPases.

Cystatin B As a Tissue and Urinary Biomarker of Bladder Cancer Recurrence and Disease Progression

Purpose: Using proteomic techniques, we sought to identify novel protein biomarkers in tissue and urine from patients with transitional cell carcinoma (TCC). Experimental Design: Urinary and tissue proteomes were analyzed and differentially expressed proteins were identified by mass spectrometry. One of the proteins, cystatin B, was further analyzed in TCC tissue by immunohistochemistry and in urine by semiquantitative Western blot analysis. Results: Cystatin B tissue staining intensity significantly increased concordantly with TCC grade (P = 0.0008). Elevated urinary cystatin B levels correlated with increasing tumor grade (P = 0.062) and stage (P = 0.0047). Patients with elevated levels of cystatin B had a shorter mean ± SE time to disease recurrence (12 ± 1.82 months) compared with patients who had low levels (28.8 ± 2.26 months; P = 0.0047). Similarly, patients with elevated cystatin B levels had a shorter time to grade/stage progression compared with patients with low urinary cystatin B (P = 0.0007). By multivariate Cox regression analysis, an elevated cystatin B level was the most significant variable predicting disease recurrence (hazard ratio, 3.8; 95% confidence interval, 1.5-9.5; P = 0.0049) and grade/stage progression (hazard ratio, 10.4; 95% confidence interval, 1.6-201.5; P = 0.0104). Conclusions: Cystatin B is elevated in tissue and urine of bladder cancer patients. Cystatin B urine levels are positively correlated with tumor grade, stage, and shorter time to disease recurrence and progression. Consequently, cystatin B may be useful as a novel predictive biomarker in TCC of the bladder.

Regulation of cell proliferation by the antizyme inhibitor: evidence for an antizyme-independent mechanism

The antizyme inhibitor was discovered as a protein that binds to the regulatory protein antizyme and inhibits the ability of antizyme to interact with the enzyme ornithine decarboxylase (ODC). Blocking antizyme activity subsequently leads to increased intracellular levels of ODC and increased ODC enzymatic activity. We now report that antizyme inhibitor is a positive modulator of cell growth. Overexpression of antizyme inhibitor in NIH-3T3 mouse fibroblasts or in AT2.1 Dunning rat prostate carcinoma cells resulted in an increased rate of cell proliferation and an increase in saturation density of the cultured cells. This was accompanied by an increase in intracellular levels of the polyamine putrescine. In AT2.1 cells, antizyme inhibitor overexpression also increased the ability of the cells to form foci when grown under anchorage-independent conditions. In order to determine the role of antizyme on antizyme inhibitor activity we created an antizyme inhibitor mutant, AZIΔ117-140, which lacks the putative antizyme-binding domain. We show that this mutant fails to bind to antizyme, but remains capable of inducing increased rates of cell proliferation, suggesting that antizyme inhibitor has antizyme-independent functions. Silencing antizyme inhibitor expression leads to diminished levels of cyclin D1 and to reduced cell proliferation. Antizyme inhibitor is capable of preventing cyclin D1 degradation, and this effect is at least partially independent of antizyme. We show that wild-type antizyme inhibitor and the AZIΔY mutant are capable of direct interaction with cyclin D1 suggesting a potential mechanism for the antizyme-independent effects. Together, our data suggest a novel function for antizyme inhibitor in cellular growth control.

The Role of Cell Motility in Prostate Cancer

Cell motility is a critical determinant of prostate cancer metastasis. The current review discusses the role for cell motility in metastatic dissemination, the evidence that prostate cancer metastasis is dependent on increased cell motility and describes the molecules whose expression has been shown to correlate with the increased motility that accompanies prostate cancer progression. These include receptors for growth factors and cytokines that regulate cell motility as well as intracellular proteins that interact with actin or that regulate signal transduction associated with cell motility. Motility related modulators include both positive regulators of cell movement that are upregulated during tumor progression and suppressors of cell movement that are down-regulated during progression. Because altered expression of such genes may determine the metastatic potential of any particular prostate tumor, we conclude that the appearance or disappearance of motility-related molecules could be used to aid in the diagnosis and prognosis of human prostate cancer.

Regulation of epithelial plasticity by miR-424 and miR-200 in a new prostate cancer metastasis model

Using an in vivo cycling strategy, we selected metastatic cancer cells from the lymph nodes (LN) of mice bearing orthotopic DU145 human prostate tumors. Repeated rounds of metastatic selection (LN1–LN4) progressively increased the epithelial phenotype, resulting in a new model of tumor cell mesenchymal-epithelial transition (MET). DU145-LN4 showed increased cell-cell adhesions, higher expression of multiple epithelial markers, such as E-cadherin, EpCAM and cytokeratin 18, and reduced expression of mesenchymal markers such as vimentin. The MET in DU145-LN4 cells was accompanied by increased expression of the miR-200 family, and antimiRs to miR-200c and miR-141 induced an EMT. MET also correlated with the loss of miR-424. Ectopic transient and stable miR-424 expression induced EMT, with reduced epithelial marker expression and increased cell scattering. Our model provides evidence for spontaneous MET in vivo. We show that this cellular plasticity can be mediated through the combined action of miR-424 and the miR-200 family.

The role of EMT and MET in cancer dissemination.

Abstract Metastatic cancer cells are lethal. Understanding the molecular mechanisms that bolster the conversion from benign to malignant progression is key for treating these heterogeneous and resistant neoplasms. The epithelial-mesenchymal transition (EMT) is a conserved cellular program that alters cell shape, adhesion and movement. The shift to a more mesenchymal-like phenotype can promote tumor cell intravasation of surrounding blood vessels and emigration to a new organ, yet may not be necessary for extravasation or colonization into that environment. Lymphatic dissemination, on the other hand, may not require EMT. This review presents emerging data on the modes by which tumor cells promote EMT/MET via microRNA and prepare the pre-metastatic niche via exosomes.

Collagen XXIII Expression Is Associated with Prostate Cancer Recurrence and Distant Metastases

Purpose: We had previously identified a new transmembrane collagen, type XXIII, in metastatic rat prostate carcinoma cells. The purpose of this study was to determine the expression of collagen XXIII in human prostate cancer and investigate its relationship with disease progression. Experimental Design: We investigated collagen XXIII expression in prostate cancer tissue and did a retrospective analysis of association with prostate-specific antigen (PSA)–defined disease recurrence. The presence of collagen XXIII in prostate cancer patient urine was also assessed before and after prostatectomy. Results: Collagen XXIII protein was detected at very low levels in benign prostate tissue and was significantly increased in prostate cancer. Distant metastases exhibited significantly higher collagen XXIII levels compared with either localized prostate cancer or regional (lymph node) metastases. Patients with high collagen XXIII levels had a 2.8-fold higher risk of PSA failure with median time to failure of 8.1 months, compared with low collagen XXIII patients with a median time to failure of 5 years. Multivariate Cox regression showed that the presence of collagen XXIII was significantly associated with time to PSA recurrence, independent of other clinical variables. Collagen XXIII was also detected in prostate cancer patient urine, with reduced levels after prostatectomy, indicating potential as a noninvasive fluid biomarker. Conclusions: We present the first report demonstrating increased collagen XXIII expression in prostate cancer tissue. We show that collagen XXIII level is a significant independent predictor of PSA-defined disease recurrence, suggesting a potential role as a molecular biomarker of prostate cancer progression and metastasis.

Identification of genes regulating migration and invasion using a new model of metastatic prostate cancer

BackgroundUnderstanding the complex, multistep process of metastasis remains a major challenge in cancer research. Metastasis models can reveal insights in tumor development and progression and provide tools to test new intervention strategies.MethodsTo develop a new cancer metastasis model, we used DU145 human prostate cancer cells and performed repeated rounds of orthotopic prostate injection and selection of subsequent lymph node metastases. Tumor growth, metastasis, cell migration and invasion were analyzed. Microarray analysis was used to identify cell migration- and cancer-related genes correlating with metastasis. Selected genes were silenced using siRNA, and their roles in cell migration and invasion were determined in transwell migration and Matrigel invasion assays.ResultsOur in vivo cycling strategy created cell lines with dramatically increased tumorigenesis and increased ability to colonize lymph nodes (DU145LN1-LN4). Prostate tumor xenografts displayed increased vascularization, enlarged podoplanin-positive lymphatic vessels and invasive margins. Microarray analysis revealed gene expression profiles that correlated with metastatic potential. Using gene network analysis we selected 3 significantly upregulated cell movement and cancer related genes for further analysis: EPCAM (epithelial cell adhesion molecule), ITGB4 (integrin β4) and PLAU (urokinase-type plasminogen activator (uPA)). These genes all showed increased protein expression in the more metastatic DU145-LN4 cells compared to the parental DU145. SiRNA knockdown of EpCAM, integrin-β4 or uPA all significantly reduced cell migration in DU145-LN4 cells. In contrast, only uPA siRNA inhibited cell invasion into Matrigel. This role of uPA in cell invasion was confirmed using the uPA inhibitors, amiloride and UK122.ConclusionsOur approach has identified genes required for the migration and invasion of metastatic tumor cells, and we propose that our new in vivo model system will be a powerful tool to interrogate the metastatic cascade in prostate cancer.

Collagen XXIII: A potential biomarker for the detection of primary and recurrent non-small cell lung cancer

Background: Collagen XXIII is a transmembrane collagen previously shown to be upregulated in metastatic prostate cancer. The purpose of this study was to determine the protein expression of collagen XXIII in tumor tissues from a variety of cancers and to assess the utility of collagen XXIII as a biomarker for non–small cell lung cancer (NSCLC). Methods: A multicancer tissue microarray was used for the immunohistochemical examination of collagen XXIII protein expression in a variety of cancers. Subsequently, collagen XXIII expression was analyzed in three separate cohorts using tissue microarrays with representative tumor and control lung tissues from NSCLC patients. In addition, NSCLC patient urine samples were analyzed for the presence of collagen XXIII through Western blot. Results: Collagen XXIII was present in tissue samples from a variety of cancers. Within lung cancer tissues, collagen XXIII staining was enriched in NSCLC subtypes. Collagen XXIII was present in 294 of 333 (88%) lung adenocarcinomas and 97 of 133 (73%) squamous cell carcinomas. In urine, collagen XXIII was present in 23 of 29 (79%) NSCLC patient samples but only in 15 of 54 (28%) control samples. High collagen XXIII staining intensity correlated with shorter recurrence-free survival in NSCLC patients. Conclusions: We show the capability of collagen XXIII as a tissue and urinary biomarker for NSCLC, in which positivity in tissue or urine significantly correlates with the presence of NSCLC and high staining intensity is a significant recurrence predictor. Impact: Inclusion of collagen XXIII in a tissue- or urine-based cancer biomarker panel could inform NSCLC patient treatment decisions. Cancer Epidemiol Biomarkers Prev; 19(5); 1362–72. ©2010 AACR.