Unbiased Phenotype-Based Screen Identifies Therapeutic Agents Selective for Metastatic Prostate Cancer.

In American men, prostate cancer is the second leading cause of cancer-related death. Dissemination of prostate cancer cells to distant organs significantly worsens patients' prognosis, and currently there are no effective treatment options that can cure advanced-stage prostate cancer. In an effort to identify compounds selective for metastatic prostate cancer cells over benign prostate cancer cells or normal prostate epithelial cells, we applied a phenotype-based in vitro drug screening method utilizing multiple prostate cancer cell lines to test 1,120 different compounds from a commercial drug library. Top drug candidates were then examined in multiple mouse xenograft models including subcutaneous tumor growth, experimental lung metastasis, and experimental bone metastasis assays. A subset of compounds including fenbendazole, fluspirilene, clofazimine, niclosamide, and suloctidil showed preferential cytotoxicity and apoptosis towards metastatic prostate cancer cells in vitro and in vivo. The bioavailability of the most discerning agents, especially fenbendazole and albendazole, was improved by formulating as micelles or nanoparticles. The enhanced forms of fenbendazole and albendazole significantly prolonged survival in mice bearing metastases, and albendazole-treated mice displayed significantly longer median survival times than paclitaxel-treated mice. Importantly, these drugs effectively targeted taxane-resistant tumors and bone metastases - two common clinical conditions in patients with aggressive prostate cancer. In summary, we find that metastatic prostate tumor cells differ from benign prostate tumor cells in their sensitivity to certain drug classes. Taken together, our results strongly suggest that albendazole, an anthelmintic medication, may represent a potential adjuvant or neoadjuvant to standard therapy in the treatment of disseminated prostate cancer.

The role of EMT and MET in cancer dissemination.

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.

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

BACKGROUND: Understanding 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. METHODS: To 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. RESULTS: Our 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 ß4) 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-ß4 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. CONCLUSIONS: Our 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.

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.

α(V)ß(3) 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)ß(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)ß(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.

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.

A prognostic gene signature in advanced ovarian cancer reveals a microfibril-associated protein (MAGP2) as a promoter of tumor cell survival and angiogenesis.

Ovarian cancer is the most lethal gynecologic cancer, and this is largely related to its late diagnosis. High grade serous cancers often initially respond to chemotherapy, resulting in a better survival rate, compared to other ovarian carcinoma subtypes. We review recent work identifying a survival-associated gene expression profile for advanced serous ovarian cancer. Within this signature, the authors identified MAGP2, also known as microfibrillar associated protein 5 (MFAP5), as a highly significant indicator of survival and chemosensitivity. MAGP2 is a multifunctional secreted protein--important for elastic microfibril assembly and modulating endothelial cell behavior--with a newly identified role in cell survival. Through alpha(V)beta(3) integrin-mediated signaling, MAGP2 promotes tumor and endothelial cell survival and endothelial cell motility, providing a potential mechanistic link between MAGP2 and angiogenesis as well as patient survival.

Stromal and epithelial networks: Temporal gene expression profiling during invasive neoplasia.

The interaction between tumor cells and the stromal microenvironment is a critical factor in cancer development and progression. A recent study from the Khavari group profiled the expression changes during progression to invasion in a Ras-inducible model of human epithelial neoplasia and used network modeling to analyze the molecular interactions. Human dermis was seeded with H-Ras- and IkappaBalpha-expressing keratinocytes then grafted on to immune-deficient mice. The epithelial and stromal gene expression profiles were captured during progression from quiescent epithelial tissue to in situ neoplasia to invasive neoplasia. A subset of these altered genes was compiled into a "core tumor progression signature" (CTPS), which was shown to have clinical relevance in several cancer types. Network modeling of the CTPS revealed highly interconnected "hubs", which was dominated by extracellular matrix-related genes, including beta(1) integrin. Targeting integrin beta(1) functionality reduced Ras-driven tumorigenesis in vivo and validated the network modeling strategy for predicting genes essential to neoplasia. By integrating temporal analysis of both the epithelial and stromal compartments with network modeling of molecular interactions, this work has described an effective strategy for identifying highly interconnected targets essential to tumor development.

Differential regulation of human thymosin beta 15 isoforms by transforming growth factor beta 1.

We recently identified an additional isoform of human thymosin beta 15 (also known as NB-thymosin beta, gene name TMSB15A) transcribed from an independent gene, and designated TMSB15B. The purpose of this study was to investigate whether these isoforms were differentially expressed and functional. Our data show that the TMSB15A and TMSB15B isoforms have distinct expression patterns in different tumor cell lines and tissues. TMSB15A was expressed at higher levels in HCT116, DU145, LNCaP, and LNCaP-LN3 cancer cells. In MCF-7, SKOV-3, HT1080, and PC-3MLN4 cells, TMSB15A and TMSB15B showed approximately equivalent levels of expression, while TMSB15B was the predominant isoform expressed in PC-3, MDA-MB-231, NCI-H322, and Caco-2 cancer cells. In normal human prostate and prostate cancer tissues, TMSB15A was the predominant isoform expressed. In contrast, normal colon and colon cancer tissue expressed predominantly TMSB15B. The two gene isoforms are also subject to different transcriptional regulation. Treatment of MCF-7 breast cancer cells with transforming growth factor beta 1 repressed TMSB15A expression but had no effect on TMSB15B. siRNA specific to the TMSB15B isoform suppressed cell migration of prostate cancer cells to epidermal growth factor, suggesting a functional role for this second isoform. In summary, our data reveal different expression patterns and regulation of a new thymosin beta 15 gene paralog. This may have important consequences in both tumor and neuronal cell motility.

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.

Unfinished business: Incomplete laminin processing exposes a tumor target.

The G45 domain of laminin-332 is cleaved in normal tissues but remains in squamous cell carcinomas. Targeting this domain using a G45 antibody reduced in vivo tumor growth and invasion and suggests a role for G45 as a new therapeutic target.

Thymosin beta-NB is the human isoform of rat thymosin beta15.

Thymosin beta15 is a small actin-binding protein upregulated in highly metastatic rat prostate cancer cells, relative to low metastatic cells. We have previously established an important role for thymosin beta15 as a diagnostic marker in human prostate cancer, with potential as a prognostic indicator. We here review the data supporting increased thymosin beta15 expression in other cancer types, including breast, brain, and lung. Human NB thymosin beta is a beta-thymosin originally found in neuroblastoma. New data demonstrate that NB thymosin beta represents the human homolog of rat thymosin beta15; thus we suggest classification as human thymosin beta15. In addition to the previously described gene, thymosin beta15a, we report the discovery of a new isoform of human thymosin beta15, thymosin beta15b, which is transcribed from an independent gene on human chromosome X. The gene structure of thymosin beta15a and beta15b is conserved and the isoforms show 87% identity across the nucleotide sequence. Across the coding sequence the nucleotide differences are silent, resulting in identical proteins. Other thymosin family members have recently been shown to exert potent clinical effects. The functional data available for thymosin beta15, combined with the tumor expression pattern, suggest that thymosin beta15 may play an important role in tumor development and progression in addition to its value as a biomarker in prostate cancer.

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.

AKT1 overexpression in endothelial cells leads to the development of cutaneous vascular malformations in vivo.

BACKGROUND: Vascular malformations are clinical disorders in which endothelial cells fail to remodel and/or undergo programmed cell death, leading to abnormal persistence of blood vessels. The abnormal persistence of vessels makes therapy difficult because these lesions are resistant to interventions that are effective against hemangiomas. Akt1 is a serine-threonine protein kinase, which is a key mediator of resistance to programmed cell death. Our objective was to determine whether sustained activation of Akt1 could lead to vascular malformation in mice. OBSERVATIONS: We examined the effect of constitutive activation of Akt1 in murine endothelial cells (MS1 cells). Overexpression of active AKT1 in MS1 cells led to the development of vascular malformations, characterized by wide endothelial lumens and minimal investment of smooth muscle surrounding the vessels. The histologic features of these vascular malformations is distinct from ras-transformed MS1 cells (angiosarcoma) and suggest that differing signal abnormalities give rise to human vascular malformations vs malignant vascular tumors. CONCLUSIONS: Inhibition of Akt signaling may be useful in the treatment of vascular malformations. Examination of problematic hemangiomas and vascular malformations for the presence of activated Akt or downstream targets of Akt, such as mammalian target of rapamycin (mTOR), may predict response to treatment with Akt inhibitors or rapamycin. This study provides a potential rationale for the systemic and topical use of these inhibitors for vascular malformations and hemangiomas.

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, AZI(Delta117-140), 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 AZI(DeltaY) 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.

Type XXIII collagen, a new transmembrane collagen identified in metastatic tumor cells.

We have identified a transmembrane collagen, collagen XXIII, in rat prostate carcinoma cells. Differential display of mRNA expression in prostate carcinoma sublines with varying metastatic potential revealed overexpression of this transcript in the metastatic AT6.1 subline. cDNA cloning identified a 2733-bp transcript from AT6.1 RNA, encoding a protein of 532 amino acids, together with a 3067-bp human homologue, resulting in a 540-amino acid protein. Collagen XXIII is predicted to be a type II membrane protein consisting of an amino-terminal cytoplasmic domain, a transmembrane region, and three collagenous domains flanked by short noncollagenous domains. Collagen XXIII is a new member of the transmembrane collagen family, showing structural homology with the transmembrane collagens XIII and XXV. We present evidence that collagen XXIII is expressed as a approximately 75-kDa protein at the cell surface and that it can be cleaved by furin protease activity. Cleavage results in a approximately 60-kDa soluble protein that forms a multimeric complex and exhibits a low affinity interaction with heparin.