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Yuchen Lee et al., We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers. © 2008 Elsevier Inc., The work was supported by National Institutes of Health Grant 2 R25 CA09481

In prostate cancer, androgen blockade strategies are commonly used to treat osteoblastic bone metastases. However, responses to these therapies are typically brief, and the mechanism underlying androgen-independent progression is not clear. Here, we established what we believe to be the first human androgen receptor-negative prostate cancer xenografts whose cells induced an osteoblastic reaction in bone and in the subcutis of immunodeficient mice. Accordingly, these cells grew in castrated as well as intact male mice. We identified FGF9 as being overexpressed in the xenografts relative to other bone-derived prostate cancer cells and discovered that FGF9 induced osteoblast proliferation and new bone formation in a bone organ assay. Mice treated with FGF9-neutralizing antibody developed smaller bone tumors and reduced bone formation. Finally, we found positive FGF9 immunostaining in prostate cancer cells in 24 of 56 primary tumors derived from human organ-confined prostate cancer and in 25 of 25 bone metastasis cases studied. Collectively, these results suggest that FGF9 contributes to prostate cancer-induced new bone formation and may participate in the osteoblastic progression of prostate cancer in bone. Androgen receptor-null cells may contribute to the castration-resistant osteoblastic progression of prostate cancer cells in bone and provide a preclinical model for studying therapies that target these cells. Fil: Zhi, Gang Li. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Mathew, Paul. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Yang, Jun. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Starbuck, Michael W.. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Zurita, Amado J.. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Liu, Jie. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Sikes, Charles. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Multani, Asha S.. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Efstathiou, Eleni. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Lopez, Adriana. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Wang, Jing. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Fanning, Tina V.. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Prieto, Victor G.. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Kundra, Vikas. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Vazquez, Elba Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Troncoso, Patricia. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Raymond, Austin K.. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Logothetis, Christopher J.. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Lin, Sue-Hwa. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Maity, Sankar. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Navone, Nora M.. University Of Texas Md Anderson Cancer Center; Estados Unidos

Osteoblast cadherin (OB-cadherin, also known as cadherin-11) is a Ca 2+ -dependent homophilic cell adhesion molecule that is expressed mainly in osteoblasts. OB-cadherin is expressed in prostate cancer and may be involved in the homing of metastatic prostate cancer cells to bone. The extracellular domain of OB-cadherin may be used to inhibit the adhesion between prostate cancer cells and osteoblasts. In this report, we describe the expression of the extracellular domain of OB-cadherin as an Fc fusion protein (OB-CAD-Fc) in human embryonic kidney 293FT cells using a bicistronic retroviral vector. Coexpression of GFP and OB-CAD-Fc through the bicistronic vector permitted enrichment of OB-CAD-Fc-expressing cells by fluorescence-activated cell sorting. Recombinant OB-CAD-Fc proteins were secreted into cell medium, and about 0.85 mg of purified OB-CAD-Fc protein was purified from 1 l of the conditioned medium using immobilized protein A-affinity chromatography. The purified OB-CAD-Fc was biologically active because it supported the adhesion of PC3 cells and L cells transduced with OB-cadherin. The availability of OB-CAD-Fc offers opportunities to test whether OB-CAD-Fc can be used to inhibit OB-cadherin-mediated prostate cancer bone metastasis in vivo or to generate antibodies for inhibiting the adhesion between prostate cancer cells and osteoblasts.

Purpose: Prostate cancer tends to metastasize to bone and induce osteoblastic lesions. We identified a soluble form of ErbB3 (sErbB3), p45-sErbB3, in bone marrow supernatant from men with prostate cancer bone metastasis and showed that p45-sErbB3 enhances bone formation. We aimed to understand clinical implications of sErbB3 by establishing an ELISA to detect sErbB3 levels in bone marrow and plasma samples. Experimental Design: We did ELISAs on marrow from 108 men [34 with androgen-dependent disease, 30 with androgen-independent disease (AI) but negative bone scan (AI/BS−), and 44 with AI and positive bone scan (AI/BS+)], sequential marrow from 5 men during treatment, plasma from 52 men before and after docetaxel treatment, and plasma from 95 men ages ≥70 years old without prostate cancer. Results: Some men with clinically detectable bone metastasis had high sErbB3 levels. Within the AI/BS− group, higher sErbB3 levels seemed to yield lower rates of bone metastasis. In the AI/BS+ group, detectable bone metastases took longer to appear in men with higher sErbB3 levels than in men with lower sErbB3 levels (median, 82 versus 41 months). However, high sErbB3 levels did not confer survival benefit after metastasis development. Among men with metastatic progression in bone, docetaxel treatment reduced plasma sErbB3 (P < 0.0001) but did not affect bone-specific alkaline phosphatase (P = 0.206) or prostate-specific antigen (P = 0.906). sErbB3 was also detected in men without prostate cancer. Conclusions: The apparent correlation between higher sErbB3 levels and longer time to bone metastasis suggests that sErbB3 participates in progression in bone of prostate cancer.

ErbB3 is a transmembrane growth factor receptor that has been implicated in the pathogenesis of human cancer. After finding that a truncated form of ErbB3 was present and upregulated in metastatic prostate cancer cells in lymph nodes and bone, we explored the pathophysiological functions of this unusual form of ErbB3 in the context of mouse calvaria as well as osteoblasts in vitro and the femur microenvironment in vivo. Here we demonstrate that prostate cancer cells expressed an alternatively spliced transcript that encodes a 45-kDa glycosylated protein (p45-sErbB3). The recombinant p45-sErbB3 purified from conditioned medium stimulated calvarial bone formation and induced osteoblast differentiation. Overexpression of p45-sErbB3 in the osteolytic prostate cancer cell line PC-3 converted its phenotype from bone lysing to bone forming upon injection into the femurs of immunodeficient mice. Further, we detected sErbB3 in plasma samples from patients with castration-resistant prostate cancer with bone metastasis. These observations establish that p45-sErbB3 is a structurally and functionally unique gene product of ErbB3 and suggest that p45-sErbB3 is likely one of the factors involved in the osteoblastic bone metastases of prostate cancer.

Increased retinal vasopermeability contributes to diabetic retinopathy, the leading cause of blindness in working-age adults. Despite clinical progress, effective therapy remains a major need. Vasoinhibins, a family of peptides derived from the protein hormone prolactin (and inclusive of the 16-kDa fragment of prolactin), antagonize the proangiogenic effects of VEGF, a primary mediator of retinal vasopermeability. Here, we demonstrate what we believe to be a novel function of vasoinhibins as inhibitors of the increased retinal vasopermeability associated with diabetic retinopathy. Vasoinhibins inhibited VEGF-induced vasopermeability in bovine aortic and rat retinal capillary endothelial cells in vitro. In vivo, vasoinhibins blocked retinal vasopermeability in diabetic rats and in response to intravitreous injection of VEGF or of vitreous from patients with diabetic retinopathy. Inhibition by vasoinhibins was similar to that achieved following immunodepletion of VEGF from human diabetic retinopathy vitreous or blockage of NO synthesis, suggesting that vasoinhibins inhibit VEGF-induced NOS activation. We further showed that vasoinhibins activate protein phosphatase 2A (PP2A), leading to eNOS dephosphorylation at Ser1179 and, thereby, eNOS inactivation. Moreover, intravitreous injection of okadaic acid, a PP2A inhibitor, blocked the vasoinhibin effect on endothelial cell permeability and retinal vasopermeability. These results suggest that vasoinhibins have the potential to be developed as new therapeutic agents to control the excessive retinal vasopermeability observed in diabetic retinopathy and other vasoproliferative retinopathies.

Advanced prostate cancer is dominated by bone-forming osseous metastases. Understanding the biology behind this striking clinical manifestation is the key to its effective treatment. A clinical trial using a bone-targeting radiopharmaceutical agent, strontium 89, combined with chemotherapy showed increased survival time among patients with progression of prostate cancer in bone, suggesting that therapeutic strategies focused on treating the tumor in bone are effective. We and others thus hypothesize that interactions between prostate cancer cells and the bone microenvironment play a role in the progression of prostate cancer in bone. Clinical trials and basic science investigations aiming to understand such interactions have been carried out in parallel. In the laboratory studies, human bone marrow specimens have been collected for identification of proteins involved in the bidirectional interactions between prostate cancer cells and bone. In addition, specimens from bone biopsies of the cancer lesions have been used to generate xenografts in animals to establish animal models for testing therapeutic strategies. Clinical trials using agents to inhibit the stromal-prostate cancer interactions (e.g., docetaxel/imatinib or thalidomide) have been done. Analyses of the specimens from these trials provided support of our hypothesis and future development of diagnosis and therapy strategies.

Prostate cancer metastasis is a unique disease. The propensity of prostate cancer to metastasize to bone and the prognostic significance of bone metastasis suggest that effective treatment of bone metastasis may provide clinical benefits. Both osteoblasts and osteoclasts have been shown to play a central role in the interactions between the metastatic prostate cancer cells and bone. Although most prostate cancer bone metastasis is osteoblastic, it remains unclear which cell type is initially involved in the process. Other components in the bone, such as the endothelium and stroma, may also play important roles in this process. The osteoblastic feature of prostate cancer bone metastasis has led to therapies focused on targeting osteoblast activity. Clinical trials targeting osteoblasts use radiopharmaceuticals (strontium-89 and samarium-153), the endothelin A receptor inhibitor atrasentan, or the vitamin D analog calcitriol. Agents that target osteoclasts include bisphosphonates; those that target endothelial cells include thalidomide and bevacizumab. Although these clinical trials for bone metastasis may provide effective treatments, novel concepts of how prostate cancer cells selectively metastasize to bone may advance our understanding and provide improved treatments for this difficult clinical problem. We propose a refined "seed" and "soil" view of metastasis. We speculate that in prostate cancer bone metastasis, the seed may comprise the so-called cancer stem cells, whereas the soil may comprise an onco-niche, ie, a unique microenvironment, that facilitate the growth and survival of cancer stem cells. If so, targeting cancer stem cells or the onco-niche may offer another way to improve treatment of prostate cancer bone metastasis.

Alix (ALG-2-interacting protein X), a cytoplasmic adaptor protein involved in endosomal sorting and actin cytoskeleton assembly, is required for the maintenance of fibroblast morphology. As Alix has sequence similarity to adhesin in Entamoeba histolytica, and we observed that Alix is secreted, we determined whether extracellular Alix affects fibroblast morphology. Here, we demonstrate that secreted Alix is deposited on the substratum of non-immortalized WI38 fibroblasts. Antibody binding to extracellular Alix retards WI38 cell adhesion and spreading on fibronectin and vitronectin. Alix knockdown in WI38 cells reduces spreading and fibronectin assembly, and the effect is partially complemented by coating recombinant Alix on the cell substratum. Immortalized NIH/3T3 fibroblasts deposit less Alix on the substratum and have defects in alpha5beta1-integrin functions. Coating recombinant Alix on the culture substratum for NIH/3T3 cells promotes alpha5beta1-integrin-mediated cell adhesions and fibronectin assembly, and these effects require the aa 605-709 region of Alix. These findings demonstrate that a sub-population of Alix localizes extracellularly and regulates integrin-mediated cell adhesions and fibronectin matrix assembly.

Most statistical approaches summarize the probe-level expression data into gene-level measures, which then are used for downstream statistical analyses. However, there are some limitations in using the gene level data for analysis, such as nonhomogeneous probe effects and the interaction effect (e.g., alternative splicing). In this paper, we consider a two-stage binomial test with a weighted probe rank approach to determine differentially expressed genes. Using a series of benchmark gene array datasets, we show the two-stage binomial test approach yielded a higher positive predictivity and a higher sensitivity than the conventional RMA, GCRMA, Dchip, and ANOVA approaches. In data application, the two-stage binomial test identified a subset of genes strongly related to cell proliferation in the prolactin study, and a subset of genes associated with lymph node metastasis in the breast cancer dataset. In addition, by exploring the proportion of probes with expression changes and the probe expression plot, the two-stage binomial test helped detect an alternative splicing form of the prolactin gene in the prolactin study. In the breast cancer dataset, the approach also identified one potential alternative splicing gene.

The propensity for prostate cancer to metastasize to bone led us and others to propose that bidirectional interactions between prostate cancer cells and bone are critical for the preferential metastasis of prostate cancer to bone. We identified previously a secreted isoform of ErbB3 (p45-sErbB3) in bone marrow supernatant samples from men with prostate cancer and bone metastasis and showed by immunohistochemical analysis of human tissue specimens that p45-sErbB3 was highly expressed in metastatic prostate cancer cells in bone. Here, we show that p45-sErbB3 stimulated mouse calvaria to secrete factors that increased the invasiveness of prostate cancer cells in a Boyden chamber invasion assay. Using gene array analysis to identify p45-sErbB3–responsive genes, we found that p45-sErbB3 up-regulated the expression of osteonectin/SPARC, biglycan, and type I collagen in calvaria. We further show that recombinant osteonectin increased the invasiveness of PC-3 cells, whereas osteonectin-neutralizing antibodies blocked this p45-sErbB3–induced invasiveness. These results indicate that p45-sErbB3 enhances the invasiveness of PC-3 cells in part by stimulating the secretion of osteonectin by bone. Thus, p45-sErbB3 may mediate the bidirectional interactions between prostate cancer cells and bone via osteonectin. [Cancer Res 2007;67(14):6544–8]

Prostate cancer is the most common cancer among men in the United States. Advanced prostate cancer has a particular propensity to metastasize to bone, where it produces predominantly osteoblastic lesions and local bone formation. The tropism for bone is thought to be due in part to specific interactions between the prostate cancer cells and cells present in the bone environment, particularly the bone marrow endothelial cells and osteoblasts. Such interactions involve numerous signaling pathways that could serve as targets for new therapeutic agents. Because androgen directly influences the proliferation and metastasis of prostate cancer cells, the current first-line treatment for metastatic prostate cancer is androgen deprivation therapy. Subsequent therapies include chemotherapy and radiation therapy. New molecular therapies are being developed to target specific steps in the metastatic process. However, as yet none of these therapies has radically improved survival. Nonetheless, it is hoped that with better understanding of the biology of the disease, combination therapy that addresses multiple pathways that support the progression of prostate cancer in bone could significantly improve the survival and quality of life of men with prostate cancer.

Lethal prostate cancer (PCa) is clinically characterized by bone-homing and bone- forming metastases, and bone is frequently the initial organ involved in castrate-resistant progression. These clinical observations led investigators to develop bone-tropic radiopharmaceuticals to treat men with advanced PCa. The ability of the bone-homing alpha emitter (RAD-223) to prolong progression free and overall survival establishes the therapeutic relevance of targeting bone 1. The long accepted paradigm of PCa progression has been dominated by the model of evolution from androgen dependence to androgen independence under the selective pressure of castration. Thus, Src family kinases (SFKs) were investigated experimentally and clinically for the treatment of putative “androgen independent” PCa, as SFKs play a role in both tumor growth and bone turnover. The roles of SFKs in migration, invasion, survival of tumor cells, and osteoclast and osteoblast function in tumor microenvironment were supported by numerous clinical and laboratory observations 2, 3. Despite promising phase I /II reports of dasatinib, a multikinase and potent SFK inhibitor, in men with advanced PCa 4, 5, a phase III trial failed to yield the expected survival advantage 6. The result of this trial highlights the difficulties in translating data from cell line models to biologically heterogeneous clinical PCa and in transitioning from phase I/II to phase III clinical trials. The efficacy of dasatinib was not observed likely due to persistent AR signaling, which remains the primary driver of progression in bone by the transition from paracrine to intracrine androgen signaling 7. Based on these considerations, we are testing the efficacy of an androgen biosynthesis inhibitor in combination with dasatinib to better understand the relevance of mechanism(s). A co–clinical investigational strategy is being applied to better define biomarkers to predict clinical efficacy. These studies also highlight the importance of preclinical work that utilizes models that better reflect the heterogeneity of PCa. This problem is partly overcome by patient- derived xenografts (PDX) 8, 9 10. These models reflect many aspects of PCa in bone and can be used to identify therapy targets and better identify both mechanisms of drug efficacy and development of resistance. Studies in PDX have led to identification of FGF signaling as a therapy target 11. Additional studies in PDX implicated the tumor microenvironment in the striking bone responses observed with cabozantinib 13. These observations led us to propose a model of prostate cancer progression founded on the hypothesis that cancer progresses from being an endocrine-driven cancer (Dihydrotestosterone-dependent) to paracrine/intracrine driven (microenvironment-dependent), and then finally to a cell autochthonous phase 12. Continued studies should lead to decisions on therapy treatment that are based on an understanding of markers that predict disease progression. This new paradigm will also result in more effective therapy combinations. References 1. Parker C, Sartor O. Radium-223 in prostate cancer. N Engl J Med. 2013;369: 1659-1660. 2. Park SI, Zhang J, Phillips KA, et al. Targeting SRC family kinases inhibits growth and lymph node metastases of prostate cancer in an orthotopic nude mouse model. Cancer Res. 2008;68: 3323-3333. 3. Lee YC, Huang CF, Murshed M, et al. Src family kinase/abl inhibitor dasatinib suppresses proliferation and enhances differentiation of osteoblasts. Oncogene. 2010;29: 3196-3207. 4. Yu EY, Massard C, Gross ME, et al. Once-daily dasatinib: expansion of phase II study evaluating safety and efficacy of dasatinib in patients with metastatic castration-resistant prostate cancer. Urology. 2011;77: 1166-1171. 5. Araujo JC, Mathew P, Armstrong AJ, et al. Dasatinib combined with docetaxel for castration-resistant prostate cancer: results from a phase 1-2 study. Cancer. 2012;118: 63-71. 6. Araujo JC, Trudel GC, Saad F, et al. Docetaxel and dasatinib or placebo in men with metastatic castration-resistant prostate cancer (READY): a randomised, double-blind phase 3 trial. Lancet Oncol. 2013;14: 1307-1316. 7. Efstathiou E, Titus M, Tsavachidou D, et al. Effects of abiraterone acetate on androgen signaling in castrate-resistant prostate cancer in bone. J Clin Oncol. 2012;30: 637-643. 8. Aparicio A, Tzelepi V, Araujo JC, et al. Neuroendocrine prostate cancer xenografts with large-cell and small-cell features derived from a single patient's tumor: morphological, immunohistochemical, and gene expression profiles. Prostate. 2011;71: 846-856. 9. Roychowdhury S, Iyer MK, Robinson DR, et al. Personalized oncology through integrative high-throughput sequencing: a pilot study. Sci Transl Med. 2011;3: 111ra121. 10. Li ZG, Mathew P, Yang J, et al. Androgen receptor-negative human prostate cancer cells induce osteogenesis in mice through FGF9-mediated mechanisms. J Clin Invest. 2008;118: 2697-2710. 11. Corn PG, Wang F, McKeehan WL, Navone N. Targeting fibroblast growth factor pathways in prostate cancer. Clin Cancer Res. 2013;19: 5856-5866. 12. Logothetis CJ, Gallick GE, Maity SN, et al. Molecular classification of prostate cancer progression: foundation for marker-driven treatment of prostate cancer. Cancer Discov. 2013;3: 849-861. 13. Varkaris A. et al. Abstract, Prostate Cancer Foundation 2013. Citation Format: Christopher Logothetis, Gary E. Gallick, Sue-Hwa Lin, Nora Navone. The role of bone microenvironment in the lethal progression of prostate cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr IA11. doi:10.1158/1538-7445.CHTME14-IA11

Prostate cancer (PCa) is the most common malignancy in men. Although mortality from PCa has been declining over the past decade, metastasis can substantially shorten survival time and remains a major challenge in maintaining quality of life for survivors. PCa cells preferentially metastasize to bone and typically result in osteoblastic lesions. In the late stages of disease, however, osteolytic lesions are observed. The mechanisms of PCa bone metastasis are still unclear, but relationships between the PCa cells and the bone tissue elements are suspected of being more complex than initially thought. Far from being an innocent bystander, the bone participates actively in the metastatic process and provides the cancer cells with growth factors and a fertile environment. Among the various cells in the bone environment, osteoblasts have a central role through their bidirectional interactions with the PCa cells. This review discusses the possible mechanisms of PCa bone metastasis and highlights the essential role of osteoblasts in the metastasis of PCa to bone.

BACKGROUND. The purpose of the study was to evaluate the efficacy and safety of a bone-targeted regimen consisting of zoledronate, thalidomide, and interferon-γ in patients with renal cell carcinoma and bone metastases. METHODS. Eligible patients had radiographic evidence of bone metastasis. Impending pathologic fractures or spinal cord compressions must have been controlled by surgery or radiation therapy before enrollment. Zoledronate (4 mg) was given intravenously every 4 weeks, thalidomide (300 mg) was given orally once a day, and interferon-γ (100 μg) was given subcutaneously once a week. Patients were evaluated for time to skeletal-related events, the appearance of calcification in osteolytic metastases, and levels of the bone formation/resorption markers. RESULTS. Fifteen patients were treated between November 2002 and November 2003; 12 had previously undergone surgery, radiation, or embolization for their bone metastases; 11 had more than 3 sites of bone involvement; and 9 also had nonosseous metastases in the lung, liver, lymph node, pancreas, or adrenal gland. The median time to progression was 8.3 weeks (range, 2.1–48 weeks). The median time to a skeletal-related event was 12.0 weeks (range, 3.9–46.4 weeks). Two patients discontinued treatment because of adverse drug reactions (1 deep venous thrombosis and 1 myocardial infarction). Two patients experienced pain improvement and developed calcification in osseous metastases; these patients also showed favorable changes in bone marker levels. CONCLUSIONS. In this pilot study a bone-targeted regimen combining zoledronate, thalidomide, and interferon-γ was well tolerated and might provide clinical benefit for a small subset of patients with renal cell carcinoma and bone metastases. Cancer 2006. © 2006 American Cancer Society.

Angiogenesis plays a key role in promoting tumorigenesis and metastasis. Several antiangiogenic factors have been shown to inhibit tumor growth in animal models. Understanding their mechanism of action would allow for better therapeutic application. 16-kDa prolactin (PRL), a NH2-terminal natural breakdown fragment of the intact 23-kDa PRL, exerts potent antiangiogenic and antitumor activities. The signaling mechanism involved in 16-kDa PRL action in endothelial cells remains unclear. One of the actions of 16-kDa PRL is to attenuate the production of nitric oxide (NO) through the inhibition of inducible NO synthase (iNOS) expression in endothelial cells. To delineate the signaling mechanism from 16-kDa PRL, we examined the effect of 16-kDa PRL on interleukin IL-1β–inducible iNOS expression, which is regulated by two parallel pathways, one involving IFN regulatory factor 1 (IRF-1) and the other nuclear factor-κB (NF-κB). Our studies showed that 16-kDa PRL specifically blocked IRF-1 but not NF-κB signaling to the iNOS promoter. We found that IL-1β regulated IRF-1 gene expression through stimulation of p38 mitogen-activated protein kinase (MAPK), which mediated signal transducer and activator of transcription 1 (Stat1) serine phosphorylation and Stat1 nuclear translocation to activate the IRF-1 promoter. 16-kDa PRL effectively inhibited IL-1β–inducible p38 MAPK phosphorylation, resulting in blocking Stat1 serine phosphorylation, its subsequent nuclear translocation and activation of the Stat1 target gene IRF-1. Thus, 16-kDa PRL inhibits the p38 MAPK/Stat1/IRF-1 pathway to attenuate iNOS/NO production in endothelial cells.

Activation of endothelial nitric oxide synthase (eNOS) and subsequent nitric oxide production (NO) are events that mediate the effect of important angiogenic, vasopermeability, and vasorelaxation factors, including vascular endothelial growth factor (VEGF), bradykinin (BK), and acetylcholine (ACh). The N-terminal 16-kDa fragment of prolactin (16KPRL) acts on endothelial cells to inhibit angiogenesis both in vivo and in vitro. Here, we show that 16K-PRL inhibits VEGFinduced eNOS activation in endothelial cells. Inhibition of eNOS activation may mediate the antiangiogenic properties of 16K-PRL, because the NO donor (Z)-1-[2-(2-aminoethyl)N-(2-ammonio-ethyl)amino]diazen-1-ium-1,2-diolate (DETANONOate) prevented 16K-PRL from blocking the VEGFinduced proliferation of endothelial cells. In addition, 16K-PRL inhibited eNOS activation by BK and blocked the BK-evoked transient increase in intracellular Ca 2 in endothelial cells. This finding suggests that 16K-PRL interferes with the mobilization of intracellular Ca 2 , thereby inhibiting the Ca 2 -dependent activation of eNOS. Blockage of eNOS activation can lead to inhibition of vasodilation. Consistently, 16K-PRL inhibited BK-induced relaxation of coronary vessels in isolated perfused guinea pig hearts. Moreover, 16K-PRL inhibited eNOS activation induced by ACh, and this action resulted in the inhibition of both ACh-evoked relaxation of coronary vessels in isolated perfused rat hearts and ACh-induced, endothelium-dependent relaxation of rat aortic segments. In conclusion, 16K-PRL can block the Ca 2 mediated activation of eNOS by three different vasoactive substances, and this action results in the inhibition of both angiogenesis and vasorelaxation. (Endocrinology 145: 5714–5722, 2004)

SummaryInsulin is viewed as a positive regulator of fatty acid synthesis by increasing fatty acid synthase (FAS) mRNA transcription. We uncover a new mechanism by which insulin acutely reduces hepatic FAS activity by inducing phosphorylation of the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) and its interaction with FAS. Ceacam1 null mice (Cc1−/−) show loss of insulin’s ability to acutely decrease hepatic FAS activity. Moreover, adenoviral delivery of wild-type, but not the phosphorylation-defective Ceacam1 mutant, restores the acute effect of insulin on FAS activity in Cc1−/− primary hepatocytes. Failure of insulin to acutely reduce hepatic FAS activity in hyperinsulinemic mice, including L-SACC1 transgenics with liver inactivation of CEACAM1, and Ob/Ob obese mice, suggests that the acute effect of insulin on FAS activity depends on the prior insulinemic state. We propose that this mechanism acts to reduce hepatic lipogenesis incurred by insulin pulses during refeeding.

In this paper, we have characterized the structure, evolutionary origin, and function of rat and human carcinoembryonic antigen-related cell adhesion molecule1 (CEACAM1) multifunctional Ig-like cell adhesion proteins that are expressed by many epithelial tissues. Restriction enzyme digestion reverse transcriptase-PCR analysis identified three cDNAs encoding novel CEACAM1 N-domains. Comparative sequence analysis showed that human and rat CEACAM1 N-domains segregated into two groups differing in similarity to rat CEACAM1(a)-4L and human CEACAM1. Sequence variability analysis indicated that both human and rat N-domains possessed two variable regions, and one contained a major adhesive epitope. Recombination analysis showed that the group of rat but not human N-domains with high sequence similarity was derived at least in part by recombination. Binding assays revealed that three monoclonal antibodies with strong reactivity for the CEACAM1(a)-4L N-domain showed no reactivity with CEACAM1(b)-4S, an allele with a different N-domain sequence. CEACAM1(b)-4S displayed adhesive activity efficiently blocked by a synthetic peptide corresponding to the adhesive epitope in CEACAM1(a)-4L. Blocking analysis also showed that the adhesive epitope for rat CEACAM1 was located downstream from the equivalent human and mouse epitopes. Glycosylation analysis demonstrated O-linked sugars on rat CEACAM1(b)-4S from COS-1 cells. However, this was not the alteration responsible for the lack of monoclonal antibody reactivity. When considered together with previous studies, our findings suggest an inverse relationship between functionality and amino acid sequence similarity to CEACAM1. Like IgG, the N-domain of CEACAM1 appears to tolerate 10-15% sequence diversification without loss of function but begins to show either altered specificity or diminished functionality at higher levels.

Bone is the second most common site of metastases in human cancer. At least two-thirds of the approximately 553,400 Americans who die from cancer each year have bone metastases (Greenlee et al., 2001). Prostate, breast, and lung cancers account for at least 80% of the skeletal metastases. Tumors arising in the prostate and breast are particularly prone to disseminate to bone; up to 85% of patients with these cancers have evidence of bone metastases at autopsy. Carcinomas of the lung, thyroid, and kidney also commonly spread to the bone (30–40%). However, tumors of the gastrointestinal tract rarely metastasize to the bone (5%) (Galasko, 1986).

Microtubules play a central role in coordinating various cellular functions that are orchestrated by their interaction with molecular motors. Anticancer drugs that target microtubule dynamics have been shown to be effective in cancer treatment. However, the effect of microtubule motor-associated molecules on cancer cell proliferation is not clear. Here, we investigated the role of NudC, a nuclear movement protein associated with the microtubule motor dynein, on prostate tumorigenesis. Recombinant adenovirus expressing NudC (Ad-NudC) was used to examine the effects of NudC on the tumorigenicity of prostate cancer cells. Expression of NudC in LNCaP cells inhibited their anchorage-independent growth in a soft agar colony assay. Expression of NudC in DU145 or PC-3 cells inhibited tumor growth in a subcutaneous xenograft model. At the cellular level, expression of NudC in DU145 and PC-3 cells inhibited cell proliferation at 48 h after Ad-NudC infection. FACS analysis of cell cycle distribution showed that 50-60% of Ad-NudC-infected PC-3 cells have a G2/M-phase DNA content compared to about 16-19% in Ad-Luciferase (Ad-Luc)-infected control cells, suggesting that NudC overexpression resulted in aberrant cell cycle progression. Immunofluorescence microscopy revealed a significant increase in cells with a single enlarged nucleus and cells exhibiting multiple nuclei, along with a concomitant increase in cell size in Ad-NudC-infected cells. These results suggest that NudC overexpression led to a block in cell division of prostate cancer cells, and that Ad-NudC may provide a new anticancer drug approach targeting the function of a microtubule motor-associated protein.

The 16-kDa fragment of prolactin (16-kDa PRL), derived from proteolytic cleavage of 23-kDa PRL, was shown to have antiangiogenic activity. Previous studies have shown that recombinant 16-kDa PRL produced from bacteria often contained endotoxins, which are cytotoxic to endothelial cells, and varied in its biological activity due to changes in its refolding from inclusion bodies. These problems limited the use of recombinant 16-kDa PRL. To improve the generation of recombinant 16-kDa PRL, we expressed 16-kDa PRL in Sf9 insect cells using a baculoviral expression system. The signal sequence of the human PRL gene and codons for seven histidines were added to the N- and C-termini, respectively, of the 16-kDa PRL cDNA construct. Recombinant 16-kDa PRL was detected in both the cell pellet and the medium. About 0.28 mg purified protein was isolated from the cell pellet of 4 x 10(7) infected cells using nickel affinity chromatography. Sixteen kilodalton PRL was posttranslationally modified with apparent molecular weights of 16 and 18 kDa on SDS-PAGE. The level of 18-kDa protein was significantly reduced after digestion with peptidyl-N-glycosidase, suggesting that the heterogeneity was due to glycosylation of 16-kDa PRL. N-terminal sequence analysis confirmed the fact that both proteins were human 16-kDa PRL and the signal sequences were cleaved at the same position as that of human PRL. Consistent with its role as an angiogenesis inhibitor, purified recombinant 16-kDa PRL inhibits the proliferation of endothelial cells with a potency similar to that previously reported for the protein generated in Escherichia coli. This 16-kDa PRL expressed in Sf9 cells is a useful reagent for functional studies and for the purification and identification of its receptor.

NudC, a nuclear movement protein that associates with dynein, was originally cloned as a mitogen-inducible early growth response gene. NudC forms a biochemical complex with components of the dynein/dynactin complex and is suggested to play a role in translocation of nuclei in proliferating neuronal progenitors as well as in migrating neurons in culture. Here, we show that NudC plays multiple roles in mitosis and cytokinesis in cultured mammalian cells. Altering NudC levels by either small interfering RNA-mediated gene silencing or adenovirus-mediated overexpression resulted in multinucleated cells and cells with persistent intercellular connections and disorganized midzone and midbody matrix. These phenotypes suggest a failure in cytokinesis in NudC altered cells. Further, a key mitotic enzyme, polo-like kinase, is mislocalized from the centrosomes and the midbody in NudC altered cells. Gene silencing of nud-1, the Caenorhabditis elegansortholog of NudC, led to a loss of midzone microtubules and the rapid regression of the cleavage furrow, which resulted in one-celled embryos containing two nuclei. The loss of midzone microtubule organization owing to silencing of the NudC/nud-1 gene in two systems, coupled with the loss of Plk1 from mitotic structures in mammalian cells, provide clues to the cytokinesis defect and the multinucleation phenotype. Our findings suggest that NudC functions in mitosis and cytokinesis, in part by regulating microtubule organization at the midzone and midbody.

The 16-kDa prolactin (PRL), derived from the proteolytic cleavage of wild-type 23-kDa PRL, has been shown to have antiangiogenic activity. Such an antiangiogenic activity may have an effect on tumor growth in vivo. Here we examined the effect of 23-kDa and 16-kDa PRL on tumor growth, and the potential of using recombinant 16-kDa human PRL for prostate cancer therapy. The effects of 23-kDa PRL and 16-kDa PRL on the tumorigenicity of prostate cancer cells in vivo were studied. Using an adenovirus transfer vector to achieve high efficiency 23-kDa and 16-kDa PRL transfection in DU145 and PC-3 human prostate carcinoma cell lines, we demonstrated that expression of 16-kDa PRL in the prostate cancer cells markedly reduced their ability to form tumors in a xenograft animal model. These studies established that the 16-kDa PRL has antitumor activity in vivo, presumably as a result of its antiangiogenic effect. Interestingly, 23-kDa PRL showed a weak and transient suppression of prostate tumor growth. The weak antitumor activity of 23-kDa PRL may be because of the production of 16-kDa PRL from 23-kDa PRL by the tumor cells. Thus, the apparent effect of 23-kDa PRL on the growth of DU145 and PC-3 cells in vivo may result from the combined effects of 23-kDa PRL and 16-kDa PRL. These results suggest that the 16-kDa PRL has potential as a treatment agent in prostate cancer.

CEACAM1 is a cell adhesion molecule. Alternative splicing of the Ceacam1 gene generated two isoforms of CEACAM1, i.e. CEACAM1-L (long form) and CEACAM1-S (short form). We have shown that CEACAM1-L plays critical roles in prostate cancer progression. We propose to determine the function of CEACAM1 in prostate development, prostate homeostasis and prostate tumorigenesis by using gene targeting technologies. Specifically, we plan to generate mice with a targeted deletion of the CEACAM1 cytoplasmic domain. We have constructed a Ceacam1 targeting vector in which exons 7-9 of Ceacam1 gene was flanked by loxP sites for the generation of conditional knockout of the Ceacam1 gene. Embryonic stem cell clones containing the recombinant gene allele were established and injected into blastocysts for germ line transmission of the targeting construct; We have succeeded in producing mice harboring the conditional Ceacam1 knockout allele. In this extended period, we generated mice with homozygote conditional Ceacam1 allele and characterized the expression of CEACAM1 protein. In addition, we have crossed the homozygote conditional Ceacam1 mice with transgenic mice containing probasin-driven cre to generate mice with prostate-specific deletion of Ceacam1 gene. Initial analysis of 3 month old mouse prostates showed that deletion of Ceacam1-L in the mouse prostates led to epithelial hyperplasia.

We have previously shown that C-CAM1-based gene therapy effectively suppressed prostate tumor growth in nude mice xenograft models. In this study, we examined the effects of combining C-CAM1-based therapy and TNP-470, a potent angiogenesis inhibitor, on prostate cancer in a xenografted tumor model. The direct cytotoxic effects of Ad-C-CAM1 (recombinant adenovirus containing C-CAM1 cDNA) and TNP-470 on DU145 cells in vitro were determined by microculture tetrazolium assay. The in vivo antitumor effects of either agent alone were studied in a DU145 xenografted tumor model. Cells were infected with Ad-C-CAM1 or the control virus at multiplicities of infection (m.o.i.) of 5 or 10 and then inoculated onto nude mice 48 h later. TNP-470 (0, 17 or 35 mg/kg) was given 15, 17 and 19 days after inoculation. Combined treatments in vivo were carried out to determine whether there were synergistic antitumor effects. Both Ad-C-CAM1 and the control virus were minimally toxic to DU145 in vitro. There was evident dose-dependent suppression of xenografted tumor growth by either Ad-C-CAM1 or TNP-470. By the median-effect analysis, combination of the two agents generated strong synergistic antitumor effects as shown by marked tumor suppression as compared to either treatment alone. The novel strategy may have clinical implications for the treatment of prostate cancer.

p95 is a putative signal transduction protein of approximately 95 kDa that contains multiple tyrosine residues that are conserved from yeast to human, a Src phosphorylation consensus sequence and a proline-rich C-terminus that binds SH3-domains. Previous studies have established that mammalian p95 is physically associated with proteins that regulate apoptotic induction and cell transformation; however, it is unclear whether p95 is a positive or negative regulator in these processes. Moreover, a p95 partner protein has been localized at both focal adhesions and actin-cytoskeletons in rat astrocytes. However, there is no evidence that mammalian p95 has roles in regulating cell adhesion or morphology. In this study, we examined the effects of p95 on the anchorage-independent growth and tumorigenicity of malignant HeLa cells, and on the growth and morphology of non-transformed NIH3T3 cells. In HeLa cells, p95 overexpression promoted detachment-induced apoptosis (anoikis), inhibited detachment of viable cells from substratum and reduced tumorigenicity. In NIH3T3 cells, p95 overexpression promoted flat cell morphology and slowed cell proliferation, whereas p95 downregulation had opposite effects. These findings indicate that the mammalian p95 is a positive regulator in apoptotic signaling and a negative regulator in cell transformation. They also suggest that p95 has roles in regulating cell adhesion and morphology.

Metastatic Prostate Cancer (PCa) is the second leading cause of cancer related-deaths in men in the United States. Understanding the molecular events critical to PCa metastasis should lead to novel therapies for treatment of metastatic disease. To assess molecular alterations required for increased PCa cell migration, a critical step in metastasis, multiple rounds of selection of PC3 and DU145 PCa cells were performed using a modified Boyden chamber assay. Highly migratory cells (termed PC3-Mig-3 and DU145-Mig-3) were obtained, a phenotype that has remained stable. PC3-Mig-3 and DU145-Mig-3 cells were also increased in invasiveness, decreased in adhesion and proliferation; thus acquiring additional properties of metastatic cells. Following orthotopic injection into nude mice, migratory variants were significantly increased in number of lymph node metastases (P Citation Format: Tanushree Chatterji, Jian H. Song, Nila U. Parikh, Chien-Jui Cheng, Sue-Hwa Lin, Gary E. Gallick. Differential phosphorylation of focal adhesion kinase and activation of Yes kinase are associated with increased metastatic potential of prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4056. doi:10.1158/1538-7445.AM2014-4056

Introduction: Advanced colorectal cancer (CRC) remains lethal and difficult to treat with over 140,000 new cases a year in the United States. One contributing factor that has emerged is the rapid development of cancers in small, sessile adenomas(polyps) (less than 1cm), which might be overlooked. Results: Using whole genome and whole exome sequence (WGS) analyses of four pairs of colorectal adenoma (early CRC) tissue samples along with their normal counterparts, we observe dramatic genomic instability in two of the samples, suggesting that these two adenomas could be on the brink of overt CRC. We proceeded to analyze a further 20 samples through RNA sequence and/or immunohistochemical analysis. The two polyps are associated with hyper mutated profile of multiple genes including Carcinoembryonic antigen (CEA) and CEACAM6 in one of the samples. Analyses of the expression levels of CEA and TGF-β signaling pathway members in 20 early adenomas and normal colon tissues demonstrate a marked increase in CEA expression in 25% of adenoma samples linked to a concomitant loss of TGF-β signaling. We proceeded to mechanistic studies examining CEA and it role in inactivating the tumor suppressive arm of transforming growth factor-β (TGF-β) signaling, providing crucial new information for CEA in promoting aggressive CRC. We found that CEA interacts directly with the transforming growth factor-β (TGF-β) receptor 1 (TBR1) with inhibition of downstream TGF-β tumor suppressor signaling through its B3 domain. Moreover, we previously found that deletion of Smad3/4 adaptor β2SP results in loss of TGF-β signaling, with dramatic and spontaneous formation of adenomas as well as CRC. Ectopic expression of CEA also induces CRC metastases, potentially indirectly through activation of STAT3 and stimulating production of IL-6, IL-10 and others, in addition to inactivating TGF-β tumor suppressor signaling. Conclusions: Our data suggest that enhanced CEA levels and oncogenic mutations in the colon adenomas can be used as prognostic markers for early detection of aggressive CRC and the B3 domain of CEA can be targeted by monoclonal antibody/peptide inhibitors/small molecule inhibitors targeting CEA and CRC cell proliferation. Citation Format: Avijit Majumdar, Jian Chen, Sue Hwa Lin, Lior Katz, Gottumukkala Subba Raju, Kirti Shetty, Jon White, Xifeng Wu, Asif Rashid, John S. McMurray, Kenna R. Shaw, Xiaoping Su, Brian Weston, Selvi Thirumurthi, Aiwu Ruth He, Lopa Mishra. Genome & exome analysis of early colon cancers reveals new targets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4703. doi:10.1158/1538-7445.AM2014-4703

Angiomotin (AMOT) is a member of the motin family that consists of three members, AMOT, AMOTL1, and AMOTL2, which function as either positive or negative regulators of cell growth depending on interacting proteins and cellular context. The role of AMOT in prostate cancer (PCa) has not been studied. AMOT is expressed as two isoforms, AMOTp80 and AMOTp130. The AMOTp130 isoform has an extended 409 aa N-terminal domain that is absent in AMOTp80. Both AMOTp80 and AMOTp130 are expressed in LNCaP and C4-2B4, but at a very low level in PC3-mm2 PCa cells. We found that expression of AMOTp130 and AMOTp80 have distinct functions in PCa cells. While AMOTp80 functions as a tumor promoter by enhancing PCa cell proliferation, AMOTp130 did not. We further show that AMOTp80 affects the Hippo tumor suppressor pathway by inhibiting LATS1 and YAP phosphorylation, resulting in nuclear translocation of YAP. Once in the nucleus, YAP increases transcription of the target protein BMP4. Moreover, inhibition of BMP receptor activity by LDN-193189 abrogates AMOTp80-mediated proliferation. These observations suggest that AMOTp80 induces the expression of BMP4, which functions in an autocrine manner to promote PCa cell proliferation. Together, our studies identify AMOTp80-MST-LATS1-YAP-BMP4 as a novel signal pathway that regulates PCa cell proliferation. Citation Format: Angelica Ortiz, Hao Zeng, Sue-Hwa Lin. Angiomotin regulates the Hippo pathway to increase prostate cancer cell growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3307. doi:10.1158/1538-7445.AM2014-3307

e16046 Background: Sipuleucel-T was the first cellular immunotherapy shown to prolong the life of patients with advanced prostate cancer. It was of interest to determine who were those patients mos...

390 Background: Cancer is a heterogeneous disease. Studying cancer heterogeneity may enable us to elucidate the various subtypes of a particular cancer, and improve the diagnosis, prognostication, and therapy of cancer. Nonseminomatous germ cell tumor (NSGCT) is a prime example of a heterogeneous disease. We determined potential lethality of NSGCT by characterizing the histological makeup of primary testicular tumors, the tumor burden, psychosocial issues, and surgical factors. Methods: From November 1997 to October 2012, 142 consecutive patients diagnosed with a NSGCT of the testis were evaluated for this study. The log-rank test was used to identify prognostic factors in univariate Kaplan-Meier analysis. A multivariate Cox regression model was used to evaluate predictive factors for overall survival. Results: Patient and tumor characteristics: median (range) age 25 (12 to 53); pathology – embryonal carcinoma 15%, teratoma 6%, and mixed NSGCT 79%; stage – I 46%, II 32%, and III 21%. Ten (7%) patients died from their NSGCT. Seven (5%) patients had incurable NSGCT. Multivariate analysis showed that presence of seminoma (p=0.007), presence of yolk sac tumor (p=0.019), clinical stage (p=0.027), and psychosocial issues (p=0.04), have unfavorable prognostic significance. Conclusions: Certain subtypes of NSGCT containing yolk sac tumor and seminoma are inherently chemotherapy resistant and potentially lethal. Psychosocial factors can delay the diagnosis and treatment of patients with such tumors and adversely affect their clinical outcome. Results of this study need to be validated in another data base or a prospective clinical trial.

Previous studies have established that the cell–cell adhesion molecule-1 (CEACAM1, previously known as C-CAM1) functions as a tumor suppressor in prostate cancer and is involved in the regulation of prostate growth and differentiation. However, the molecular mechanism that modulates CEACAM1 expression in the prostate is not well defined. Since the growth of prostate epithelial cells is androgen-regulated, we investigated the effects of androgen and the androgen receptor (AR) on CEACAM1 expression. Transient transfection experiments showed that the AR can enhance the Ceacam1 promoter activity in a ligand-dependent manner and that the regulatory element resides within a relatively short ( − 249 to − 194 bp) segment of the 5-flanking region of the Ceacam1 gene. This androgen regulation is likely through direct AR-promoter binding because a mutant AR defective in DNA binding failed to upregulate reporter gene expression. Furthermore, electrophoretic mobility shift assays demonstrated that the AR specifically binds to this sequence, and mutation analysis of the potential ARE sequences revealed a region within the sequence that was required for the AR to activate the Ceacam1 gene. Therefore, the regulation of Ceacam1 gene expression by androgen may be one of the mechanisms by which androgen regulates prostatic function. © 2001 Elsevier Science Ireland Ltd. All rights reserved.

The transgenic adenocarcinoma of mouse prostate (TRAMP) model, in which various grades of prostate intraepithelial neoplasia (PIN) and prostate cancer with metastases can be reproducibly generated, is a paradigm for prostate disease progression. We have previously shown that C-CAM, an adhesion molecule, can suppress the growth of prostate cancer. In this report, we describe immunohistochemical characterization of differential expression of C-CAM at various stages of prostate tumorigenesis in the TRAMP model.We sampled prostate specimens and periaortic lymph nodes from TRAMP mice. Indirect immunohistochemical staining with a polyclonal anti-C-CAM antibody was performed on the formalin-fixed, paraffin-embedded specimens. After castration at 12 weeks of age, the TRAMP mice developed androgen-independent prostate cancer (AIPC) and lymph node metastasis at 18 to 24 weeks of age. Samples from these castrated mice were also analyzed.C-CAM protein was expressed in the normal prostate epithelia of non-transgenic and TRAMP mice as well as in low-grade PINs in TRAMP mice. Expression was uniform on the luminal surfaces of these epithelia. C-CAM expression was noticeably reduced and the staining pattern heterogeneous in some high-grade PINs. C-CAM staining was generally absent in prostate cancer and metastatic lymph nodes. Androgen independent prostate cancer and its metastatic tumors generated in castrated TRAMP mice were also C-CAM negative.C-CAM expression correlates with the differentiation states of prostate epithelia and is down regulated early in prostate tumorigenesis in the TRAMP model.

CD66a (human homolog of rat cell-cell adhesion molecule, also known as biliary glycoprotein) is a cell surface protein of the immunoglobulin family. CD66a has been shown to mediate homotypic cell adhesion. Aside from this, no other functions of this molecule have been demonstrated. We have observed previously that CD66a protein expression is lost in most prostate tumors, suggesting that the down-regulation of CD66a is associated with the abnormal growth of prostate cells. CD66a is homologous (65% identity) to rat cell-cell adhesion molecule, which has been shown to have tumor-suppressive activity. This homology suggests the possibility that CD66a might also be a tumor suppressor. In this report, we show that restoring CD66a expression in DU145 human prostate cancer cells by adenovirus (Ad)-mediated gene transfer dramatically altered the malignant phenotype of these cells, as evidenced by their reduced ability to form tumors in a xenograft animal model. This result suggests that loss of CD66a protein plays an important role in the development of prostate cancer, and that restoring CD66a expression might provide an effective treatment for prostate cancer. We further explored the possibility of using Ad vectors to deliver CD66a as a potential therapeutic agent for prostate cancer. Direct injection of Ad-CD66a, an Ad vector carrying the CD66a gene, into DU145 tumors in mice significantly suppressed the growth of these tumors. This antitumor activity of CD66a was found to be dose-dependent. These results suggest that CD66a has tumor-suppressive activity and that Ad-CD66a is a potential therapeutic agent for prostate cancer treatment.

C-CAM is an epithelial cell adhesion molecule with two major splice variants that differ in the length of the cytoplasmic domain. C-CAM1 (long (L)-form) strongly suppresses the tumorigenicity of human prostate carcinoma cells. In contrast, C-CAM2 (short (S)-form) does not exhibit tumor-suppressive activity. In the present study we have investigated the functional significance of L-form and S-form C-CAM in rat prostate by examining their expression and distribution in different prostate lobes and their response to androgen deprivation. RNase protection assays with a probe for both C-CAM isoforms detected high levels of C-CAM messages in the rat dorso-lateral prostate (DLP). L- and S-form proteins, localized by indirect immunofluorescence using isoform-specific antipeptide antibodies, were co-expressed on the apical surface of prostate epithelial cells in normal DLP. Androgen depletion did not significantly change the steady state levels of C-CAM message and protein expression in the DLP, although there was a change in the pattern of protein expression in these lobes. In contrast, C-CAM isoform messages and proteins were undetectable in normal ventral prostate (VP) but increased markedly in this lobe in response to castration, producing isoform ratios similar to those in DLP. These results demonstrate that coordinate expression of C-CAM isoforms is maintained in the VP following androgen depletion and suggest that androgen suppresses C-CAM expression in VP but not in DLP. These results suggest that balanced expression of L- and S-form C-CAM is important for normal prostate growth and differentiation.

People carrying a mutant BRCA2 gene are susceptible to breast, ovarian, pancreatic and other tumors. Many facets of BRCA2 have been studied, including its mutation in human cancers, its role in mouse embryogenesis and its RNA expression in different tissues and different mouse embryogenesis stages. However, there has been very little characterization of BRCA2 protein. We investigated the biochemical and biological properties of BRCA2 by using a monoclonal antibody we generated against the N-terminal portion of BRCA2. We discovered that the detection of BRCA2 by immunoblot analysis was sensitive to the temperature used to denature the samples before gel electrophoresis. BRCA2 was easily detectable when samples were denatured at low temperature instead of boiling. Although the precise mechanism underlying this observation is not clear yet, this finding will significantly improve our ability to study BRCA2. We examined the expression of BRCA2 using an immunoblot analysis protocol modified according to this observation. We showed that BRCA2 was presented in every human cell lines examined, including Capan-1, which expressed a truncated BRCA2 due to a BRCA2 frameshift mutation. We also showed that the expression of BRCA2 was cell-cycle regulated. Our results suggest that BRCA2 has an important role in cell growth regulation.

C-CAM1 is an epithelial adhesion molecule of immunoglobulin supergene family and has been implicated in the growth suppression of prostate cancer cells. Here we show that C-CAM1 can also suppress the tumorigenicity of breast cancer cells. These observations suggest that C-CAM1 may be a general growth suppressor in epithelial cells. In addition, we have identified the cytoplasmic domain, but not the extracellular adhesion domain, of C-CAM1 as critical for the growth suppression. Thus, the adhesion and the growth suppression functions of C-CAM1 are independent of each other. Furthermore, mutation at the tyrosine phosphorylation site in the cytoplasmic domain of C-CAM1 did not obliterate C-CAM1's growth suppression function, suggesting that tyrosine phosphorylation is not involved in the signal transduction pathway leading to cell growth suppression. These studies provide the structural basis for future development of therapeutics that may selectively activate C-CAM1's growth suppression function.

e22047 Background: CTC are believed to require EMT to metastasize. Cells undergoing EMT may lack or express low levels of epithelial markers such as EpCAM and cytokeratins (CK) and experience a cadherin switch from epithelial to mesenchymal. However, most current detection methods only rely on epithelial markers for enrichment, resulting in failure to recognize other CTC phenotypes. We previously showed that PCa cells expressing the mesenchymal (osteoblast-lineage) marker Cad11 are more likely to home to and colonize bone (Cancer Res 2010). Whether Cad11+ PCa CTC lose CK expression is uncertain. Since Cad11 is an EMT marker relevant to the bone tropism of PCa, we characterized its expression in CTC from patients (pts) with bone metastasis. Methods: Blood from PCa pts (n=86) with different extents of bone involvement was analyzed employing Biocept’s microfluidic CTC technology. For cell capture, we used a PCa CTC-optimized cocktail of antibodies that included epithelial, mesenchymal and putative stem cell markers. Captured cells were scored based on standard stain criteria (CK+/CD45−/DAPI+) and Cad11 expression. To ascertain malignant identity of CK−/CD45−/DAPI+cells, we used FISH to characterize androgen receptor [AR], Myc and PTEN status. Results: Among 69 pts with detectable CK+ CTC, 39% had at least 1 cell co-expressing Cad11. Very rare CK−Cad11− CTC displaying different degrees of aneuploidy were also observed, but only in 4 pts. No single CK−Cad11+ FISH “abnormal” cell was seen. FISH profiling of individual CK+ CTC revealed an association between Myc gain and PTEN loss (P=0.00004). We also found positive correlations between AR and PTEN amplification (r=0.55) as well as AR amplification and Myc loss (r=0.56; both P-6). Interestingly, CK−Cad11+cells (range, 1-910) were observed in 45 pts, all with a “normal” pattern of AR, Myc and PTEN expression. Conclusions: Complete loss of epithelial marker expression in CTC (identified by concurrent gene alterations) is a very infrequent event in PCa pts with established bone metastasis. Simultaneous individual CTC profiling of gene gains/losses may provide insights into PCa biology. Evaluation of the identity of CK−Cad11+ cells is ongoing.

Integrins have been shown to play a critical role in cell survival. We recently demonstrated that integrins are constitutively activated in highly metastatic prostate cancer cell PC3-mm2. Activation of integrins increases their binding affinity with extracellular matrix, resulting in increased migration and cancer metastasis. Talin is a 270 kDa cytoskeletal protein that has been shown to regulate integrin activity. Previous reports showed that the S425 residue in the N-terminal domain of talin was phosphorylated by Cdk5 to increase talin stability. In this study, we determined whether talin S425 phosphorylation regulates β1 integrin activation. We found that β1 integrin activation in prostate tumor cells correlates with talin S425 phosphorylation but not total talin levels. To examine the role of talin-pS425 in β1 integrin activation, endogenous talin-1 in PC3-mm2 cells was knocked down by shRNA, followed by re-expression of a phosphorylation-deficient S425A or phosphorylation-mimicking S425D talin-1 mutant. Re-expressing phosphorylation-deficient S425A-talin-1 in talin-1-knockdown PC3-mm2 cells showed reduced activation of β1 integrins, and decreased migration and invasion, compared to cells re-expressing wild type talin-1. In contrast, re-expressing the phosphorylation-mimicking S425D mutant in talin-1-knockdown PC3-mm2 cells restored β1 integrin activation and increased migratory and invasive properties of these cells. Inhibition of Cdk5 by roscovitine, a Cdk5 inhibitor, decreases talin-1 S425 phosphorylation and also inhibits downstream integrin signaling, including FAK and Src phosphorylation. Treatment of PC3-mm2 cells with roscovitine decreases β1 integrin activation and migration of PC3-mm2 cells. These results suggest that phosphorylation of talin-1 at S425 plays a role in promoting β1 integrin activation, leading to increased metastatic potential of prostate tumor cells. Citation Format: Jung-Kang Jin, Yu-Chen Lee, Sue-Hwa Lin, Gary E. Gallick. Talin-1 phosphorylation promotes invasion and migration of prostate tumor cells through activation of β1 integrins. [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 3783. doi:10.1158/1538-7445.AM2013-3783

Introduction: Advanced colorectal cancer (CRC) remains the second most common cause of cancer mortality worldwide. Carcinoembryonic antigen (CEA) represents one of the few biomarkers approved by the U.S. Food and Drug Administration (FDA). Ectopic expression of CEA induces CRC metastases, potentially indirectly through activation of STAT3, stimulating production of IL-6, IL-10 and others. Yet until recently a clear mechanism of CEA in tumorigenesis has remained elusive. Our study demonstrated that CEA inactivates the tumor suppressive arm of transforming growth factor-β (TGF-β) signaling, providing crucial new mechanistic information for CEA in promoting aggressive CRC. Moreover, we previously found that deletion of Smad3/4 adaptor β2SP results in loss of TGF-β signaling, with dramatic and spontaneous formation of CRC, presenting a strong mouse model of CRC. We hypothesized that CEA interacts through a specific site targeting the TGF-β pathway potentially TGF-βRI leading to aggressive colorectal cancers. Impeding this interaction by using specific antibodies or peptide inhibitors may be used as a therapeutic agent to treat advanced CRC. Methods & Results: We observed that CEA interacts directly with the transforming growth factor-β (TGF-β) receptor 1 (TGF-βR1) and causes inhibition of downstream TGF-β tumor suppressor signaling. Targeting CEA with either an anti-CEA specific antibody or siRNA-mediated CEA silencing restores the tumor suppressive properties of TGF-β signaling. Detailed analysis of this interaction revealed the D7 fragment of CEA responsible for binding with TGF-β receptor I and capable of inhibiting tumor suppressor function of TGF-β signaling as evident by the ability of D7 fragment to reduce phospho -Smad3 level upon TGF-β stimulation. In human CRC, increased CEA levels are associated with loss of TGF-β signaling. Immunohistochemical analysis of the CRC revealed induction of phospho STAT3 level compared to normal colon tissue. To impede the CEA mediated suppression of growth inhibitory TGF-β signaling we are currently using antibodies which block this interaction and reinforce tumor suppressor TGF-β signaling. Conclusions: Our results demonstrate that CEA induces CRC metastasis through interacting with TGF-βR1 and suppressing growth inhibitory TGF-β signaling pathway which is mediated by the D7 fragment of CEA. Inhibiting the interaction of CEA and TGF-βR1 using peptide inhibitors/antibodies /small molecule inhibitors will be very useful in preventing CRC cell growth and proliferation. Thus the study holds promise to generate novel therapeutic agents for the treatment of lethal metastatic CRC. Citation Format: Avijit Majumdar, Ying Li, Sue-Hwa Lin, Lopa Mishra. CEA: A therapeutic target for the treatment of advanced colorectal cancer. [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 4335. doi:10.1158/1538-7445.AM2013-4335

Antipeptide antibodies have become indispensable tools in modern biochemistry and molecular biology. Unfortunately, not all antipeptide antibodies react with their target proteins. The reasons why certain antipeptide antibodies fail to do so are not always clear, although it is commonly assumed that conformational difference between the peptide antigens and the corresponding sequences in proteins accounts for most failures. Here, we report detailed characterization of an antipeptide mAb which reacted avidly with the peptide antigen but did not react with the same sequence in a protein. ELISA analysis using analogs of the antigen peptide revealed that this mAb did not react with a C-terminus-extended analog of the antigen peptide and reacted poorly with a peptide amide analog of the antigen peptide. These results suggest that the mAb recognizes an epitope including the C-terminal-free carboxyl group of the peptide. This analysis also revealed that the epitope recognized by this mAb was located in the C-terminal pentapeptide, RY-IRS. Four amino acid side chains (R,I,R, and S) in this pentapeptide were shown by alanine-scanning to be critical for antibody recognition. Analysis of the polyclonal antisera raised against this peptide revealed that antibodies reacting with this unique carboxyl-containing epitope are most abundant. This unexpected finding has since been shown in several other cases in this laboratory, suggesting that generation of antibodies that recognize carboxyl-containing artificial epitopes may be rather common. we also found that the use of a peptide amide (instead of peptide acid) antigen did not prevent a similar problem; in this case, the C-terminal amide became part of the epitope. Based on these findings, we suggest a method for enhancing the probability of isolating protein-reactive mAbs.