Potential roles for bcl-2 in the pathogenesis, progression, and treatment of prostate cancer

The most common molecular alterations observed in prostate cancer are increased bcl-2 protein expression and mutations in p53. Understanding the molecular alterations associated with prostate cancer are critical for successful treatment and designing new therapeutic interventions. Hormone-ablation therapy remains the most effective nonsurgical treatment; however, most patients will relapse with hormone-independent, refractory disease. This study addresses how hormone-ablation therapy may increase bcl-2, develops a transgenic model to elucidate the role of bcl-2 multistep prostate carcinogenesis, and assesses how bcl-2 may confer resistance to cell death induction using adenoviral wild-type p53 gene therapy. ^ Two potential androgen response elements were identified in the bcl-2 promoter. Bcl-2 promoter luciferase constructs were transfected into the hormone- sensitive LNCaP prostate cell line. In the presence of dihydrotestosterone, the activity of one bcl-2 promoter luciferase construct was repressed 40% compared to control cells grown in charcoal-stripped serum. Additionally, it was demonstrated that both bcl-2 mRNA and protein were downregulated in the LNCaP cells grown in the presence DHT. This suggests that DHT represses bcl-2 expression through possible direct and indirect mechanisms and that hormone-ablation therapy may actually increases bcl-2 protein. ^ To determine the role of bcl-2 in prostate cancer progression in vivo, probasin-bcl-2 mice were generated where human bcl-2 was targeted to the prostate. Increased bcl-2 expression rendered the ventral prostate more resistant to apoptosis induction following castration. When the probasin-bcl-2 mice were crossed with TRAMP mice, the latency to tumor formation was decreased. The expression of bcl-2 in the double transgenic mice did not affect the incidence of metastases. The double transgenic model will facilitate the study of in vivo effects of specific genetic lesions during the pathogenesis of prostate cancer. ^ The effects of increased bcl-2 protein on wild-type adenoviral p53-mediated cell death were determined in prostatic cell lines. Increased bcl-2 protected PC3 and DU145 cell lines, which possess mutant p53, from p53-mediated cell death and reductions in cell viability. Bcl-2 did not provide the same protective effect in LNCaP cell line, which expresses wild-type p53. This suggests that the ability of bcl-2 to protect against p53-mediated cell death is dependent upon the endogenous status of p53. ^

*Ras proteins and human tumor cell radiosensitivity

Ras proteins (H-, N-, K4A-, and K4B) are associated with cellular resistance to ionizing radiation (IR) and, consequently, may provide a potential target for radiosensitization strategies in cancer treatment. Several approaches have been used to compromise Ras activity and enhance IR-induced cell killing; however, these techniques either target proteins in addition to Ras or only target one member of the Ras family. In this study, I have used an adenovirus (AV1Y28) that expresses a single-chain antibody fragment directed against Ras proteins to investigate the mechanism(s) responsible for Ras-mediated radiation resistance. AV1Y28 enhanced the radiosensitivity of a number of human tumor cell lines without affecting the radiosensitivity of normal human fibroblasts. Whereas AV1Y28-mediated sensitization was independent of ras gene mutational status, it was dependent on active Ras proteins suggesting that AV1Y28 may be useful against a broad range of tumors. AV1Y28-mediated cell killing was not the result of redistributing cells into a more radiosensitive phase of the cell cycle and did not enhance IR-induced apoptosis. Given that Ras proteins transduce environmental signals to the nucleus, the effect of AV1Y28 on the IR-inducible transcription factor NF-κB were determined. Although AV1Y28 inhibited IR-induced NF-κB through the suppression of IKK, additional work established that NF-κB did not play a role in AV1Y28-mediated radiosensitization. However, a novel component of the signaling pathway responsible for IR-induced NF-κB was identified. Previous studies had suggested a relationship between mutant ras genes and IR-induced G2 delay; therefore the effects of AV1Y28 on the progression of cells from G2 to M after IR were determined. Pretreatment of cells with AV1Y28 prevented the IR-induced G2 arrest. AV1Y28-mediated abrogation of IR-induced G2 arrest correlated with those cell line lines that were sensitized by AV1Y28. Moreover, a significant increase in cells undergoing mitotic catastrophe was found after IR in AV1Y28 treated cells. The abrogation of G2 arrest by AV1Y28 was the result of maintaining the active form of cdc2, an inducer of mitosis, after exposure to IR. This study identified the mechanism of AV1Y28-mediated radiosensitization and has provided insight into the signal transduction pathways responsible for Ras-mediated radiation resistance. ^

Dynamic contrast agent -enhanced magnetic resonance imaging assessment of tumor microvasculature

Dynamic contrast agent-enhanced magnetic resonance imaging (DCE MRI) data, when analyzed with the appropriate pharmacokinetic models, have been shown to provide quantitative estimates of microvascular parameters important in characterizing the angiogenic activity of malignant tissue. These parameters consist of the whole blood volume per unit volume of tissue, v b, transport constant from the plasma to the extravascular, extracellular space (EES), k1 and the transport constant from the EES to the plasma, k2. Parameters vb and k1 are expected to correlate with microvascular density (MVD) and vascular permeability, respectively, which have been suggested to serve as surrogate markers for angiogenesis. In addition to being a marker for angiogenesis, vascular permeability is also useful in estimating tumor penetration potential of chemotherapeutic agents. ^ Histological measurements of the intratumoral microvascular environment are limited by their invasiveness and susceptibility to sampling errors. Also, MVD and vascular permeability, while useful for characterizing tumors at a single time point, have shown less utility in longitudinal studies, particularly when used to monitor the efficacy of antiangiogenic and traditional chemotherapeutic agents. These limitations led to a search for a non-invasive means of characterizing the microvascular environment of an entire tumor. ^ The overall goal of this project was to determine the utility of DCE MRI for monitoring the effect of antiangiogenic agents. Further applications of a validated DCE MRI technique include in vivo measurements of tumor microvascular characteristics to aid in determining prognosis at presentation and in estimating drug penetration. DCE MRI data were generated using single- and dual-tracer pharmacokinetic models with different molecular-weight contrast agents. The resulting pharmacokinetic parameters were compared to immunohistochemical measurements. The model and contrast agent combination yielding the best correlation between the pharmacokinetic parameters and histological measures was further evaluated in a longitudinal study to evaluate the efficacy of DCE MRI in monitoring the intratumoral microvascular environment following antiangiogenic treatment. ^

Interferon-β gene therapy inhibits tumor formation and causes regression of established tumors in immune-deficient mice

Despite the potential of type 1 interferons (IFNs) for the treatment of cancer, clinical experience with IFN protein therapy of solid tumors has been disappointing. IFN-β has potent antiproliferative activity against most human tumor cells in vitro in addition to its known immunomodulatory activities. The antiproliferative effect, however, relies on IFN-β concentrations that cannot be achieved by parenteral protein administration because of rapid protein clearance and systemic toxicities. We demonstrate here that ex vivo IFN-β gene transduction by a replication-defective adenovirus in as few as 1% of implanted cells blocked tumor formation. Direct in vivo IFN-β gene delivery into established tumors generated high local concentrations of IFN-β, inhibited tumor growth, and in many cases caused complete tumor regression. Because the mice were immune-deficient, it is likely that the anti-tumor effect was primarily through direct inhibition of tumor cell proliferation and survival. Based on these studies, we argue that local IFN-β gene therapy with replication-defective adenoviral vectors might be an effective treatment for some solid tumors.

Thrombospondin-2: A potent endogenous inhibitor of tumor growth and angiogenesis

Recent evidence suggests a potential role for thrombospondin-2 (TSP-2), a matricellular glycoprotein, in the regulation of primary angiogenesis. To directly examine the biological effect of TSP-2 expression on tumor growth and angiogenesis, human A431 squamous cell carcinoma cells, which do not express TSP-2, were stably transfected with a murine TSP-2 expression vector or with vector alone. A431 cells expressing TSP-2 did not show an altered growth rate, colony-forming ability, or susceptibility to induction of apoptosis in vitro. However, injection of TSP-2-transfected clones into the dermis of nude mice resulted in pronounced inhibition of tumor growth that was significantly stronger than the inhibition observed in A431 clones stably transfected with a thrombospondin-1 (TSP-1) expression vector, and combined overexpression of TSP-1 and TSP-2 completely prevented tumor formation. Extensive areas of necrosis were observed in TSP-2-expressing tumors, and both the density and the size of tumor vessels were significantly reduced, although tumor cell expression of the major tumor angiogenesis factor, vascular endothelial growth factor, was maintained at high levels. These findings establish TSP-2 as a potent endogenous inhibitor of tumor growth and angiogenesis.

NF-κB activation provides the potential link between inflammation and hyperplasia in the arthritic joint

The transcription factor NF-κB is a pivotal regulator of inflammatory responses. While the activation of NF-κB in the arthritic joint has been associated with rheumatoid arthritis (RA), its significance is poorly understood. Here, we examine the role of NF-κB in animal models of RA. We demonstrate that in vitro, NF-κB controlled expression of numerous inflammatory molecules in synoviocytes and protected cells against tumor necrosis factor α (TNFα) and Fas ligand (FasL) cytotoxicity. Similar to that observed in human RA, NF-κB was found to be activated in the synovium of rats with streptococcal cell wall (SCW)-induced arthritis. In vivo suppression of NF-κB by either proteasomal inhibitors or intraarticular adenoviral gene transfer of super-repressor IκBα profoundly enhanced apoptosis in the synovium of rats with SCW- and pristane-induced arthritis. This indicated that the activation of NF-κB protected the cells in the synovium against apoptosis and thus provided the potential link between inflammation and hyperplasia. Intraarticular administration of NF-kB decoys prevented the recurrence of SCW arthritis in treated joints. Unexpectedly, the severity of arthritis also was inhibited significantly in the contralateral, untreated joints, indicating beneficial systemic effects of local suppression of NF-κB. These results establish a mechanism regulating apoptosis in the arthritic joint and indicate the feasibility of therapeutic approaches to RA based on the specific suppression of NF-κB.

PTGF-β, a type β transforming growth factor (TGF-β) superfamily member, is a p53 target gene that inhibits tumor cell growth via TGF-β signaling pathway

Identification and characterization of p53 target genes would lead to a better understanding of p53 functions and p53-mediated signaling pathways. Two putative p53 binding sites were identified in the promoter of a gene encoding PTGF-β, a type β transforming growth factor (TGF-β) superfamily member. Gel shift assay showed that p53 bound to both sites. Luciferase-coupled transactivation assay revealed that the gene promoter was activated in a p53 dose- as well as p53 binding site-dependent manner by wild-type p53 but not by several p53 mutants. The p53 binding and transactivation of the PTGF-β promoter was enhanced by etoposide, a p53 activator, and was largely blocked by a dominant negative p53 mutant. Furthermore, expression of endogenous PTGF-β was remarkably induced by etoposide in p53-positive, but not in p53-negative, cell lines. Finally, the conditioned medium collected from PTGF-β-overexpressing cells, but not from the control cells, suppressed tumor cell growth. Growth suppression was not, however, seen in cells that lack functional TGF-β receptors or Smad4, suggesting that PTGF-β acts through the TGF-β signaling pathway. Thus, PTGF-β, a secretory protein, is a p53 target that could mediate p53-induced growth suppression in autocrinal as well as paracrinal fashions. The finding made a vertical connection between p53 and TGF-β signaling pathways in controlling cell growth and implied a potential important role of p53 in inflammation regulation via PTGF-β.

Synthesis and in vivo murine evaluation of Na4[1-(1′-B10H9)-6-SHB10H8] as a potential agent for boron neutron capture therapy

Reaction of the normal isomer of [B20H18]2− and the protected thiol anion, [SC(O)OC(CH3)3]−, produces an unexpected isomer of [B20H17SC(O)OC(CH3)3]4− directly and in good yield. The isomer produced under mild conditions is characterized by an apical–apical boron atom intercage connection as well as the location of the thiol substituent on an equatorial belt adjacent to the terminal boron apex. Although the formation of this isomer from nucleophilic attack of the normal isomer of [B20H18]2− has not been reported previously, the isomeric assignment has been unambiguously confirmed by one-dimensional and two-dimensional 11B NMR spectroscopy. Deprotection of the thiol substituent under acidic conditions produces a protonated intermediate, [B20H18SH]3−, which can be deprotonated with a suitable base to yield the desired product, [B20H17SH]4−. The sodium salt of the resulting [B20H17SH]4− ion has been encapsulated in small, unilamellar liposomes, which are capable of delivering their contents selectively to tumors in vivo, and investigated as a potential agent for boron neutron capture therapy. The biodistribution of boron was determined after intravenous injection of the liposomal suspension into BALB/c mice bearing EMT6 mammary adenocarcinoma. At low injected doses, the tumor boron concentration increased throughout the time-course experiment, resulting in a maximum observed boron concentration of 46.7 μg of B per g of tumor at 48 h and a tumor to blood boron ratio of 7.7. The boron concentration obtained in the tumor corresponds to 22.2% injected dose (i.d.) per g of tissue, a value analogous to the most promising polyhedral borane anions investigated for liposomal delivery and subsequent application in boron neutron capture therapy.

Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines

Induction of wild-type p53 in the ECV-304 bladder carcinoma cell line by infection with a p53 recombinant adenovirus (Ad5CMV-p53) resulted in extensive apoptosis and eventual death of nearly all of the cells. As a strategy to determine the molecular events important to p53-mediated apoptosis in these transformed cells, ECV-304 cells were selected for resistance to p53 by repeated infections with Ad5CMV-p53. We compared the expression of 5,730 genes in p53-resistant (DECV) and p53-sensitive ECV-304 cells by reverse transcription–PCR, Northern blotting, and DNA microarray analysis. The expression of 480 genes differed by 2-fold or more between the two p53-infected cell lines. A number of potential targets for p53 were identified that play roles in cell cycle regulation, DNA repair, redox control, cell adhesion, apoptosis, and differentiation. Proline oxidase, a mitochondrial enzyme involved in the proline/pyrroline-5-carboxylate redox cycle, was up-regulated by p53 in ECV but not in DECV cells. Pyrroline-5-carboxylate (P5C), a proline-derived metabolite generated by proline oxidase, inhibited the proliferation and survival of ECV-304 and DECV cells and induced apoptosis in both cell lines. A recombinant proline oxidase protein tagged with a green fluorescent protein at the amino terminus localized to mitochondria and induced apoptosis in p53-null H1299 non-small cell lung carcinoma cells. The results directly implicate proline oxidase and the proline/P5C pathway in p53-induced growth suppression and apoptosis.

CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth

CYR61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible immediate–early gene. Acting as an extracellular, matrix-associated signaling molecule, CYR61 promotes the adhesion of endothelial cells through interaction with the integrin αVβ3 and augments growth factor-induced DNA synthesis in the same cell type. In this study, we show that purified CYR61 stimulates directed migration of human microvascular endothelial cells in culture through an αVβ3-dependent pathway and induces neovascularization in rat corneas. Both the chemotactic and angiogenic activities of CYR61 can be blocked by specific anti-CYR61 antibodies. Whereas most human tumor-derived cell lines tested express CYR61, the gastric adenocarcinoma cell line RF-1 does not. Expression of the CYR61 cDNA under the regulation of a constitutive promoter in RF-1 cells significantly enhances the tumorigenicity of these cells as measured by growth in immunodeficient mice, resulting in tumors that are larger and more vascularized than those produced by control RF-1 cells. Taken together, these results identify CYR61 as an angiogenic inducer that can promote tumor growth and vascularization; the results also suggest potential roles for CYR61 in physiologic and pathologic neovascularization.

Polyclonal structure of intestinal adenomas in ApcMin/+ mice with concomitant loss of Apc+ from all tumor lineages

When tumors form in intestinal epithelia, it is important to know whether they involve single initiated somatic clones. Advanced carcinomas in humans and mice are known to be monoclonal. However, earlier stages of tumorigenesis may instead involve an interaction between cells that belong to separate somatic clones within the epithelium. The clonality of early tumors has been investigated in mice with an inherited predisposition to intestinal tumors. Analysis of Min (multiple intestinal neoplasia) mice chimeric for a ubiquitously expressed cell lineage marker revealed that normal intestinal crypts are monoclonal, but intestinal adenomas frequently have a polyclonal structure, presenting even when very small as single, focal adenomas composed of at least two somatic lineages. Furthermore, within these polyclonal adenomas, all tumor lineages frequently lose the wild-type Apc allele. These observations can be interpreted by several models for clonal interaction within the epithelium, ranging from passive fusion within regions of high neoplastic potential to a requirement for active clonal cooperation.

High-sensitivity array analysis of gene expression for the early detection of disseminated breast tumor cells in peripheral blood

Early detection is an effective means of reducing cancer mortality. Here, we describe a highly sensitive high-throughput screen that can identify panels of markers for the early detection of solid tumor cells disseminated in peripheral blood. The method is a two-step combination of differential display and high-sensitivity cDNA arrays. In a primary screen, differential display identified 170 candidate marker genes differentially expressed between breast tumor cells and normal breast epithelial cells. In a secondary screen, high-sensitivity arrays assessed expression levels of these genes in 48 blood samples, 22 from healthy volunteers and 26 from breast cancer patients. Cluster analysis identified a group of 12 genes that were elevated in the blood of cancer patients. Permutation analysis of individual genes defined five core genes (P ≤ 0.05, permax test). As a group, the 12 genes generally distinguished accurately between healthy volunteers and patients with breast cancer. Mean expression levels of the 12 genes were elevated in 77% (10 of 13) untreated invasive cancer patients, whereas cluster analysis correctly classified volunteers and patients (P = 0.0022, Fisher's exact test). Quantitative real-time PCR confirmed array results and indicated that the sensitivity of the assay (1:2 × 108 transcripts) was sufficient to detect disseminated solid tumor cells in blood. Expression-based blood assays developed with the screening approach described here have the potential to detect and classify solid tumor cells originating from virtually any primary site in the body.

Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts

Prostate stem-cell antigen (PSCA) is a cell-surface antigen expressed in normal prostate and overexpressed in prostate cancer tissues. PSCA expression is detected in over 80% of patients with local disease, and elevated levels of PSCA are correlated with increased tumor stage, grade, and androgen independence, including high expression in bone metastases. We evaluated the therapeutic efficacy of anti-PSCA mAbs in human prostate cancer xenograft mouse models by using the androgen-dependent LAPC-9 xenograft and the androgen-independent recombinant cell line PC3-PSCA. Two different anti-PSCA mAbs, 1G8 (IgG1κ) and 3C5 (IgG2aκ), inhibited formation of s.c. and orthotopic xenograft tumors in a dose-dependent manner. Furthermore, administration of anti-PSCA mAbs led to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies suggest PSCA as an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-PSCA mAbs for the treatment of local and metastatic prostate cancer.

Nitric oxide-induced cytostasis and cell cycle arrest of a human breast cancer cell line (MDA-MB-231): Potential role of cyclin D1

DETA-NONOate, a nitric oxide (NO) donor, induced cytostasis in the human breast cancer cells MDA-MB-231, and the cells were arrested in the G1 phase of the cell cycle. This cytostatic effect of the NO donor was associated with the down-regulation of cyclin D1 and hypophosphorylation of the retinoblastoma protein. No changes in the levels of cyclin E or the catalytic partners of these cyclins, CDK2, CDK4, or CDK6, were observed. This NO-induced cytostasis and decrease in cyclin D1 was reversible for up to 48 h of DETA-NONOate (1 mM) treatment. DETA-NONOate (1 mM) produced a steady-state concentration of 0.5 μM of NO over a 24-h period. Synchronized population of the cells exposed to DETA-NONOate remained arrested at the G1 phase of the cell cycle whereas untreated control cells progressed through the cell cycle after serum stimulation. The cells arrested at the G1 phase after exposure to the NO donor had low cyclin D1 levels compared with the control cells. The levels of cyclin E and CDK4, however, were similar to the control cells. The decline in cyclin D1 protein preceded the decrease of its mRNA. This decline of cyclin D1 was due to a decrease in its synthesis induced by the NO donor and not due to an increase in its degradation. We conclude that down-regulation of cyclin D1 protein by DETA-NONOate played an important role in the cytostasis and arrest of these tumor cells in the G1 phase of the cell cycle.

Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways

Lethal factor is a protease, one component of Bacillus anthracis exotoxin, which cleaves many of the mitogen-activated protein kinase kinases (MEKs). Given the importance of MEK signaling in tumorigenesis, we assessed the effects of anthrax lethal toxin (LeTx) on tumor cells. LeTx was very effective in inhibiting mitogen-activated protein kinase activation in V12 H-ras-transformed NIH 3T3 cells. In vitro, treatment of transformed cells with LeTx caused them to revert to a nontransformed morphology, and inhibited their abilities to form colonies in soft agar and to invade Matrigel without markedly affecting cell proliferation. In vivo, LeTx inhibited growth of ras-transformed cells implanted in athymic nude mice (in some cases causing tumor regression) at concentrations that caused no apparent animal toxicity. Unexpectedly, LeTx also greatly decreased tumor neovascularization. These results demonstrate that LeTx potently inhibits ras-mediated tumor growth and is a potential antitumor therapeutic.