Michael L. Cher

Original Articles: Prostate Cancer: The Use and Accuracy of Cross-Sectional Imaging and Fine Needle Aspiration Cytology for Detection of Pelvic Lymph Node Metastases Before Radical Prostatectomy

The role of cross-sectional pelvic imaging with computerized tomography or magnetic resonance imaging and fine needle aspiration in the assessment of pelvic lymph nodes in patients with prostate cancer is undefined. To address this issue we used formal decision analysis, comparing an imaging arm to a no imaging arm. Patient utility values were calculated, and test parameters and complication rates were extracted from the literature. Imaging was superior to no imaging only when the pretest probability of pelvic lymph node metastases was high. The most important parameter was the sensitivity of cross-sectional imaging for lymphadenopathy. When the sensitivity was 36%, which was the baseline figure derived from the literature, the probability of lymph node metastases required for imaging to be beneficial overall was 32%. We also performed a retrospective review of magnetic resonance imaging examinations at our institution in 174 patients with newly diagnosed prostate cancer and pathological confirmation of nodal status. The sensitivity for detecting nodal metastases was 25%. With this figure, the estimated probability of nodal metastases required to make imaging beneficial would be 45%, which is possible to achieve with highly selective clinical criteria. With a policy of imaging only in select patients the marginal cost is

Loss of Let-7 Up-Regulates EZH2 in Prostate Cancer Consistent with the Acquisition of Cancer Stem Cell Signatures That Are Attenuated by BR-DIM

794 per patient benefited (aborted radical prostatectomy because of nodal metastases detected with fine needle aspiration) compared to

Maspin expression inhibits osteolysis, tumor growth, and angiogenesis in a model of prostate cancer bone metastasis

50,661 per patient benefited if all patients are imaged. Thus, cross-sectional pelvic imaging before radical prostatectomy, solely for the purpose of detecting pelvic lymph node metastases, is not justified routinely. However, it is worthwhile on the basis of patient use values and cost-effectiveness in a select group of patients at high risk for nodal metastases.

Antitumor and Antimetastatic Activities of Docetaxel Are Enhanced by Genistein through Regulation of Osteoprotegerin/Receptor Activator of Nuclear Factor-κB (RANK)/RANK Ligand/MMP-9 Signaling in Prostate Cancer

The emergence of castrate-resistant prostate cancer (CRPC) contributes to the high mortality of patients diagnosed with prostate cancer (PCa), which in part could be attributed to the existence and the emergence of cancer stem cells (CSCs). Recent studies have shown that deregulated expression of microRNAs (miRNAs) contributes to the initiation and progression of PCa. Among several known miRNAs, let-7 family appears to play a key role in the recurrence and progression of PCa by regulating CSCs; however, the mechanism by which let-7 family contributes to PCa aggressiveness is unclear. Enhancer of Zeste homolog 2 (EZH2), a putative target of let-7 family, was demonstrated to control stem cell function. In this study, we found loss of let-7 family with corresponding over-expression of EZH2 in human PCa tissue specimens, especially in higher Gleason grade tumors. Overexpression of let-7 by transfection of let-7 precursors decreased EZH2 expression and repressed clonogenic ability and sphere-forming capacity of PCa cells, which was consistent with inhibition of EZH2 3′UTR luciferase activity. We also found that the treatment of PCa cells with BR-DIM (formulated DIM: 3,3′-diindolylmethane by Bio Response, Boulder, CO, abbreviated as BR-DIM) up-regulated let-7 and down-regulated EZH2 expression, consistent with inhibition of self-renewal and clonogenic capacity. Moreover, BR-DIM intervention in our on-going phase II clinical trial in patients prior to radical prostatectomy showed upregulation of let-7 consistent with down-regulation of EZH2 expression in PCa tissue specimens after BR-DIM intervention. These results suggest that the loss of let-7 mediated increased expression of EZH2 contributes to PCa aggressiveness, which could be attenuated by BR-DIM treatment, and thus BR-DIM is likely to have clinical impact.

Matrix Metalloproteinase Activity and Osteoclasts in Experimental Prostate Cancer Bone Metastasis Tissue

Emerging evidence indicates that tumor-associated proteolytic remodeling of bone matrix may underlie the capacity of tumor cells to colonize and survive in the bone microenvironment. Of particular importance, urokinase-type plasminogen activator (uPA) has been shown to correlate with human prostate cancer (PC) metastasis. The importance of this protease may be related to its ability to initiate a proteolytic cascade, leading to the activation of multiple proteases and growth factors. Previously, we showed that maspin, a serine protease inhibitor, specifically inhibits PC-associated uPA and PC cell invasion and motility in vitro. In this article, we showed that maspin-expressing transfectant cells derived from PC cell line DU145 were inhibited in in vitro extracellular matrix and collagen degradation assays. To test the effect of tumor-associated maspin on PC-induced bone matrix remodeling and tumor growth, we injected the maspin-transfected DU145 cells into human fetal bone fragments, which were previously implanted in immunodeficient mice. These studies showed that maspin expression decreased tumor growth, reduced osteolysis, and decreased angiogenesis. Furthermore, the maspin-expressing tumors contained significant fibrosis and collagen staining, and exhibited a more glandular organization. These data represent evidence that maspin inhibits PC-induced bone matrix remodeling and induces PC glandular redifferentiation. These results support our current working hypothesis that maspin exerts its tumor suppressive role, at least in part, by blocking the pericellular uPA system and suggest that maspin may offer an opportunity to improve therapeutic intervention of bone metastasis.

Lycopene and soy isoflavones in the treatment of prostate cancer.

Bone metastasis is very common in advanced prostate cancer. Docetaxel has been shown to improve survival in patients with metastatic prostate cancer. However, treatment with docetaxel is associated with a certain degree of toxicity. Genistein, derived from soybeans, has been found to inhibit cancer cell growth without toxicity. We have recently reported that genistein could potentiate the antitumor activity of chemotherapeutic agents both in vitro and in vivo. However, the molecular mechanism of this novel effect of genistein has not been fully elucidated. In this study, we found that genistein significantly potentiated the antitumor, anti-invasive, and antimetastatic activities of docetaxel both in culture and in severe combined immunodeficient (SCID)-human model of experimental prostate cancer bone metastasis. We further conducted microarray analysis, real-time reverse transcription-PCR, Western blot analysis, small interfering RNA and cDNA transfection, matrix metalloproteinase-9 (MMP-9) activity assay, and invasion assay. We found that the expression of osteoprotegerin (OPG) was induced by genistein and inhibited by docetaxel, whereas genistein significantly down-regulated the expression and secretion of receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) and inhibited osteoclast formation. Moreover, genistein down-regulated the expression and activity of MMP-9, which was induced by docetaxel treatment, and inhibited invasion of PC-3 cells. These results suggest that the observed potentiation of antitumor activity of docetaxel by genistein in the SCID-human model of experimental bone metastasis could be mediated by regulation of OPG/RANK/RANKL/MMP-9 signaling, resulting in the inhibition of osteoclastic bone resorption and prostate cancer bone metastasis. From these results, we conclude that genistein could be a promising nontoxic agent to improve the treatment outcome of metastatic prostate cancer with docetaxel.

In vitro and In vivo Molecular Evidence for Better Therapeutic Efficacy of ABT-627 and Taxotere Combination in Prostate Cancer

Previously, we and others showed that broad spectrum pharmaceutical inhibition of matrix metalloproteinase (MMP) activity reduces intraosseous tumor burden and bone degradation in animal models of bone metastasis. Herein, we used specific assays to measure net enzymatic activities of individual MMPs during colonization of bone by prostate cancer cells. PC3 cells were injected into the marrow of human fetal femurs previously implanted in SCID mice. Net MMP-9 activity in bone tissues peaked 2 weeks after injection, coinciding with a wave of osteoclast recruitment. In contrast, MMP-2 and MT1-MMP activity did not change. In vitro, co-culture of PC3 cells with bone tissue led to activation of pro-MMP-9 and increases in secreted net MMP-9 activity. Activation of pro-MMP-9 was prevented by metalloprotease inhibitors but not by inhibitors of other classes of proteases. Ribozyme suppression of MMP-9 expression in PC3 cells did not affect pro-MMP-9 activation or net MMP-9 activity and did not affect the phenotype of bone tumors. siRNA targeting of MMP-9 expression in preosteoclasts in vitro demonstrated that tumor-induced preosteoclast motility was dependent on MMP-9 expression. These data suggest that osteoclast-derived MMP-9 may represent a potential therapeutic target in bone metastasis and provide a rationale for the development of MMP-9-specific inhibitors.

A Potential Oncogenic Activity of Platelet-Derived Growth Factor D in Prostate Cancer Progression

Abstract Dietary intake of lycopene and soy has been associated with a lower risk of prostate cancer. In vitro studies with lycopene and genistein, a soy isoflavone, have shown induction of apoptosis and inhibition of cell growth in androgen-sensitive (LNCaP) and androgen–independent (PC3 and VeCaP) prostate cancer cell lines. In a previous Phase II clinical trial in prostate cancer patients, we observed prostate-specific antigen (PSA) stabilization with soy isoflavone intake. In this Phase II clinical trial, we investigated the efficacy of lycopene alone or in combination with soy isoflavones on serum PSA levels in men with prostate cancer. To be eligible for the study, men with prostate cancer had to have rising serum PSA following local therapy or while on hormone therapy. Study population included 71 eligible patients who had 3 successive rising PSA levels or a minimum PSA of 10 ng/ml at 2 successive evaluations prior to starting therapy. Subjects were randomly assigned to receive a tomato extract capsule containing 15 mg of lycopene alone (n = 38) or together with a capsule containing 40 mg of a soy isoflavone mixture (n = 33) twice daily orally for a maximum of 6 mo. One patient on the lycopene arm did not receive therapy due to his inability to ingest the study pill. There was no decline in serum PSA in either group qualifying for a partial or complete response. However, 35 of 37 (95%) evaluable patients in the lycopene group and 22 of 33 (67%) evaluable patients in the lycopene plus soy isoflavone group achieved stable disease described as stabilization in serum PSA level. The data suggest that lycopene and soy isoflavones have activity in prostate cancer patients with PSA relapse disease and may delay progression of both hormone-refractory and hormone-sensitive prostate cancer. However, there may not be an additive effect between the 2 compounds when taken together. Future studies are warranted to further investigate the efficacy of lycopene and soy isoflavones in prostate cancer as well as the mechanism of potential negative interaction between them.

Inhibition of human prostate cancer growth, osteolysis and angiogenesis in a bone metastasis model by a novel mechanism-based selective gelatinase inhibitor

Bone is the key metastatic site for prostate cancer. Endothelin 1 (ET-1) produced abundantly by prostate cancer cells binds to its receptor present on bone marrow stromal cells and favors osteoblastic response during bone metastases of prostate cancer. This suggests that interrupting ET-1 interaction with its endothelin A (ET(A)) receptor could be useful for inhibiting prostate cancer bone metastasis and, as such, may enhance the therapeutic activity of docetaxel (Taxotere), the most commonly used drug for the treatment of metastatic prostate cancer. Therefore, the goal of our study was to obtain preclinical data supporting our hypothesis that the combined use of ET(A) receptor antagonist (ABT-627; Atrasentan) with Taxotere will be superior in inducing apoptosis in vitro and inhibiting tumor growth in vivo in a SCID-hu model of experimental bone metastasis induced by C4-2b prostate cancer cells. In vitro studies were done on a panel of prostate cancer cell lines to understand the molecular basis of combination therapy, and we found that the combination was more effective in the inhibition of cell viability and induction of apoptosis in LNCaP and C4-2b cells (androgen receptor positive) but not in PC-3 cells. These results were correlated with inactivation of Akt/nuclear factor-kappaB and its target genes. For in vivo studies, the therapeutic regimen was initiated when the tumor began showing signs of growth and treatment was continued for 5 weeks. Tumor volume and serum prostate-specific antigen were used as terminal index to evaluate the therapeutic advantage of combination therapy relative to a single regimen and untreated control. At termination, we found a 90% reduction in tumor volume by combination treatment relative to the untreated control group. Most importantly, the antitumor activity was associated with the down-regulation of molecular markers in tumor tissues that were similar to those observed in vitro.

MAPPING OF REGIONS OF PHYSICAL DELETION ON CHROMOSOME 16q IN PROSTATE CANCER CELLS BY FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

The platelet-derived growth factor (PDGF) proteins are potent stimulators of cell proliferation/transformation and play a major role in cell-cell communication. For over two decades, PDGFs were thought to exist as three dimeric polypeptides (the homodimers AA and BB and the heterodimer AB). Recently, however, the PDGF C and D chains were discovered in a BLAST search of the expressed sequence tag databases. The PDGF CC and DD dimers have a unique two-domain structure with an NH2-terminal CUB (compliment subcomponents C1r/C1s, Uegf, and Bmp1) domain and a COOH-terminal PDGF/vascular endothelial growth factor domain. Whereas secreted PDGF AA, BB, and AB readily activate their cell surface receptors, it was suggested that extracellular proteolytic removal of the CUB domain is required for the PDGF/vascular endothelial growth factor domain of PDGF CC and DD to activate PDGF receptors. In the present study, we examined the processing of latent PDGF D into its active form and the effects of PDGF D expression on prostate cancer progression. We show that LNCaP cells auto-activate latent PDGF DD into the active PDGF domain, which can induce phosphorylation of the β-PDGF receptor and stimulates LNCaP cell proliferation in an autocrine manner. Additionally, LNCaP-PDGF D-conditioned medium induces migration of the prostate fibroblast cell line 1532-FTX, indicating LNCaP-processed PDGF DD is active in a paracrine manner as well. In a severe combined immunodeficient mouse model, PDGF DD expression accelerates early onset of prostate tumor growth and drastically enhances prostate carcinoma cell interaction with surrounding stromal cells. These demonstrate a potential oncogenic activity of PDGF DD in the development and/or progression of prostate cancer.