[Abstract unavailable

BACKGROUND Prostate tumors shed circulating tumor cells (CTCs) into the blood stream. Increased evidence shows that CTCs are often present in metastatic prostate cancer and can be alternative sources for disease profiling and prognostication. Here we postulate that CTCs expressing genes related to epithelial-mesenchymal transition (EMT) are strong predictors of metastatic prostate cancer. METHODS A microfiltration system was used to trap CTCs from peripheral blood based on size selection of large epithelial-like cells without CD45 leukocyte marker. These cells individually retrieved with a micromanipulator device were assessed for cell membrane physical properties using atomic force microscopy. Additionally, 38 CTCs from eight prostate cancer patients were used to determine expression profiles of 84 EMT-related and reference genes using a microfluidics-based PCR system. RESULTS Increased cell elasticity and membrane smoothness were found in CTCs compared to noncancerous cells, highlighting their potential invasiveness and mobility in the peripheral circulation. Despite heterogeneous expression patterns of individual CTCs, genes that promote mesenchymal transitioning into a more malignant state, including IGF1, IGF2, EGFR, FOXP3, and TGFB3, were commonly observed in these cells. An additional subset of EMT-related genes (e.g., PTPRN2, ALDH1, ESR2, and WNT5A) were expressed in CTCs of castration-resistant cancer, but less frequently in castration-sensitive cancer. CONCLUSIONS The study suggests that an incremental expression of EMT-related genes in CTCs is associated with metastatic castration-resistant cancer. Although CTCs represent a group of highly heterogeneous cells, their unique EMT-related gene signatures provide a new opportunity for personalized treatments with targeted inhibitors in advanced prostate cancer patients.

Metastatic malignant melanoma is an extremely aggressive cancer, with no currently viable therapy. 4-Allyl-2-methoxyphenol (eugenol) was tested for its ability to inhibit proliferation of melanoma cells. Eugenol but not its isomer, isoeugenol (2-methoxy-4-propenylphenol), was found to be a potent inhibitor of melanoma cell proliferation. In a B16 xenograft study, eugenol treatment produced a significant tumor growth delay (p = 0.0057), an almost 40% decrease in tumor size, and a 19% increase in the median time to end point. More significantly, 50% of the animals in the control group died from metastatic growth, whereas none in the treatment group showed any signs of invasion or metastasis. Eugenol was well tolerated as determined by measurement of bodyweights. Examination of the mechanism of the antiproliferative action of eugenol in the human malignant melanoma cell line, WM1205Lu, showed that it arrests cells in the S phase of the cell cycle. Flow cytometry coupled with biochemical analyses demonstrated that eugenol induced apoptosis. cDNA array analysis showed that eugenol caused deregulation of the E2F family of transcription factors. Transient transfection assays and electrophoretic mobility shift assays showed that eugenol inhibits the transcriptional activity of E2F1. Overexpression of E2F1 restored about 75% of proliferation ability in cultures. These results indicate that deregulation of E2F1 may be a key factor in eugenol-mediated melanoma growth inhibition both in vitro and in vivo. Since the E2F transcription factors provide growth impetus for the continuous proliferation of melanoma cells, these results suggest that eugenol could be developed as an E2F-targeted agent for melanoma treatment.

Purpose A subpopulation of pancreatic adenocarcinoma (PDAC) cells is thought to be inherently resistant to chemotherapy or to give rise to tumor cells that become resistant during treatment. Here we determined the role of CD44 expression and its isoforms as a marker and potential target for tumor cells that give rise to invasive and gemcitabine resistant tumors. Experimental Design RT-PCR, Western blotting and DNA sequencing was used to determine CD44 isoform and expression levels. Flow cytometry was used to sort cells on the basis of their CD44 expression level. CD44 expression was knocked down using shRNA. Tumorigenic properties were determined by clonogenic and Matrigel assays, immunohistochemistry, tumor growth in vivo using luciferase imaging and by tumor weight. Results We identified an invasive cell population that gives rise to gemcitabine resistant tumors. These cancer cells express a high level of CD44 standard isoform and have an EMT phenotype (CD44s/EMT). In vivo, CD44s/EMT engraft and expand rapidly and give rise to tumors that express high levels of CD44 isoforms that contain multiple exon variants. CD44 low expressing cells show continued sensitivity to gemcitabine in vivo and knockdown of CD44 in CD44s/EMT cells increases sensitivity to gemcitabine and decreases invasiveness. Conclusion PDAC cells expressing high levels of CD44s with a mesenchymal-like phenotype were highly invasive and developed gemcitabine resistance in vivo. Thus, initial targeting CD44 or reversing the CD44 high phenotype may improve therapeutic response.

Inheritance of a mutant allele of the breast cancer susceptibility gene BRCA1 confers increased risk of developing breast and ovarian cancers. Likewise, inheritance of a mutant allele of the retinoblastoma susceptibility gene (RB1) results in the development of retinoblastoma and/or osteosarcoma, and both alleles are often mutated or inactivated in sporadic forms of these and other cancers. We now demonstrate that the product of the RB1 gene, Rb, regulates the expression of the murine Brca1 and human BRCA1 genes through its ability to modulate E2F transcriptional activity. The Brca1 gene is identified as an in vivo target of E2F1 in a transgenic mouse model. The Brca1 promoter contains E2F DNA-binding sites that mediate transcriptional activation by E2F1 and repression by Rb. Moreover, ectopic expression of cyclin D1 and Cdk4 can stimulate the Brca1 promoter in an E2F-dependent manner, and this is inhibited by coexpression of the p16INK4a cyclin-dependent kinase inhibitor. The human BRCA1 promoter also contains a conserved E2F site and is similarly regulated by E2F1 and Rb. This functional link between the BRCA1 and Rb tumor suppressors may provide insight into the mechanism by which BRCA1 inactivation contributes to cancer development.The mechanism by which loss of BRCA1 function leads to breast and ovarian cancer is unclear. A general role for BRCA1 in cell growth control is suggested by BRCA1's growth-regulated and ubiquitous expression pattern (1-4). Involvement of BRCA1 in DNA repair, replication, and transcriptional regulation have all been suggested. The BRCA1 protein associates with the RAD51 DNA repair factor as well as the BRCA2 and BARD1 proteins (5, 6). These proteins colocalize in nuclear foci, termed "dots," that dissipate upon DNA damage and may reappear at replication structures containing PCNA (7). BRCA1 is also found in complexes containing RNA polymerase II and has a carboxyl-terminal acidic region that can function as a transcriptional activation domain (8 -10). Several recent reports have demonstrated that BRCA1 physically associates with the p53 tumor suppressor protein and functions as a transcriptional coactivator for p53 (11,12). BRCA1 can induce apoptosis and this activity is enhanced by coexpression of p53 (11,13,14). This correlates with the ability of BRCA1 to significantly augment transcriptional activation of the bax gene by p53 (11). BRCA1 can also induce apoptosis through the p53-independent stimulation of GADD45 expression and the activation of the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway (14).Inactivation of the Rb tumor suppressor, through mutation of the RB1 gene or deregulation of cyclin D-associated kinase activity, is a common event in many cancers (15). Recent data suggests that the resultant activation of E2F transcription factors contributes to tumor development (16 -18). The Rb-E2F pathway regulates the expression of many genes whose products are required for DNA synthesis and cell cycle progression. Transcriptional activation of at le...

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17beta-estradiol, is present in human blood and urine. Here we show for the first time that 2-ME significantly inhibited the growth of normal prostate epithelial cells and androgen-dependent LNCaP and androgen-independent DU145 prostate cancer cells. This growth inhibition was accompanied by a twofold increase in the G(2)/M population, with a concomitant decrease in the G(1) population, as shown by cell-cycle analysis. 2-ME treatment affected the cell-cycle progression of prostate cancer cells specifically by blocking cells in the G(2) phase. Immunoblot analysis of the key cell-cycle regulatory proteins in the G(2)/M phase showed a 14-fold increase in the expression of p21 and an eightfold increase in the expression of p34 cell division cycle 2 (cdc2). We also found an accumulation of phosphorylated cdc2 after 2-ME treatment. Furthermore, Wee 1 kinase was detectable after 2-ME treatment. 2-ME treatment also led to an increase in the activity of caspase-3, followed by apoptosis, as shown by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick end-labeling and fluorescein isothiocyanate-poly(ADP-ribose) polymerase assay. Estrogen receptor levels did not change after treatment with 2-ME. Examination of the signaling pathways that mediate 2-ME-induced apoptosis showed reduction in the level of p53 expression and its DNA-binding activity. Given the fact that p53 mutations are common in patients with metastatic prostate cancer, our finding that 2-ME-mediated growth inhibition of human prostate cancer cells occurred in a p53-independent manner has considerable clinical significance. These findings, combined with the limited toxicity of 2-ME, may have significant implications for alternative treatment of advanced prostate cancer.

Purpose: Development of prostate cancer prevention strategies is an important priority to overcome high incidence, morbidity, and mortality. Recently, we showed that Nexrutine, an herbal extract, inhibits prostate cancer cell proliferation through modulation of Akt and cAMP-responsive element binding protein (CREB)^mediated signaling pathways. However, it is unknown if Nexrutine can be developed as a dietary supplement for the prevention of prostate cancer. In this study, we used the transgenic adenocarcinoma of mouse prostate (TRAMP) model to examine the ability of Nexrutine to protectTRAMP mice from developing prostate cancer. Experimental Design: Eight-week-oldTRAMP mice were fed with pelleted diet containing 300 and 600 mg/kg Nexrutine for 20 weeks. Efficacy of Nexrutine was evaluated by magnetic resonance imaging at 18 and 28 weeks of progression and histologic analysis of prostate tumor or tissue at the termination of the experiment. Tumor tissue was analyzed for modulation of various signaling molecules. Results: We show that Nexrutine significantly suppressed palpable tumors and progression of cancer in the TRAMP model. Expression of total and phosphorylated Akt, CREB, and cyclin D1 was significantly reduced in prostate tissue from Nexrutine intervention group compared with tumors from control animals. Nexrutine also inhibited cyclin D1 transcriptional activity in androgen-independent PC-3 cells. Overexpression of kinase dead Akt mutant or phosphorylationdefective CREB inhibited cyclin D1transcriptional activity. Conclusions: The current study shows that Nexrutine-mediated targeting of Akt/CREBî nduced activation of cyclin D1prevents the progression of prostate cancer. Expression of CREB and phosphorylated CREB increased in human prostate tumors compared with normal tissue, suggesting their potential use as prognostic markers.Prostate cancer is the second leading cause of cancer-related deaths in men and expected to lead to f27,350 deaths in 2006 (1). African American men have the highest incidence of prostate cancer in the world, whereas Asian men native to their countries who consume a low-fat, high-fiber diet have the lowest risk (2). Epidemiologic studies suggest that a reduced risk of cancer is associated with the consumption of a phytochemical-rich diet that includes fruits and vegetables (3). Evidence suggests that prostate cancer progresses from normal epithelium to proliferative inflammatory atrophy, to low-grade prostatic intraepithelial neoplasia (PIN), and to high-grade PIN that eventually progresses to the more aggressive-metastatic and clinically evident prostate cancer (ref. 4 and references therein). Such preneoplastic lesions have been found in young men in their 20s and are common in men in their 50s (5). However, clinically detectable prostate cancer does not generally manifest itself until the 60s. In addition, the occurrence of precancerous lesions is more prevalent (about 1 in three men) than the incidence of carcinoma (about one in nine men; ref. 6). Therefore, the develop...

Purpose Near equal rates of incidence and mortality emphasize the need for novel targeted approaches for better management of pancreatic cancer patients. Inflammatory molecules NFκB and Stat3 are overexpressed in pancreatic tumors. Inhibition of one protein allows cancer cells to survive using the other. The goal of the present study is to determine whether targeting Stat3/NFκB cross talk with a natural product Nexrutine (Nx) can inhibit inflammatory signaling in pancreatic cancer. Experimental design HPNE, HPNE-Ras, BxPC3, Capan-2, MIA PaCa-2 and AsPC-1 cells were tested for growth, apoptosis, Cox-2, NFκB and Stat3 level in response to Nx treatment. Transient expression, gel shift, ChIP was used to examine transcriptional regulation of Cox-2. Stat3 knockdown was used to decipher Stat3/NFκB cross talk. Histopathological and immunoblotting evaluation was performed on BK5-Cox2 transgenic mice treated with Nx. In vivo expression of prostaglandin receptor EP4 was analyzed in a retrospective cohort of pancreatic tumors using a TMA. Results Nx treatment inhibited growth of pancreatic cancer cells through induction of apoptosis. Reduced levels and activity of Stat3, NFκB and their cross talk led to transcriptional suppression of Cox-2 and subsequent decreased levels of PGE2 and PGF2. Stat3 knockdown studies suggest Stat3 as negative regulator of NFκB activation. Nx intervention reduced the levels of NFκB, Stat3 and fibrosis in vivo. Expression of prostaglandin receptor EP4 that is known to play a role in fibrosis was significantly elevated in human pancreatic tumors. Conclusions Dual inhibition of Stat3-NFκB by Nx may overcome problems associated with inhibition of either pathway.