Showing papers by "Kenneth J. Pienta published in 2004"

Androgen-independent prostate cancer is a heterogeneous group of diseases: lessons from a rapid autopsy program.

Abstract: Understanding the biology of prostate cancer metastasis has been limited by the lack of tissue for study. We studied the clinical data, distribution of prostate cancer involvement, morphology, immunophenotypes, and gene expression from 30 rapid autopsies of men who died of hormone-refractory prostate cancer. A tissue microarray was constructed and quantitatively evaluated for expression of prostate-specific antigen, androgen receptor, chromogranin, synaptophysin, MIB-1, and alpha-methylacylCoA-racemase markers. Hierarchical clustering of 16 rapid autopsy tumor samples was performed to evaluate the cDNA expression pattern associated with the morphology. Comparisons were made between patients as well as within the same patient. Metastatic hormone-refractory prostate cancer has a heterogeneous morphology, immunophenotype, and genotype, demonstrating that "metastatic disease" is a group of diseases even within the same patient. An appreciation of this heterogeneity is critical to evaluating diagnostic and prognostic biomarkers as well as to designing therapeutic targets for advanced disease.

Skeletal localization and neutralization of the SDF-1(CXCL12)/CXCR4 axis blocks prostate cancer metastasis and growth in osseous sites in vivo.

Abstract: To delineate the role of SDF-1 and CXCR4 in metastatic prostate cancer (CaP), positive correlations were established between SDF-1 levels and tumor metastasis. Neutralization of CXCR4 limited the number and the growth of intraosseous metastasis in vivo. Together, these in vivo metastasis data provide critical support that SDF-1/CXCR4 plays a role in skeletal metastasis. Introduction: Previously we determined that the stromal-derived factor-1 (SDF-1)/CXCR4 chemokine axis is activated in prostate cancer (CaP) metastasis to bone. To delineate the role of SDF-1/CXCR4 in CaP, we evaluated SDF-1 levels in a variety of tissues and whether neutralization of SDF-1 prevented metastasis and/or intraosseous growth of CaPs. Materials and Methods: SDF-1 levels were established in various mouse tissues by ELISA, immunohistochemistry, and in situ hybridization. To assess the role of SDF-1/CXCR4 in metastasis, bone metastases were established by administering CaP cells into the left cardiac ventricle of nude animals in the presence or absence of neutralizing CXCR4 antibody. The effect of SDF-1 on intraosseous growth of CaP cells was determined using intratibial injections and anti-CXCR4 antibodies and peptides. Results: There was a positive correlation between the levels of SDF-1 and tissues in which metastatic CaP lesions were observed. SDF-1 levels were highest in the pelvis, tibia, femur, liver, and adrenal/kidneys compared with the lungs, tongue, and eye, suggesting a selective effect. SDF-1 staining was generally low or undetectable in the center of the marrow and in the diaphysis. SDF-1 mRNA was localized to the metaphysis of the long bones nearest to the growth plate where intense expression was observed near the endosteal surfaces covered by osteoblastic and lining cells. Antibody to CXCR4 significantly reduced the total metastatic load compared with IgG control-treated animals. Direct intratibial injection of tumor cells followed by neutralizing CXCR4 antibody or a specific peptide that blocks CXCR4 also decreased the size of the tumors compared with controls. Conclusions: These data provide critical support for a role of SDF-1/CXCR4 in skeletal metastasis. Importantly, these data show that SDF-1/CXCR4 participate in localizing tumors to the bone marrow for prostate cancer.

Whole genome scanning identifies genotypes associated with recurrence and metastasis in prostate tumors

Abstract: Prostate cancer is the most commonly diagnosed non-cutaneous neoplasm among American males and is the second leading cause of cancer-related death. Prostate specific antigen screening has resulted in earlier disease detection, yet approximately 30% of men will die of metastatic disease. Slow disease progression, an aging population and associated morbidity and mortality underscore the need for improved disease classification and therapies. To address these issues, we analyzed a cohort of patients using array comparative genomic hybridization (aCGH). The cohort comprises 64 patients, half of whom recurred postoperatively. Analysis of the aCGH profiles revealed numerous recurrent genomic copy number aberrations. Specific loss at 8p23.2 was associated with advanced stage disease, and gain at 11q13.1 was found to be predictive of postoperative recurrence independent of stage and grade. Moreover, comparison with an independent set of metastases revealed approximately 40 candidate markers associated with metastatic potential. Copy number aberrations at these loci may define metastatic genotypes.

Eligibility and Outcomes Reporting Guidelines for Clinical Trials for Patients in the State of a Rising Prostate-Specific Antigen: Recommendations From the Prostate-Specific Antigen Working Group

Abstract: Purpose To define methodology to show clinical benefit for patients in the state of a rising prostate-specific antigen (PSA). Results Hypothesis. A clinical states framework was used to address the hypothesis that definitive phase III trials could not be conducted in this patient population. Patient Population. The Group focused on men with systemic (nonlocalized) recurrence and a defined risk of developing clinically detectable metastases. Models to define systemic versus local recurrence, and risk of metastatic progression were discussed. Intervention. Therapies that have shown favorable effects in more advanced clinical states; meaningful biologic surrogates of activity linked with efficacy in other tumor types; and/or effects on a target or pathway known to contribute to prostate cancer progression in this state can be considered for evaluation. Outcomes. An intervention-specific posttherapy PSA-based outcome definition that would justify further testing should be described at the outset. Reporting. T...

Overexpression, Amplification, and Androgen Regulation of TPD52 in Prostate Cancer

Abstract: Gains in the long arm of chromosome 8 (8q) are believed to be associated with poor outcome and the development of hormone-refractory prostate cancer Based on a meta-analysis of gene expression microarray data from multiple prostate cancer studies (D R Rhodes et al, Cancer Res 2002;62:4427-33), a candidate oncogene, Tumor Protein D52 (TPD52), was identified in the 8q21 amplicon TPD52 is a coiled-coil motif-bearing protein, potentially involved in vesicle trafficking Both mRNA and protein levels of TPD52 were highly elevated in prostate cancer tissues Array comparative genomic hybridization and amplification analysis using single nucleotide polymorphism arrays demonstrated increased DNA copy number in the region encompassing TPD52 Fluorescence in situ hybridization on tissue microarrays confirmed TPD52 amplification in prostate cancer epithelia Furthermore, our studies suggest that TPD52 protein levels may be regulated by androgens, consistent with the presence of androgen response elements in the upstream promoter of TPD52 In summary, these findings suggest that dysregulation of TPD52 by genomic amplification and androgen induction may play a role in prostate cancer progression

Apoptosis of circulating tumor cells in prostate cancer patients.

Abstract: Background The prescence of circulating tumor cells (CTCs) in the peripheral blood of cancer patients and their frequency has been correlated with disease status. Methods In this study, CTCs were characterized by flow cytometry and fluorescence microscopy after immunomagnetic enrichment from 7.5-ml blood samples collected from patients with prostate cancer in evacuated blood-draw tubes that contained an anticoagulant and a preservative. Events were classified as tumor cell candidates if they expressed cytokeratin, lacked CD45, and stained with the nucleic acid dye 4,6-diamidino-2-phenylindole. Results In the blood of prostate cancer patients, only few of these events were intact cells. Other CTC events appeared as damaged cells or cell fragments by microscopy. By flow cytometry, these events stained variably with 4,6-diamidino-2-phenylindole and frequently expressed the apoptosis-induced, caspase-cleaved cytokeratin 18. Similar patterns of cell disintegration were observed when cells of the prostate line LNCaP were exposed to paclitaxel before spiking the cells into normal blood samples. Conclusions The different observed stages of tumor cell degradation or apoptosis varied greatly between patients and were not found in blood of normal donors. Enumeration of CTCs and identification of CTCs undergoing apoptosis may provide relevant information to evaluate the response to therapy in cancer patients. © 2004 Wiley-Liss, Inc.

Dynamic process of prostate cancer metastasis to bone

Abstract: Prostate cancer metastasis to the bone occurs at high frequency in patients with advanced disease, causing significant morbidity and mortality. Over a century ago, the "seed and soil" theory was proposed to explain organ-specific patterns of metastases. Today, this theory continues to be relevant as we continue to discover factors involved in the attraction and subsequent growth of prostate cancer cells to the bone. These include the accumulation of genetic changes within cancer cells, the preferential binding of cancer cells to bone marrow endothelial cells, and the release of cancer cell chemoattractants from bone elements. A key mediator throughout this metastatic process is the integrin family of proteins. Alterations in integrin expression and function promote dissociation of cancer cells from the primary tumor mass and migration into the blood stream. Once in circulation, integrins facilitate cancer cell survival through interactions between other cancer cells, platelets, and endothelial cells of the target bone. Furthermore, dynamic changes in integrins and in integrin-associated signal transduction aid in the extravasation of cancer cells into the bone and in expansion to a clinically relevant metastasis. Thus, we will review the critical roles of integrins in the process of prostate cancer bone metastasis, from the escape of cancer cells from the primary tumor, to their survival in the harsh "third microenvironment" of the circulation, and ultimately to their attachment and growth at distant bone sites.

Cellular interactions in the tropism of prostate cancer to bone

Abstract: At autopsy >or=80% of prostate cancers have established macrometastases in marrow containing bone. The mechanism(s) to explain this remarkable level of bone involvement remain to be elucidated. We examined the adhesive and invasive behavior of prostate cancer cells to osteoblastic and human bone marrow endothelial cells (HBME-1) in an attempt to explain the tropism of prostate cells for bone. We found an inverse relationship between adhesion and prostate cell tumorigenicity and metastatic potential. Relative cell adhesion of P69 between cell lines was 1.74-fold (95% confidence interval [CI] = 1.15-2.64) and 1.58-fold (95% CI = 0.94-2.68) greater at 1 hr and 2 hr, respectively, than LNCaP that was essentially equivalent to C4-2 cells when using an osteoblastic cell line, D1 as the substrate. Similar results were acquired when HBME-1 were used as substratum. There was a marked increase in adhesion of the poorly tumorigenic cell line P69 as compared to the cancer cells to HBME-1. P69 adhesion was 2.78-fold (95% CI = 1.87-4.84) and 2.0-fold (95% CI = 1.43-2.80) greater at 1 hr and 2 hr, respectively when compared to LNCaP or C4-2 cells. D1 cells, a bone homing osteoblastic precursor, behaved contrary to the metastatic, bone-colonizing C4-2 cell line and bound best to other bone cells but not as well as a non-homing fetal bone marrow-derived cell line, D2. Invasion of prostate cancer cells through HBME-1 lawns was examined at 8 hr and 16 hr. In contrast to the adhesion studies, the invasion of the more aggressive C4-2 cells was 3.46-fold (95% CI = 1.18-10.17) and 2.65-fold (95% CI = 1.26-5.56) greater at 8 hr and 16 hr, respectively than LNCaP cells. Similarly, LNCaP cell invasion was 1.73-fold (95% CI = 0.69-4.37) and 2.35-fold (95% CI = 1.41-3.93) greater at 8 hr and 16 hr, respectively than P69 cells at the invasion of HBME-1 monolayers. At 8 hr, C4-2 cells had 6.0-fold (95% CI = 2.63,13.65) higher invasive potential than P69 cells. Phage display biopanning of LNCaP cells versus C4-2 cells in vitro using 4 separate techniques repeatedly identified the same peptide in support of minimal cell surface changes associated with the ability of C4-2 cells to metastasize to bone. As integrins are vital to cell adhesion and migration, we examined the integrin subunit expression in the prostate cell lines. The expression of integrin subunits is much higher in the nontumorigenic cell line, P69, whereas the differences in integrin expression between LNCaP and C4-2 are negligible. Only alpha(2) and beta(5) integrin subunits increase from LNCaP to C4-2. Given that C4-2 cells spontaneously metastasize to bone in vivo and LNCaP cells do not, these studies imply that the ability of a metastatic prostate cancer cell to colonize the bone is not completely dependent upon the ability of the cancer cell to adhere to either osteoblastic cells or to the bone marrow endothelial cell lining. Therefore, the initial interaction between the bone endothelium or stroma and prostate cells is not accurately referred to as a tropic or homing response. The invasion assay results indicate that the invasive potential of the cell more accurately reflects the bone colonizing potential of a prostate cancer cell. It is likely that bidirectional paracrine interactions, subsequent to marrow adhesion, between prostate cancer cells and the bone microenvironment are what determine the successful colonization of the bone by prostate cancer cells. Further, functional changes in surface proteins that are involved in invasion are likely to occur without major changes in levels of cell surface protein expression. Functional integrin association, substratum usage and outside in signaling are more likely to predict metastatic behavior.

Evidence of porcine and human endothelium activation by cancer-associated carbohydrates expressed on glycoproteins and tumour cells.

Abstract: It is well established that after metastatic cancer cells escape the primary tumour and enter the circulation, their interactions with microvascular endothelium of a target organ constitute an essential rate-limiting step in haematogenous cancer metastasis. However, the physiological and biochemical processes supporting neoplastic cell arrest and retention in the microcirculation are still poorly understood. In this study, we present experimental evidence that microvascular endothelium of metastasis-prone tissues undergoes activation in response to desialylated cancer-associated carbohydrate structures such as Thomsen-Friedenreich (TF) antigen (Galbeta1-3GalNAc) expressed on circulating glycoproteins and neoplastic cells. The metastasis-associated endothelium activation, manifested by marked increase in endothelial cell surface galectin-3 expression, causes gradual decrease in cancer cell velocities (from 72 x 10(2)+/- 33 x 10(2) microm s-1 to 7.6 x 10(2)+/- 1.9 x 10(2) microm s-1, mean +/-s.d.) accompanied by a corresponding increase in the percentage of rolling cells (from 3.3%+/- 1.2% to 24.3%+/- 3.6%, mean +/-s.d.), and results in human breast and prostate carcinoma cell arrest and retention in the microvasculature. This process, which could be of high importance in haematogenous cancer metastasis, was inhibited efficiently by an anti-TF antigen function-blocking antibody. Carbohydrate-mediated endothelial activation could be a process of physiological significance as it probably occurs in the interactions between a variety of circulating constituents and the vessel wall.

In vivo real-time imaging of TGF-β-induced transcriptional activation of the RANK ligand gene promoter in intraosseous prostate cancer

Abstract: BACKGROUND Current animal models of prostate cancer (CaP) bone metastasis do not allow measurement of either tumor growth in bone over time or activation of gene promoters in intraosseous tumors. To develop these methods, we used bioluminescent imaging (BLI) to determine if expression of receptor activator of NF-κB ligand (RANKL), a pro-osteoclastogenic factor that promotes CaP bone metastases, is modulated by the bone matrix protein transforming growth factor-β (TGF-β) in vivo. METHODS C4-2B human CaP cells were treated with TGF-β in vitro and RANKL mRNA and protein production were measured by polymerase chain reaction (PCR) and ELISA, respectively. Then C4-2B cells stably transfected with the RANKL promoter driving luciferase (lux) were injected intra-tibially into severe combined immundeficient (SCID) mice. Tumors were subjected to BLI every 2 weeks for 6 weeks and serum prostate specific antigen (PSA) was measured using ELISA. Vehicle (V), 1,25 dihydroxyvitamin D (VitD), or TGF-β was administered to mice with established tumors and BLI to measure RANKL promoter activity was performed. Tumors were then subjected to immunohistochemistry for lux and assayed for RANKL mRNA levels. RESULTS TGF-β induced RANKL protein and mRNA expression and activated the RANKL promoter activity in a dose-dependent manner in vitro. BLI demonstrated an increase in intraosseous tumor size over time, which correlated with serum PSA levels. Administration of TGF-β and VitD to mice with established intraosseous tumors increased lux activity compared to V. Intratibial tumor RANKL mRNA expression paralleled the increased promoter activity. Immunohistochemistry confirmed the presence of lux in the intraosseous tumors. CONCLUSIONS These results demonstrate the ability to measure intraosseous tumor growth over time and gene promoter activation in an established intraosseous tumor in vivo and also demonstrate that TGF-β induces activates the RANKL promoter. These results provide a novel method to explore the biology of CaP bone metastases. © 2004 Wiley-Liss, Inc.

Expression mapping at 12p12-13 in advanced prostate carcinoma.

Abstract: We have previously mapped a putative prostate cancer tumor-suppressor gene to a 1-2 Mb region of 12p12-13. Initial work to identify the tumor suppressor at this locus focused on candidates previously implicated in malignancy; however, mutational and methylation analyses failed to identify significant genomic events. An alternative approach is to use expression analysis to prioritize the genes within the region of interest. This experimental design is based on the hypothesis that tumor-suppressor genes demonstrate decreased expression in tumors compared to normals. Herein, we narrow the region of interest using deletion mapping data and employ expression analysis to prioritize the genes in the minimal deleted region. Highly informative polymorphic markers spanning our region were used to assess for loss of heterozygosity in 99 tumor and normal DNA pairs. The minimal region of deletion was determined to be approximately 500 kb bounded by D12S391 and A002Q26. Publically available databases place 7 genes within this minimal deletion region. An additional 3 genes lie just outside this minimal deletion region and could possibly be inactivated by deletion of promoter, 3'-untranslated region sequences or alternative splice variants. Relative levels of expression of these 10 candidate genes were determined in 6 normal prostates, 5 local prostate tumors, 9 prostate lymph node metastases, 6 prostate cancer cell lines and 12 prostate cancer xenografts using quantitative RT-PCR. DUSP16, FLJ10298 and BCLG were significantly downregulated in both clinical tumors and cultured prostate cancer tissue, indicating that one or all may be critical to initiation or progression of prostate carcinoma.

Cancer cells homing to bone: the significance of chemotaxis and cell adhesion.

Abstract: Cancer cell metastasis is a complex process involving several well-characterized steps. To metastasize successfully, tumor cells must first detach from a primary mass, enter the blood circulation or lymphatics. Subsequently, tumors must home to a particular organ, adhere to the endothelium lining the capillaries of that organ, exit the circulation where they must adhere to organ-specific extra-cellular matrix (ECM) components to begin new growth in an foreign environment (Geldof, 1997). Although the metastatic pattern of some cancers may be explained by anatomy, such as the pattern of efferent venous and lymphatic drainage, anatomy does not account for the metastatic pattern of all cancers (Nicolson et al., 1984). This suggests that other factors contribute significantly to the metastatic fate of cancer cells.

Circulating tumor cells (ctc’s): apoptotic assessment in prostate cancer patients

Abstract: A method for detecting and enumerating apoptotic cancer cells in a mixed cell population by obtaining a biological specimen, the presence of said cancer cells in said population being indicative of a diseases state, is provided. The method comprises obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing said cancer cells; preparing an immunomagnetic sample wherein said specimen is mixed with magnetic particles coupled to a biospecific ligand which reacts specifically with said cancer cells, to the substantial exclusion of other sample components; contacting said immunomagnetic sample with at least one biospecific reagent which labels said cancer cells; and characterizing said cancer cells into one or more apoptotic states from the group consisting of enzymatic analysis, biochemical analysis, morphological analysis and combination thereof, the presence and number of said apoptotic state being indicative of said disease state.