Paul Thelen

EXPRESSION OF METALLOPROTEINASE 2 AND 9 AND THEIR INHIBITORS IN RENAL CELL CARCINOMA

Degradation of the extracellular matrix is necessary for invasion and metastasis by cancer cells. Two gelatinolytic matrix metalloproteinase enzymes, MMP-2 and MMP-9, are supposed to be key enzymes in this process. The purpose of this study was to correlate the presence of MMP-2, MMP-9 and their inhibitors with the tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 RNA using reverse transcriptase PCR technique with tumor stage in 17 samples of renal cell carcinoma. The ratio of tissues expressing MMP-2 and MMP-9 to those expressing TIMP-1 and TIMP-2 was defined to be 1 in normal kidney tissue. This MMP:TIMP ratio was significantly increased to 2.43 (standard deviation, SD = 0.8) in locally confined renal cell carcinoma and to 4.86 (SD = 1.1) in advanced carcinoma (p <0.01). In primary tumor cell lines the ratio of MMP:TIMP expression was 3.44 (SD = 0.6). These data suggest that the balance of MMP-2 and MMP-9 to TIMP-1 and TIMP-2 expression is an essential factor in the aggressiveness of renal cell carcinoma.

Matrix Metalloproteinases and Their Inhibitors

Matrix metalloproteinases (MMPs) are a group of 16 enzymes that are capable of degrading extracellular matrix components. Their catalytic function is dependent on a zinc ion in the active center. MMPs are separated in three groups: gelatinases (type IV-collagenases), stromelysins, and interstitial collagenases. Their physiological and pathological significance is to modulate the extracellular matrix-e. g., in embryogenesis, in the ovarian cycles, or in inflammatory diseases such as rheumatoid arthritis or fibrosis of the liver or kidney (1,2).

Blockade of the type I IGF receptor expression in human prostate cancer cells inhibits proliferation and invasion, up-regulates IGF binding protein-3, and suppresses MMP-2 expression

The type I insulin‐like growth factor receptor (IGF‐IR) is involved in tumour cell proliferation, invasion, and cancer cell survival. Several studies indicate that the IGF axis contributes to prostate cancer pathogenesis, but there is no consensus regarding the relative expression of the IGF‐IR in benign and malignant prostate epithelium. In this study, endogenous IGF‐IR gene expression was reduced in stably transfected PC‐3 cells by employing an antisense RNA strategy which resulted in significant suppression of both PC‐3 cell invasion and proliferation in vitro. Furthermore, it was demonstrated that a direct correlation exists between the inhibition of IGF‐IR gene expression and either up‐regulation of IGF binding protein (BP)‐3 or down‐regulation of matrix metalloproteinase (MMP)‐2 expression in androgen‐independent PC‐3 cells. Moreover, inhibition of IGF‐IR gene expression in transfected PC‐3 cells leads to an enhanced rate of spontaneous apoptosis. In addition, expression analyses by quantitative RT‐PCR on RNA from laser microdissected matched normal prostate and prostate tumour samples revealed that IGF‐IR gene expression was up‐regulated in nine of 12 prostate cancers, whereas IGFBP‐3 gene expression was down‐regulated in all 12 prostate carcinomas analysed. These results indicate an important role for IGF‐IR and IGFBP‐3 in the homeostasis of prostate carcinoma cells and provide a further basis for targeting IGF‐IR or IGFBP‐3 gene expression in order to improve understanding of the IGF‐IR‐activated signalling pathways and as a potential treatment for prostate cancer. Copyright

Bax Inhibitor-1 Is Overexpressed in Prostate Cancer and Its Specific Down-Regulation by RNA Interference Leads to Cell Death in Human Prostate Carcinoma Cells

To analyze differential gene expression of putative prostate tumor markers we compared the expression levels of more than 400 cancer-related genes using the cDNA array technique in a set of capsule-invasive prostate tumor and matched normal prostate tissue. The overexpression of Bax inhibitor-1 (BI-1) in prostate carcinoma and prostate cancer cell lines was confirmed by using Northern blot and Western blot analyses. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) on intact RNAs from 17 paired laser-captured microdissected epithelial tissue samples confirmed up-regulated BI-1 expression in 11 of 17 prostate tumors. In addition, it was demonstrated that BI-1 expression is down-regulated in stromal cells as compared to matched normal epithelial cells of the prostate. In situ hybridization experiments on prostate sections also revealed that BI-1 expression is mainly restricted to epithelial cells. Furthermore, quantitative RT-PCR on RNAs derived from five benign prostate hyperplasia (BPH) samples showed no significant difference in BI-1 expression as compared to normal epithelial prostate tissue. To determine the function of BI-1 in vitro, human PC-3, LNCaP, and DU-145 prostate carcinoma cells were transfected with small interfering double-strand RNA (siRNA) oligonucleotides against the BI-1 gene leading to a specific down-regulation of BI-1 expression. Furthermore, transfection of PC-3, LNCaP, and DU-145 cells with BI-1 sequence-specific siRNAs caused a significant increase in spontaneous apoptosis in all cell lines. Taken together, our results indicate that the human BI-1 gene contains the potential to serve as a prostate cancer expression marker and as a potential target for developing therapeutic strategies for prostate cancer.

Vascular endothelial growth factor expression, angiogenesis, and necrosis in renal cell carcinomas

Rapidly growing tumors often develop necrosis. In the present study the expression of vascular endothelial growth factor (VEGF) was investigated and compared to microvessel density and necrosis of renal cell carcinomas. In the tumor-host interface the microvessel density was significantly increased compared to central tumor areas. Tumor necrosis was associated with a decrease of microvessel density and an increase of the VEGF protein expression within the perinecrotic rim. VEGF protein was focally upregulated in vital tumor tissue. An increase of the apoptotic rate of endothelia and vital tumor tissue in tumors with necrosis could not be detected. VEGF(121,165) mRNA was decreased in proliferatively active carcinomas compared to less proliferative tumors. Multicellular renal cell cancer spheroids as a model of chronic hypoxia developed central apoptosis but no necrosis. VEGF was upregulated in the spheroid. Tumor microvessels expressed matrix metalloproteinase -2 and -9 and an incomplete pericyte covering in comparison to tumor-free tissue indicating immature active angiogenesis. We conclude that highly proliferative renal cell carcinomas outgrow their vascular supply and develop chronic hypoxia inducing a decrease of proliferation and an increase of VEGF expression. However, chronic hypoxia does not cause significant necrosis or apoptosis. Tumor necrosis is more likely induced by acute hypoxia due to immature microvessels. Furthermore, VEGF expression associated with concomitant tumor necrosis may help identify renal cell carcinomas susceptible to antiangiogenic therapy.

DNA methylation biomarkers of prostate cancer: confirmation of candidates and evidence urine is the most sensitive body fluid for non-invasive detection.

A prostate cancer (PCa) biomarker with improved specificity relative to PSA is a public health priority. Hypermethylated DNA can be detected in body fluids from PCa patients and may be a useful biomarker, although clinical performance varies between studies. We investigated the performance of candidate PCa DNA methylation biomarkers identified through a genome‐wide search.

Expression of the T-cell chemoattractant chemokine lymphotactin in Crohn's disease.

Recruitment of lymphocytes is a prominent feature of the inflammatory process in Crohns disease (CD). The present study was undertaken to investigate the expression of the novel lymphocyte-specific chemoattractant lymphotactin (Lptn) as a potential regulatory factor for the recruitment of T cells in CD. The expression of Lptn mRNA was quantified in resection specimens of patients with CD in comparison to normal controls without signs of inflammation by real-time quantitative reverse transcriptase-polymerase chain reaction and localized by nonradioactive in situ hybridization. Furthermore, the phenotype of cells expressing Lptn mRNA was characterized. In contrast to normal controls Lptn mRNA was significantly increased in tissue samples affected by CD. Cells expressing Lptn were identified as T cells, mast cells, and unexpectedly dendritic cells. Lptn mRNA was found to be up-regulated on stimulation with phorbol-12-myristate-13-acetate and concanavalin A in T cells isolated from peripheral blood, which could be prevented by dexamethasone, cyclosporine A, and FK506. A similar regulation mechanism could be identified for the Lptn receptor GPR-5 in peripheral T cells. In addition, Lptn mRNA expression could be induced in mature monocyte-derived dendritic cells. The results indicate that local expression of Lptn by activated T cells and to a lesser extent by mast cells and dendritic cells represents a key regulator for lymphocyte trafficking and maintenance of the inflammatory process observed in CD, which might be partly mediated through an autocrine/paracrine pathway of activated T cells.

Expression and functional analysis of Bax inhibitor-1 in human breast cancer cells.

Recently, deregulated expression of the anti‐apoptotic protein Bax inhibitor‐1 (BI‐1) has been shown in several human cancers. In this report, we show that BI‐1 is expressed at various levels in six different human breast cancer cell lines. In order to investigate the function of BI‐1 in oestrogen‐dependent MCF‐7, T‐47D and oestrogen‐independent MDA‐MB‐231 breast cancer cells, the RNA interference technique was used to knock down BI‐1 expression specifically. Suppression of BI‐1 expression caused a significant increase in spontaneous apoptosis in MDA‐MB‐231 cells, whereas MCF‐7 and T‐47D cells remained almost unaffected. Furthermore, BI‐1 expression analysis using a cancer profiling array showed up‐regulation of BI‐1 expression in cancer samples of breast, uterus and ovary, whereas down‐regulated BI‐1 expression was identified in stomach, colon, kidney, lung and rectal cancer. In addition, immunohistochemical studies using a BI‐1‐specific antibody on human breast cancer specimens also revealed that BI‐1 is expressed in the majority of cases. Moreover, to analyse whether BI‐1 expression is oestrogen receptor‐dependent, tumour cells were treated with oestradiol, ICI and tamoxifen: this showed no significant changes in BI‐1 expression. Taken together, our results demonstrate that BI‐1 expression is differentially deregulated in different cancers and that BI‐1 plays an important role in preventing certain breast cancer cells from undergoing apoptosis. Thus, the development of novel therapeutic strategies based on targeting BI‐1 gene expression in breast cancer could be restricted to selected individual cancer types. Copyright

Leupaxin, a Novel Coactivator of the Androgen Receptor, Is Expressed in Prostate Cancer and Plays a Role in Adhesion and Invasion of Prostate Carcinoma Cells

In the present study, we demonstrate that leupaxin mRNA is overexpressed in prostate cancer (PCa) as compared with normal prostate tissue by using cDNA arrays and quantitative RT-PCR analyses. Moderate to strong expression of leupaxin protein was detected in approximately 22% of the PCa tissue sections analyzed, and leupaxin expression intensities were found to be significantly correlated with Gleason patterns/scores. In addition, different leupaxin expression levels were observed in PCa cell lines, and at the subcellular level, leupaxin was usually localized in focal adhesion sites. Furthermore, mutational analysis and transfection experiments of LNCaP cells using different green fluorescent protein-leupaxin constructs demonstrated that leupaxin contains functional nuclear export signals in its LD3 and LD4 motifs, thus shuttling between the cytoplasm and the nucleus. We could also demonstrate for the first time that leupaxin interacts with the androgen receptor in a ligand-dependent manner and serves as a transcriptional activator of this hormone receptor in PCa cells. Down-regulation of leupaxin expression using RNA interference in LNCaP cells resulted in a high rate of morphological changes, detachment, spontaneous apoptosis, and a reduction of prostate-specific antigen secretion. In contrast, knockdown of leupaxin expression in androgen-independent PC-3 and DU 145 cells induced a significant decrease of both the invasive capacity and motility. Our results therefore indicate that leupaxin could serve as a potential progression marker for a subset of PCa and may represent a novel coactivator of the androgen receptor. Leupaxin could function as a putative target for therapeutic interventions of a subset of advanced PCa.

Expression and function of protein phosphatase PP2A in malignant testicular germ cell tumours

Testicular germ cell tumours (TGCT) represent the most common malignancy in young males. We reported previously that two prototype members of the mitogen‐activated protein kinase (MAPK) family, the MAPK ERK kinase (MEK) and extracellular signal‐regulated kinase (ERK), are inactive in malignant testicular germ cells and become active after drug stimulation, leading to apoptosis of tumour cells. In this study, we asked whether the protein phosphatase PP2A, a known inhibitor of the MEK–ERK pathway, participates in the proliferation and/or apoptosis of primary TGCT (n = 48) as well as two TGCT cell lines (NTERA and NCCIT). Quantitative RT–PCR, immunohistochemistry, western blot analyses and phosphatase assay indicate that primary TGCT as well as TGCT cell lines express PP2A and that PP2A is active in TGCT cell lines. The inhibition of PP2A by application of two PP2A inhibitors, cantharidic acid (CA) and okadaic acid (OA), results in a significant increase in caspase‐3‐mediated apoptosis of TGCT cell lines. Thereby, PP2A inhibition was accompanied by phosphorylation and activation of MEK and ERK. Functional assays using the MEK inhibitor PD98059 demonstrated that the phosphorylation of MEK and ERK was required for the induction of caspase‐3‐mediated apoptosis of malignant germ cells. Thus, our data suggest that inhibition of PP2A mediates its apoptosis‐inducing effect on TGCT through activation of the MEK–ERK signalling pathway that leads to caspase‐3‐mediated apoptosis of tumour cells. In addition our results support previous observations that PP2A exerts an anti‐apoptotic effect on malignant tumour cells. Copyright