Marie-Pierre Podgorniak

Stromal cells from human benign prostate hyperplasia produce a growth‐inhibitory factor for LNCaP prostate cancer cells, identified as interleukin‐6

To understand specific interactions between stromal cells and epithelial cells in benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma, we developed stromal‐cell cultures from normal human prostate (PNX) and BPH (BH101), composed of fibroblasts and myofibroblasts. Their role in epithelial‐cell growth was studied using the established cancer cell lines LNCaP, PC3 and DU 145 and an SV40 large T‐immortalized normal epithelial‐cell line, PNTIA, in double‐diffusion co‐culture chambers. PNTIA was stimulated by PNX (x 1.6) and more strongly by BH101 stromal cells (x 2.7). Conversely, LNCaP growth decreased by 50% in the presence of BH101 stromal cells (stromal/epithelial ratio: 10). A BH101‐conditioned medium (CM), obtained in serum‐free conditions, induced 90% inhibition of [3H]thymidine incorporation of the LNCaP androgen‐sensitive cell line. Two other androgen‐independent prostate cancer cell lines were either insensitive to BH101 CM (PC3) or slightly inhibited (40% for DU145). BH101 produced large amounts of IL‐Iβ, IL‐6 and IL‐8. HPLC gel filtration enabled separation of an inhibitory fraction which contained IL‐6. IL‐6 was demonstrated to be responsible for the strong inhibitory effect since an IL‐6‐neutralizing antibody abolished this inhibition, which was reproduced by human recombinant IL‐6. Recombinant IL‐6 growth inhibition was observed only on LNCaP prostate cancer androgen‐sensitive cells.

Epstein-Barr Virus (EBV) Genome and Expression in Breast Cancer Tissue: Effect of EBV Infection of Breast Cancer Cells on Resistance to Paclitaxel (Taxol)

ABSTRACT The Epstein-Barr virus (EBV) has been detected in subsets of breast cancers. In order to elaborate on these observations, we quantified by real-time PCR (Q-PCR) the EBV genome in biopsy specimens of breast cancer tissue as well as in tumor cells isolated by microdissection. Our findings show that EBV genomes can be detected by Q-PCR in about half of tumor specimens, usually in low copy numbers. However, we also found that the viral load is highly variable from tumor to tumor. Moreover, EBV genomes are heterogeneously distributed in morphologically identical tumor cells, with some clusters of isolated tumor cells containing relatively high genome numbers while other tumor cells isolated from the same specimen may be negative for EBV DNA. Using reverse transcription-PCR, we detected EBV gene transcripts: EBNA-1 in almost all of the EBV-positive tumors and RNA of the EBV oncoprotein LMP-1 in a smaller subset of the tissues analyzed. Moreover, BARF-1 RNA was detected in half of the cases studied. Furthermore, we observed that in vitro EBV infection of breast carcinoma cells confers resistance to paclitaxel (taxol) and provokes overexpression of a multidrug resistance gene (MDR1). Consequently, even if a small number of breast cancer cells are EBV infected, the impact of EBV infection on the efficiency of anticancer treatment might be of importance.

EMMPRIN promotes angiogenesis through hypoxia-inducible factor-2α–mediated regulation of soluble VEGF isoforms and their receptor VEGFR-2

Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) is thought to promote tumor angiogenesis mostly through its protease-inducing function and more recently by its ability to increase tumor cell expression of vascular endothelial growth factor (VEGF). In this study, we present evidence that EMMPRIN can promote angiogenesis by a direct effect on endothelial cells through a paracrine regulation of the VEGF/VEGF-receptor (VEGFR) system. Using human microvascular endothelial cell line-1 endothelial cells, we show that EMMPRIN selectively increased the soluble VEGF isoforms (121 and 165), but not the matrix-bound VEGF 189 form. In addition, EMMPRIN up-regulated the expression of VEGFR-2 without an effect on VEGFR-1. This increase in VEGFR-2 was responsible for the observed EMMPRIN stimulation of the migratory and tube formation capacity of endothelial cells. EMMPRINs effects, which were matrix metalloproteinase and urokinase-type plasminogen activator independent, were mediated primarily through hypoxia-inducible factor-2alpha expression, also up-regulated by EMMPRIN. VEGFR-2 increase was also observed in vivo in a mouse model of xenograph tumors overexpressing EMMPRIN. These results suggest that in addition to increasing protease production, EMMPRIN may contribute to the formation of a reactive stroma also through the up-regulation of hypoxia-inducible factor-2alpha, VEGFR-2, and the soluble forms of VEGF in endothelial cells, thus directly regulating the angiogenic process.

Characterization of in vitro culture of HIV-negative Kaposi's sarcoma-derived cells. In vitro responses to alfa interferon

We established long-term cultures from skin tumors of nine patients suffering from classical Kaposi’s sarcoma (KS). Spindle cells obtained after enzymatic digestion were cultured on gelatin- or fibronectin-coated flasks in DMEM with 15% fetal calf serum, aFGF and heparin. Immunohistochemical staining was positive for MHC class I, laminin, type IV collagen, vimentin, α smooth muscle actin (20–40% of cells), caldesmon (20%), calponin (20–40%) and smooth muscle myosin (20–40%), and was negative for common leukocyte antigen, CD4, LFA1, CD34 and cytokeratin. Around 20% of cells up to the third passage in culture expressed the endothelial markers CD36, BMA 120 but were negative for UEA and Fc von Willebrand. Smooth muscle proteins were detected with immunoblotting. Using the polymerase chain reaction, human herpes virus 8 (HHV8) sequences were detected in primary cultures of three out of seven cell lines but were rapidly lost during in vitro passaging. KS-derived cells did not proliferate in serum-free medium, had a normal karyotype and did not grow in soft agar medium. Tumors formed in nude mice injected with KS-derived cells. The tumors were composed of mouse cells and were highly vascularized. Our results suggest that KS-derived cells are heterogeneous: the majority of cells have either a smooth muscle cell or a fibroblastic phenotype. Another minor cell compartment was composed of endothelium-derived cells. KS cells do not possess the characteristics of transformed cells in vitro and may be composed of polyclonal activated cells. Recombinant α interferon (rIFN) slightly inhibited the growth of KS-derived cells and increased the expression of MHC class I antigens. While cells were resistant to natural killer (NK) cell-mediated cytotoxicity, they became sensitive to rIFN-primed NK cells. Thus, the antitumor potential of rIFN against KS in vivo could result from immunomodulatory rather than from direct antiproliferative effects.

Soluble Isoforms of Vascular Endothelial Growth Factor Are Predictors of Response to Sunitinib in Metastatic Renal Cell Carcinomas

Background Angiogenesis is the target of several agents in the treatment of malignancies, including renal cell carcinoma (RCC). There is a real need for surrogate biomarkers that can predict selection of patients who may benefit from antiangiogenic therapies, prediction of disease outcome and which may improve the knowledge regarding mechanism of action of these treatments. Tyrosine kinase inhibitors (TKI) have proven efficacy in metastatic RCC (mRCC). However, the molecular mechanisms underlying the clinical response to these drugs remain unclear. Methodology/Principal Findings The present study aimed to identify molecular biomarkers associated with the response to sunitinib, a Tyrosine kinase inhibitor. To evaluate this relationship, primary tumors from 23 metastatic RCC patients treated by sunitinib were analyzed for a panel of 16 biomarkers involved in tumor pathways targeted by sunitinib, using real-time quantitative reverse-transcriptase PCR. Nine of the 23 patients (39%) responded to sunitinib. Among transcripts analyzed, only the levels of vascular endothelial growth factor (VEGF) soluble isoforms (VEGF121 and VEGF165) were associated with the response to sunitinib (P = 0.04 for both). Furthermore, the ratio of VEGF soluble isoforms (VEGF121/VEGF165) was significantly associated with prognosis (P = 0.02). Conclusions This preliminary study provides a promising tool that might help in the management of metastatic RCC, and could be extended to other tumors treated by TKI.

SIAH-1 interacts with CtIP and promotes its degradation by the proteasome pathway.

SIAH-1 and SIAH-2 are the human members of an evolutionary highly conserved E3 ligase family. SIAH-1 is a p53 and p21Waf−1/Cip−1 induced gene during apoptosis and tumor suppression. In stable-transfected clones of MCF-7 cells, SIAH-1 overexpression was associated with apoptosis, mitotic alterations and p21Waf−1/Cip−1 induction of expression. Using a two-hybrid screening, we identified here the transcriptional corepressor CtBP-interacting protein (CtIP) as a SIAH-1-interacting protein. CtIP has been proposed as a regulator of p21Waf−1/Cip−1 gene transcription through a protein complex involving BRCA1. We demonstrate that SIAH-1 associates with CtIP both in vitro and in vivo. This interaction led to CtIP degradation by the ubiquitin–proteasome pathway. As expected, SIAH-1 induced p21Waf−1/Cip−1 transcription in Jurkat-T cell. Surprisingly, a SIAH protein deleted of its RING finger, SIAH-1ΔN, which is able to interact with CtIP but does not promote its degradation, also induced transcription from the p21Waf−1 promoter in a similar extent as did SIAH-1. Our results suggest that p21Waf−1/Cip−1 induction by SIAH-1 could not be mediated by CtIP degradation.

Quantification of VEGF isoforms and VEGFR transcripts by qRT-PCR and their significance in acute myeloid leukemia

Vascular endothelial growth factor (VEGF) and its receptors are known to play an important role in normal and pathological hematopoiesis but the prognostic impact of VEGF isoform transcripts in acute myeloid leukemia (AML) has not been addressed. We conducted a single-institution prospective study to analyze the impact of these angiogenic factors and the expression of their receptors on the survival of adult patients newly diagnosed with AML. We investigated the levels of VEGF transcript isoforms VEGF121, -145, -165, -189 and -206 and their receptors, VEGFR-1 and VEGFR-2, using quantitative reverse transcriptase polymerase chain reaction assays in peripheral blood mononuclear cells (PBMCs) of 67 consecutive AML patients at diagnosis. VEGF total protein was measured for comparison with mRNA levels in PBMCs. The VEGF121 splice variant transcript in AML PBMCs was significantly higher than in the normal controls. VEGF transcripts were quantified in all samples while its protein was detected in 42/67 (63%) of AML samples. High levels of VEGF121, VEGF165 transcripts and VEGF protein in AML were significantly related to a worse prognosis when analyzing overall survival (p<0.0001, p=0.019 and p=0.012, respectively) or event-free survival (p<0.0001, p=0.010 and p=0.047) using univariate analysis. In multivariable analysis only VEGF121 expression remained an independent prognostic factor for either event-free survival or overall survival [aHR=8.83 (3.48-22.4), p<0.0001, and aHR=9.52 (3.41-26.6), p<0.0001]. No prognostic value was observed for the other isoforms and the two receptors. Our findings show that the level of VEGF121 mRNA in circulating cells from AML patients is a strong independent prognostic parameter, which could be useful in the management of unselected AML patients.

Estramustine resistance correlates with tau over-expression in human prostatic carcinoma cells

Estramustine (EM) is an anti‐microtubule drug used in the treatment of hormone‐refractory advanced prostate cancer. Since microtubules are the targets for EM cytotoxicity, we investigated the effects of EM on the microtubule‐associated protein tau to determine what role it may play in drug resistance. We have compared tau expression in human prostate cancer cells (DU145) and an EM‐resistant derived cell line (E4). Reverse transcriptase polymerase chain reaction has established that tau is expressed in both cell lines but increased 1.9‐fold in E4 compared with DU145 cells. This result was confirmed at the protein level by Western blotting. Tau is a phosphoprotein, most of its reported phosphorylation sites being serine or threonine residues. We have shown, however, that tau is also phosphorylated at tyrosine residues in DU145 cells and that the phosphotyrosine level of tau is significantly increased in E4 cells. Moreover, DU145 cells exposed to short term micromolar drug concentrations enter a phase of microtubule depolymerization, display an increased level of tau phosphorylation and follow a pattern similar to that observed in EM‐resistant E4 cells. EM is therefore able to induce a very rapid change in the post‐translational state of tau. Our results show that the acquisition of EM resistance in E4 cells, which is accompanied by changes at the tubulin level, is also associated with important changes in tau expression and phosphorylation. Int. J. Cancer 77:626–631, 1998.

Highly Sensitive Multivariable Assay Detection of Melanocytic Differentiation Antigens and Angiogenesis Biomarkers in Sentinel Lymph Nodes With Melanoma Micrometastases

OBJECTIVES To evaluate the prognostic value of melanocytic differentiation antigens and angiogenesis biomarkers in sentinel lymph nodes (SLNs) with melanoma micrometastases. DESIGN Prognostic study of an inception cohort. SETTING Academic research. Patients Between July 1, 1999, and July 31, 2002, all patients who had primary cutaneous or mucosal melanomas that have a Breslow depth of 1.5 mm or greater, ulceration, or Clark level IV or V, or had SLN biopsies. MAIN OUTCOME MEASURES By the use of quantitative reverse transcription-polymerase chain reaction, the expression of the following was analyzed in SLNs: 2 melanocytic differentiation antigens (tyrosinase [P17646] and melanoma antigen recognized by T cells [MART-1; Q16655]) and genes involved in angiogenesis (VEGF [NM_001025366] and VEGFR2 [AF035121]), lymphangiogenesis (VEGFC [NM_005429], VEGFR3 [X68203], LYVE1 [NM_016164], and PROX1 [002763]), and invasion (uPA [NM_002658], PAI1 [NM_00602], and EMMPRIN [L10240]). Outcome measures were the association of these melanocytic differentiation antigens and angiogenesis biomarkers with clinicopathologic characteristics of patients, and an evaluation of the prognostic value for relapse-free survival and overall survival. RESULTS Ninety-one patients were included, with a median follow-up period of 41 months. Micrometastases were present in 15% (14 of 91) of patients. Tyrosinase (P < .001), MART-1 (P < .001), vascular endothelial growth factor 121 (VEGF(121)) (P = .007), and PAI1 (P = .02) expression was significantly associated with micrometastasis. In univariate analysis, histologic findings and tyrosinase and MART-1 expression were significantly associated with relapse-free survival. Tyrosinase and MART-1 expression was associated with overall survival. A multiple Cox proportional hazards regression model identified negative histologic findings and tyrosinase expression that exceeded 27 copies/copy of TATA box-binding protein (third quartile) as significantly associated with an increased risk of relapse or death. CONCLUSIONS Quantitative assessment of melanocytic differentiation antigens in SLNs, which has prognostic value, is more specific than qualitative assessment. Prognosis may be more effectively predicted by the combination of quantitative assessment of melanocytic differentiation antigens in SLNs with histologic assessment. A significant association was found between the presence of micrometastases and the expression of angiogenesis biomarkers.

Identification of a novel biomarker signature associated with risk for bone metastasis in patients with renal cell carcinoma.

When renal cell carcinoma (RCC) metastasizes to bone (a frequent site of systemic spread of this cancer) it becomes highly resistant to radiation therapy and chemotherapy. A better understanding of the biology of bone metastasis in RCC may permit to identify biomarkers for early detection of subclinical disease and better stratification of patients prior to treatment. We therefore investigated in this study, using a multiplex real-time RT-PCR assay, the expression of a panel of 16 biomarkers involved in angiogenesis and tumor invasion; the panel was applied to primary tumors and normal tissues obtained from clear-cell RCC patients with and without bone metastases. We identified a novel combination of biomarkers associated with the risk of bone metastasis. Among the transcripts of the genes studied, VEGFR-1, VEGFR-2, HIF-1alpha, uPA , and PA I-1 overexpression in tumor tissues was significantly associated with the presence of bone metastasis (p=0.02, p=0.02, p<0.0001, p=0.04, and p=0.03, respectively). No differences were found in the expression of these transcripts in the corresponding normal tissues. This preliminary study provides a promising tool that may help in the management of RCC patients with bone metastasis. Indeed, these predictive markers could be useful to identify subclinical disease, improve staging, and guide treatment decisions.