Adipocyte-conditioned medium induces resistance of breast cancer cells to lapatinib.

BACKGROUND: The existence of a cross-talk between peritumoral adipocytes and cancer cells has been increasingly investigated. Several studies have shown that these adipocytes protect tumor cells from the effect of anticancer agents. METHODS: To investigate a potential protective effect of adipocyte-conditioned medium on HER2 positive breast cancer cells exposed to tyrosine kinase inhibitors (TKI) such as lapatinib, we analyzed the sensitivity of HER2 positive breast cancer models in vitro and in vivo on SCID mice in the presence or absence of adipocytes or adipocyte-conditioned medium. RESULTS: Conditioned medium from differentiated adipocytes reduced the in vitro sensitivity of the HER2+ cell lines BT474 and SKBR3 to TKI. Particularly, conditioned medium abrogated P27 induction in tumor cells by lapatinib but this was observed only when conditioned medium was present during exposure to lapatinib. In addition, resistance was induced with adipocytes derived from murine NIH3T3 or human hMAD cells but not with fibroblasts or preadipocytes. In vivo studies demonstrated that the contact of the tumors with adipose tissue reduced sensitivity to lapatinib. Soluble factors involved in this resistance were found to be thermolabile. Pharmacological modulation of lipolysis in adipocytes during preparation of conditioned media showed that various lipolysis inhibitors abolished the protective effect of conditioned media on tumor cells, suggesting a role for adipocyte lipolysis in the induction of resistance of tumor cells to TKI. CONCLUSIONS: Overall, our results suggest that contact of tumor cells with proximal adipose tissue induces resistance to anti HER2 small molecule inhibitors through the production of soluble thermolabile factors, and that this effect can be abrogated using lipolysis inhibitors.

Host immune genetic variations influence the risk of developing acute myeloid leukaemia: results from the NuCLEAR consortium.

The purpose of this study was to conduct a two-stage case control association study including 654 acute myeloid leukaemia (AML) patients and 3477 controls ascertained through the NuCLEAR consortium to evaluate the effect of 27 immune-related single nucleotide polymorphisms (SNPs) on AML risk. In a pooled analysis of cohort studies, we found that carriers of the IL13rs1295686A/A genotype had an increased risk of AML (PCorr = 0.0144) whereas carriers of the VEGFArs25648T allele had a decreased risk of developing the disease (PCorr = 0.00086). In addition, we found an association of the IL8rs2227307 SNP with a decreased risk of developing AML that remained marginally significant after multiple testing (PCorr = 0.072). Functional experiments suggested that the effect of the IL13rs1295686 SNP on AML risk might be explained by its role in regulating IL1Ra secretion that modulates AML blast proliferation. Likewise, the protective effect of the IL8rs2227307 SNP might be mediated by TLR2-mediated immune responses that affect AML blast viability, proliferation and chemorresistance. Despite the potential interest of these results, additional functional studies are still warranted to unravel the mechanisms by which these variants modulate the risk of AML. These findings suggested that IL13, VEGFA and IL8 SNPs play a role in modulating AML risk.

Tissue-nonspecific alkaline phosphatase is an anti-inflammatory nucleotidase.

Tissue-nonspecific alkaline phosphatase (TNAP) is necessary for skeletal mineralization by its ability to hydrolyze the mineralization inhibitor inorganic pyrophosphate (PPi), which is mainly generated from extracellular ATP by ectonucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Since children with TNAP deficiency develop bone metaphyseal auto-inflammations in addition to rickets, we hypothesized that TNAP also exerts anti-inflammatory effects relying on the hydrolysis of pro-inflammatory adenosine nucleotides into the anti-inflammatory adenosine. We explored this hypothesis in bone metaphyses of 7-day-old Alpl+/- mice (encoding TNAP), in mineralizing hypertrophic chondrocytes and osteoblasts, and non-mineralizing mesenchymal stem cells (MSCs) and neutrophils, which express TNAP and are present, or can be recruited in the metaphysis. Bone metaphyses of 7-day-old Alpl+/- mice had significantly increased levels of Il-1ß and Il-6 and decreased levels of the anti-inflammatory Il-10 cytokine as compared with Alpl+/+ mice. In bone metaphyses, murine hypertrophic chondrocytes and osteoblasts, Alpl mRNA levels were much higher than those of the adenosine nucleotidases Npp1, Cd39 and Cd73. In hypertrophic chondrocytes, inhibition of TNAP with 25 µM of MLS-0038949 decreased the hydrolysis of AMP and ATP. However, TNAP inhibition did not significantly modulate ATP- and adenosine-associated effects in these cells. We observed that part of TNAP proteins in hypertrophic chondrocytes was sent from the cell membrane to matrix vesicles, which may explain why TNAP participated in the hydrolysis of ATP but did not significantly modulate its autocrine pro-inflammatory effects. In MSCs, TNAP did not participate in ATP hydrolysis nor in secretion of inflammatory mediators. In contrast, in neutrophils, TNAP inhibition with MLS-0038949 significantly exacerbated ATP-associated activation and secretion of IL-1ß, and extended cell survival. Collectively, these results demonstrate that TNAP is a nucleotidase in both hypertrophic chondrocytes and neutrophils, and that this nucleotidase function is associated with autocrine effects on inflammation only in neutrophils.

Monoclonal antibody therapy in multiple myeloma.

The therapeutic landscape of multiple myeloma (MM) has evolved spectacularly over the past decade with the discovery and validation of proteasome inhibitors and immunomodulatory agents as highly active agents, both in front-line therapy as well as in the relapse and maintenance settings. Although previous attempts to apply available monoclonal antibodies (Mabs) to the treatment of patients with MM has until recently been disappointing, novel targets specifically explored in the context of MM have recently lead to the first approvals of Mabs for the treatment of patients with MM. We have performed a literature search to identify preclinical targeting of MM, including in vitro and in vivo models using monoclonal antibodies, as well as clinical trials of monoclonal antibodies in patients with MM. Sources used were peer-reviewed publications, congress abstracts and on-line clinical trials data (such as clinicaltrials.gov). Several targets have been evaluated in preclinical models and a growing number of agents are being evaluated in clinical trials, as single agents or in combination and under various antibody formats. Two agents, targeting for the first time CD38 and SLAMF7, respectively, have recently been approved for the treatment of patients with MM. The recent approval of these two antibodies is expected to have a strong impact on treatment modalities and outcome in patients with MM, including both transplant eligible and elderly patients.

Cell proliferation and drug sensitivity of human glioblastoma cells are altered by the stable modulation of cytosolic 5'-nucleotidase II.

Cytosolic 5'-nucleotidase II (cN-II) has been reported to be involved in cell survival, nucleotide metabolism and in the cellular response to anticancer drugs. With the aim to further evaluate the role of this enzyme in cell biology, we stably modulated its expression the human glioblastoma cell ADF in which the transient inhibition of cN-II has been shown to induce cell death. Stable cell lines were obtained both with inhibition, obtained with plasmids coding cN-II-targeting short hairpin RNA, and stimulation, obtained with plasmids coding Green Fluorescence Protein (GFP)-fused wild type cN-II or a GFP-fused hyperactive mutant (GFP-cN-II-R367Q), of cN-II expression. Silenced cells displayed a decreased proliferation rate while the over expressing cell lines displayed an increased proliferation rate as evidenced by impedance measurement using the xCELLigence device. The expression of nucleotide metabolism relevant genes was only slightly different between cell lines, suggesting a compensatory mechanism in transfected cells. Cells with decreased cN-II expression were resistant to the nucleoside analog fludarabine confirming the involvement of cN-II in the metabolism of this drug. Finally, we observed sensitivity to cisplatin in cN-II silenced cells and resistance to this same drug in cN-II over-expressing cells indicating an involvement of cN-II in the mechanism of action of platinum derivatives, and most probably in DNA repair. In summary, our findings confirm some previous data on the role of cN-II in the sensitivity of cancer cells to cancer drugs, and suggest its involvement in other cellular phenomenon such as cell proliferation.

The purine analog fludarabine acts as a cytosolic 5'-nucleotidase II inhibitor.

For several years the IMP/GMP-preferring cytosolic 5'-nucleotidase II (cN-II) has been considered as a therapeutic target in oncology. Indeed, various reports have indicated associations between cN-II expression level and resistance to anticancer agents in several cancer cell lines and in patients affected with neoplasia, mainly by hematologic malignancies. In this paper we present evidence showing that, among the commonly used cytotoxic nucleoside analogs, fludarabine can act as a cN-II inhibitor. In vitro studies using the wild type recombinant cN-II demonstrated that fludarabine inhibited enzymatic activity in a mixed manner (Ki 0.5 mM and Ki' 9 mM), whereas no inhibition was observed with clofarabine and cladribine. Additional experiments with mutant recombinant proteins and an in silico molecular docking indicated that this inhibition is due to an interaction with a regulatory site of cN-II known to interact with adenylic compounds. Moreover, synergy experiments between fludarabine and 6-mercaptopurine in human follicular lymphoma (RL) and human acute promyelocytic leukemia (HL-60) cells transfected with control or cN-II-targeting shRNA-encoding plasmids, showed synergy in control cells and antagonism in cells with decreased cN-II expression. This is in line with the hypothesis that fludarabine acts as a cN-II inhibitor and supports the idea of using cN-II inhibitors in association with other drugs to increase their therapeutic effect and decrease their resistance.

The acridone derivative MBLI-87 sensitizes breast cancer resistance protein-expressing xenografts to irinotecan.

The breast cancer resistance protein ABCG2 confers cellular resistance to irinotecan (CPT-11) and its active metabolite SN-38. We utilised ABCG2-expressing xenografts as a model to evaluate the ability of a non-toxic ABCG2 inhibitor to increase intracellular drug accumulation. We assessed the activity of irinotecan in vivo in SCID mice: irinotecan completely inhibited the development of control pcDNA3.1 xenografts, whilst only delaying the growth of ABCG2-expressing xenografts. Addition of MBLI-87, an acridone derivative inhibitor, significantly increased the irinotecan effect against the growth of ABCG2-expressing xenografts. In vitro, MBLI-87 was as potent as GF120918 against ABCG2-mediated irinotecan efflux, and additionally was specific for ABCG2. A significant sensitisation to irinotecan was achieved despite the fact that doses remained well below the maximum tolerated dose (due to the rather limited solubility of MBLI-87). This suggested that MBLI-87 is an excellent candidate to prevent drug efflux by ABCG2, without altering plasma concentrations of irinotecan and SN-38 after IP (intra-peritoneal) injections. This could constitute a useful strategy to improve drug pharmacology, to facilitate drug penetration into normal tissue compartments protected by ABCG2, and potentially to reverse drug resistance in cancer cells.

[Class III beta tubulin expression in nonsmall cell lung cancer]. / beta-tubuline de classe III et cancer du poumon non à petites cellules.

INTRODUCTION: Several investigators have addressed the relationship between expression of class III beta-tubulin and outcome in patients with nonsmall cell lung cancer (NSCLC) treated with tubulin-binding agents (TBA). STATE OF ART: High expression of class III beta-tubulin has been found to be correlated with low response rates and reduced survival in advanced NSCLC patients treated with taxane/vinorelbine--containing regimens. Two studies have shown that patients receiving paclitaxel whose tumours expressed high levels of class III beta-tubulin had a lower response rate and shorter survival, whereas this variable was not found to be predictive in patients receiving regimens without TBA. Conversely, analysis of samples from operable NSCLC patients in the JBR-10 trial showed that cisplatin/vinorelbine chemotherapy seemed to overcome the negative prognostic effect of high class III beta-tubulin expression and that the greatest benefit from chemotherapy was observed in patients with high class III beta-tubulin expression. PERSPECTIVES: In advanced NSCLC, high betaIII tubulin expression may prompt selection of taxane-free regimens as the preferred initial treatment approach. The epothilones may fulfil such a role in the treatment of NSCLC. CONCLUSIONS: betaIII tubulin expression is emerging as a valuable biomarker for taxane resistance in advanced disease, as well as offering prognostic information for outcomes among patients with earlier stage disease.

A fluorescent biomarker of the polyamine transport system to select patients with AML for F14512 treatment.

The polyamine transport system (PTS), hyperactive in cancer cells, can constitute a gate to deliver F14512, a novel spermine epipodophyllotoxin conjugate recently selected for clinical development in AML phase I. We investigated in vitro the high antiproliferative effect of F14512 against 13 leukemia cell lines, and demonstrated a statistically significant correlation with the level of PTS activity, using a novel fluorescent marker F96982. This labelling protocol was then adapted for clinical applications for blood, bone marrow and AML samples with CD45 gating. Within the patient samples, the PTS activity varied significantly in AML cells, as compared to normal lymphocytes. In conclusion, the identification of PTS-positive AML with F98982 probe offers new perspectives to select patients prone to respond to F14512.

Genetic polymorphisms associated with outcome in multiple myeloma patients receiving high-dose melphalan.

High-dose melphalan (HDM) is an essential component in the treatment of patients with multiple myeloma (MM). Few data are available regarding genetic polymorphisms associated with patient outcome or toxicity in this setting. To identify such polymorphisms, we performed a retrospective analysis, genotyping single nucleotide polymorphisms (SNPs) with the arrayed primer extension (APEX) technology in 169 patients having received HDM for MM. We analyzed 209 SNPs in 95 genes involved in drug metabolism, DNA repair, cell cycle and apoptosis. SNPs in ABCB1, CYP3A4 and TP53BP2 were associated with response to VAD induction therapy (P<0.01). SNPs in ALDH2, GSTT2 and BRCA1 were associated with response to HDM (P<0.01). Polymorphisms in CYP1A1, RAD51 and PARP were associated with disease progression whereas polymorphisms in ALDH2 and CYP1A1 were correlated with OS. Polymorphisms in BRCA1, CDKN1A and XRCC1 were associated with the occurrence of severe mucositis after HDM. These results suggest that SNPs of genes involved in drug metabolism or DNA repair could be used to distinguish MM patient subgroups with different toxicity/efficacy profiles.

Impact of clinical practice guidelines on the management for carcinomas of unknown primary site: a controlled "before-after" study.

BACKGROUND: In 2002, the French Federation of Comprehensive Cancer Centers published clinical practice guidelines (CPGs) for the management of carcinomas of unknown primary (CUP). METHODS: A controlled "before-after" study at two centers (experimental in Lyon, France and control in Edmonton, Canada) to assess the impact of CPGs on CUP management. Fifty-CUP patients treated in 2000-2001, i.e. before CPG publication, and 50 patients treated in 2003-2004, were analyzed for both centers. RESULTS: In both groups, compliance for diagnostic workup was the same before or after CPGs publication. Non-adenocarcinoma histology and performance status (PS) < 2 were independent factors for CPGs compliance. In the experimental group, 75% of patients underwent inappropriate investigations. The proportion of patients from this group with unfavourable clinicopathologic entity and PS < or = 1, who received cisplatin-based chemotherapy did not significantly change (2000-2001: 27% vs. 2003-2004: 37.5%; P = 0.45). However, most patients treated in the pre period received organ-specific regimens, while most patients treated in the post period received taxane or gemcitabine-based regimens. Patients from the control group generally received taxane/carboplatin. CONCLUSIONS: Our study show that simply distributing CUP CPGs did not change practice and underline the necessity to disseminate and implement CPGs, both to oncologists and organ-specialist physicians.

Development of gene therapy in association with clinically used cytotoxic deoxynucleoside analogues.

The clinical use of cytotoxic deoxynucleoside analogues is often limited by resistance mechanisms due to enzymatic deficiency, or high toxicity in nontumor tissues. To improve the use of these drugs, gene therapy approaches have been proposed and studied, associating clinically used deoxynucleoside analogues such as araC and gemcitabine and suicide genes or myeloprotective genes. In this review, we provide an update of recent results in this area, with particular emphasis on human deoxycytidine kinase, the deoxyribonucleoside kinase from Drosophila melanogaster, purine nucleoside phosphorylase from Escherichia coli, and human cytidine deaminase. Data from literature clearly show the feasibility of these systems, and clinical trials are warranted to conclude on their use in the treatment of cancer patients.

A microarray to measure repair of damaged plasmids by cell lysates.

DNA repair mechanisms constitute major defences against agents that cause cancer, degenerative disease and aging. Different repair systems cooperate to maintain the integrity of genetic information. Investigations of DNA repair involvement in human pathology require an efficient tool that takes into account the variety and complexity of repair systems. We have developed a highly sensitive damaged plasmid microarray to quantify cell lysate excision/synthesis (ES) capacities using small amounts of proteins. This microsystem is based on efficient immobilization and conservation on hydrogel coated glass slides of plasmid DNA damaged with a panel of genotoxic agents. Fluorescent signals are generated from incorporation of labelled dNTPs by DNA excision-repair synthesis mechanisms at plasmid sites. Highly precise DNA repair phenotypes i.e. simultaneous quantitative measures of ES capacities toward seven lesions repaired by distinct repair pathways, are obtained. Applied to the characterization of xeroderma pigmentosum (XP) cells at basal level and in response to a low dose of UVB irradiation, the assay showed the multifunctional role of different XP proteins in cell protection against all types of damage. On the other hand, measurement of the ES of peripheral blood mononuclear cells from six donors revealed significant diversity between individuals. Our results illustrate the power of such a parallelized approach with high potential for several applications including the discovery of new cancer biomarkers and the screening of chemical agents modulating DNA repair systems.

Differential allelic expression in leukoblast from patients with acute myeloid leukemia suggests genetic regulation of CDA, DCK, NT5C2, NT5C3, and TP53.

mRNA expression levels of certain genes have shown predictive value for the outcome of cytarabine-treated AML-patients. We hypothesized that genetic variants play a role in the regulation of the transcription of these genes. We studied leukoblasts from 82 patients with acute myeloid leukemia and observed various extent and frequency of differential allelic expression in the CDA, DCK, NT5C2, NT5C3, and TP53 genes. Our attempts to identify the causative regulatory single nucleotide polymorphisms by a bioinformatics approach did not succeed. However, our results indicate that genetic variations are at least in part responsible for the differences in overall expression levels of these genes.

Monoclonal antibodies in clinical oncology.

Monoclonal antibodies have yet considerably modified the field of clinical oncology. The growing knowledge of key cellular pathways in tumor induction and progression, targeted therapies represent an increasing proportion of new drugs entering clinical trials. Some molecules such as trastuzumab, rituximab, alemtuzumab, cetuximab are now widely used in clinical practice. These antibodies are now tested in different indications alone or in combination with standard chemotherapy. They are also developed for the treatment of inflammatory diseases (rituximab). Numerous others antibodies are currently in pre-clinical and clinical development phases for several malignancies including renal carcinoma, melanoma, lymphomas, leukaemia, breast, ovarian and colorectal cancer. An alternative approach is to conjugate the monoclonal antibody to a toxin, a cytotoxic agent, or a radioisotope. In other cases these antibodies aim to modify the tumour microenvironment through inhibition of angiogenesis or enhancing host immune response against cancer. If the molecule targeted by the antibodies is clearly identified, most often the precise mechanism of action of these immunoglobulins is not fully understood. They can have direct effects in inducing apoptosis or programmed cell death. They can block growth factor receptors, efficiently arresting proliferation of tumor cells. Indirect effects include recruiting cells that exert cytotoxicity, such as monocytes and macrophages (ADCC). Monoclonal antibodies also bind complement, leading to toxicity known as complement dependent cytotoxicity (CDC). The side effects associated with these new treatments were in part foreseeable depending on the affected cell or function. But new or surprising side effects emerged from clinical studies. We present an overview of the monoclonal antibodies used in clinical oncology or currently in development phases. We particularly focus on recent development including new indications, clinical trial results and specific side effects of monoclonal antibodies used in the treatment of cancer.

[Translational research and Cancer Plan]. / Recherche translationnelle et plan Cancer.

The French Cancer Plan 2003-2007 has made translational research central to its research programme, to ensure the care-research continuum and the quickest application possible for the most recent discoveries, for the patients' benefit. This is a new field of research, still little-known or ill-understood. A working group, composed of physicians and researchers from academic research and industrial research, sought to define translational research in cancerology and define the issues at stake in it. Translational research needs to develop in close connection with the patients in order to enable a bi-directional flow of knowledge from cognitive research toward medical applications and from observations made on patients toward cognitive research. Placed under the aegis of the French National Cancer Institute and Leem Research, the group has put forth a strategy for implementing translational research in cancerology in France to make it attractive, competitive and efficient and to foster the development of public-private partnerships.

Heterogeneity of protein kinase C beta(2) expression in lymphoid malignancies.

AIMS: Protein kinase C (PKC) beta is an important regulator of lymphoid survival and its expression has been shown to be altered in lymphomas. The aim was to determine the expression of PKC beta(2) in various subtypes of lymphoproliferative diseases by immunohistochemistry. METHODS AND RESULTS: One hundred and forty archival samples representing various subtypes of lymphoproliferative diseases were analysed. Certain subtypes, such as mantle cell, lymphocytic or follicular lymphoma, were found to express PKC beta(2) in > 90% of the samples. In follicular lymphomas, the follicular lymphomatous areas were constantly labelled, whereas residual germinal centres remained negative. In follicular hyperplasia, PKC beta(2)+ cells were found in the mantle and marginal zones. Most angioimmunoblastic T-cell lymphomas, lymphoblastic T-cell lymphomas and marginal zone/mucosa-associated lymphoid tissue (MALT) lymphomas were labelled with anti-PKC betaII antibody, but the pattern of expression was more heterogeneous in these subtypes. A minority of diffuse large B-cell lymphomas were stained and most plasma cell malignancies were negative. None of the cases of Hodgkin's disease and anaplastic large cell lymphoma expressed PKC beta(2). CONCLUSIONS: PKC beta(2) expression varies significantly among lymphoproliferative diseases. In our series, the highest level of expression was found in mantle cell lymphomas and chronic lymphocytic lymphoma.

A20/TNFAIP3, a new estrogen-regulated gene that confers tamoxifen resistance in breast cancer cells.

The zinc-finger protein A20/TNFAIP3, an inhibitor of nuclear factor-kappaB (NF-kappaB) activation, has been shown to protect MCF-7 breast carcinoma cells from TNFalpha-induced apoptosis. As estrogen receptor (ER) status is an important parameter in the development and progression of breast cancer, we analysed the effect of 17beta-estradiol (E2) treatment on the expression of A20. We found that A20 is a new E2-regulated gene, whose expression correlates with ER expression in both cell lines and tumor samples. With the aim of investigating the impact of A20 expression on MCF-7 cells in response to ER ligands, we established stably transfected-MCF-7 cells overexpressing A20 (MCF-7-A20). These cells exhibited a phenotype of resistance to the 4-hydroxy-tamoxifen cytostatic and pro-apoptotic actions and of hyper-response to E2. Dysregulations in bax, bcl2, bak, phospho-bad, cyclin D1, cyclin E2, cyclin D2 and cyclin A2 proteins expression were shown to be related to the resistant phenotype developed by the MCF-7-A20 cells. Interestingly, we found that A20 was also overexpressed in MVLN and VP tamoxifen-resistant cell lines. Furthermore, high A20 expression levels were observed in more aggressive breast tumors (ER-negative, progesterone receptor-negative and high histological grade). These overall findings strongly suggest that A20 is a key protein involved in tamoxifen resistance, and thus represents both a new breast cancer marker and a promising target for developing new strategies to prevent the emergence of acquired mechanisms of drug resistance in breast cancer.