Skjalg Bruheim

Mechanism of chemoresistance mediated by miR-140 in human osteosarcoma and colon cancer cells

In this study, high-throughput microRNA (miRNA) expression analysis revealed that the expression of miR-140 was associated with chemosensitivity in osteosarcoma tumor xenografts. Tumor cells ectopically transfected with miR-140 were more resistant to methotrexate and 5-fluorouracil (5-FU). Overexpression of miR-140 inhibited cell proliferation in both osteosarcoma U-2 OS (wt-p53) and colon cancer HCT 116 (wt-p53) cell lines, but less so in osteosarcoma MG63 (mut-p53) and colon cancer HCT 116 (null-p53) cell lines. miR-140 induced p53 and p21 expression accompanied with G1 and G2 phase arrest only in cell lines containing wild type of p53. Histone deacetylase 4 (HDAC4) was confirmed to be one of the important targets of miR-140. The expression of endogenous miR-140 was significantly elevated in CD133+hiCD44+hi colon cancer stem-like cells that exhibit slow proliferating rate and chemoresistance. Blocking endogenous miR-140 by locked nucleic acid-modified anti-miR partially sensitized resistant colon cancer stem-like cells to 5-FU treatment. Taken together, our findings indicate that miR-140 is involved in the chemoresistance by reduced cell proliferation through G1 and G2 phase arrest mediated in part through the suppression of HDAC4. miR-140 may be a candidate target to develop novel therapeutic strategy to overcome drug resistance.

Systematic Evaluation of Three microRNA Profiling Platforms: Microarray, Beads Array, and Quantitative Real-Time PCR Array

Background A number of gene-profiling methodologies have been applied to microRNA research. The diversity of the platforms and analytical methods makes the comparison and integration of cross-platform microRNA profiling data challenging. In this study, we systematically analyze three representative microRNA profiling platforms: Locked Nucleic Acid (LNA) microarray, beads array, and TaqMan quantitative real-time PCR Low Density Array (TLDA). Methodology/Principal Findings The microRNA profiles of 40 human osteosarcoma xenograft samples were generated by LNA array, beads array, and TLDA. Results show that each of the three platforms perform similarly regarding intra-platform reproducibility or reproducibility of data within one platform while LNA array and TLDA had the best inter-platform reproducibility or reproducibility of data across platforms. The endogenous controls/probes contained in each platform have been observed for their stability under different treatments/environments; those included in TLDA have the best performance with minimal coefficients of variation. Importantly, we identify that the proper selection of normalization methods is critical for improving the inter-platform reproducibility, which is evidenced by the application of two non-linear normalization methods (loess and quantile) that substantially elevated the sensitivity and specificity of the statistical data assessment. Conclusions Each platform is relatively stable in terms of its own microRNA profiling intra-reproducibility; however, the inter-platform reproducibility among different platforms is low. More microRNA specific normalization methods are in demand for cross-platform microRNA microarray data integration and comparison, which will improve the reproducibility and consistency between platforms.

Antiproliferative activity, mechanism of action and oral antitumor activity of CP-4126, a fatty acid derivative of gemcitabine, in in vitro and in vivo tumor models

SummaryGemcitabine is a deoxycytidine (dCyd) analog with activity in leukemia and solid tumors, which requires phosphorylation by deoxycytidine kinase (dCK). Decreased membrane transport is a mechanism of resistance to gemcitabine. In order to facilitate gemcitabine uptake and prolong retention in the cell, a lipophilic pro-drug was synthesized (CP-4126), with an elaidic fatty acid esterified at the 5′position. CP-4126 was tested in cell lines resistant to cytarabine, another dCyd analog or gemcitabine. Activity of gemcitabine and the derivative was comparable in the parent cell lines, while in dCK deficient cells all compounds were inactive. However, inhibition of nucleoside transport increased the IC50 for gemcitabine up to 200-fold, but not for CP-4126, underlining the independence of a nucleoside transporter. For in vivo evaluation, nude mice bearing a human xenograft were treated intraperitoneally every third day for five doses at the maximal tolerated dose. In melanoma, sarcoma, lung, prostate, pancreatic and breast cancer xenografts, gemcitabine and CP-4126 were equally and highly effective; in four other xenografts moderately but equally active. In contrast to gemcitabine, CP-4126 could be administered orally, with a schedule and dose dependent toxicity and antitumor activity. In a colon cancer xenograft, antitumor activity of orally administered CP-4126 was equal to the intraperitoneally administered drug. In conclusion, CP-4126 is membrane transporter independent. Intraperitoneally administered CP-4126 was as effective as gemcitabine in several xenografts and CP-4126 is tolerated when orally administered. CP-4126 seems to be a promising new anticancer drug.

Gene Expression Profiles Classify Human Osteosarcoma Xenografts According to Sensitivity to Doxorubicin, Cisplatin, and Ifosfamide

Purpose: In osteosarcoma, aggressive preoperative and postoperative multidrug chemotherapy given to all patients has improved patient survival rate to the present level of ∼60%. However, no tumor marker is available that reliably can identify those patients who will or will not respond to chemotherapy. Experimental Design: In an attempt to find leads to such markers, we have obtained microarray gene expression profiles from a panel of 10 different human osteosarcoma xenografts and related the results to their sensitivity to ifosfamide, doxorubicin, and cisplatin. Results: The expression data identified genes with highly significant differential expression between poor and good responder xenografts to the three different drugs: 85 genes for doxorubicin, 74 genes for cisplatin, and 118 genes for ifosfamide. Technical validation with quantitative reverse transcription-PCR showed good correlation with the microarray expression data. Gene Ontology–guided analysis suggested that properties of the poorly responsive xenografts were resistance to undergo programmed cell death and, particularly for ifosfamide, a drive toward dedifferentiation and increased tumor aggressiveness. Leads toward metabolic alterations and involvement of mitochondrial pathways for apoptosis and stress response were more prominent for doxorubicin and cisplatin. Finally, small interfering RNA–mediated gene silencing of IER3 and S100A2 sensitized the human osteosarcoma cell line OHS to treatment with 4-hydroperoxyifosfamide. Conclusions: The expression profiles contained several novel biomarker candidates that may help predict the responsiveness of osteosarcoma to doxorubicin, cisplatin, and ifosfamide. The potential of selected candidates will be further validated on clinical specimens from osteosarcoma patients. (Clin Cancer Res 2009;15(23):7161–9)

Human osteosarcoma xenografts and their sensitivity to chemotherapy

Despite the increased survival rates of osteosarcoma patients attributed to adjuvant chemotherapy, at least one third of the patients still die due to their disease. Further improvements in the management of osteosarcoma may rely on a more individualised treatment strategy, as well as on the introduction of new drugs. To aid in the preclinical evaluation of new candidate substances against osteosarcoma, we have established 11 human osteosarcoma xenograft lines and characterised them with regard to response to five different reference drugs. Doxorubicin, cisplatin methotrexate, ifosfamide and lomustine were effective in 3/11, 3/11, 1/10, 5/11 and 4/11 of the xenografts, respectively. Five xenografts were resistant to all compounds tested. We also assessed the mRNA expression levels of the xenografts for the O6-Methylguanine DNA Methyltransferase (MGMT), DNA topoisomerase II-(Topo II)-α, Gluthathione-S-transferase (GST)-π, Multidrug-resistance related protein (MRP) 1 and Multidrug-resistance (MDR) 1 genes. There was an inverse correlation between the transcript levels of GST-π and doxorubicin growth inhibition (r= −0.66; p<0.05), and between the transcript levels of MGMT and the effect of lomustine (r= −0.72; p<0.01), whereas the expression of MRP1 and cisplatin growth inhibition was positively correlated (r=0.82; p<0.005. This panel of xenografts should constitute a good tool for pharmacological and molecular studies in osteosarcoma.

Antitumour activity of oral E7080, a novel inhibitor of multiple tyrosine kinases, in human sarcoma xenografts.

E7080 is an inhibitor of multiple tyrosine kinases, several of which have pro‐angiogenic properties, including receptors for VEGF, FGF, SCF and PDGF. We undertook our study to evaluate the preclinical activity of E7080 in human sarcomas. The antitumour activity of orally administered E7080 was tested in ten human tumour xenografts representing different sarcoma histotypes. Concomitant changes in microvessel density were assayed by immunohistochemistry to CD31. Immunohistochemistry was also used to assess the expression of kinases that E7080 is known to inhibit. The MTS assay was applied to determine effects on tumour cell viability in vitro. At the Q1D5 × 2 schedule, E7080 (30 mg/kg) was active (T/C<40%) in 7/10 xenografts. The effects were accompanied by marked decrease in microvessel densities. Given at the Q1D5 × 4 schedule, E7080 (30, 10, 3 mg/kg) showed antitumour activity in a dose dependent manner in two different xenografts. E7080 growth inhibition did not correlate with the expression of VEGFR1‐3, PDGFRA, PDGFRB, FGFR1 or KIT on tumour cells but was significantly correlated with expression of VEGFR2 on tumour microvessels. In vitro E7080 did not show potent effects on tumour cell viability in four different sarcoma cell lines, with IC50 values ≥10 μM. In conclusion, E7080 showed broad in vivo antitumour activity in sarcoma, mainly attributable to angiogenesis inhibition. E7080 was also active in xenografts resistant to one or more clinically relevant reference drugs given at MTD (doxorubicin, cisplatin or ifosfamide). The present results encourage further investigation of a potential role of E7080 in sarcoma therapy in the clinic.

Radiosynthesis and Biodistribution of a Prosthetic Group (18F-FENMA) Conjugated to Cyclic RGD Peptides

We have recently reported a new N-methylaminooxy-based prosthetic group for the site-selective introduction of ¹⁸F-fluorine under mild acidic aqueous conditions into model peptides functionalized with a Michael acceptor moiety. To further investigate the utility of this methodology, the radiosynthesis of two cyclic RGD peptides was carried out, and in vivo biodistribution and microPET studies were performed in tumor-bearing mice. A cyclic RGD peptide was functionalized with the Michael acceptors trans-β-nitrostyrene carboxylic acid and 3-vinylsulfonylpropionic acid. Radiolabeling was then performed with the prosthetic group O-(2-(2-[¹⁸F]fluoroethoxy)ethyl)-N-methylhydroxylamine (¹⁸F-FENMA) yielding the ¹⁸F-conjugates in moderate yields (8.5-12%). Biodistribution, blocking, and microPET imaging studies were performed in a mouse xenograft model. The vinylsulfonyl-modified conjugate demonstrated good in vitro plasma stability. Biodistribution and microPET studies revealed excellent tumor uptake with low background in key organs and renal elimination as the predominant route of excretion. Blocking studies with coinjected nonlabeled RGD peptide confirmed the in vivo specificity for the integrin α(v)β₃. On the other hand, ¹⁸F-FENMA-nitrostyrene-RGD, although stable at conjugation pH 5, was found to rapidly degrade at physiological pH through loss of the ¹⁸F-prosthetic group.

The novel HSP90 inhibitor NVP-AUY922 shows synergistic anti-leukemic activity with cytarabine in vivo.

HSP90 is a molecular chaperone essential for stability, activity and intracellular sorting of many proteins, including oncoproteins, such as tyrosine kinases, transcription factors and cell cycle regulatory proteins. Therefore, inhibitors of HSP90 are being investigated for their potential as anti-cancer drugs. Here we show that the HSP90 inhibitor NVP-AUY922 induced degradation of the fusion oncoprotein FOP2-FGFR1 in a human acute myeloid leukemia (AML) cell line, KG-1a. Concordantly, downstream signaling cascades, such as STAT1, STAT3 and PLCγ were abrogated. At concentrations that caused FOP2-FGFR1 degradation and signaling abrogation, NVP-AUY922 treatment caused significant cell death and inhibition of proliferation of KG-1a cells in vitro. In an animal model for AML, NVP-AUY922 administrated alone showed no anti-leukemic activity. However, when NVP-AUY922 was administered in combination with cytarabine, the two compounds showed significant synergistic anti-leukemic activity in vivo. Thus NVP-AUY922 and cytarabine combination therapy might be a prospective strategy for AML treatment.