Angelika M. Burger

Comparison of 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17DMAG) and 17-allylamino-17-demethoxygeldanamycin (17AAG) in vitro: effects on Hsp90 and client proteins in melanoma models

The heat shock protein Hsp90 is a potential target for drug discovery of novel anticancer agents. By affecting this protein, several cell signaling pathways may be simultaneously modulated. The geldanamycin analog 17AAG has been shown to inhibit Hsp90 and associated proteins. Its clinical use, however, is hampered by poor solubility and thus, difficulties in formulation. Therefore, a water-soluble derivative was desirable and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17DMAG) is such a derivative. Studies were carried out in order to evaluate the activity and molecular mechanism(s) of 17DMAG in comparison with those of 17-allylamino-demethoxygeldanamycin (17AAG). 17DMAG was found to be more potent than 17AAG in a panel of 64 different patient-derived tumor explants studied in vitro in the clonogenic assay. The tumor types that responded best included mammary cancers (six of eight), head and neck cancers (two of two), sarcomas (four of four), pancreas carcinoma (two of three), colon tumors (four of eight for 17AAG and six of eight for 17DMAG), and melanoma (two of seven). Bioinformatic comparisons suggested that, while 17AAG and 17DMAG are likely to share the same mode(s) of action, there was very little similarity with standard anticancer agents. Using three permanent human melanoma cell lines with differing sensitivities to 17AAG and 17DMAG (MEXF 276L, MEXF 462NL and MEXF 514L), we found that Hsp90 protein was reduced following treatment at a concentration associated with total growth inhibition. The latter occurred in MEXF 276L cells only, which are most sensitive to both compounds. The depletion of Hsp90 was more pronounced in cells exposed to 17DMAG than in those treated with 17AAG. The reduction in Hsp90 was associated with the expression of erbB2 and erbB3 in MEXF 276L, while erbB2 and erbB3 were absent in the more resistant MEXF 462NL and MEXF 514L cells. Levels of known Hsp90 client proteins such as phosphorylated AKT followed by AKT, cyclin D1 preceding cdk4, and craf-1 declined as a result of drug treatment in all three melanoma cell lines. However, the duration of drug exposure needed to achieve these effects was variable. All cell lines showed increased expression of Hsp70 and activated cleavage of PARP. No change in PI3K expression was observed and all melanoma cells were found to harbor the activating V599E BRAF kinase mutation. The results of our in vitro studies are consistent with both 17AAG and 17DMAG acting via the same molecular mechanism, i.e. by modulating Hsp90 function. Since 17DMAG can be formulated in physiological aqueous solutions, the data reported here strongly support the development of 17DMAG as a more pharmaceutically practicable congener of 17AAG.

Down-regulation of T1A12/mac25, a novel insulin-like growth factor binding protein related gene, is associated with disease progression in breast carcinomas.

To define genes that are essential to the initiation and progression of breast cancer we utilized subtractive hybridization and differential display cloning techniques and isolated over 950 cDNAs from breast cell-lines derived from matched normal and tumor tissue. Of these, 102 cDNAs were characterized by DNA sequencing and Northern blot analysis. GenBank searches showed that one of these genes, T1A12 is identical to mac25, an insulin-like growth factor-binding protein related gene. Antibodies generated against the C-terminal region of the T1A12/mac25 protein were used to investigate its expression in 60 primary breast tissues. Sections of 12 benign, 16 ductal carcinoma in situ and 32 infiltrating ductal carcinoma specimens were examined. Strong immunoperoxidase staining was observed in luminal epithelial cells of normal lobules and ducts, in apocrine cells of cysts and fibroadenomas. Moderate to weak protein expression was found in hyperplastic and DCIS cells, but no specific staining was detected in invasive carcinoma cells. FISH mapping using a PAC clone localized the T1A12/mac25 gene to 4q12-13. Microsatellite length polymorphism was studied using markers for 4q in paired normal and tumor breast tissues. Thirty-three per cent (10/30) of the samples were found to be polymorphic with D4S189 and D4S231 microsatellite markers and LOH was detected in 50% (5/10) of these informative samples. Our data indicate that T1A12/mac25 expression is abrogated during breast cancer progression concomitant with loss of heterozygosity on chromosome 4q. T1A12/mac25 may therefore have a tumor suppressor-like function and its expression could indicate a disease with a more favorable status, having a better prognosis.

Differential inhibition of tumor angiogenesis by tie2 and vascular endothelial growth factor receptor-2 dominant-negative receptor mutants

Tumor growth is angiogenesis‐dependent. Current evidence suggests that vascular endothelial growth factor (VEGF), a major regulator of embryonic and hypoxia‐mediated angiogenesis, is necessary for tumor angiogenesis. VEGF is expressed in tumor cells in vivo, and its tyrosine kinase receptors VEGFR‐1 and VEGFR‐2 are up‐regulated in the tumor endothelium. A second endothelial cell–specific ligand/receptor tyrosine kinase system, consisting of the tie2 receptor, its activating ligand angiopoietin‐1 and the inhibitory ligand angiopoietin‐2, has been characterized. We have examined 6 human primary breast‐cancer samples and 4 murine breast‐cancer cell lines (M6363, M6378, M6444, M6468), transplanted into nude mice, by in situ hybridization and/or Northern analysis. Expression of angiopoietin‐1, angiopoietin‐2 and tie2 was compared to VEGF and VEGFR‐2 expression. Human tumors expressed VEGFR‐2 and tie2 but varied considerably in VEGF and angiopoietin‐1/‐2 expression. In the murine tumor models, we observed high heterogeneity of receptor and ligand expression. M6363 and M6378 tumors were analyzed in detail because they showed different expression of components of the tie2/angiopoietin signaling system. M6363 tumors expressed VEGF, VEGFR‐2 and angiopoietin‐2 but not tie2 or angiopoietin‐1, suggesting activation of VEGFR‐2 and inhibition of tie2 signaling pathways, whereas M6378 tumors expressed VEGF, VEGFR‐2, tie2 and angiopoietin‐1 but little angiopoietin‐2, suggesting activation of both VEGFR‐2 and tie2 signaling pathways. In vivo studies using truncated dominant‐negative tie2 and VEGFR‐2 mutants revealed inhibition of M6363 tumor growth by 15% (truncated tie2) and 36% (truncated VEGFR‐2), respectively. In contrast, M6378 tumor growth was inhibited by 57% (truncated tie2) and 47% (truncated VEGFR‐2), respectively. These findings support the hypothesis that tumor angiogenesis is dependent on VEGFR‐2 but suggest that, in addition, tie2‐dependent pathways of tumor angiogenesis may exist. For adequate application of angiogenesis inhibitors in tumor patients, analysis of prevailing angiogenesis pathways may be a prerequisite.

Pre-clinical evaluation of a methotrexate–albumin conjugate (MTX-HSA) in human tumor xenografts in vivo†

Methotrexate covalently bound to human serum albumin in a 1:1 molar ratio (MTX‐HSA) is a new macromolecular drug which is currently being studied in phase I clinical trials by the German Association for Medical Oncology (AIO) Phase I/II study group. Previous studies have shown that MTX‐HSA differs favorably from unbound MTX in terms of plasma half‐life time, tumor accumulation of albumin and uptake mechanisms into cancer cells. To achieve optimal drug efficacy, repeated treatment cycles were necessary. To evaluate the anti‐tumor activity of MTX‐HSA and MTX in pre‐clinical in vivo models, we selected 7 solid human tumor xenografts growing s.c. in nude mice and administered drug either i.p. or i.v. weekly for 3 weeks. The maximal tolerated dose (MTD) of MTX‐HSA in nude mice was 12.5 mg/kg given i.p. on days 1, 8 and 15, whereas the MTD for free MTX was 100 mg/kg given i.v. MTX‐HSA was significantly more active (p > 0.01) than MTX in 3 models. In the soft tissue sarcoma SXF 1301, MTX‐HSA effected complete remission/cure after a single injection, whereas free MTX resulted in short‐lasting, partial tumor regression. In the prostate‐cancer model PRXF PC3M, MTX‐HSA produced growth inhibition of 92.8% of control or an optimal test/control (T/C) of 7.2% compared to a T/C of 20.8% for MTX (p = 0.05). In the osteosarcoma model SXF 1410, optimal T/C values were 10.2% and 14.5%, respectively (p = 0.025). In lung cancers LXFE 409 and LXFL 529, bladder cancer BXF 1258 and breast cancer MAXF 449, both compounds were inactive. The improved therapeutic effects seen in 3 xenograft models under MTX‐HSA treatment are promising and might be due to specific accumulation of the compound in solid tumors owing to their enhanced permeability and retention effect. Thus, clinical development of MTX‐HSA will continue and sarcomas as well as prostate cancers will be included as potential target tumors for upcoming clinical phase II trials.

Genotyping of NAD(P)H:quinone oxidoreductase (NQO1) in a panel of human tumor xenografts: relationship between genotype status, NQO1 activity and the response of xenografts to mitomycin C chemotherapy in vivo

Pharmacogenetic analysis of polymorphisms in drug metabolizing enzymes is currently generating considerable interest as a means of individualizing patient therapy. Recent studies have suggested that patients that are homozygous for a polymorphic variant (a C to T transition at position 609 of the cDNA sequence) of the enzyme NAD(P)H:quinone oxidoreductase (NQO1) may be resistant to Mitomycin C (MMC). Genotyping of a panel of 54 human tumor xenografts by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), classified tumors as wild type (40/54), heterozygotes (11/54), and homozygous mutants (3/54). Previously, 37 of these tumors had been characterized in terms of their response to MMC in vivo, and in this study, a further nine tumor xenografts have been characterized in terms of their response to MMC. No correlation could be found between the NQO1 polymorphic status of xenografts and their response to MMC in vivo. In terms of genotype/phenotype relationships, NQO1 activity in tumors genotyped as wild type, heterozygotes, and homozygous mutants were 311.1 +/- 421.9 (N = 40), 76.9 +/- 109.5 (N = 11), and 0.2 +/- 0.17 (N = 3) nmol/min/mg, respectively. Genotyping of patients may provide a useful initial step in identifying patients who are unlikely to benefit from quinone-based chemotherapy. In the case of MMC, however, the work presented here demonstrates that genotyping of individuals with respect to NQO1 is unlikely to be beneficial in terms of predicting tumor responses to MMC.

The differentiation status of primary gonadal germ cell tumors correlates inversely with telomerase activity and the expression level of the gene encoding the catalytic subunit of telomerase

BackgroundThe activity of the ribonucleoprotein enzyme telomerase is detectable in germ, stem and tumor cells. One major component of telomerase is human telomerase reverse transcriptase (hTERT), which encodes the catalytic subunit of telomerase. Here we investigate the correlation of telomerase activity and hTERT gene expression and the differentiation status of primary testicular germ cell tumors (TGCT).MethodsTelomerase activity (TA) was detected by a quantitative telomerase PCR ELISA, and hTERT mRNA expression was quantified by online RT-PCR in 42 primary testicular germ cell tumors. The control group consisted of benign testicular biopsies from infertile patients.ResultsHigh levels of telomerase activity and hTERT expression were detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content. In contrast, differentiated teratomas and testicular control tissue without germ cells (Sertoli-cell-only syndrome) showed no telomerase activity and only minimal hTERT expression.ConclusionsThese findings demonstrate an inverse relationship between the level of telomerase activity and hTERT mRNA expression and the differentiation state of germ cell tumors. Quantification of telomerase activity and hTERT mRNA expression enables a new molecular-diagnostic subclassification of germ cell tumors that describes their proliferation potential and differentiation status.

Telomerase in Cancer Diagnosis and Therapy

Curing cancers is one of the most challenging tasks of modern medicine. The major problem is the heterogeneity of human tumours and thus finding a ‘universal’ target for cancer treatment. The discovery that the expression of the enzyme telomerase is a hallmark of immortality and cancer, and that it is found in the majority (>85%) of human tumours but is repressed in most normal cells, has therefore caused considerable excitement. These observations led to the design of potential telomerase inhibitors and ideas about targeting telomerase in the clinic. To date, several classes of telomerase inhibitory agents have been identified and are in preclinical development. However, the approach has not yet been tested clinically.Because of the proposed function of telomerase, and the understanding that replicative cell senescence or cell death result from progressive telomere shortening during successive cell divisions, even complete enzyme inhibition will not produce immediate cell death. Designing clinical trials for promising telomerase inhibitors requires consideration of the novel mechanism of action of these drugs. A lag period between initiation of treatment and occurrence of effects is likely, and thus anti-telomerase therapy might best be given in adjuvant treatment protocols after initial tumour debulking therapy and in combination with other cytostatic agents.The available knowledge of telomerase biology and its association with human tumours suggests that telomerase inhibition might prove a valuable addition to current cancer treatment regimens.

The standardized mistletoe preparation Lektinol has antitumoral potencies

Extracts of Viscum albumL have been used for decades for non-specific stimulation of the immune system in cancer therapy. Mistletoe lectins have been identified as the active components, with cytotoxic and immunomodulatory activities. New experimental data demonstrate that the special extract preparation Lektinol® (Madaus AG, Cologne, Germany), standardized for bioactive mistletoe lectin (ML), has antitumoral potencies in vitro and in animal tumor models. In vitro studies on human tumor cell lines and xenografts showed Lektinol to be highly cytotoxic (ie toward breast, lung, prostate and renal cell cancers). The in vivo antitumoral effects of Lektinol were examined in different subcutaneously growing murine neoplasms following repeated intraperitoneal treatment of 0.3-3-30-300 ng ML/kg. Marked tumor growth inhibition was observed with Renca renal carcinoma, C8 colon 38, and F9 testicular teratoma. The antimetastatic effects of Lektinol were investigated in the B16 melanoma model in mice. Following a single intravenous injection of the melanoma cells, the daily treatment with 3-30-150 ng ML/kg significantly reduced the formation of lung metastases. In parallel, Lektinol enhanced several immune parameters (ie the number of MAC-1+ mononuclear cells and CD4+8+ thymocytes in the tumor-bearing animals). In a further study, the effects of locally administered Lektinol were evaluated in the MB49 urinary bladder carcinoma model in mice. After a single instillation of the tumor cells, Lektinol was given repeatedly by intravesical administration of 3-30 ng ML/0.1 ml/animal. Lektinol showed a distinct effect on survival ratio, growth of primary bladder tumors and the formation of multiple metastases.