ABSTRACT
BACKGROUND: Frequent failure and severe side effects of current sarcoma therapy warrants new therapeutic approaches. The small-molecule MDM2 antagonist Nutlin-3a activates the p53 pathway and efficiently induces apoptosis in tumours with amplified MDM2 gene and overexpression of MDM2 protein. However, the majority of human sarcomas have normal level of MDM2 and the therapeutic potential of MDM2 antagonists in this group is still unclear. We have investigated if Nutlin-3a could be employed to augment the response to traditional therapy and/or reduce the genotoxic burden of chemotherapy. METHODS: A panel of sarcoma cell lines with different TP53 and MDM2 status were treated with Nutlin-3a combined with Doxorubicin, Methotrexate or Cisplatin, and their combination index determined. RESULTS: Clear synergism was observed when Doxorubicin and Nutlin-3a were combined in cell lines with wild-type TP53 and amplified MDM2, or with Methotrexate in both MDM2 normal and amplified sarcoma cell lines, allowing for up to tenfold reduction of cytotoxic drug dose. Interestingly, Nutlin-3a seemed to potentiate the effect of classical drugs as Doxorubicin and Cisplatin in cell lines with mutated TP53, but inhibited the effect of Methotrexate. CONCLUSION: The use of Nutlin in combination with classical sarcoma chemotherapy shows promising preclinical potential, but since clear biomarkers are still lacking, clinical trials should be followed up with detailed tumour profiling.
Subject(s)
Antineoplastic Agents/pharmacology , Cytotoxins/pharmacology , Imidazoles/pharmacology , Piperazines/pharmacology , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Synergism , Humans , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Osteosarcomas are the most common primary malignant tumor of bone, and almost all conventional osteosarcomas are high-grade tumors with complex karyotypes. We have examined DNA copy number changes in 36 osteosarcoma tumors and 20 cell lines using microarray-based comparative genomic hybridization. The most frequent minimal recurrent regions of gain identified in the tumor samples were in 1q21.2-q21.3 (78% of the samples), 1q21.3-q22 (78%), and 8q22.1 (72%). Minimal recurrent regions in 10q22.1-q22.2 (81%), 6q16.1 (67%), 13q14.2 (67%), and 13q21.1 (67%) were most frequently lost. A small region in 3q13.31 (2.1 Mb) containing the gene limbic system-associated membrane protein (LSAMP) was frequently deleted (56%). LSAMP has previously been reported to be a candidate tumor suppressor gene in other cancer types. The deletion was validated using fluorescence in situ hybridization, and the expression level and promoter methylation status of LSAMP were investigated using quantitative real-time reverse transcription PCR and methylation-specific PCR, respectively. LSAMP showed low expression compared to two normal bone samples in 6/15 tumors and 5/9 cell lines with deletion of 3q13.31, and also in 5/14 tumors and 3/11 cell lines with normal copy number or gain. Partial or full methylation of the investigated CpG island was identified in 3/30 tumors and 7/20 cell lines. Statistical analyses revealed that loss of 11p15.4-p15.3 and low expression of LSAMP (both P = 0.011) were significantly associated with poor survival. Our results show that LSAMP is a novel candidate tumor suppressor gene in osteosarcomas.
Subject(s)
Bone Neoplasms/genetics , Cell Adhesion Molecules, Neuronal/genetics , Comparative Genomic Hybridization/methods , Genes, Tumor Suppressor , Oligonucleotide Array Sequence Analysis/methods , Osteosarcoma/genetics , Adolescent , Adult , Aged , Bone Neoplasms/metabolism , Cell Adhesion Molecules, Neuronal/metabolism , Cell Line, Tumor , Child , Chromosome Aberrations , Cluster Analysis , CpG Islands/genetics , DNA Methylation , Data Interpretation, Statistical , Female , GPI-Linked Proteins , Gene Dosage , Gene Expression Profiling , Humans , In Situ Hybridization, Fluorescence , Kaplan-Meier Estimate , Male , Middle Aged , Osteosarcoma/metabolism , Reproducibility of Results , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The MDM2-antagonist Nutlin 3A can efficiently induce apoptosis in osteosarcoma cell lines with amplified MDM2. However, Nutlin-based therapy could be even more important in more common sarcoma types where this aberration is frequent. The well- and de-differentiated liposarcomas have complex marker chromosomes, consistently including multiple copies of the MDM2 locus. Since amplification seems to be a primary aberration in these tumors, whereas amplification in osteosarcoma generally is a progression marker, the underlying biological mechanisms may be different. We have therefore investigated the molecular response to Nutlin treatment in several liposarcoma cell lines with such markers, as well as a panel of other sarcoma cell lines. We report that Nutlin efficiently stabilized p53 and induced downstream p53 dependent transcription and apoptosis in liposarcoma cells with amplified MDM2 in vitro. Some effect of Nutlin was also observed on cell lines without amplified MDM2 but with wt TP53, but no apoptosis was induced. The MDM4 protein, reported to interfere with the reactivation of p53, was undetectable in cells with amplified MDM2. Thus, Nutlin represents a promising new therapeutic principle for the treatment of an increasing group of sarcomas.
