RESUMO
BACKGROUND: Resistance to chemotherapy is common in gastroesophageal cancer. Mechanisms of resistance are incompletely characterised and there are no predictive biomarkers in clinical practice for cytotoxic drugs. We used new cell line models to characterise novel chemotherapy resistance mechanisms and validated them in tumour specimens to identify new targets and biomarkers for gastroesophageal cancer. METHODS: Cell lines were selected for resistance to oxaliplatin, cisplatin and docetaxel and gene expression examined using Affymetrix Exon 1.0 ST arrays. Leads were validated by qRT-PCR and HPLC of tumour metabolites. Protein expression and pharmacological inhibition of lead target SPHK1 was evaluated in independent cell lines, and by immunohistochemistry in gastroesophageal cancer patients. RESULTS: Genes with differential expression in drug resistant cell lines compared to the parental cell line they were derived from, were identified for each drug resistant cell line. Biological pathway analysis of these gene lists, identified over-represented pathways, and only 3 pathways - lysosome, sphingolipid metabolism and p53 signalling- were identified as over-represented in these lists for all three cytotoxic drugs investigated. The majority of genes differentially expressed in chemoresistant cell lines from these pathways, were involved in metabolism of glycosphingolipids and sphingolipids in lysosomal compartments suggesting that sphingolipids might be important mediators of cytotoxic drug resistance in gastroeosphageal cancers . On further investigation, we found that drug resistance (IC50) was correlated with increased sphingosine kinase 1(SPHK1) mRNA and also with decreased sphingosine-1-phosphate lysase 1(SGPL1) mRNA. SPHK1 and SGPL1 gene expression were inversely correlated. SPHK1:SGPL1 ratio correlated with increased cellular sphingosine-1-phosphate (S1P), and S1P correlated with drug resistance (IC50). High SPHK1 protein correlated with resistance to cisplatin (IC50) in an independent gastric cancer cell line panel and with survival of patients treated with chemotherapy prior to surgery but not in patients treated with surgery alone. Safingol a SPHK1 inhibitor, was cytotoxic as a single agent and acted synergistically with cisplatin in gastric cancer cell lines. CONCLUSION: Agents that inhibit SPHK1 or S1P could overcome cytotoxic drug resistance in gastroesophageal cancer. There are several agents in early phase human trials including Safingol that could be combined with chemotherapy or used in patients progressing after chemotherapy.
Assuntos
Aldeído Liases/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Lisofosfolipídeos/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Esfingosina/análogos & derivados , Neoplasias Gástricas/genética , Aldeído Liases/biossíntese , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Lisofosfolipídeos/biossíntese , Masculino , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , RNA Neoplásico/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Esfingosina/biossíntese , Esfingosina/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismoRESUMO
Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the most frequently occurring histological subtypes of breast cancer, accounting for 80-90% and 10-15% of the total cases, respectively. At the time of diagnosis and surgical resection of the primary tumour, most patients do not have clinical signs of metastases, but bone micrometastases may already be present. Our aim was to develop a novel preclinical ILC model of spontaneous bone micrometastasis. We used murine invasive lobular breast carcinoma cells (KEP) that were generated by targeted deletion of E-cadherin and p53 in a conditional K14cre;Cdh1((F/F));Trp53((F/F)) mouse model of de novo mammary tumour formation. After surgical resection of the growing orthotopically implanted KEP cells, distant metastases were formed. In contrast to other orthotopic breast cancer models, KEP cells readily formed skeletal metastases with minimal lung involvement. Continuous treatment with SD-208 (60 mg/kg per day), an orally available TGFß receptor I kinase inhibitor, increased the tumour growth at the primary site and increased the number of distant metastases. Furthermore, when SD-208 treatment was started after surgical resection of the orthotopic tumour, increased bone colonisation was also observed (versus vehicle). Both our in vitro and in vivo data show that SD-208 treatment reduced TGFß signalling, inhibited apoptosis, and increased proliferation. In conclusion, we have demonstrated that orthotopic implantation of murine ILC cells represent a new breast cancer model of minimal residual disease in vivo, which comprises key steps of the metastatic cascade. The cancer cells are sensitive to the anti-tumour effects of TGFß. Our in vivo model is ideally suited for functional studies and evaluation of new pharmacological intervention strategies that may target one or more steps along the metastatic cascade of events.
