Background: LDH (lactate dehydrogenase), AFP (alpha-fetoprotein) and β-HCG (human chorionic gonadotropin) are used in diagnosis and follow-up of testicular germ cell cancer (TGCC) patients. Our aim was to investigate the association between levels of miR-371a-3p, miR-373-3p and miR-367-3p and clinical features in metastatic TGCC. Methods: relative levels of miR-371a-3p, miR-373-3p and miR-367-3p were evaluated in serum of metastatic TGCC patients. A prospectively included and a retrospectively selected cohort were studied (total patient number = 109). Blood samples were drawn at start of chemotherapy and during follow-up. Serum microRNA (miR) levels were determined using the ampTSmiR test. Results: at start of chemotherapy, miR-371a-3p, miR-373-3p and miR-367-3p levels were positively correlated to LDH. The median level of these miRs was higher in patients who developed a relapse after complete biochemical remission (n = 34) than in those who had complete durable remission (n = 60). Higher levels of miR-367-3p were found in patients with refractory disease (n = 15) compared to those who had complete response. miR levels decreased during the first week of chemotherapy in patients with complete response and stayed below threshold after one year of treatment. Conclusion: high miR levels at start of chemotherapy are associated with worse clinical outcome and can assist in early diagnosing of relapses.
Testicular cancer is the most common cancer type among young men. Despite highly effective cisplatin-based chemotherapy, around 20% of patients with metastatic disease will still die from the disease. The aim of this study was to explore the use of kinase inhibitors to sensitize testicular cancer cells to cisplatin treatment. Activation of kinases, including receptor tyrosine kinases and downstream substrates, was studied in five cisplatin-sensitive or-resistant testicular cancer cell lines using phospho-kinase arrays and Western blotting. The phospho-kinase array showed AKT and S6 to be among the top phosphorylated proteins in testicular cancer cells, which are part of the PI3K/AKT/mTORC pathway. Inhibitors of most active kinases in the PI3K/AKT/mTORC pathway were tested using apoptosis assays and survival assays. Two mTORC1/2 inhibitors, AZD8055 and MLN0128, strongly enhanced cisplatin-induced apoptosis in all tested testicular cancer cell lines. Inhibition of mTORC1/2 blocked phosphorylation of the mTORC downstream proteins S6 and 4E-BP1. Combined treatment with AZD8055 and cisplatin led to reduced clonogenic survival of testicular cancer cells. Two testicular cancer patient-derived xenografts (PDX), either from a chemosensitive or-resistant patient, were treated with cisplatin in the absence or presence of kinase inhibitor. Combined AZD8055 and cisplatin treatment resulted in effective mTORC1/2 inhibition, increased caspase-3 activity, and enhanced tumor growth inhibition. In conclusion, we identified mTORC1/2 inhibition as an effective strategy to sensitize testicular cancer cell lines and PDX models to cisplatin treatment. Our results warrant further investigation of this combination therapy in the treatment of patients with testicular cancer with high-risk relapsed or refractory disease.
Metastatic testicular cancer (TC) is highly sensitive to cisplatin-based chemotherapy. However, patients with advanced disease in the poor prognosis group only have a 50% 5-year survival resulting from chemoresistance. Previous data showed that both the PI3K/Akt pathway and the MDM2/p53 axis are involved in cisplatin resistance of TC cells. In this study we aim to establish and characterize TC patient-derived xenografts (PDX) in order to investigate PI3K/Akt and/or MDM2 inhibition as possible treatment options for TC. Excised and minced (8mm3) primary TC tissue was implanted subcutaneously into male NSG mice to obtain a first generation PDX model (F1). Animals were sacrificed when tumor volume exceeded 1500 mm3. The tumor was harvested and minced. Small pieces (8mm3) were implanted in a second generation (F2), as well as stored in FCS-5% DMSO to establish a biobank. In a panel of TC cell lines, combination treatment of Akt inhibition with or without cisplatin or Nutlin-3a was performed. Cleaved caspase-3 staining was assessed to measure apoptosis, and western blot was used to determine the effect of different treatments on sub-cellular localization and phosphorylation status of MDM2. We have established 3 TC PDX models. Histology of the primary tumors included the following subtypes: mixed germ cell tumors with embryonal- and yolk sac carcinoma and teratoma components. Engraftment efficacy was 100% and tumor growth initiated within 4-5 weeks after implantation. Interestingly, we successfully implanted a biopsy taken from a metastatic lesion of a patient presenting with progressive refractory disease after receiving standard chemotherapy. Histology of the different PDX generations was comparable to the patient material, although a loss of the teratoma component was observed. In our cell line panel, Akt inhibition using MK2206 sensitized the acquired cisplatin resistant cell line TeraCP towards cisplatin treatment, whereas an additive effect was observed in other cell lines. Combination of Akt and MDM2 inhibition was highly synergistic in apoptosis induction in all cell lines. No relation was observed between the synergistic effect of Akt combined with MDM2 inhibition and sub-cellular localization or phosphorylation levels of MDM2. Taken together, we have successfully established 3 TC PDX models, including a chemo-resistant model. The biobank is currently being expanded. Combined Akt and MDM2 inhibition resulted in synergistic induction of apoptosis, and these combinations or other novel therapies will be tested in established TC PDX models. Supported by Dutch Cancer Society grant RUG 2014-6691 and CONACYT grant 381543 Citation Format: Gerda de Vries, Ximena Rosas-Plaza, Marcel A. van Vugt, Albert J.H. Suurmeijer, Jourik A. Gietema, Steven de Jong. Development of testicular cancer patient derived xenograft models to test combination therapies targeting PI3K/Akt and MDM2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3861. doi:10.1158/1538-7445.AM2017-3861
Testicular cancer (TC) is the most common solid tumour in young men. While cisplatin-based chemotherapy is highly effective in TC patients, chemoresistance still accounts for 10% of disease-related deaths. Pre-clinical models that faithfully reflect patient tumours are needed to assist in target discovery and drug development. Tumour pieces from eight TC patients were subcutaneously implanted in NOD scid gamma (NSG) mice. Three patient-derived xenograft (PDX) models of TC, including one chemoresistant model, were established containing yolk sac tumour and teratoma components. PDX models and corresponding patient tumours were characterised by H&E, Ki-67 and cyclophilin A immunohistochemistry, showing retention of histological subtypes over several passages. Whole-exome sequencing, copy number variation analysis and RNA-sequencing was performed on these TP53 wild type PDX tumours to assess the effects of passaging, showing high concordance of molecular features between passages. Cisplatin sensitivity of PDX models corresponded with patients’ response to cisplatin-based chemotherapy. MDM2 and mTORC1/2 targeted drugs showed efficacy in the cisplatin sensitive PDX models. In conclusion, we describe three PDX models faithfully reflecting chemosensitivity of TC patients. These models can be used for mechanistic studies and pre-clinical validation of novel therapeutic strategies in testicular cancer.
<p>mTORC1/2 downstream pathway evaluation in Tera cells</p>
<p>mTORC1/2 inhibition in combination with cisplatin in TC PDX models</p>
<p>PDGFRbeta protein expression in TC cell lines</p>
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