University of Groningen
Mechanistic and translational studies to improve cisplatin sensitivity of testicular cancer
de Vries, Gerda
DOI:
10.33612/diss.135496604
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Publication date: 2020
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de Vries, G. (2020). Mechanistic and translational studies to improve cisplatin sensitivity of testicular cancer. https://doi.org/10.33612/diss.135496604
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CHAPTER 1
General introduction
General Introduction
GENERAL INTRODUCTION
Testicular cancer (TC) is a rare cancer accounting for approximately 1% of all cancers in men1.
In the Netherlands, approximately 800-850 patients are diagnosed with testicular cancer each year2. While these numbers are low, testicular cancer is the most common solid tumor affecting
young men between 20-40 years of age3. Over 95% of testicular cancers are germ cell tumors4.
Germ cell tumors are mostly found in the reproductive organs, the testes or ovaries. Other tumors of the testes include tumors arising from stromal tissue, including Leydig cell tumors and Sertoli cell tumors and lymphoma. Testicular germ cell tumors can be divided in two classes, seminomas and non- seminomas, each accounting for about 50%. The studies presented in this thesis concern non- seminomas, which are a heterogeneous group of tumors with varying stages of differentiation including embryonal carcinoma, yolk-sac carcinoma, choriocarcinoma and teratoma. A mixed histology of these subtypes is common, and even seminoma components may be present in mixed non-seminoma tumors.
Overall cure rates of TC are high, even for metastatic patients, which is the result from the intrinsically high sensitivity of TC cells to cisplatin-based chemotherapy. Five-year survival of non- seminoma patients is around 90%5. Patients with metastatic TC however have different survival
outcomes depending on the tumor type and prognosis group. Current risk stratification of TC patients was established in 1997 by the International Germ Cell Consensus Classification (IGCCC), which is a prognostic staging system for patients with metastatic disease6. The IGCCC classification
stratifies patients into good, intermediate and poor prognosis groups based on levels of different tumor biomarkers and clinical features such as the location of the metastasis. Five year survival rate for the good prognosis group is 92%, for intermediate prognosis TC patients 80%, and for the poor prognosis group 48% respectively6.
Treatment of patients with testicular cancer depends on multiple factors such as disease stage, tumor subtype and whether the tumor has metastasized. All patients undergo surgical removal of the affected testicle. For patients with disease confined to the testicle this is most often followed by surveillance or one course of adjuvant chemotherapy. In case of tumor spreading to lymph nodes and/or other regions, additional treatment with chemotherapy is instituted. This treatment strategy is highly efficient, curing more than 90% of patients4. A small subset of patients however,
has refractory disease or experience disease relapse. Besides high dose chemotherapy, no alternative treatment options are available.
The most important chemotherapeutic drug used in treatment of TC patients is cisplatin. When cisplatin is taken up by cells, it interacts with DNA leading to different forms of cisplatin-DNA adducts. These are mainly intrastrand cross-links (>90%), but also interstrand cross-links, protein- DNA cross-links and DNA monoadducts are formed7,8. Cisplatin-DNA cross-links interfere with
DNA replication, which results in the formation of DNA double stranded breaks (DSBs). In response to DNA DSBs, DNA repair is initiated, in conjunction with activation of the DNA damage response (DDR), followed by p53 activation and induction of cell cycle arrest or apoptosis. In TC,
activation of the DDR leads to rapid induction of apoptosis with a major role for wild type p53. Cisplatin treatment results in enhanced expression of the FAS death receptor, a transcriptional target of p53, and subsequently activation of the extrinsic apoptosis pathway via the interaction between FAS and FAS ligand9,10. In addition, the intrinsic apoptosis pathway is activated upon
cisplatin treatment by p53-mediated transcription of pro-apoptotic proteins PUMA and NOXA11– 13. Crosstalk between the extrinsic and intrinsic apoptosis pathways further strengthens the
apoptotic response. FAS receptor activation results in caspase-8 mediated cleavage of BID which in turn activates the intrinsic apoptosis pathway14.
While TC is in general a curable disease, around 10% of patients have refractory disease or relapse after initial treatment4. Several mechanisms underlying cisplatin resistance have been identified,
suggesting no uniform cause of resistance. These resistance mechanisms include hyperactivation of the PI3K/AKT pathway driven by several receptor tyrosine kinases15–19, and suppression of
p53 activity by its negative regulator MDM210,20. Despite research into mechanisms of cisplatin
resistance and the discovery of novel druggable targets, no alternative treatments have made it to the clinic.
Preclinical testing of new therapeutic agents or combination strategies in testicular cancer is mainly performed in cell lines or cell line xenograft models. While cell lines can be great tools for target discovery and mechanistic studies, they also have important limitations when it comes to preclinical drug testing. For TC, the number of cell lines available is limited, with around 20 cell lines described in literature21,22. In addition, not all TC subtypes are well represented in these cell
line models, because most of them are of the EC subtype. Another limitation of using cell line models, especially in the context of TC, is the lack of tumor heterogeneity as most non-seminoma tumors are mixed tumors, a characteristic that will not be recapitulated by cell lines. Pre-clinical models that faithfully reflect the patient tumor are needed to assist in target discovery and development of more active drugs. To overcome the limitations presented by cell line models and cell line-based xenografts, much preclinical research is now being performed using patient derived xenograft (PDX) models. Advantages of PDX models include the histological preservation of the tumor when serially transplanted in different generations of mice, and the molecular resemblance to the original tumor looking at genomic features and expression levels23–28. For TC
there are only 30 established PDX models29,30, and only one systematic report is available of their
establishment31.
AIM OF THE THESIS
The studies presented in this thesis are aimed at discovering better systemic treatment options for patients with metastatic testicular cancer suffering from cisplatin resistant disease. Resistance mechanisms and novel combination therapies are discussed, as well as the development of an in vivo TC PDX model, used for pre-clinical testing of the novel combination therapies.
THESIS OUTLINE
In chapter 2 we provide an overview of current topics in TC. A general introduction on TC, including current treatment strategies, and a summary of mechanisms of cisplatin sensitivity and resistance are given. Genetic alterations as well novel therapeutics in clinical development of TC are described. Finally, pre-clinical TC models are described that can be used to study resistance mechanisms and pre-clinical drug efficacy.
Previously, it was described that PI3K or AKT inhibition sensitized cisplatin-resistant TC cells to cisplatin32. Furthermore, small molecule inhibitors of MDM2 like nutlin-3a, targeting the interaction
between MDM2 and p53, have been shown to sensitize TC cells to cisplatin treatment10. In chapter
3 and 4 of this thesis we investigate the potential of novel combinatorial treatment approaches for TC patients not responding to cisplatin-containing chemotherapy. Because most TC patients are young adults when undergoing chemotherapy, they are at risk of developing late side effects. These treatment related toxicities are an important issue for testicular cancer survivors33. Using
low-dose chemotherapy, or even eliminating chemotherapy from the treatment regimen would be an attractive alternative in reducing long-term treatment side effects. Hyperactivity of the PI3K/AKT/mTOR pathway is identified in chapter 3. To that end, several kinase inhibitors of the PI3K/AKT/mTOR pathway are screened for their potential to sensitize testicular cancer cell lines to cisplatin-induced apoptosis. The most effective kinase inhibitor is selected and further evaluated using two TC PDX models. In chapter 4 we focus on the MDM2/p53 axis, which was previously identified to be involved in cisplatin resistance10. Here we investigate two combinatorial
treatment approaches, one combination consists of an MDM2 inhibitor with cisplatin, and the second is a combination of an MDM2 inhibitor with an mTORC1/2 inhibitor in an attempt to find a therapeutic strategy that does not include a classic chemotherapeutic agent. Mechanistically, we have investigated whether mTOR inhibition affects transcription or protein stability of p53 and transcriptional targets MDM2 and p21. In addition, p53 transcriptional activity after MDM2 inhibition or cisplatin treatment is investigated. Finally, treatment efficacy of both combination treatments is tested in two of our PDX models.
In chapter 5 we describe the development and characterization of testicular cancer patient derived xenograft (PDX) models. Patient tumor samples were collected between March 2016 and March 2019 and implanted subcutaneously in immunodeficient mice. A total number of eight tumor samples was implanted, out of which we have successfully established three PDX models. These three models are characterized at the protein and molecular level for three subsequent passages. Finally, sensitivity of these models to cisplatin and two experimental targeted drugs is evaluated.
One mechanism of cisplatin resistance has previously been attributed to increased expression of p21 in cisplatin resistant TC cell lines32. In chapter 6 we assess the involvement of p21 in
cisplatin resistance. Several genetic methods to modulate p21 levels are used to measure the effect on cisplatin-induced apoptosis. With these cell line models the effect of absent, reduced
or overexpressed p21 levels on response to cisplatin is determined using apoptosis and survival assays, and cell cycle analysis.
Finally, a summary of the studies presented in this thesis can be found in chapter 7, including implications, discussions and future directions arising from this thesis.
REFERENCES
Ferlay, J. et al. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int. J. Cancer 136, E359–E386 (2015).
Integraal Kankercentrum Nederland. Nederlandse Kankerregistratie [Internet]. Available at: https://www. cijfersoverkanker.nl/. (Accessed: 21st August 2019)
Rajpert-De Meyts, E., McGlynn, K. A., Okamoto, K., Jewett, M. A. S. & Bokemeyer, C. Testicular germ cell tumours. Lancet 387, 1762–1774 (2016).
Adra, N. & Einhorn, L. H. Testicular Cancer Update. Clin. Adv. Hematol. Oncol. 15, 386–396 (2017). Ylönen, O., Jyrkkiö, S., Pukkala, E., Syvänen, K. & Boström, P. J. Time trends and occupational variation in the incidence of testicular cancer in the Nordic countries. BJU Int. 122, 384–393 (2018).
Mead, G. M. & Stenning, S. P. The international germ cell consensus classification: A new prognostic factor-based staging classification for metastatic germ cell tumours. Clin. Oncol. 9, 207–209 (1997). Eastman, A. Characterization of the adducts produced in DNA by cis- diamminedichloroplatinum(II) and cis-dichloro(ethylenediamine)platinum(II). Biochemistry 22, 3927–3933 (1983).
Yang, D. & Wang, A. H.-J. Structural studies of interactions between anticancer platinum drugs and DNA. Prog. Biophys. Mol. Biol. 66, 81–111 (1996).
Spierings, D., De Vries, E., Vellenga, E. & De Jong, S. Loss of drug-induced activation of the CD95 apoptotic pathway in a cisplatin-resistant testicular germ cell tumor cell line. Cell Death Differ. 10, 808–822 (2003). Koster, R., Timmer-Bosscha, H., Bischoff, R., Gietema, J. A. & de Jong, S. Disruption of the MDM2–p53 interaction strongly potentiates p53-dependent apoptosis in cisplatin-resistant human testicular carcinoma cells via the Fas/FasL pathway. Cell Death Dis. 2, e148 (2011).
Mueller, T. et al. Failure of activation of caspase-9 induces a higher threshold for apoptosis and cisplatin resistance in testicular cancer. Cancer Res. 63, 513–521 (2003).
Gutekunst, M. et al. p53 hypersensitivity is the predominant mechanism of the unique responsiveness of testicular germ cell tumor (TGCT) cells to cisplatin. PLoS One 6, e19198 (2011).
Gutekunst, M. et al. Cisplatin hypersensitivity of testicular germ cell tumors is determined by high constitutive Noxa levels mediated by Oct-4. Cancer Res. 73, 1460–1469 (2013).
Green, D. R. & Llambi, F. Cell Death Signaling. Cold Spring Harb. Perspect. Biol. 7, a006080 (2015). Di Vizio, D. et al. Loss of the tumor suppressor gene PTEN marks the transition from intratubular germ cell neoplasias (ITGCN) to invasive germ cell tumors. Oncogene 24, 1882– 1894 (2005).
Chen, K. S. et al. EGF receptor and mTORC1 are novel therapeutic targets in nonseminomatous germ cell tumors. Mol. Cancer Ther. 17, 1079–1089 (2018).
Juliachs, M. et al. The PDGFRβ–AKT pathway contributes to CDDP-acquired resistance in testicular germ cell tumors. Clin. Cancer Res. 20, 658–667 (2014).
Juliachs, M. et al. Effectivity of pazopanib treatment in orthotopic models of human testicular germ cell tumors. BMC Cancer 13, 382 (2013).
Selfe, J. et al. IGF1R signalling in testicular germ cell tumour cells impacts on cell survival and acquired cisplatin resistance. J. Pathol. 244, 242–253 (2018).
Bauer, S. et al. Therapeutic potential of Mdm2 inhibition in malignant germ cell tumours. Eur. Urol. 57, 679–687 (2010).
Rahman, N. A. & Huhtaniemi, I. T. Testicular cell lines. Mol. Cell. Endocrinol. 228, 53–65 (2004).
Andrews, P. W., Bronson, D. L., Benham, F., Strickland, S. & Knowles, B. B. A comparative study of eight cell lines derived from human testicular teratocarcinoma. Int. J. Cancer 26, 269–280 (1980).
General Introduction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Bertotti, A. et al. A molecularly annotated platform of patient-derived xenografts (‘xenopatients’) identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal cancer. Cancer Discov. 1, 508–523 (2011).
Sivanand, S. et al. A validated tumorgraft model reveals activity of dovitinib against renal cell carcinoma. Sci. Transl. Med. 4, 137ra75 (2012).
Alkema, N. G. et al. Biobanking of patient and patient-derived xenograft ovarian tumour tissue: Efficient preservation with low and high fetal calf serum based methods. Sci. Rep. 5, 14495 (2015).
Liu, J. F. et al. Establishment of patient-derived tumor xenograft models of epithelial ovarian cancer for preclinical evaluation of novel therapeutics. Clin. Cancer Res. 23, 1263–1273 (2017).
Kageyama, K. et al. Establishment of an orthotopic patient-derived xenograft mouse model using uveal melanoma hepatic metastasis. J. Transl. Med. 15, 145 (2017).
Zhang, C., Awasthi, N., Schwarz, M. A. & Schwarz, R. E. Establishing a peritoneal dissemination xenograft mouse model for survival outcome assessment of experimental gastric cancer. J. Surg. Res. 182, 227–234 (2013).
Byrne, A. T. et al. Interrogating open issues in cancer precision medicine with patient-derived xenografts. Nat. Rev. Cancer 17, 254–268 (2017).
Burger, A. M. & Fiebig, H. H. Screening using animal systems. in Anticancer Drug Development (ed. Baguley, B.C., Kerr, D. J.) 285–299 (Academic Press, 2002).
Piulats, J. M. et al. Orthoxenografts of testicular germ cell tumors demonstrate genomic changes associated with cisplatin resistance and identify PDMP as a resensitizing agent. Clin. Cancer Res. 24, 3755–3766 (2018).
Koster, R. et al. Cytoplasmic p21 expression levels determine cisplatin resistance in human testicular cancer. J. Clin. Invest. 120, 3594–3605 (2010).
Haugnes, H. S. et al. Long-term and late effects of germ cell testicular cancer treatment and implications for follow-up. J. Clin. Oncol. 30, 3752–3763 (2012).
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