Mechanistic and translational studies to improve cisplatin sensitivity of testicular cancer
de Vries, Gerda
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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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Summary and General
Discussion
SUMMARY
Testicular cancer (TC) is one of the most common solid tumors in men between 20-40 years of age and the incidence is rising worldwide1. TC can be divided in two types, seminomas
and non-seminomas, each accounting for approximately 50% of cases2. Seminomas resemble
undifferentiated spermatogonia with low metastatic potential. Non-seminomas can be further divided into four subtypes with varying stages of differentiation, ranging from undifferentiated embryonal carcinoma (EC), to more differentiated extra-embryonic components like yolk sac carcinomas (YSC) and choriocarcinomas (CC), and highly differentiated teratomas3. Tumors with
a mixture of histological components are common and classified as non-seminoma. Treatment for patients with metastatic testicular cancer usually consists of orchiectomy followed by cisplatin-based chemotherapy. TC represents one of the few solid tumor types that, even when metastasized, can be cured by cisplatin-based chemotherapy. Cure rates in metastatic patients treated with cisplatin-based chemotherapy are around 80% and salvage therapy cures another ~10% of patients4. However, a subset of patients with metastatic disease will die as a result of
chemoresistance. For this subset of patients there are no alternative treatment options available. Pre-clinical studies of TC have identified several druggable targets, including VEGFR, PDGFR and KIT5. Subsequently, several small clinical trials have been performed in an attempt to find better
treatment options for refractory and chemoresistant TC patients6. Unfortunately, none of these
strategies has been very successful. Accordingly, there is a need to deepen our knowledge on TC disease characteristics and resistance mechanisms to find novel therapeutic targets. Therefore, the research presented in this thesis aims to identify new therapeutic targets and treatment strategies for TC.
In chapter 2 we provide an overview of our current knowledge on several aspects related to TC, including conventional and novel treatments, resistance mechanisms and novel pre-clinical models. We describe that TCs are highly aneuploid and the 12p isochromosome is the most frequent chromosomal abnormality.
The mutation rate is of TC is low, with recurrent mutations in KIT and KRAS observed only at low frequency in seminomas. Overall cure rates are high, even in a metastatic setting, as a result of exquisite cisplatin sensitivity of TCs. Factors contributing to the observed cisplatin sensitivity include defective DNA damage repair and a hypersensitive apoptotic response to DNA damage. Nonetheless, around 10-20% of TC patients with metastatic disease cannot be cured by cisplatin-based chemotherapy. Resistance mechanisms include downregulation of OCT4 and failure to induce PUMA and NOXA, elevated levels of MDM2, and hyperactivity of the PI3K/AKT/mTOR pathway. Several pre-clinical approaches have proven successful in overcoming cisplatin resistance, including therapeutic targeting of PARP, MDM2 or AKT/mTOR combined with cisplatin. Nevertheless, results of clinical trials investigating targeted drugs as single agent have been disappointing so far. Finally, patient-derived xenograft models hold potential for mechanistic studies and pre-clinical validation of novel therapeutic strategies in TC. Although clinical trials investigating targeted agents have been disappointing, these pre-clinical successes fuel the need for further investigation in clinical setting.
Mutation-driven activation of members from the PI3K/AKT/mTOR pathway, among other pro-survival pathways, is observed in many cancers7,8. However, almost no mutations in PI3K/AKT/
mTOR pathway components or upstream receptor tyrosine kinases are found in either cisplatin-sensitive or resistant TCs9–11. Nonetheless, it has previously been described that PI3K or AKT
inhibition sensitizes cisplatin-resistant TC cells to cisplatin12. In addition, specific receptor tyrosine
kinase (RTK) activity has been investigated in TC cell lines, identifying IGF1R and PDGFRβ as therapeutic targets13,14. However, the involvement of other upstream kinases, causing activation of
the PI3K/AKT pathway, or the involvement of other intracellular kinases in resistance mechanisms against cell death were not evaluated in depth. In chapter 3 we aimed to investigate 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.
A rather unique feature of TC is the presence of wild type TP53 in most tumors15–19, which is
frequently mutated in other types of cancer. In response to chemotherapy, p53 is activated by the DNA damage response (DDR). As a consequence, p53 regulates the transcription of genes involved in cell cycle arrest, DNA repair or apoptosis through the extrinsic and intrinsic apoptotic pathways20. Activity of p53 is regulated by MDM2, an E3 ubiquitin ligase. MDM2 binds
to the transactivation domain of p53, thereby preventing activation of p5321. In addition, MDM2
constantly ubiquitinates p53, thereby targeting it for proteasomal degradation22. MDM2 itself
is a transcriptional target of p53, and these two proteins are linked through an autoregulatory negative feedback loop maintaining low p53 levels under physiological conditions. Disruption of the MDM2-p53 interaction has been of interest as a therapeutic strategy to activate p53 and thereby inducing a p53-dependent apoptotic response. Small molecule inhibitors of MDM2, like nutlin-3a, targeting the interaction between MDM2 and p53, have been shown to sensitize TC cells to cisplatin treatment23,24. In chapter 4, in light of cisplatin-related toxicities, we compared
the effectiveness of combined nutlin-3a and cisplatin to a combination of targeted drugs only. A panel of TC cell lines were treated with inhibitors of MDM2 (nutlin-3a, RG7388), AKT (MK2206)
and mTORC1/2 (everolimus, AZD8055 and INK128) and cisplatin. We found that MDM2 inhibitors sensitized TC cells to mTORC1/2 inhibitors and cisplatin. Nutlin-3a combined with AZD8055 or cisplatin induced caspase-3 and PARP cleavage and reduced clonogenic outgrowth. Using chromatin immunoprecipitation, enriched p53 binding to promoter regions of the FAS, PUMA and NOXA genes was observed in response to nutlin-3a or cisplatin. BH3 profiling and siRNA-mediated silencing identified PUMA and NOXA as key pro-apoptotic proteins in TC cell lines. Interestingly, addition of the BH3 mimetics ABT-737 and A-1210477 to nutlin-3a/AZD8055 and nutlin-3a/cisplatin completely shifted the balance towards apoptosis. In vivo efficacy of RG7388/AZD8055 or RG7388/cisplatin therapies was tested in two TP53 wild type PDX models. RG7388/cisplatin treatment reduced tumor growth in PDX TC1 and induced p53 activation and caspase-3 cleavage. In conclusion, we demonstrate that targeting MDM2 in combination with chemotherapy can be very effective in TC models. These results warrant further investigation of this drug combination, potentially with the addition of BH3 mimetics, in the treatment of TC. In chapter 3 and 4 we use TC PDX models to test our combination treatment strategies in vivo. These models are a more accurate reflection of the patient tumor compared to cell line xenograft models. In chapter 5 we describe the establishment and characterization of 3 TC PDX models. 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 characterized 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. One of the mechanisms behind cisplatin resistance previously implicated for TC is high expression levels of p21 protein, mainly located in the cytoplasm25. P21 functions as a regulator of G1 and
S phase progression, providing a link between the DNA damage response and cell cycle arrest. Cytoplasmic localized p21 has oncogenic properties, inhibiting apoptosis and promoting cell proliferation, while nuclear localized p21 is considered a tumor suppressor26. In chapter 6 we
evaluated the role of p21 as a determinant of cisplatin resistance using several genetic approaches to modulate p21 protein levels in TC cell lines. Reduced expression was accomplished by knock down of p21 and OCT4 using either transient siRNA or stably introducing shRNA plasmids targeting p21. In addition, a TC cell line with a homozygous knock out of CDKN1A, which encodes p21, was created using CRISPR/Cas9. For overexpression of p21, TC cell lines expressing an inducible sgRNA targeting p21 were generated using dCas9/CRISPR-mediated gene regulation. Using these models, the effects of reduced or increased p21 levels on response to cisplatin were
determined using apoptosis and survival assays and cell cycle analysis. Downregulation of p21 or OCT4 protein levels, as well as knock out of CDKN1A, did not change the sensitivity of TC cells towards cisplatin treatment. In addition, sensitivity towards cisplatin treatment was not affected by overexpression of p21 in TC cells. In summary, the present study does not suggests that p21 and OCT4 play a significant role in cisplatin sensitivity of TC cell models.
GENERAL DISCUSSION AND FUTURE PERSPECTIVES
Cisplatin resistance
The research presented in this thesis aims to identify new therapeutic targets and treatment strategies for TC. As cisplatin resistance is still an important issue and refractory testicular cancer a significant cause of death in this relatively young age group, we focused on therapeutic strategies to overcome cisplatin resistance or cisplatin related toxicities. Cisplatin resistance of TC is considered to be multifactorial27 and knowledge of these alterations is required in order to
advance drug evaluation and validation. A number of cisplatin resistance mechanisms have been identified and described in chapters 2, 3 and 4. These resistance mechanisms can be classified as pre-target, on-target, post-target and off-target.
Pre-target resistance
These resistance mechanisms represent alterations that operate prior to binding of cisplatin to DNA, for example in drug uptake or efflux. For TC however, no pre-target resistance mechanisms have yet been identified.
On-target resistance
Alterations directly related to cisplatin-DNA adducts are discussed in chapter 2, in which we have conducted a literature review. It has been described that sensitivity to cisplatin in TC is related to a diminished capacity to repair cisplatin-DNA adducts28. Conversely, TC cells resistant to cisplatin
may have enhanced ability to resolve cisplatin-DNA adducts or gained the ability to cope with unrepaired lesions. Cisplatin-induced intrastrand cross-links are mostly repaired by the nucleotide excision repair (NER) pathway29. However, it has been shown that HMGB4, which is preferentially
expressed in testes, blocks NER of cisplatin-DNA adducts and potentiates the sensitivity of TC cells to cisplatin therapy. In line with this notion, loss of HMGB4 expression by using CRISPR-Cas9 gene editing in TC cells elicits resistance to cisplatin30. Besides NER, homologous recombination
(HR) is involved in repairing interstrand cross-links. Reduced proficiency of HR contributes to high cisplatin sensitivity in TC cell lines. Cisplatin sensitivity of a panel of TC cell lines with different levels of sensitivity correlates with the interstrand cross-link repair proficiency of TC cells. In addition, sensitivity to PARP inhibition also correlates with the level of proficiency of TC cells to repair interstrand cross-links31. Even though differences in HR deficiency exist between
cisplatin-sensitive and -resistant TC cell lines, all TC cell lines are shown to be more HR deficient compared to a cisplatin-resistant osteosarcoma cell line. This observation explains why both cisplatin-sensitive and - resistant TC cell lines are sensitized to cisplatin treatment by PARP inhibition31. This report
is the only one showing that a higher level of DNA damage repair is associated with cisplatin resistance in TC, whereas most studies have only linked reduced DNA damage repair to cisplatin sensitivity30–34. Thus, more research should be conducted to establish the exact role of the DNA
damage response in determining cisplatin sensitivity among TC, for example whether increased expression of proteins involved in HR cause acquired or intrinsic cisplatin resistance in TC. Finally, mismatch repair (MMR) deficiency and microsatellite instability (MSI) have been linked to cisplatin resistance in TC cell lines and TC patients35,36. Possibly also related to chemoresistance of secondary
malignancies after treatment for testicular cancer. However, the molecular mechanisms remain unclear and need to be further investigated to really establish the role of MMR deficiency in cisplatin resistance of TC.
Post-target resistance
This type of resistance involves mechanisms downstream of cisplatin-mediated DNA damage that are discussed in chapter 2, 3 and 4. Increased activity of pro-survival pathways and reduced activity of apoptotic pathways can contribute to chemoresistance. While TC tumors are mostly highly sensitive to cisplatin-based chemotherapy, resistance to treatment does occur. Indeed, hyperactivation of a pro-survival pathway, namely the PI3K/AKT/mTOR pathway, has been implicated in TC13,14. To determine in depth which upstream kinases cause activation of the PI3K/
AKT/mTOR pathway or which intracellular kinases are involved in resistance mechanisms, we screened a panel of cisplatin-sensitive and -resistant TC cell lines in chapter 3. By using phospho-arrays, we determined the phosphorylation status of kinases and their downstream targets. Based on these results, we screened a number of kinase inhibitors alone and in combination with cisplatin, using apoptosis induction as read-out of sensitization. The best candidates to sensitize TC models to cisplatin treatment proved to be mTORC1/2 inhibitors. Inhibition of mTORC1/2 strongly enhanced cisplatin-induced apoptosis in sensitive and resistant TC cell lines as well as in the tested PDX models. One of the reasons why mTORC1/2 inhibitors are more efficient then AKT inhibitors or mTORC1 inhibitors could be the absence of feedback signaling, either from S6K1/ IRS-1 to AKT or mTORC2 to AKT. In addition, both AKT inhibitors and mTORC1 inhibitors induce autophagy via mTORC1 inhibition. We show that in our TC cell lines, blocking autophagy increases apoptosis levels, pointing towards a protective effect of autophagy in this context. However, despite the induction of autophagy, the mTORC1/2 inhibitors still sensitize our TC cells to cisplatin-induced apoptosis. The exact mechanisms of sensitization, including the role for autophagy, are not completely understood and need to be further investigated. Furthermore, the reason why the PI3K/AKT/mTOR pathway is hyperactive in TC remains unclear, although diverse causes have been reported. Di Vizio et al. discovered that the majority of TC tumors are characterized by loss of PTEN, a tumor suppressor that negatively regulates PI3K signaling37. Mutational deregulation
of the PI3K/AKT/mTOR pathway has been identified11,38,39, as well as overexpression of several
receptor tyrosine kinases (RTK) involved in activation of the PI3K/AKT/mTOR pathway in TC5.
Another driver behind hyperactivity of the PI3K/AKT/mTOR pathway could be an adaptation by TC cells to disconnect cell growth, survival and metabolism from external growth stimuli to become reliant on cell intrinsic stimuli. Altered cellular metabolism is essential for tumor cells to support tumor growth and proliferation. In addition, sensitivity to different chemotherapeutics has been related to altered metabolism in different cell lines, including lung and ovarian cancer cell lines40. As cellular metabolism acts as an important mediator of chemotherapy resistance,
targeting specific components involved in metabolic rewiring (e.g. pentose phosphate pathway, facilitating the biosynthesis of macromolecules and maintaining redox homeostasis, or other ROS inducing agents) has been shown to overcome chemotherapy resistance40. For TC, studies linking
altered metabolism to cisplatin resistance are not available and may provide a novel research opportunity.
Another mechanism of cisplatin resistance in TC cells involves the MDM2/p53 axis, involved in growth arrest and apoptotic signaling. It has previously been shown that p53 function is hampered by the interaction with MDM2, especially in cisplatin resistant cells where higher cisplatin concentrations are needed to interfere with the MDM2/p53 interaction24. Therefore,
MDM2 appears an interesting therapeutic target to activate wild type p53. Indeed, small molecule inhibitors of MDM2 (e.g. nutlin-3) targeting the MDM2-p53 interaction are shown to sensitize TC cells to cisplatin treatment23,24. In chapter 4 we examined the small molecule inhibitor RG7388, a
second generation MDM2 inhibitor, in combination with cisplatin and observe similar apoptosis-enhancing effects as previously observed with nutlin-324. In addition, we investigate the feasibility
of MDM2 inhibition combined with inhibition of the pro-survival PI3K/AKT/mTOR in an attempt to find a treatment strategy excluding chemotherapy, a combination that proves highly efficient in vitro. These observations are not fully reproduced in vivo, where anti-tumor responses are only observed in the cisplatin-sensitive PDX model TC1 treated with the combination RG7388/ cisplatin, but no effect is observed for this combination in the cisplatin-resistant PDX model TC4. PDX TC4 is derived from a patient refractory to cisplatin treatment. One of the reasons that may have contributed to the lack of response in PDX TC4 could be underdosing. The concentration of cisplatin used in PDX model TC4 (4 mg/kg) is the maximum dose tolerated by NSG mice in terms of treatment related toxicities, however this model is highly resistant to cisplatin treatment. Furthermore, minimal response to RG7388 as indicated by low levels of p53 activation suggests that the dose of RG7388 (75 mg/kg) might also have been too low for this PDX model. However, it might well be that there are other mechanisms causing the cisplatin-resistance of this model. Our data indicates that the apoptotic response induced by either of the combination treatments is activated via the extrinsic and intrinsic apoptosis pathways. However, the exact mechanism behind p53-mediated activation of pro-and anti-apoptotic proteins in TC cell lines remains unclear. We show that silencing of pro-apoptotic proteins PUMA and NOXA represses the induction of apoptosis in our cell lines. However, this effect is larger in the cisplatin sensitive cell line Tera compared to cisplatin resistant cell lines TeraCP and Scha. These findings suggest that cisplatin-resistant cell lines rely less on p53-mediated induction of PUMA and NOXA in response to nutlin-3a or cisplatin. BH3 profiling of our TC cell lines does not point to specific anti-apoptotic proteins balancing the anti-apoptotic response. Nonetheless, we do show that the addition of BH3 mimetics to single drug treatment, or to one of the combination treatments, induces massive apoptosis in our cell lines. These data show us that BH3 mimetics might be a novel way to overcome treatment resistance by removing the break on apoptosis. Several BH3 mimetics are in clinical development and the BCL-2 inhibitor venetoclax has been FDA approved for the treatment of chronic lymphocytic leukemia. The next step would be to test these BH3 mimetics in TC PDX models.
Alternative resistance mechanisms
This involves alterations that are not directly related to cisplatin-mediated DNA damage. One example of an off-target resistance mechanism, discussed in chapter 2, is the loss of OCT4 expression which has been demonstrated by several studies in vitro and in vivo25,41–43. These
studies suggest that cisplatin treated tumors with EC histology who are losing OCT4 expression become cisplatin resistant, either via lower NOXA levels or via differentiation of TC cells. Important to mention is that other TC subtypes like YSC and CC do not express OCT4, while these are not necessarily less sensitive to cisplatin. Therefore, the question remains whether OCT4 itself is a critical determinant of cisplatin sensitivity, or other factors related to OCT4 and pluripotency, for example the pro-apoptotic protein NOXA or micro-RNA clusters miR-371-373 and miR-106/302, determine responsiveness to cisplatin. Noteworthy, an upregulation of miR-371-373, suggested to interfere with wild type p53 signaling, was observed in cisplatin resistant EC cell lines compared to their cisplatin sensitive counterparts44,45.
Another alternative process playing a role in cisplatin resistance is epigenetics of TC. Recent results suggest that increased DNA methylation is associated with cisplatin resistance of TC46.
Cisplatin-sensitive seminomas for example, are hypomethylated, while EC tumors that have a relatively higher incidence of cisplatin resistance show intermediate DNA methylation, and more differentiated tumors like YSC, CC and teratomas show the highest level of DNA methylation47.
Interestingly, TC cell lines are hypersensitive to the second generation DNA methylation inhibitor guadecitabine, both in vitro and in vivo. In addition, pretreatment of cisplatin resistant TC cells with this inhibitor re-sensitizes cells to cisplatin and is associated with p53 activation48. Mechanistically,
hypersensitivity of TC cells to DNA methylation inhibitors has been linked to high expression of the DNA methyltransferase DNMT3B49.
Very recently, a tight association between cisplatin resistance, decreased histone H3K27 methylation, and decreased expression of members of the polycomb repressive complex 2 (PRC2) has been discovered46. Tri-methylation at the 27th lysine residue of histone 3 (H3K27),
catalyzed by the multiprotein complex PRC2, is a hallmark of heterochromatin regions that maintain transcriptional repression of nearby genes. Using RNA sequencing and gene set enrichment analysis, it has been shown that acquired resistant TC cell lines display a significant enrichment of genes normally repressed by H3K27 methylation and PRC2 compared to their sensitive counterparts. The observed gene enrichment correlates with decreased protein expression of specific PRC1/2 components (BMI1 and EZH2) and decreased H3K27 methylation46.
Besides H3K27 and PRC2, enrichment of other gene sets included those related to epithelial to mesenchymal transition, radiation response and graft vs. host rejection. Interestingly, treatment of cisplatin resistant TC cells with the histone lysine demethylase inhibitor GSK-J4 results in increased H3K27 methylation and consequently increased cisplatin sensitivity46. Thus, restoration
of the PRC2 complex function might have potential as a novel treatment approach. These data suggest that epigenetic reprogramming of TC cells is associated with cisplatin resistance. A more complete understanding of the epigenetic state of cisplatin-sensitive and -resistant TC is needed, including the identification of crucial components by means of genetic screening for example. Besides the resistance mechanisms highlighted above, additional mechanisms of resistance could be present. To investigate the resistance phenotype in depth, one could for example use functional genomic screening, a tool that is used to determine the relationship between
phenotype and genotype by identifying molecular events underlying drug resistance. CRISPR-Cas9 knockout screens create specific gene knockouts and then determine which genes confer drug-resistance. These screens can be performed quickly and at high-throughput, which makes it possible to screen the entire genome at once. However, in light of the low mutation rate of TC tumors gene knock down or overexpression might better reflect drug activity. Alternatives of the CRISPR-Cas9 system include CRISPRi and CRISPRa, which can modulate gene expression by using an inactive Cas9 (dCas9) bound to a transcriptional repressor (CRISPRi) or activator (CRISPRa) domain. A combined screening approach has been developed using a melanoma cell line, where CRISPRi and CRISPRa are used in parallel to identify those genes involved in resistance to the BRAF inhibitor vemurafinib50.
Novel therapeutic options for TC patients
First line cisplatin-based chemotherapy will fail in 10-15% of TC patients with metastatic disease, including refractory and relapsed patients51. These patients receive salvage treatment with long
term remission in approximately 50% of the cases52. A prospective global trial (the TIGER trial) is
currently performed in TC patients with relapsed or refractory disease, to address the question whether standard-dose or high-dose salvage chemotherapy provides better outcomes when used as initial salvage treatment. Besides salvage treatment regimens, no alternative treatment options are available at the moment, highlighting the need for clinical trials investigating novel therapies. One question remaining is: How can we use the knowledge gained in this thesis to help refractory TC patients to better treatments?
In chapter 2, we have described a number of clinical trials that were performed in refractory and chemoresistant TC patients. All these clinical trials investigated different targeted therapies as a single agent. Unfortunately, none of them showed promising results. In this thesis we have described two novel drug targets for TC, namely mTORC1/2 and MDM2. So far both these targets have not been investigated in clinical trials in TC. We argue that, following our pre-clinical results presented in chapters 3 and 4, future clinical trials should start investigating these novel therapies in combination with cisplatin in an effort to overcome cisplatin resistance in TC patients.
Besides the two novel combination therapies investigated in this thesis, other drugs are worth clinical investigation as well. First, as TC tumors have been characterized as relatively HR deficient, PARP inhibitors combined with cisplatin may provide a therapeutic opportunity. Currently, two phase II trials are evaluating the potential of PARP inhibitors in relapsed or refractory TC, either as single agent (NCT02533765, active) or combined with gemcitabine and carboplatin (NCT02860819, active and recruiting). Second, as cisplatin resistant TC has been found to be hypermethylated, and TC cell lines are hypersensitive to the second generation DNA methylation inhibitor guadecitabine, therapeutic efficacy of guadecitabine might be extended to refractory TC patients. A phase I clinical trial studied the use of guadecitabine in combination with cisplatin in a group of refractory TC patients, looking at safety and clinical activity. Preliminary results were recently published at the ASCO 2020 meeting, showing that guadecitabine was safe and tolerable and showed clinical activity in 4 out of 14 patients53. Other potentially interesting epigenetic
modifiers could be the so-called non-nucleoside demethylating agents that selectively inhibit individual DNA methyltransferases (DNMTs)54. As discussed above, hypersensitivity of TC cells to
DNA methylation inhibitors have been linked to high expression of DNMT3B49. Nanaomycin A
selectively inhibits DNMT3B by binding to its catalytic site55. In other cancer types nanaomycin
A resulted in re-expression of RASSF1A and anti-proliferative effects55. RASSF1A is frequently
inactivated in human tumor cells including TC, which normally functions as a tumor suppressor modulating DNA repair and Hippo pathway activity56–58. The downstream effector of the Hippo
pathway YAP/TAZ interacts with SMAD2/3, a main transcriptional regulator of the TGF-β signaling pathway. It has been shown that absence of RASSF1A is important to allow the association between YAP1 and SMAD2/3, resulting in the nuclear translocation of SMAD2/3. In contrast, expression of RASSF1A leads to retention of SMAD2/3 to the cytoplasm and loss of TGF-β oncogenic signaling57.
Interestingly, RASSF1A silencing has been associated with cisplatin resistance in TC58. In line
with those findings, the expression of RASSF1A by nanaomycin A treatment potentially re-sensitizes refractory TC patients to cisplatin-based chemotherapy. So far, no reports are available on nanaomycin A efficacy in TC models, but this specific DNMT3B inhibitor might be preferred over the global demethylating agent guadecitabine. Furthermore, it needs to be determined whether TGF-β signaling plays a role in cisplatin resistance of TC models and whether this can be influenced by (re-)expression of RASSF1A.
Models for pre-clinical drug evaluation
In chapter 5, we have described the establishment and characterization of three TC PDX models. Two models were obtained from cisplatin-sensitive patients, and one model was obtained from a cisplatin-resistant patient. These models were used for pre-clinical drug testing in chapters 3 and 4. The number of models we have established is, however, limited and needs to be expended. Furthermore, the scope of histological subtypes present in our collection of PDX models is limited, including YSC with some immature teratoma components. Histological subtypes CC and EC are not reflected by our PDX models. In addition, it would be beneficial to acquire more cisplatin-resistant PDX models to assist in treatment evaluation and future pre-clinical drug testing. PDX models are also used as ‘avatars’ for individual patients. Using personalized PDX avatars makes it possible to test the efficacy of diverse treatments or combinations and customize treatment to the individual patient accordingly. Promising, a study by Izumchenko et al. showed an 87% association between drug response in PDX models and corresponding patients59. However, there
are some limitations to the use of avatar PDX models, including the lack of a functional immune system, differences in metabolisms and clonal selection potentially explaining the negative association in 13% of Izumchenko’s study. In addition, take rates and establishment rates might limit their use for patients with limited time. Therefore, a more realistic role for PDX models is to be used in mechanistic studies and pre-clinical validation of novel therapeutic strategies.
As pre-clinical drug screening in PDX models is costly and time consuming, it would be most efficient to use them in the final stage of treatment evaluation. Screening of treatment efficacy, after cell line-based experimental work, could for example be done using TC organoid cultures, after which the in vivo validation can be performed in PDX models. Organoids are 3D cultures with organ-like structures and some organ-specific cell types, structure and functionality. Like PDX tumors, tumor organoids can be derived from primary tumor material and are believed to better reflect the patient tumor compared to cell lines models60. In addition, drug screens will be
much faster and cheaper using organoids compared to PDX models. However, so far no organoids have been established of testicular cancer tissue. Testicular organoids from healthy human adult tissue have been developed by seeding primary testicular cells into hanging transwell inserts. A scaffold free culture system was compared to a scaffold-based system in which the testicular microenvironment was formed by a decellularized testicular matrix on top of an agarose layer. Although lacking histological similarities to human testis, testicular organoids were capable of reorganizing into spheroid structures, producing testosterone and secreting cytokines regardless of scaffold presence. Moreover, germ cells were proliferative for up to four weeks61,62. Research
efforts should be made towards the development of TC organoids that together with PDX models can be used for pre-clinical drug evaluation.
Challenging cisplatin resistance mechanisms
Previously, cytoplasmic p21 was introduced as a key determinant of cisplatin resistance in TC25.
Cytoplasmic localized p21 has oncogenic properties, inhibiting apoptosis and promoting cell proliferation, while nuclear localized p21 is considered a tumor suppressor26. It was shown that
localization of p21 in TC cells was regulated by AKT, which phosphorylates p21 and thereby traps it to the cytoplasm where it inhibits apoptosis. Consequently, inhibition of p21 phosphorylation by using a PI3K or AKT inhibitor re-sensitized TC cells to cisplatin treatment.
In chapter 6, we have repeated some key experiments from the study by Koster et al,25 in order
to validate their findings. To this end, we make use of several approaches to modify p21 protein levels by creating genetically modified TC cell lines and determine the effect of p21 modulation on apoptosis induction and cell survival after cisplatin treatment. Surprisingly, none of the approaches to either down- or upregulate p21 protein levels alteres cisplatin sensitivity. Some differences exist between the experiments performed earlier and the ones presented in this thesis, including different types of assays and culture conditions. For example, we treated our TC cell lines with cisplatin and performed cell survival assays in different types of culture media. Interestingly, these data shows us that cisplatin sensitivity partly depends on type of culture media, hence it may influence experimental outcomes. This indicates that the influence of p21 on chemosensitivity is not a significant mechanism in vitro and that the extent of its role as a determinant of chemosensitivity should be reconsidered.
CONCLUSIONS
In conclusion, this thesis provides new leads for patients with refractory or cisplatin resistant testicular cancer for future combination therapies involving targeted drugs against mTORC1/2, MDM2 and the chemotherapeutic cisplatin. Furthermore, we developed and extensively characterized three novel PDX models that may be of use for the therapeutic development and pre-clinical validation of novel therapeutic strategies in testicular cancer.
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