Testicular germ cell tumors (TGCTs) are a heterogeneous group of tumors that occur primarily in children and young men and are the most common tumor types in men aged 20-40 years. Based on histology, TGCTs are classified as seminoma (SE-TGCT) or non-seminoma (NSE-TGCT) tumors (1,2). The latter can be further classified as undifferentiated embryonal carcinoma (EC), choriocarcinoma, yolk sac tumor, differentiated teratoma or mixed tumors (a mixture of two or more components) (3-5). TGCTs develop from precancerous germ cell tumors in the renal tubule, mainly because of the failure of lymphocytes to mature properly during fetal or postnatal development. It progresses to aggressive TGCTs such as seminomas and non-seminomas after puberty, and environmental and genetic risk factors are significant factors in the susceptibility to TGCTs (6). Studies have shown that most testicular germ cell tumors originate from germ cell neoplasia in situ, which is thought to be due to the stagnation and transformation of the original germ cells. Seminomas have the same characteristics as germ cells or primordial germ cell formation in situ, whereas non-seminomas exhibit differential differentiation. Neoplasms and embryonic cell carcinomas are pluripotent and are thought to be responsible for the histological heterogeneity and mixed pathology of testicular germ cell tumors (7).

Platinum-based therapies are often used as first-line treatment for TGCTs in children and adults. TGCTs have a very high cure rate and are highly sensitive to cisplatin chemotherapy (8,9). The sensitivity of TGCTs to cisplatin may correlate with two key reactions: Inadequate repair of cisplatin-induced DNA damage and a hypersensitive apoptotic response (10). Various sources of endogenous and exogenous damage constantly assault the genome, among which DNA double-strand breaks (DSBs) are the most cytotoxic DNA lesions (11-13). To maintain chromatin stability, cells have developed a complex system of biochemical pathways called the DNA damage response (DDR) (14). DNA repair mechanisms have an indispensable role in cisplatin-induced cytotoxicity. DNA repair systems, including nucleotide excision repair (NER), homologous recombination (HR), nonhomologous end joining (NHEJ) and mismatch repair (15).

For metastatic disease, the overall disease-free survival rate of TGCTs is ~80%. Even in patients with advanced metastatic disease, complete remission can be achieved with systemic therapy and secondary removal of residual mass. However, ~10-20% of advanced TGCTs are resistant to platinum-based chemotherapy and have a poor prognosis or recurrence, and the treatments for these patients are poorly understood. Studies have shown that cisplatin resistance is mainly related to the apoptotic pathway, tumor cell cycle regulation-related factors, DNA methylation and DNA damage repair pathways. Among them, the regulated death pathway is mainly related to murine double minute 2 (MDM2)/p53, octamer-binding transcription factor 4 (OCT4)/phorbol-12-myristate-13-acetate-induced protein 1 (NOXA), platelet-derived growth factor receptor (PDGFR)/PI3K/AKT and insulin-like growth factor 1 receptor (IGF1R)/AKT, and certain related microRNA (miRNA) pathways. The main cell cycle regulatory factors were cyclin-dependent kinase (CDK)4/6 inhibitors and CDK2 inhibitor p21. During DNA methylation, hypermethylation can promote cisplatin resistance; therefore, since vir-like N6-methyladenosine (m⁶A) methyltransferase associated (VIRMA) is positively correlated with m⁶A in TGCTs, it stimulates the methylation process and thus cisplatin resistance. However, the DNA hypomethylating agent 5-azacytidine (5-aza) and the demethylating agent guadecitabine (SGI-110) promoted cisplatin sensitivity and inhibited cisplatin resistance. In the process of DNA damage repair, cisplatin resistance is positively correlated with NHEJ inhibition and increased HR, and a decrease in DNA damage response protein p53-binding protein 1 (53BP1) and DNA-dependent protein kinases (DNA-PKCs) can inhibit NHEJ and promote HR, thus promoting cisplatin resistance.

This review describes the sensitivity of TGCTs to cisplatin (also referred to as CDDP), the underlying mechanisms related to CDDP and the potential therapeutic agents for cisplatin-resistant TGCTs.

In cisplatin-sensitive TGCT cells, cisplatin interacts with the DNA base to produce different forms of cisplatin-DNA adducts, of which in-chain crosslinking (>90%) is the dominant DNA adduct (15,16). The NER pathway can repair intra-chain crosslinking that can destroy DNA structures induced by cisplatin, and intra-chain crosslinking can be recognized by proteins associated with DNA repair (17). Certain proteins also act as inhibitors. For instance, high-mobility-group (HMG) proteins can inhibit NER by binding to DNA adducts. HMG box protein 4 (HMGB4) enhances the sensitivity of TGCTs to cisplatin by blocking the excision repair of cisplatin-DNA adducts (18). Furthermore, numerous essential proteins involved in NER have low expression levels in TGCTs, including xeroderma pigmentosum group A (XPA), xeroderma pigmentosum group F (XPF) and excision repair cross-complementing 1 (ERCC1) (19). A study showed that high levels of XPF and ERCC could decrease the sensitivity to cisplatin in TGCT cell lines, indicating that XPF and ERCC are rate-limiting factors for the repair of cisplatin-DNA cross-links in TGCTs, which contributes to remarkable cisplatin sensitivity (20). Furthermore, homologous recombination repair defects of DSB also cause high cisplatin sensitivity in TGCT cell lines (18). Of note, a related study showed that compared to normal testes, poly(ADP-ribose) polymerase (PARP) is overexpressed in TGCT and is involved in the repair of DNA single-strand breaks by base excision repair (21). Meanwhile, PARP inhibitors can promote the response of resistant EC cells to cisplatin by reducing their ability to repair damage, providing a potential therapeutic approach for resistant TGCTs (22).

The high sensitivity of TGCTs to cisplatin is due to the various apoptotic pathways induced by cisplatin, in which wild-type p53 plays a major role (23). p53 is frequently mutated in cancer as a tumor suppressor, but is not mutated in TGCTs, retains its wild-type configuration and is activated following exposure to chemotherapeutic agents (8). Cisplatin treatment increases the expression of p53 transcriptional target FAS death receptors and subsequently activates exogenous apoptotic pathways through the interaction of FAS with its ligand (24,25). It has also been shown that cisplatin treatment can upregulate the pro-apoptotic protein p53 upregulated modulator of apoptosis (PUMA) and NOXA (26). As a target protein of p53, NOXA is a Bcl-2 family protein that belongs to a subclass of BH3-only proteins that induce apoptosis via p53-dependent and/or p53-independent mechanisms (27), thus playing a crucial role in cisplatin-induced apoptosis of TGCTs cells (28). In addition, high expression of the pluripotent factor OCT4 (also known as POU5F1) is positively correlated with NOXA, leading to a hypersensitive apoptotic reaction in EC cells (23). OCT4 is also related to the regulation of p21 expression and p21 is associated with cisplatin sensitivity both in vivo and in vitro (28). Furthermore, crosstalk between exogenous and intrinsic apoptotic pathways may further enhance the apoptotic response (10) (Fig. 1).

Cisplatin resistance is a major clinical challenge and a thorough understanding of the mechanisms underlying the development of TGCT resistance will help improve the efficacy of TGCT therapy. Several mechanisms of cisplatin resistance have been identified, mainly related to apoptotic pathways, tumor cell cycle regulation (Fig. 2), methylation and DNA repair systems (Fig. 3). Specific mechanisms are outlined and potential treatments that currently exist are discussed in this chapter.

In the past 50 years, significant progress has been made in the treatment of TGCTs, particularly advanced diseases, and the cure rate has increased from 25% in the 1970s to nearly 80% (86). Radical orchiectomy combined with chemotherapy is currently considered the standard treatment (87,88). As TGCT is highly sensitive to chemotherapy, particularly cisplatin chemotherapy, the combination of cisplatin, etoposide and bleomycin or ifosfamide remains the first-line treatment (10,89,90). These combination chemotherapy regimens have shown high efficacy in the treatment of early and advanced TGCT, resulting in a significant increase in the 5-year survival rate to >95% (91,92). However, owing to the long-term side effects of platinum chemotherapy, patients still experience long-term toxicity after being cured (93,94). Approximately 20-30% of patients cannot be cured by standard treatment, particularly those who develop cisplatin resistance, and the prognosis is still unfavorable (95,96). Patients with recurrent TGCT are usually treated with high-dose chemotherapy, but this treatment results in severe adverse effects and cytotoxicity (97-99). Therefore, there is an urgent need to develop new, less toxic drugs to overcome this challenge and to improve the health status and quality of life of patients.

Numerous studies have shown that epigenetic drugs can induce tumor suppressor gene expression, making cancer sensitive to chemotherapeutic drugs again (100-102). Of note, TGCT tumorigenesis is closely associated with epigenetic and drug resistance mechanisms. Therefore, epigenetic drugs may be a viable treatment option for TGCT treatment (103-105). Numerous agents have been tested in TGCT cell lines and animal models with promising results (103,106-108). DNA methylation is a key epigenetic regulatory mechanism that silences gene expression by adding methyl groups to promoter regions of genes through the action of DNA methyltransferase (DNMT). This phenomenon often leads to loss of tumor suppressor gene function (109-112). DNA methylation is mainly mediated by DNMT1, whereas DNA remethylation is mainly mediated by DNMT3A and DNMT3B (113). Studies have shown that chemical DNA demethylation can restore the expression of silencing genes, e.g., using a first-generation DNMT inhibitor (DNMTI) 5-aza. The cytotoxic activity of 5-aza as a ribonucleoside is based on its wide range of effects on DNA methylation patterns, cellular transcriptomes and gene expression profiles. It can bind to DNA and RNA and is primarily used to treat myelodysplastic syndromes (114,115). A previous study demonstrated that 5-aza could contribute to decreased cisplatin resistance in a seminoma cell line (116). Oing et al (107) investigated the effects of 5-aza on two different EC cell lines (NCCIT carrying TP53 mutations and 2102Ep carrying wild-type TP53), using isogenic resistant sublines 2102EP-R and NCCIT-R to establish an acquired cisplatin resistance model system in vivo and in vitro. They found that nanomolar doses of EC cells are highly sensitive to 5-aza regardless of cisplatin sensitivity and that 5-aza at nanomolar concentrations can overcome cisplatin resistance and induce an intense and long-lasting apoptotic response in EC cells (107). DNMTIs such as 5-aza and decitabine (DAC) have been approved by the US Food and Drug Administration (FDA), and breakthroughs have been made in the treatment of hematological cancers by blocking the activity of DNMT (117). DNMTIs have also shown potential in preclinical experimental studies for the treatment of TGCT (106,118). Beyrouthy et al (118) in their experiments with cisplatin-resistant embryonic cancer cells pretreated with 5-aza, found that the drug not only affected resistant cells, but also through the induction of p53 target genes or through corresponding promoter methylation to the expression of other genes (such as MGMT, RASSF1A and HOXA-9), to restore sensitivity to cisplatin chemotherapy. Collectively, these results suggest that 5-aza can promote apoptosis and overcome cisplatin resistance in non-cysteine germ cell tumor cells. As mentioned previously, 5-aza is a first-generation DNMTI. Notably, SGI-110, a second-generation DNMTI, has been used to treat refractory TGCTs (106). Issa et al (119) evaluated the effects of SGI-110 (a demethylating agent) on EC cells and in cisplatin-resistant non-seminoma testicular cancer animal models and found that cisplatin-resistant cells and tumors derived from EC were highly sensitive to SGI-110, which showed potential antitumor effects in EC (106). Notably, very low doses of SGI-110 as a single agent eliminated the progression of cisplatin-resistant EC tumors (106). In addition, they conducted a Phase I trial combining SGI-110 with cisplatin in patients with relapsed cisplatin-resistant TGCT (120), indicating that the combination of SGI-110 and cisplatin displayed good efficacy in patients with platinum-refractory germ cell cancer (120). Furthermore, Lobo et al (105) tested a newly synthesized flavonoid-derived compound, MLo1302, in TGCT cell lines and found that it significantly reduced tumor cell activity and induced apoptosis and cell cycle arrest by reducing DNMT expression.

In addition to DNMTIs, histone deacetylase inhibitors (HDACIs) related to histone modifications have also shown promising results in preclinical studies (103,121,122). Lobo et al (103) used FDA-approved HDACIs, Belinostat and Panobinostat, and observed that these agents reduced cell viability and induced cell cycle arrest and apoptosis in cisplatin-sensitive and cisplatin-resistant TGCT cell lines. In addition, Steinemann et al (122,123) used a novel dual-mode compound called animacroxam, which was conjugated to an HDAC-inhibiting component and a cytoskeletal-disrupting component, to show significant anti-proliferation, cell cycle arrest and apoptosis-inducing effects in TGCT cell lines, and no nonspecific cytotoxicity was observed. Of note, animacroxam also reduced glucose uptake in TGCT and inhibited the expression of glycolytic enzymes, leading to a collapse in glycolytic energy production (122,123). Nettersheim et al (124) used the HDACI romidepsin and found that it was highly toxic to cisplatin-resistant TGCT cells. They also showed that combined treatment with the glucocorticoid dexamethasone further enhanced the expression of romidepsin effector factors and reduced TGCT cell activity more significantly than monotherapy (124). Notably, the bromodomain inhibitor JQ1, which acts by interfering with the function of bromodomain and extra terminal (BET) (125), increased apoptosis and growth arrest in TGCT cells, reduced tumor size and vascular density in mice and was more effective in combination with romidepsin (108). Burmeister et al (126) treated cisplatin-resistant TGCT cells with dual HDAC and BET inhibitors, resulting in decreased cell viability and impairment of the cell cycle. Furthermore, in the TGCT xenograft model, the dual inhibitor significantly reduced the tumor burden.

Other targeted agents have also shown potential for the treatment of cisplatin-resistant TGCT. TLS protects cells against DNA damage. Recently, Lengert et al (127) found that TLS inhibitors may overcome the resistance of TGCTs to cisplatin, and the targets can be diverse. MG-132 (carbobenzoxyl-L-leucyl-L-leucyl-L-leucine) is a peptide aldehyde that effectively blocks the proteolytic activity of the 26S proteasome complex and prevents TLS in human cancer cells but not in normal cells (128). However, the exact mechanism through which MG-132 inhibits TLS remains elusive. However, MG-132 showed significant cytotoxicity in cisplatin-resistant TGCT cell lines in the nanomolar range (127). In addition, Hasibeder et al (129) demonstrated the potential of the phytoestrogens Belamcanda chinensis extract and tectorigenin to inhibit TGCT.

Combination therapy is a promising therapeutic strategy. For cisplatin-resistant TGCTs, triple therapy with gemcitabine, oxaliplatin and paclitaxel is the standard of care (130). In addition, the combination of palbociclib and cisplatin greatly reduced TGCT cell viability in vitro and in zebrafish embryos and the drug combination also played a positive role in cell recovery after toxic damage (51). In addition, it has been reported that inhibitors specifically targeting PARP, MDM2 or AKT/mTOR combined with cisplatin have been shown to successfully overcome cisplatin resistance, which was also demonstrated in patient-derived xenograft models (10). Therefore, these agents may be promising candidates for the treatment of TGCT, particularly cisplatin-resistant TGCT; however, their application in patients with TGCT still requires further clinical studies and validation (Table II).

Cisplatin is the first-line drug for TGCT; therefore, it is necessary to study the mechanisms of its sensitivity and resistance.

In the mechanism of TGCT sensitivity to cisplatin, the expression of the NER essential proteins XPA, XPF and ERCC1 was significantly associated with TGCT sensitivity to cisplatin. In addition, defects in DSB homologous recombination repair also lead to high sensitivity of TGCT cell lines to cisplatin. Furthermore, the expression of the apoptosis-related factor p53 and its related proteins such as NOXA and OCT4 is also closely related to cisplatin sensitivity.

In the mechanism of TGCT resistance to cisplatin, studies have shown that cisplatin-resistant cells are mainly associated with the apoptotic pathway (24), DNA methylation, tumor cell cycle regulation-related factors and DNA damage repair pathways.

In the apoptotic signaling pathway, MDM2 can mediate the ubiquitination and further degradation of p53, thereby inhibiting apoptosis and cell cycle arrest and promoting cisplatin resistance (24). PDGF phosphorylates PDGFR, thereby promoting the PDGFR/PI3K/AKT signaling pathway, resulting in enhanced AKT phosphorylation. Enhanced phosphorylation of p21 and MDM2 by AKT promotes the degradation of p53. In addition, decreased expression of OCT4 could result in a decrease in NOXA, which inhibits apoptosis and promotes cisplatin resistance (23). By contrast, inhibition of FOXO3 expression leads to the suppression of PUMA and FAS, thereby inhibiting the process of programmed death (35-37). Similarly, upregulation of IGFR promotes AKT phosphorylation and inhibits apoptosis (49). Regarding the mechanism of tumor cell cycle-related regulatory factors, p-AKT could lead to the enhancement of p21 phosphorylation, which leads to an increase in cytoplasmic p21, and high expression of cytoplasmic p21 could inhibit cell apoptosis and promote drug resistance. Cytoplasmic p21 is a new therapeutic target or biomarker that also plays an important role in chemotherapy resistance in other tumor cells. For instance, nuclear protein 1 contributes to chemotherapy resistance in breast cancer by inducing Akt-mediated phosphorylation and the subsequent cytoplasmic relocalization of p21(131). In addition, cytoplasmic p21 is a potential predictor of cisplatin sensitivity in ovarian cancer (132). Therefore, whether it is possible to regain sensitivity to chemotherapy by inhibiting the expression of p21 in the cytoplasm or altering its localization needs to be further investigated.

In addition, studies have shown that increased methylation can promote cisplatin resistance, and VIRMA, METTL3 and IGF2BP1 can promote the upregulation of m⁶A and thus promote drug resistance, whereas DNMT1 inhibitors 5-aza and SGI-110 can inhibit methylation and lead to cisplatin sensitivity, which can be used as a potential treatment for resistant TGCTs. However, its practicability requires further investigation. In the DNA repair system, 53BP1 was far from HR. Upregulation of HR and downregulation of NHEJ were positively correlated with cisplatin resistance, and the downregulation of 53BP1 and DNA-PKCs may promote cisplatin resistance (3,133).

In the TCGT NT2/D1 cell line, the CDK4/6 inhibitor palbocinib enhanced CDK4/6 protein expression in NT2/D1 cells, thereby inhibiting the cytotoxic effect of cisplatin and inducing cisplatin resistance by influencing tumor cell cycle regulation-related factors (51). However, the specific mechanisms involved require further investigation. For instance, whether an increase in CDK4/6 protein expression directly leads to cisplatin resistance or whether other factors lead to cisplatin resistance requires further exploration (51). In addition, palbocinb reduced RB phosphorylation in TGCTs, thus promoting apoptosis and inhibiting cisplatin resistance. Whether RB phosphorylation is inhibited in NT2/D1 cells to promote cisplatin resistance should be investigated in the future (134). MG-132 has been proven to promote apoptosis in refractory TGCTs and it can treat resistant TGCTs alone or in combination with cisplatin; however, its mechanism of action remains elusive (127). Study has shown that p53 promotes apoptosis in cisplatin-resistant TGCTs. Notably, MDM2-mediated ubiquitination and degradation of p53 can result in the downregulation of p53 and inhibition of apoptosis, contributing to cisplatin resistance (135). Therefore, it is speculated that MG-132, a proteasome inhibitor, promotes apoptosis by inhibiting the ubiquitination and degradation of p53, thereby inhibiting cisplatin resistance, which requires further investigation (136).

In DNA repair systems, REV7 can bind to 53BP1 to promote NHEJ, and depletion of REV7 leads to the accumulation of DNA double-strand breaks and activation of cell apoptosis, thereby inhibiting cisplatin resistance (137). However, study has shown that cisplatin resistance is negatively related to NHEJ and that REV7 upregulation could promote NHEJ, which inhibits cisplatin resistance (71). However, study has shown that REV7 depletion can also inhibit cisplatin resistance, and whether this is a contradiction requires further discussion. In addition, the DNA repair-related factor XPA is highly expressed in TGCTs, and its association with cisplatin resistance in TGCTs should be further studied (138). In cisplatin-sensitive and drug-resistant TGCT cells, there were differential expressions of the L1TD1, GAL, NTF3, NANOG, WNT6, POU5F1, IGFBP2, IGFBP7, PCP4, ZFP42, ID2, TRIB3 and SLC40A1 genes (139). However, whether their corresponding proteins are differentially expressed has not yet been studied, and whether these proteins can be used as potential therapeutic targets needs to be studied (134).

In cisplatin-resistant TGCT therapy, studies have shown that methylation is associated with cisplatin-resistant TGCT; therefore, DNMTIs such as 5-aza and DAC SGI-110 can restore sensitivity to cisplatin-resistant chemotherapy by inhibiting methylation. In addition, drugs such as HDACIs, belinostat and panobinostat can exert therapeutic effects by inducing apoptosis. Furthermore, TLS inhibitors such as MG-132 and lomidoxin can enhance the toxic effects on cisplatin-resistant TGCT cells. Although the efficacy of certain drugs has been demonstrated in patient-derived xenotransplantation models, their use in patients with TGCT still requires further clinical research and validation.

In addition, similar to the mechanism of cisplatin resistance in other tumors, such as in ovarian yolk sac tumor (oYST), long-term cisplatin exposure resulted in a 7-fold increase in the IC₅₀ concentration in resistant cells. Drug-resistant cells showed significantly increased expression of prominin-1 (CD133), ATP-binding cassette subfamily G member 2 and aldehyde dehydrogenase 3 subtype A1 (ALDH3A1), decreased gene and promoter methylation, increased expression of ALDH1A3 and increased overall ALDH enzyme activity (140). The study showed that high expression of ALDH1A3 and elevated activity of ALDH were detected in cisplatin-resistant TCGT cells, which is identical to oYST (141). However, in TGCTs, reduced methylation promotes cisplatin resistance, in contrast to that in oYST. However, the role of other factors in the cisplatin resistance mechanism of TGCTs remains elusive and may be considered a potential factor. In medulloblastoma, anti-miR-31 enhances cisplatin resistance through the PI3K/AKT and NF-κB pathways (142). Similarly, in glioblastoma, circPTN (a newly discovered circular RNA) contributes to cisplatin resistance through PI3K/AKT signaling via the miR-3-542p/PIK3R3 pathway (143). The PI3K/AKT pathway also affects cisplatin resistance in TGCTs; therefore, it is worth exploring whether miR-31 and circPTN also play a role in cisplatin resistance in TGCTs. A previous study showed that PD-L1 is highly expressed in TGCTs (144). Studies have shown that in non-small cell lung cancer (NSCLC), NSCLC cell-derived programmed death-ligand 1 (PD-L1)-containing exosomes promote cell stemness and increase the resistance of NSCLC cells to cisplatin (145), and high expression of PD-L1 in cancer cells drives immune-independent, cell-intrinsic functions, leading to resistance to DNA-damaging therapies, such as cisplatin (146). In NSCLC, the increase in CDK5 could lead to the downregulation of F-box only protein 22, which leads to the downregulation of PD-L1 ubiquitination, and the increase in PD-L1 levels leads to cisplatin resistance (146). In medulloblastoma, CDK5 and PD-L1 also play a role in cisplatin resistance; therefore, the role of CDK5 in cisplatin resistance of TGCTs is also worth discussing (146).