The P53 pathway is the most important cellular pathway to maintain genomic and cellular integrity, both in embryonic and non-embryonic cells. Stress signals induce its activation, initiating autophagy or cell cycle arrest to enable DNA repair. The persistence of these signals causes either senescence or apoptosis. Over 50% of all solid tumors harbor mutations in TP53 that inactivate the pathway. The remaining cancers are suggested to harbor mutations in genes that regulate the P53 pathway such as its inhibitors Mouse Double Minute 2 and 4 (MDM2 and MDM4, respectively). Many reviews have already been dedicated to P53, MDM2, and MDM4, while this review additionally focuses on the other factors that can deregulate P53 signaling. We discuss that P14ARF (ARF) functions as a negative regulator of MDM2, explaining the frequent loss of ARF detected in cancers. The long non-coding RNA Antisense Non-coding RNA in the INK4 Locus (ANRIL) is encoded on the same locus as ARF, inhibiting ARF expression, thus contributing to the process of tumorigenesis. Mutations in tripartite motif (TRIM) proteins deregulate P53 signaling through their ubiquitin ligase activity. Several microRNAs (miRNAs) inactivate the P53 pathway through inhibition of translation. CCCTC-binding factor (CTCF) maintains an open chromatin structure at the TP53 locus, explaining its inactivation of CTCF during tumorigenesis. P21, a downstream effector of P53, has been found to be deregulated in different tumor types. This review provides a comprehensive overview of these factors that are known to deregulate the P53 pathway in both somatic and embryonic cells, as well as their malignant counterparts (i.e., somatic and germ cell tumors). It provides insights into which aspects still need to be unraveled to grasp their contribution to tumorigenesis, putatively leading to novel targets for effective cancer therapies.

Germ cell tumors (GCTs) are considered to be highly curable; however, there are major differences in the outcomes related to histology and anatomical localization. GCTs originating from the testis are, overall, sensitive to platinum-based chemotherapy, whereas GCTs originating from the mediastinum show a worse response, which remains largely unexplained. Here, we address the differences among GCTs from two different anatomical locations (testicular versus mediastinal/extragonadal), with a specific focus on the role of the P53 pathway. It was recently shown that GCTs with TP53 mutations most often localize to the mediastinum. To elucidate the underlying mechanism, TP53 knock-out lines were generated in cisplatin-sensitive and -resistant clones of the representative 2102Ep cell line (wild-type TP53 testicular GCT) and NCCIT cell line (hemizygously mutated TP53, mutant TP53 mediastinal GCT). The full knock-out of TP53 in 2102Ep and resistant NCCIT resulted in an increase in cisplatin resistance, suggesting a contributing role for P53, even in NCCIT, in which P53 had been reported to be non-functional. In conclusion, these results suggest that TP53 mutations contribute to the cisplatin-resistant phenotype of mediastinal GCTs and, therefore, are a potential candidate for targeted treatment. This knowledge provides a novel model system to elucidate the underlying mechanism of clinical behavior and possible alternative treatment of the TP53 mutant and mediastinal GCTs.

PURPOSE Cisplatin is the main systemic treatment modality for male type II germ cell tumors (GCTs). Although generally very effective, 5%-10% of patients suffer from cisplatin-resistant disease. Identification of the driving mechanisms of resistance will enable improved risk stratification and development of alternative treatments. METHODS We developed and characterized cisplatin-resistant GCT cell line models and compared their molecular characteristics with patient samples with cisplatin resistance and/or a poor clinical outcome. Subsequently, the association between the overlapping genetic features and clinical data was assessed. Finally, we used Cox regression to determine the prognostic relevance of these features within the currently used risk classification. RESULTS Gain of chromosome 3p25.3 was detected in all cisplatin-resistant cell lines, and copy number of this region correlated with the level of resistance (R = 0.96, P = 1.5e-04). Gain of this region was detected at low frequencies in primary tumors and at higher frequencies in relapsed and/or cisplatin-resistant tumors. Chromosome 3p25.3 gain was associated with shorter progression-free survival and overall survival, with the strongest association observed in nonseminomas excluding pure teratomas. 3p25.3 gain was more frequently observed in tumors with yolk sac tumor histology and predicted adverse outcome independent of the International Germ Cell Cancer Collaborative Group risk classification and the presence of TP53/ MDM2 alterations. CONCLUSION On the basis of both in vitro analyses and clinical data, we found 3p25.3 to be strongly associated with cisplatin resistance and poor clinical outcome in male type II GCTs. Using genomic profiling, 3p25.3 status could help to improve risk stratification in male patients with type II GCT. Further characterization of this locus and underlying mechanisms of resistance is warranted to guide development of novel treatment approaches for cisplatin-resistant disease.

MicroRNAs (miRNAs) are short, non-coding RNAs involved in translation regulation. Dysregulation has been identified in cancer cells. miRNAs can be secreted and detectable in body fluids; therefore, they are potential liquid-biopsy biomarkers. The miR-371a-3 cluster members are an example, monitoring the presence of malignant germ cell tumors based on patient serum/plasma analyses. However, a large variety of isolation techniques on sample types (serum vs. plasma) are reported, hampering interstudy comparisons. Therefore, we analyzed the impact of using the miRNeasy Serum/Plasma Kit (cell-free total RNA purification) Qiagen extraction kit and the TaqMan anti-miRNA bead-capture procedure of ThermoFisher for miRNA isolation. Ten normal male matched serum and plasma samples and seventeen testicular germ cell tumor patient serum samples were investigated. The Qiagen kit requires a higher input volume (200 µL vs. 50 µL), resulting in higher sensitivity. Serum and plasma comparison demonstrated high similarity in miRNA levels. Titration experiments showed that the bead-capture procedure is superior in cases of lower starting volumes (<100 µL). This study highlights the strengths and limitations of two different isolation protocols, relevant for in vivo analysis with small starting volumes. In summary, miRNA detection levels results varied little between plasma and serum, whereas for low volumes the bead capture isolation method is preferable.