Key points

Introduction

Testicular cancer represents the most common form of cancer in men of Caucasian ancestry aged 15 to 40 years. In addition to the different histologic growth patterns (isolated vs mixed histology), testicular cancer can be divided into seminomatous or nonseminomatous tumors, which are clinically distinct in terms of therapy, surveillance requirements, and prognosis. Nonseminomatous germ cell tumors (GCTs) contain 1, 2, or several proportions of the following major histologic subtypes: embryonal carcinoma, yolk sac tumor, choriocarcinoma, or teratoma.

These microscopic differences are mirrored by differences in biological phenotype, in terms of metastatic potential, uptake of radioisotopes in medical imaging, drug sensitivity, and radioresistance. The cellular and molecular mechanisms that underpin each histologic subtype are also distinct. In general, patients with testicular cancer are present with symptoms of local disease or less commonly with signs of metastatic spread (eg, respiratory symptoms in case of lung metastases or localized pain in case of bone metastases).

Serum-based tumor markers (including alpha-fetoprotein [AFP], human chorionic gonadotropin [HCG], and lactate dehydrogenase [LDH]) have an established role in diagnosis and prognostic risk stratification of patients with testicular cancer. However, these biomarkers are limited by relatively high false-negativity rates (only 60% through all testicular cancer subtypes can be detected) at the time of presentation coupled by the potential for false-positive detection associated with nonspecific alterations related to other diseases (eg, AFP increase associated with liver diseases). In particular, the proportion of patients with seminomas and embryonal carcinomas presenting with elevated tumor markers is low, as AFP is predominantly associated with yolk sac tumors and HCG with choriocarcinoma histology.

It is therefore clear that improved detection and risk stratification in early-stage disease, and the use of biomarkers to guide decision making around the use of adjuvant therapy and local surgical resection would have considerable clinical utility.

The biological importance of microRNAs (miRNAs) was identified in the early 1990s, when several groups reported the regulation of developmental processes in worms by small non–protein-coding RNA molecules. MiRNAs are small (approximately 20 nucleotides in length) RNA molecules that do not translate into peptides or proteins, but regulate the expression of larger messenger RNAs (mRNAs) by sequence complementarity, and block protein translation or accelerate mRNA degradation.

In the past 20 years, this class of small regulatory RNAs has been associated with the pathogenesis of cancer and the mechanisms that govern metastatic spread. They have been implicated in all of the hallmark processes of cancer, including but not restricted to genome instability, cancer stem cells, and response to anticancer therapies. In the context of cancer initiation and progression, miRNAs can either act as tumor suppressors or oncogenic drivers, depending on the regulated genes, the cellular context, and the cancer type. In this review, we provide an overview of the molecular role of miRNAs specifically in the context of testicular cancer and we discuss their potential utility as diagnostic and prognostic biomarkers in the clinical setting.

Introduction

Testicular cancer represents the most common form of cancer in men of Caucasian ancestry aged 15 to 40 years. In addition to the different histologic growth patterns (isolated vs mixed histology), testicular cancer can be divided into seminomatous or nonseminomatous tumors, which are clinically distinct in terms of therapy, surveillance requirements, and prognosis. Nonseminomatous germ cell tumors (GCTs) contain 1, 2, or several proportions of the following major histologic subtypes: embryonal carcinoma, yolk sac tumor, choriocarcinoma, or teratoma.

These microscopic differences are mirrored by differences in biological phenotype, in terms of metastatic potential, uptake of radioisotopes in medical imaging, drug sensitivity, and radioresistance. The cellular and molecular mechanisms that underpin each histologic subtype are also distinct. In general, patients with testicular cancer are present with symptoms of local disease or less commonly with signs of metastatic spread (eg, respiratory symptoms in case of lung metastases or localized pain in case of bone metastases).

Serum-based tumor markers (including alpha-fetoprotein [AFP], human chorionic gonadotropin [HCG], and lactate dehydrogenase [LDH]) have an established role in diagnosis and prognostic risk stratification of patients with testicular cancer. However, these biomarkers are limited by relatively high false-negativity rates (only 60% through all testicular cancer subtypes can be detected) at the time of presentation coupled by the potential for false-positive detection associated with nonspecific alterations related to other diseases (eg, AFP increase associated with liver diseases). In particular, the proportion of patients with seminomas and embryonal carcinomas presenting with elevated tumor markers is low, as AFP is predominantly associated with yolk sac tumors and HCG with choriocarcinoma histology.

It is therefore clear that improved detection and risk stratification in early-stage disease, and the use of biomarkers to guide decision making around the use of adjuvant therapy and local surgical resection would have considerable clinical utility.

The biological importance of microRNAs (miRNAs) was identified in the early 1990s, when several groups reported the regulation of developmental processes in worms by small non–protein-coding RNA molecules. MiRNAs are small (approximately 20 nucleotides in length) RNA molecules that do not translate into peptides or proteins, but regulate the expression of larger messenger RNAs (mRNAs) by sequence complementarity, and block protein translation or accelerate mRNA degradation.

In the past 20 years, this class of small regulatory RNAs has been associated with the pathogenesis of cancer and the mechanisms that govern metastatic spread. They have been implicated in all of the hallmark processes of cancer, including but not restricted to genome instability, cancer stem cells, and response to anticancer therapies. In the context of cancer initiation and progression, miRNAs can either act as tumor suppressors or oncogenic drivers, depending on the regulated genes, the cellular context, and the cancer type. In this review, we provide an overview of the molecular role of miRNAs specifically in the context of testicular cancer and we discuss their potential utility as diagnostic and prognostic biomarkers in the clinical setting.

Molecular aspects of microRNA biogenesis

Human miRNAs originated from the cell nucleus, and are encoded by genes commonly assembled in clusters. On activation by various transcription factors, RNA polymerase II transcribes the gene information to a primary miRNA (“pri-miRNA”) molecule, which in turn is processed into a precursor form (“pre-miRNA”) by the ribonuclease (RNase) III enzyme Drosha in association with the binding protein Pasha (DGCR8). The hairpin structure of pre-miRNA allows the nuclear export into the cytoplasm by the transporter exportin-5 and its associated protein GTPase Ran. In the cytoplasm, the RNase III endonuclease Dicer cleaves the hairpin loop and produces small, 19 to 22 nucleotides long miRNAs. Mature miRNAs in the cytoplasm then interact with complementary target mRNAs within the RNA-induced silencing complex, and regulate mRNA stability or protein translation. Fig. 1 summarizes the process of miRNA biogenesis. It has been estimated that more than one-third of human protein-coding genes are controlled by miRNAs.

Illustration of miRNA biogenesis.

MicroRNAs in testicular cancer

Current hypotheses assume that GCTs develop from noninvasive intratubular germ cell neoplasia unclassified (IGCNU), also known as carcinoma in situ (CIS), cells that are for whatever reasons prevented from maturation. Under normal conditions, germ cells migrate after embryogenesis into the genital ridge to become gonocytes and later on spermatogonia. Particular underlying and yet unknown triggers lead to the development of CIS cells. They often persist until puberty and then start to proliferate, presumably as a consequence of endocrine signals, which might result in the progression to seminomatous or nonseminomatous tumors. CIS cells mimic embryonic stem cells in their pluripotent properties, which enable the conversion to multiple lineages, including germ cell lineage, embryonal carcinoma, somatic lineage teratoma, and yolk sac tumors or choriocarcinoma ( Fig. 2 ).

MiRNA involvement in different types of GCTs.

By controlling protein-coding genes involved in cancer development, miRNAs may affect every aspect of testicular cancer pathogenesis from initiation to progression to metastatic spread. Early in 2006, Voorhoeve and colleagues identified miRNA-372 and miRNA-373 as oncogenic drivers in testicular cancer by using high-throughput screening with a library of miRNA expression vectors. They found that expression levels of miR-372 and miR-373 determine the fate of proliferation and apoptosis under the stress induced by rat sarcoma viral oncogene homolog (RAS) in the wild-type p53 background. Further analysis showed that these 2 miRNAs directly target large tumor suppressor kinase 2 (LATS2) to abrogate the cell cycle arrest initiated by p53-p21-CDK signaling, and consequently increase proliferation of testicular germ cells. Similarly, Gillis and colleagues reported in 2007 that the miRNA-371 to 373 cluster, including miR-371a, miR-371b, miR-372, and miR373, are significantly upregulated in type II and III (ie, testicular germ cell tumors of adolescents and adults can be subdivided into seminomas and nonseminomas, all referred to as type II GCTs, whereas the other type [type III germ cell tumor] is the so-called spermatocytic seminoma) human GCTs. They also noted that the expression of the hsa-miR-302 to 367 cluster, including miR-302a, miR-302b, miR-302c, miR-302d, miR-302a, and hsa-miR-367, are downregulated on differentiation, suggesting functional involvement of these miRNAs in controlling stem cell differentiation in GCTs. Echoing these findings, Barroso-del Jesus and colleagues surmised that the miR-302 to 367 cluster is transcriptionally activated by Oct3/4, Sox2, and Nanog, and potentially regulates stemness in embryonic stem cells. Furthermore, Palmer and colleagues showed that the miR-371 to 373 and miR-302 clusters are overexpressed in all types of malignant GCTs, regardless of histology, tumor site, or patient age. The same group subsequently performed a global analysis of mRNAs that are downregulated in testicular cancers, and discovered an enrichment of mRNAs containing 3′UTR sequence of GCACTT, which is complementary to the seed sequence (AAGUGC) contained within the previously discussed miRNAs. This enrichment of miRNA-targeted mRNAs provides strong evidence on the functional relevance of aberrant miRNA expression in testicular cancer.

Crucially, testicular cancers appear to express a unique and consistent miRNA signature, that is, the enhanced expression of the miR-371 to 373 cluster located at chromosome 19q13. This indicates an intrinsic link between the expressions of this miRNA cluster and an essential characteristic of testicular cancers. Zhou and colleagues examined the mechanisms underlying this unique upregulation pattern of miRNAs in germ cell cancers. They identified a feedback loop between the miR-371 to 373 cluster and the Wnt/β-catenin signaling pathway, whereby miR-372 and miR-373, transcriptionally activated by Wnt signaling, inhibit DKK1, a key antagonist of Wnt/β-catenin signaling. Wnt signaling plays critical roles in regulating cell stemness, and thus it appears that the miR-371 to 373 cluster contributes to the maintenance of stem cell status. In support of this hypothesis, one study showed that the miR-371 to 373 cluster functions as a self-renewal miRNA to induce and maintain the pluripotent state of stem cells. A further study showed higher expression of miR-371 to 373 cluster miRNAs in stem cells than differentiated cells, which again supports an association between miR-371 to 373 miRNAs and stem cell status in testicular cancer.

Other miRNAs also have been reported to associate with testicular cancer. Several reports from the Chan group showed that miR-199a is downregulated in testicular cancer, largely due to hypermethylation at the miR-199a promoter region, and overexpression of miR-199a inhibits testicular cancer growth and metastasis by targeting the embryonal carcinoma antigen, podocalyxin-like protein 1 (PODXL). Male infertility has been associated with increased risk of developing testicular cancer. Lian and colleagues proposed miR-383 as a link between male infertility and testicular cancer, based on the findings of reduced miR-383 expression in testicular tissues of infertile patients, and in vitro effect of miR-383 on germ cell proliferation, probably via regulating the tumor suppressor gene, interferon regulator factor1 (IRF1). However, unlike the miR-371 to 373 cluster and the miR-302 to 367 cluster that are consistently linked with testicular cancer by multiple groups, the significance of miR-199a and miR-383 in this type of cancer remains to be verified by further studies.