The tumor suppressor gene TRC8/RNF139 is disrupted by a constitutional balanced translocation t(8;22)(q24.13;q11.21) in a young girl with dysgerminoma

Dysgerminomas are rare ovarian tumors most common in adolescent women, representing 5–10% of all malignant ovarian tumors in the first two decades of life. They arise from germ cells within the gonad and represent the ovarian counterpart of testicular seminoma[1]. Histologically and clinically dysgerminomas are classified as type II Germ Cell Tumors (GCT).

The female germ cells enter meiosis during intrauterine development (11–12 weeks of gestation), whereas for the male germ cells this only happens after the onset of puberty. This could explain the difference in incidence of the type II gonadal GCTs between females and males and the median age of clinical manifestation. In fact, the number of target cells (PGCs/gonocytes) for initiation is significantly lower in females compared with males [2].

RNF139/TRC8 (NM_007218; henceforth, TRC8) is a potential tumor suppressor gene (TSG) with similarity to PTCH [3]. Mutations in PTCH result in predisposition to basal cell carcinoma and medulloblastoma, while its inactivation leads to cell proliferation [4‐6]. TRC8 was identified in a family with the constitutional translocation t(3;8)(p14.2;q24.1), and hereditary renal cell carcinoma (RCC) and thyroid cancer[3, 7, 8]. Recently, a second, independent, family with hereditary kidney cancer was discovered that carries a cytogenetically indistinguishable translocation and a VHL mutation. [9]. Secondary loss of the wild type TRC8 allele was observed in a subset of tumor cells, consistent with tumor suppressor function. TRC8 is a membrane-bound E3 ubiquitin ligase that inhibits the growth of cells in a ubiquitylation-dependent manner [10]. Interestingly, its level and stability are modulated by cholesterol and it interacts with components of the lipid homeostatic machinery, especially INSIG and the lipid-regulated transcription factors, SREBP1/2 [11]. A frequent genetic alteration in both hereditary and sporadic RCC is mutation of VHL, which encodes the targeting subunit of another ubiquitin ligase complex responsible for degrading hypoxia inducible factors, HIF1/2 alpha. TRC8 interacts with VHL and, in fruit flies, knockdown of either gene led to a similar embryonic phenotype [12]. Thus alterations in TRC8 have the potential to affect cellular growth control and possibly its linkage to lipid homeostasis and/or hypoxia responses.

TRC8 has been identified as a target of the Translin (TSN) gene in postmeiotic male germ cells, and testis is known to contain higher levels of TRC8 mRNA than other tissues[3]. TSN, formerly known as testis brain-RNA binding protein, is found in the cytoplasm and functions as a posttranscriptional regulator of a group of genes transcribed by the transcription factor CREM-tau. Cho et al.[13] identified four new TSN target mRNAs encoding diazepam-binding inhibitor-like 5, arylsulfatase A, a tetratricopeptide repeat structure-containing protein, and TRC8. These observations suggest an important function for TRC8 in germ cells. Here, we describe a paternally inherited balanced translocation t(8;22) in a proposita with dysgerminoma. The breakpoints of the translocation are located within the LCR-B low copy repeat on chromosome 22q11.21, containing the palindromic AT-rich repeat (PATRR) involved in recurrent and non-recurrent translocations and in an AT-rich sequence inside intron 1 of the TRC8 tumor-suppressor gene at 8q24.13. This translocation raises the possibility that disruption of TRC8 may contribute to development of the proposita's dysgerminoma.

We report a girl who developed a dysgerminoma at the age of 10 years and carries a balanced translocation t(8;22)(q24.13;q11.21). The breakpoints of our proposita may be identical to those recently described by Gotter et al., [20]. In that case, the proband showed an unbalanced translocation with a supernumerary der(22) derived as a result of 3:1 meiotic malsegregation of the paternal balanced translocation 46, XY, t(8;22)(q24.13;q11.21). The proband showed an abnormal phenotype, while his father was apparently normal. Interestingly, a breakpoint within the first intron of the TRC8 gene has been described as an inherited rearrangement associated with renal cell carcinoma in two unrelated families[3, 9, 21, 22]. In our case, we have localized the breakpoint to a 700 bp region in intron 1 of the TCR8 gene, containing an AluSp and a 180 bp (AT)n simple repeat. The breakpoint is probably positioned, as in the family described by Gotter et al.[20], in a palindrome within the simple repeat. In our case and in Gotter et al.[20], the breakpoint on chromosome 22 lies within the PATRR on 22q11. The analysis of many unrelated t(11;22)(q23;q11) cases revealed that the breakpoints occur within palindromic PATRRs on 11q23 and 22q11 (PATRR11 and PATRR22). The majority of the breakpoints, including the case described by Gotter et al.[20], are localized to the centre of the PATRRs, suggesting that the palindrome mid-point is susceptible to double-strand breaks (DSBs); this would occur as the result of a cruciform extrusion promoting the DSBs that initiate stabilizing rearrangements or recombination events[23]. The PATRR22 appears to be highly unstable in the human genome[24]. Palindrome-mediated genomic instability contributes to a variety of genome rearrangements including not only constitutional translocations, but also large germ line deletions[25]. Cancer-related gross chromosomal rearrangements and genomic amplification are also reported to be associated with palindromic DNA[26, 27]. Recent studies have produced a detailed map showing that palindromes tend to cluster at specific regions, some of which undergo gene amplification[28]. Individual tumors seem to have a non-random distribution of palindromes in their genomes, and a subset of palindromic loci is associated with gene amplification. Inverted repeats or palindromes are known to generate hairpins or cruciform structures, facilitated by intrastrand pairing of complementary single-strand DNA sequences assembled in the lagging strand during DNA replication. A double-strand break (DSB) introduced in these hairpins can result in deletions or in either inter- or intrachromosomal recombination events mediated by the homologous recombination machinery[26, 28].

TRC8 encodes an endoplasmic reticulum-resident E3-ubiquitin ligase with multiple transmembrane segments. TRC8 contains a C-terminal RING-H2 domain shown to catalyze in vitro ubiquitylation reactions[10, 11]. In Drosophila and mammalian cells, over-expressed TRC8 suppressed growth[11, 12, 29]. In HEK293 cells, TRC8 induced G2/M arrest and accumulation of sub-G1 cells indicative of apoptosis[11]. These effects were dependent upon an ubiquitylation-competent RING domain, suggesting that specific substrates were critical to growth inhibition. Tumor formation in a nude mouse model was also inhibited by TRC8 in a RING-dependent manner. TRC8 contains a putative sterol-sensing domain (SSD) which in other proteins confers sensitivity to sterols, affecting either stability or trafficking between membrane compartments[3]. Moreover, knock-down of endogenous TRC8 increased levels of the lipid homeostasis transcription factors, the SREBPs and their target genes, including enzymes of cholesterol and lipid metabolism [11]. However, these effects were only evident in cells simultaneously depleted of sterols, since cholesterol-replete cells contain very low amounts of TRC8. Importantly, expression of activated SREBP-1 partially restored the growth of TRC8-inhibited cells [11], indicating that changes in lipid regulation underlie some of the growth inhibitory actions of this tumor suppressor. Since these data suggested that TRC8 activity linked growth control to the cholesterol/lipid homeostasis pathway, we analyzed two SREBP target genes, HMGCR and SCD1, in control and tumor tissue (Fig. 6). However, expression levels were depressed compared to normal ovarian tissues. This could be due to lack of proper control tissues (germ cells as opposed to whole ovary) or perhaps more likely to the presence of plentiful lipids, since the effects of TRC8 loss are most readily observed following sterol-depletion [11].

Interestingly, TRC8 expression appeared down-regulated in tumor tissue (Fig. 6) while TRC8 protein was not detectable in tumor tissue by IHC (Fig. 1). This was despite the absence of any evidence for loss, genetic alteration or promoter methylation of the remaining TRC8 allele. We surmise that additional mechanisms reducing TRC8 expression may be involved in this dysgerminoma. For example, the 3' UTR of TRC8 contains target sites for multiple microRNAs (miR), several of which are highly conserved among human, mouse, rat, dog and chicken (miR-218, -101, -27, -128, -375). In addition, the coding sites for some of these miRs are found on the aneuploid chromosomes observed in this tumor (i.e., miR-218-1 is on chromosome 4). It is possible that miRNAs are mediating the additional loss of expression for this gene. Recently, the stabilization of specific miRNAs by Translin in male germ cells has been reported[30].

Translin, formerly known as testis brain-RNA binding protein (TB-RBP), is both a DNA-binding and RNA-binding protein. As a DNA binding protein, it interacts with the breakpoint junctions of chromosomal translocations[31, 32]. Translin also mediates intracellular and intercellular mRNA transport, possibly controlling the temporal and spatial translation of specific mRNAs in postmeiotic germ cells [33‐35]. Kasai et al. [36] have proposed that human Translin may play a role in DNA recombination, repair, and metabolism, but deficiencies in somatic recombination or DNA repair are not readily detectable in mice lacking Tsn [37]. However, the marked increase in apoptosis of spermatocytes in Tsn-null mice suggests a role for TSN during meiosis, perhaps functioning as a post-transcriptional regulator in spermatocytes [37]. Recently, Cho et al. [13] identified four meiotic mRNA targets of Translin, one of which is TRC8. They hypothesized that disruption of the precise regulation of TRC8 may cause the growth retardation and the increased apoptosis in pachytene spermatocytes observed in Tsn-null mice [37]. To our knowledge, there are no studies of the role of the TSN gene in female meiosis.

TRC8 is a potent TSG involved in different tissue-specific pathways. Its haploinsufficiency may facilitate the development of CC-RCC in association with VHL mutations, or otherwise lead to increased risk for other types of tumor. Any role in dysgeminoma may relate to its interaction with translin. We envision a model whereby one copy of TRC8 is disrupted by palindrome-mediated translocation (step 1), then, in a second step, translin, miRNA, or another yet undiscovered mechanism leads to further loss of TCR8 expression, setting the stage for deregulated proliferation.