Abstract
In this issue of Thyroid, Piekielko-Witkowska et al. (6) report a new aspect of D1 biology that would be “a first” in the field of deiodinases—the potential use of D1 mRNA splicing variants as a molecular fingerprint for kidney tumors. These authors cloned and identified multiple D1 mRNA transcripts, the expression of which were dramatically reduced in samples of human clear cell renal cell carcinoma (ccRCC), the most common subtype of renal cancer. Remarkably, three new D1 mRNA variants were cloned exclusively from cancer samples, supporting their potential use as tumor markers.
Not all nucleotide sequences present in genes are encoded in the mature mRNA molecule. Different gene transcripts or mRNA molecules are generated by gene splicing, a process in which nucleotide sequences (introns) are removed from the primary RNA transcript with the remaining sequences (exons) forming the mature mRNA molecule. Through alternative splicing, the primary transcript encoded by a single gene can lead to different mature mRNA molecules that can generate multiple functional proteins. This process is observed in about 40–60% of the human genes and constitutes an important source of protein diversity, whereby a single gene can increase its coding capacity to generate proteins that are structurally and functionally distinct (7). Gene splicing is mediated by a protein–RNA complex called the spliceosome and involves the alternative processing of donor and acceptor splice sites of the RNA primary transcripts (8). This results in the loss of exons(s) (exon skipping) or the presence of intronic sequences (intron retention) in the mRNA. Accumulating evidence indicates that splicing events play an essential role in tumorigenesis. Coordinated alternative splicing in cancerous cells have been shown to regulate the post-transcriptional expression of many genes, such as tumor suppression genes, leading to the expression of oncogenic splice variants that are capable of modifying cancer development and progression (9). The recent discovery that the spliceosome is a target for novel compounds with anticancer activity has energized the field and opened multiple new therapeutic avenues (10).
Although alternative splicing gives rise to an inactive human D2 molecule of higher molecular weight (11) and a tissue- and species-specific spliced intron has been described for the D2 mRNA (12), the splicing pattern uncovered by Piekielko-Witkowska et al. (6) for DIO1 is particularly interesting. The D1-encoding DIO1 gene consists of four exons with the selenocystein-encoding UGA codon located in exon 2 and the UAG STOP codon and the SECIS element in exon 4 (13,14). In their study, Piekielko- Witkowska et al. report the cloning of seven previously unreported D1 transcripts and, most importantly, three of them (nos. 9, 10, and 11) were detected exclusively in the renal tumor samples (6). These transcripts encode putative D1 proteins of 115, 111, and 14 amino acids, respectively. Each of these lacks the exon 2 region that encodes the enzyme's active center, and they are partially frame shifted. If translated, these truncated proteins are sure to yield inactive D1 molecules. This correlates with earlier studies of this group demonstrating lowered D1 activity in ccRCC (15). The molecular basis of ccRCC is not fully understood, and a complex system of molecular markers is in place for differential diagnosis, staging, and monitoring of the prognosis and recurrence of the disease (16 –19). Thus, there is hope that these newly identified tumor-specific D1 splice variants will add significantly to the presently available ccRCC markers; future studies should clarify how they could be used to aid differential diagnosis and/or monitor tumor progression.
Looking at these and other data one can't help but wonder what are the mechanisms underlying the frequent disregulation of deiodinase expression in tumoral cells, often expressed at levels much higher than those observed in the normal or untransformed tissue or cell. For example, it is accepted now that D2 is expressed in human and rodent skeletal muscle samples at very low levels (20,21), but levels that are three to four orders of magnitude higher are observed in rhabdomyosarcoma (RMS)-13 cells (22). A similar situation is seen in normal lung tissue, in the mesothelioma cell line MSTO-211 (23), and in other tumors that express the type 3 iodothyronine deiodinase (D3) (24), an enzyme that inactivates thyroid hormone. Of note, the finding by Piekielko-Witkowska et al. (6) indicates that disregulation of deiodinase expression in tumors could be much broader than previously known, given that attention has been focused on enzyme activity and not on inactive splice variants. Piekielko-Witkowska et al. (6) suggest that their findings could be related to changes in expression of splicing factors SF2/ASF (splicing factor 2/alternative splicing factor) and hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), but more work is needed to better understand this relationship and to ascertain if other deiodinase genes could also be affected by these factors as well.
Given that in some settings thyroid hormone is known to reduce cell proliferation, does deiodinase disregulation in tumor cells affect thyroid hormone signaling and possibly interfere with tumor progression? This question stems from the modern paradigm that thyroid hormone signaling can be regulated relatively independent of plasma thyroid hormone levels, in a time- and tissue-specific fashion by the deiodinases (25). In fact, in the developing chicken growth plate, loss of D2 activity via sonic hedgehog (Shh)-induced D2 ubiquitination has been linked to parathyroid hormone-related peptide (PTHrP) expression and chondrocyte proliferation (26). These studies prompted investigators to look into other settings in which Shh is active, such as the basal cell carcinomas (BCCs), the most common human malignancy characterized by a constitutively active Shh pathway (27). In these cells, the Shh pathway induces a loss of D2 activity and concomitant expression of D3, increasing cyclin D1 and cell proliferation (28). Remarkably, BCC xenografts in nude mice grew fivefold less as a result of D3 knockdown, offering a potential new therapeutic approach to BCC.
It is clear that we need a better understanding of how deiodinase genes and their splice variants are regulated in tumor cells and also further insight on how their expression affects cell proliferation and differentiation and consequently tumor development. Work in this area is already leading to exciting breakthroughs and could potential unveil novel therapeutic approaches to stop tumor progression.