Effect of BRAF^V600E mutation on transcription and post-transcriptional regulation in a papillary thyroid carcinoma model

Papillary thyroid carcinoma (PTC) is the most commonly occurring thyroid malignancy accounting for approximately 80% of cases. Genetically PTC is defined by several alterations which cause abnormal activation of the mitogen-activated protein kinase (MAPK) pathway, the most prevalent being point mutations in the intracellular signalling kinase BRAF [1, 2]. An activating mutation in BRAF (V600E) leads to constitutive activation and hence de-regulation of MAPK pathway. Several groups have reported that PTCs harbouring mutations in BRAF have more aggressive properties, present more often with extra-thyroidal invasion and also at a more advanced stage. Histological features such as the classic PTC architecture and tall cell features are also associated with the BRAF oncogene [3, 4].

Despite this range of knowledge thyroid carcinoma still poses a significant diagnostic challenge and understanding of the complete range of genes and pathways involved in its pathogenesis is incomplete. Until recently biomarker discovery has concentrated largely on protein coding RNAs in a quest to identify cancer associated genes involved in thyroid carcinogenesis [5–7]. The recent discovery however that a large group of non-coding RNAs called microRNAs (miRNAs) may potentially play a role in cancer has led to a proliferation of miRNA related research of late including the publication of several key reviews [8–12]. These small non-coding RNAs constitute a novel class of gene regulators that function by negatively regulating gene expression by targeting mRNAs for cleavage or translational repression. Several recent studies on the regulatory functions of miRNAs suggest that they play critical roles in central processes such as development, cell proliferation and differentiation, stress resistance, metabolism and apoptosis all of which are involved in tumourigenesis [13, 15]. Not surprisingly aberrant miRNA expression has been implicated in cancer development and a hypothesis has emerged suggesting that tumours may each have a discrete "miRNA signature" [16–20]. Differential expression of miRNAs between malignant tissue and normal tissue and between different types of tumour, has been shown in several key studies, indicating that miRNAs are determinants of clinical diagnostic and prognostic significance [21, 22].

Recently several groups have utilised a range of techniques most notably northern blot analysis, cloning and oligonucleotide microarrays, in order to analyse miRNA expression profiles in different cancer types [23–26]. In this study a novel stem-looped TaqMan^® RT-PCR method was used to measure the expression levels of a panel of 160 mature miRNAs in thyroid cell lines containing BRAF mutation.

microRNAs (miRNAs) are now recognized as an additional layer of post-transcriptional control that must be considered in order to understand the complexity of gene expression in humans. miRNAs are now known as a novel class of gene regulators that are involved in cancer related processes and deregulation or aberrant expression of miRNAs may contribute to human diseases, including cancer. Calin et al showed down-regulation of miRNA-15 and miRNA-16 in a majority of chronic lymphatic leukaemia (CLL). Altered miRNA expression has also been reported in breast cancer, glioblastoma, lung cancer and colorectal cancer [29–35].

This study utilized a novel stem-looped TaqMan RT-PCR method to measure the expression levels of 160 mature miRNAs in a collection of thyroid cell lines. This method offers several distinct advantages over conventional miRNA detection methods including a higher sensitivity and specificity and a fast and simple methodology. The aim of this study was to correlate BRAF mutant status with an associated miRNA profile, with a view to elaborating the downstream regulatory effect of BRAF mutation.

The data showed a set of differentially expressed miRNAs in cell lines with BRAF V600E mutation compared to a normal thyroid cell line (Table 1). There were 15 overexpressed miRNAs and 23 underexpressed miRNAs in these cell lines compared to the normal cell line. Of these miRNAs listed in table 1 only a few have been previously associated with other cancers. One of which is miR-127, that showed down-regulation in BRAF V600E mutated cell lines. Human miR-127 is embedded in a CpG island and is silenced in several human cancers including bladder, prostate and colon cancer and has been suggested to play a role as a possible tumour suppressor gene [33]. miR-127 also an example of the potential utility of miRNA targeted therapy in cancer. Saito et al demonstrated that induction of miR-127 by with 5-aza-20-deoxycytidine and 4-phenylbutyric acid, which inhibits DNA methylation and histone deacetylase respectively, reduces expression of the oncogene BCL6 in bladder cancers [33]. Its utility as a potential cancer target has yet to be explored in other cancers however this suggests the possibility of direct targeting of miRNAs that are amplified or upregulated in patient tumours. Two other miRNAs listed in table 2 have also been described in other cancers. These include miR-200b and miR-141 which have been shown to be highly overexpressed in malignant cholangiocytes and in colon carcinoma. [18]. This suggests the possibility of miR-200b and miR-141 down-regulating possible tumour suppressor genes. Of particular interest in this study is the downregulation of miR-323 and miR-302b in BRAF mutated cell line. miRBASE target database predicted that the BRAF transcript has binding sites for miR-323 and miR-302b to potentially bind and down- regulate BRAF expression.

A number of miRNAs exhibited a difference in expression level between KAT10 cell line and normal but did not show a significant change in the BRAF transfected cell lines and normal. miRNAs specific to the PTC cell line with native mutant BRAF included, mir-10a [overexpressed with a fold change of 95 in KAT10] mir-23b [downregulated with a fold change of 2949] and mir-154 [downregulated with a fold change of 142]. It is possible these miRNAs may play a role in a later stage in tumour formation and progression; given the transfected cell line had only been exposed to mutant BRAF for a short period (3 passages) unlike KAT10 which represents a genuine PTC which has evolved with BRAT mutant as an intrinsic component. Alternatively, the difference may reflect the accumulation of several genetic insults in the process of tumourigenesis.

Recent studies in miRNA deregulation PTC found an aberrant miRNA expression profile in PTCs compared to normal thyroid tissues [36, 37]. In particular, a significant increase in mir-222, mir-221, mir-146. Our study did not find any of these miRNAs to be significantly overexpressed in the cell lines examined. miR-222 was actually found to be downregulated in the BRAF mutated cell lines. Another miRNA these two groups found to be upregulated albeit less significantly was miR-213, our study also found miR-213 to be upregulated in BRAF mutated cell lines [25 fold]. These differences may be due to the different experimental approaches; Huiling et al and Pallante et al used miRNA microarrays. They also used different chemistry and fresh tissue samples; also the RET and BRAF status of each PTC case was not disclosed.

Prediction of miRNA targets is an immediate challenge in miRNA research. Several computational approaches have been implemented for miRNA gene prediction using methods based on sequence conservation and/or structural similarity. To overcome the limitations of target prediction three of these databases MiRBase, TARGETSCAN and PICTAR were used in this study to predict possible targets of the differentially expressed miRNAs associated with BRAF mutated (Table 2). Examples of other programs developed for target identification include RNAhybrid, RNA calibrate and RNAeffective. Only a few target mRNAs have been experimentally proven and studied in vitro and not all the prediction algorithms yield overlapping lists therefore the lists may contain several false positives that may fit the criteria but do not interact with the miRNA in vitro. To overcome this, results were intersected from these databases to identify the genes commonly predicted by at least two of the algorithms. The results of the potential targets of several differentially expressed miRNAs are displayed in table 2.

It is reasonable to expect that targets of downregulated miRNAs include oncogenes or genes encoding proteins with potential oncogenic function. Indeed among putative targets, several genes with potential oncogenic functions were found include RAB35, ERBB4, MAP3K3, VASP6, KIT, FOSB, NOTCH1. Upregulated miRNAs are purported to interact with tumour suppressor genes. Those identified from this study include MAD 4 and ST6. Several extracellular membrane molecules were also identified including; myotubulin, MAD1A, Cadherins. In general overexpression of an miRNA would be associated with downregulation of its target miRNA.

Interestingly among these potential gene targets are a number of genes that have implications in thyroid carcinoma progression such as genes involved in thyroid function, cell cycle control, cell signalling, MAPKKK cascade, retinoic acid receptors, cell adhesion and cell structure associated genes. Therefore, it seems plausible that this subset of miRNAs may be important in the progression of thyroid carcinoma by working together or independently to regulate genes involved in thyroid tumourigenesis

Our group has also previously examined gene expression levels in these same cell lines. This analysis was done by evaluating genome-wide gene expression levels in BRAF mutated cell lines and matched normal thyroid cell lines using the Applied Biosystems Gene Expression Arrays. Treatment comparisons were preformed resulting in lists of differentially expressed genes (Figure 3). These gene lists were uploaded into PANTHER (Protein ANalysis THrough Evolutionary Relationships) classification system and the software statistically compared them to a reference list to look for under and over-represented molecular functions and biological process. Analysis revealed a set of genes associated with mutant BRAF expression which are involved in processes such as apoptosis, chromosomal instability, invasiveness and metastasis and proliferation. Other genes identified were shown to be involved in the insulin-like growth factor pathway (including (insulin-like growth factor binding protein) IGFBP4, (insulin receptor substrate) IRSI, GRB14, PLAG1 and IGF2) and the Wnt signaling pathway suggesting their importance in BRAF signalling.

Up and down regulation of cell adhesion and chromosome remodelling molecules were also shown. BRAF mutation has previously been shown to induce chromosome instability and decrease cell- cell interactions [38] and our data further corroborates the involvement of these processes in papillary thyroid carcinoma. Not surprisingly genes involved in the MAPK pathway, of which BRAF is an integral part, showed an increase in expression. Among these genes were DDIT, DUSP6, TFPI-2, Seladin-1, MAP3K12, and GRB14. These results provided an insight into the different pathways through which BRAF exerts its oncogenic effect and emphasised the importance of the MAPK and Wnt signalling pathways in thyroid tumour formation.

In summary this study revealed a set of miRNAs associated with expression of mutant BRAF^V600E oncogene in PTC cell lines when compared to a normal thyroid cell line. The BRAF oncogene may influence miRNA expression directly or indirectly by altering the expression patterns of genes, such as transcription factors, involved in tumour progression or indeed the genes involved in the biogenesis and synthesis of miRNAs such as those from the RISC complex and miRNA machinery. These miRNAs associated with BRAF oncogene as listed in Table 1 may contribute to tumour formation by regulating genes involved in the MAPK pathway in which BRAF plays a role.

This data suggests the potential utility of miRNA expression patterns as a molecular screen for PTC to use as a diagnostic adjuvant to already established morphological criteria. A recent study by this group examined the effect the ret/PTC 1 oncogene has on miRNA expression in PTC cell lines. ret/PTC 1 rearrangements are generally associated with classic papillary architecture and indolent behaviour in thyroid carcinoma. This study showed a distinct pattern of miRNAs associated with the ret/PTC 1 oncogene when compared to a normal thyroid cell lines [39].

Although the exact function of many miRNAs remains to be elucidated it is clear that miRNAs potentially have a broad influence over several diverse genetic pathways and that their deregulation is likely to contribute to disease including cancer. An exciting future prospect is that the miRNA patterns associated with a particular tumour may ultimately be of diagnostic and prognostic significance and contribute to the understanding of the molecular pathogenesis and gene regulatory processes of cancer.