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Human Molecular Genetics, 2003, Vol. 12, No. 14 1661-1669
DOI: 10.1093/hmg/ddg178
© 2003 Oxford University Press

Upregulation of the transcription factor TFEB in t(6;11)(p21;q13)-positive renal cell carcinomas due to promoter substitution

Roland P. Kuiper1,*, Marga Schepens1, José Thijssen1, Martien van Asseldonk1, Eva van den Berg2, Julia Bridge3, Ed Schuuring4, Eric F.P.M. Schoenmakers1 and Ad Geurts van Kessel1

1Department of Human Genetics, University Medical Center Nijmegen, PO Box 9101, 6500 HB, Nijmegen, The Netherlands, 2Department of Medical Genetics, University of Groningen, Antonius Deusinglaan 4, 9713 AW Groningen, The Netherlands, 3Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NB 68198, USA and 4Department of Pathology, University of Groningen, Hanzeplein 1, 9700 RB, Groningen, The Netherlands

Received March 4, 2003; Revised April 29, 2003; Accepted May 9, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The MITF/TFE subfamily of basic helix–loop–helix leucine-zipper (bHLH-LZ) transcription factors consists of four closely related members, TFE3, TFEB, TFEC and MITF, which can form both homo- and heterodimers. Previously, we demonstrated that in t(X;1)(p11;q21)-positive renal cell carcinomas (RCCs), the TFE3 gene on the X chromosome is disrupted and fused to the PRCC gene on chromosome 1. Here we show that in t(6;11)(p21;q13)-positive RCCs the TFEB gene on chromosome 6 is fused to the Alpha gene on chromosome 11. The AlphaTFEB fusion gene appears to contain all coding exons of the TFEB gene linked to 5' upstream regulatory sequences of the Alpha gene. Quantitative PCR analysis revealed that AlphaTFEB mRNA levels are up to 60-fold upregulated in primary tumor cells as compared with wild-type TFEB mRNA levels in normal kidney samples, resulting in a dramatic upregulation of TFEB protein levels. Additional transfection studies revealed that the TFEB protein encoded by the AlphaTFEB fusion gene is efficiently targeted to the nucleus. Based on these results we conclude that the RCC-associated t(6;11)(p21;q13) translocation leads to a dramatic transcriptional and translational upregulation of TFEB due to promoter substitution, thereby severely unbalancing the nuclear ratios of the MITF/TFE subfamily members. We speculate that this imbalance may lead to changes in the expression of downstream target genes, ultimately resulting in the development of RCC. Moreover, since this is the second MITF/TFE transcription factor that is involved in RCC development, our findings point towards a concept in which this bHLH-LZ subfamily may play a critical role in the regulation of (aberrant) renal cellular growth.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Renal cell carcinomas (RCCs) represent ~85% of all renal tumors in adults (1–3), and comprise a heterogeneous group of tumors. According to the Heidelberg classification (4) they can be divided into benign and malignant parenchymal neoplasms, of which the malignant neoplasms are predominantly represented by common (non-papillary) RCCs (~75% of all RCCs) and papillary (chromophilic) RCCs (~10% of all RCCs). The various RCC subtypes exhibit recurrent cytogenetic anomalies (4,5), including recurrent chromosomal translocations. Such chromosomal translocations are thought to be causally related to tumor development (6). In a subgroup of papillary RCCs, for example, a t(X;1)(p11;q21) is consistently observed (7). Through this translocation, the TFE3 gene on the X chromosome is fused to the PRCC gene on chromosome 1 (8). TFE3 is a member of the MITF/TFE subfamily of basic helix–loop–helix leucine-zipper (bHLH-LZ) transcription factors (also referred to as MiT subfamily) (9), which also includes TFEB, TFEC and MITF. Recent studies have indicated that the PRCC-TFE3 chimeric genes encode aberrant transcription factors with transforming capacities (10,11). In addition, it was found that PRCC can interact with the mitotic spindle checkpoint component MAD2B, and that expression of the PRCCTFE3 chimeric protein leads to abrogation of this cell cycle checkpoint in a dominant-negative fashion (3,12).

Another recurrent chromosomal translocation, t(6;11)(p21;q13), has been encountered in a subgroup of RCCs with pleiomorphic histologic features. These RCCs occur at relatively young ages and may constitute a distinct clinical entity (13,14). Here, we report that through this translocation the bHLH-LZ transcription factor TFEB gene on chromosome 6 is fused to the anonymous non protein-encoding Alpha gene on chromosome 11. Our data indicate that this gene fusion results in a dramatic increase in the expression of TFEB mRNA and protein levels in the tumor cells due to promoter substitution. As such, the TFEB gene may act as a novel RCC-related proto-oncogene.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Positional cloning of the t(6;11)(p21;q13) breakpoint on 11q13
Previously, cells from t(6;11)(p21;q13)-positive tumor 1 (Table 1) were fused with Chinese hamster A3 cells to generate somatic cell hybrids segregating the translocation-associated chromosomes (15). By using these somatic cell hybrids, the 11q13 breakpoint could be mapped within a ~200 kb genomic interval between the markers D11S4933 and D11S546. To locate the position of the breakpoints on chromosomes 6 and 11 in further detail, we selected hybrid cell lines on their presence or absence of normal chromosomes 6 and 11, and derivative chromosomes der(6) and der(11). Of these four chromosomes, clone MT1D only contained the der(6), clone MT4C the der(11) and a normal chromosome 6, and in clone MT8 both the normal chromosomes 6 and 11 were present. Subsequently, primer sets were selected between the markers D11S4933 and D11S546 and used for PCR analysis on genomic DNAs extracted from the somatic cell hybrids and human control samples. After several rounds of PCR we were able to locate the breakpoint on a 839 bp fragment within a gene with unknown function, named Alpha. This gene is ubiquitously expressed and encodes a single transcript of ~7.5 kb (16). We found no indications for RNA splicing, suggesting that the transcript is encoded by a single exon. No open reading frames (ORFs) longer than 55 amino acids could be detected within the transcript, and none of the putative ORF-deduced peptides showed homologies with known proteins and/or protein domains in any of the non-redundant protein databases (SWISS-PROT/TrEMBL). Therefore, we assume that the Alpha gene does not encode a functional protein.


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  		Table 1. Characteristics of RCC patients and tumors
 
To confirm the location of the breakpoint within the 839 bp Alpha fragment, this DNA fragment was radioactively labeled and hybridized to Southern blots containing HinfI-digested DNAs extracted from the somatic cell hybrids MT1D, MT4C and MT8. Both MT1D and MT4C showed shifts in restriction fragment sizes from 2.3 kb (MT8) to ~700 bp (MT1D) and ~2.1 kb (MT4C), respectively (not shown), thus confirming that the 11q13-associated breakpoints in both translocation derivatives are indeed located within the 839 bp Alpha fragment.

Localization of the 6p21 breakpoint within the TFEB gene
In order to identify the 6p21 translocation breakpoint-associated sequences, we set out to positionally clone a t(6;11) chimeric genomic fragment using vectorette PCR. Therefore, a library of linker-ligated HinfI-digested fragments from somatic cell hybrid MT4C (which includes the shifted ~2.1-kb HinfI fragment) was amplified using a universal vectorette primer and an Alpha gene-specific primer positioned just centromeric to the breakpoint on der(11). Following hemi-nested PCR, a ~700 bp PCR fragment was isolated, cloned and sequenced. Analysis of the obtained sequence revealed the presence of a stretch of 171 bp from the Alpha gene (including the Alpha-specific vectorette nested primer), followed by a 436 bp sequence corresponding to the TFEB gene on 6p21. Based on sequence information present in the data base, the breakpoint could be located within intron 1 of the TFEB gene, 96 bp upstream from exon 2 (Fig. 1A). The orientation of the two (fusion) genes on the der(11) chromosome appeared to be in a head-to-tail fashion. An in-frame ATG codon, preceded by a perfect Kozak consensus sequence, was found to be present within exon 2 of TFEB, indicating that the AlphaTFEB fusion transcript may encode a full-length TFEB protein. To confirm the positions of the genomic breakpoints on both der(6) and der(11), and to characterize these breakpoints in further detail, PCR analyses were performed on genomic DNAs extracted from somatic cell hybrids MT1D, MT4C and MT8, and normal human control (C) cells. Forward and reverse primers were selected on each side of the breakpoints on chromosomes 6 and 11, allowing the amplification of four (wild-type and fusion) fragments (Fig. 1B). As expected, in those samples that contained the normal chromosome 11 (MT8 and C) or the normal chromosome 6 (MT4C, MT8 and C), we were able to amplify fragments corresponding to the wild-type Alpha and TFEB genes, respectively. Furthermore, TFEBAlpha fusion fragments were only amplified from clone MT1D and AlphaTFEB fusion fragments were exclusively found in clone MT4C. Subsequent cloning and sequencing of these latter fragments confirmed their chimeric nature, and revealed that the translocation in tumor 1 coincides with a loss of 15 bp on 6p21 and an insertion of 39 bp on 11q13.



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  		Figure 1. The RCC-associated t(6;11)(p21;q13) translocation fuses the Alpha gene on chromosome 11 with the TFEB gene on chromosome 6. (A) Schematic representation of the wild-type Alpha and TFEB genes and the AlphaTFEB fusion gene present in tumor 1. The positions of the breakpoints in the tumors 1, 2 and 3 in the Alpha gene are indicated, as well as the position of the 839-bp Alpha breakpoint fragment. In the TFEB gene, the three breakpoints cluster in a 140-bp breakpoint cluster region (bcr), 95 bp upstream of exon 2. The putative ATG start codon, preserved in exon 2 of the AlphaTFEB fusion gene, and the TAG stop codon are indicated. Accession numbers of nucleotide sequences around the breakpoint regions are AJ535461; AJ535462; AJ535463; AJ535464; AJ535465; AJ535466. (B) PCR analysis on genomic DNAs from somatic cell hybrids MT1D, MT4C and MT8, and control human genomic DNA (C), using breakpoint-flanking forward (AlphaRT1f or TFEBintron1f) and reverse (AlphaRT1r or TFEBexon2r) primers in four possible combinations.

 
To further substantiate these findings, we analyzed two independent, clinically and histologically similar, renal cell carcinomas that harbor a t(6;11)(p21;q13) translocation (tumors 2 and 3; Table 1) by PCR. Also from these tumors, AlphaTFEB and TFEBAlpha genomic fragments could be amplified. Subsequent sequence analysis confirmed their chimeric nature and, again, allowed the exact location of the breakpoints. These appeared to be close to, but distinct from, each other and those observed in tumor 1, again leaving the protein coding exons intact (Fig. 1A). No base pair losses and/or insertions were detected at the breakpoints in these two tumors.

Both AlphaTFEB and TFEBAlpha are expressed in tumor cells
Northern blots containing total RNA from normal kidney, somatic cell hybrids MT8, MT1D, and MT4C, and renal tumor 3 were hybridized with probes comprising TFEB gene sequences (exons 2–9), and Alpha gene sequences (5' of the breakpoint in tumor 1), respectively. Wild-type ~7.5 kb Alpha transcripts could readily be detected in normal kidney, hybrid cell line MT8 and tumor 3, whereas wild-type TFEB transcripts (with an expected size of ~2.3 kb) could not be detected in any of these samples, even after prolonged exposures (Fig. 2A, and data not shown). Apparently, the expression of TFEB in these cells is below the detection level of our northern blots. However, in hybrid cell line MT4C and in renal tumor 3, mRNAs with sizes of ~3.2 and ~3.5 kb, respectively, could be detected using either the Alpha probe or the TFEB probe (Fig. 2A). This result indicates that AlphaTFEB fusion transcripts are indeed expressed in t(6;11)(p21;q13)-positive cells. An additional ~2.5 kb TFEB-related transcript is observed in hybrid cell line MT4C (Fig. 2A). This transcript was not detected with the Alpha probe. Two additional Alpha-related transcripts of ~3.0 and 4.3 kb, only present in tumor 3, were not detected by the TFEB probe. These transcripts may represent tumor-specific alternative splice variants. For detection of reciprocal TFEBAlpha fusion transcripts a mixture of two non-overlapping probes, each comprising ~800 bp of the Alpha gene sequence upstream of the breakpoint in tumor 1, was used. As in case of full-length TFEB transcripts, the expression of TFEBAlpha fusion transcripts was too low to be detected on northern blots (not shown). To reveal whether TFEBAlpha fusion transcripts are nevertheless expressed at low levels, we performed RT–PCR on RNAs extracted from the three somatic cell hybrids and from the primary tumors. As expected, wild-type TFEB transcripts were detected in hybrids MT8, MT4C and the primary tumors, and Alpha transcripts were found to be present in hybrid MT8 and the primary tumors (Fig. 2B). In addition, TFEBAlpha and AlphaTFEB fusion transcripts were detected in hybrids MT1D and MT4C, respectively, and both fusion transcripts were present in all three primary tumors (Fig. 2B). The sizes of the RT–PCR products differed between the hybrids and the tumors, completely in line with the observed differences in the positions of the respective breakpoints (as depicted in Fig. 1A). Thus, in all three t(6;11)(p21;q13)-positive RCCs both the normal TFEB and Alpha genes and the two reciprocal fusion genes, AlphaTFEB and TFEBAlpha, are expressed.



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  		Figure 2. Expression of Alpha, TFEB, and the fusion transcripts AlphaTFEB and TFEBAlpha in normal kidney, somatic cell hybrids MT8, MT1D and MT4C, and t(6;11)(p21;q13)-positive renal tumors. (A) northern blot analysis of Alpha- and TFEB-related transcripts using TFEB (exons 2–9) and Alpha (5' region) specific probes. (B) RT–PCR analysis on RNA from normal kidney, somatic cell hybrids, and the three t(6;11)-positive tumors. For tumors 1 and 3, primer sets used were AlphaRT1f-AlphaRT1r and TFEBexon1f-TFEBexon2/3r (two upper panels), and the AlphaRT1f-TFEBexon2/3r and TFEBintron1f-AlphaRT1r primer sets (two lower panels). For tumor 2, these were AlphaRT1f-AlphaRT1r, TFEBexon1f-TFEBexon2/3r, AlphaRT2f-TFEBintron1r and TFEBintron1f-AlphaRT2r, respectively.

 
Increased TFEB expression due to promoter substitution
The above northern blot data indicate that the t(6;11)(p21;q13) translocation results in a strong expression of TFEB-encoding transcripts. In order to quantify this expression, we performed real-time RT–PCR analysis on RNAs extracted from t(6;11)(p21;q13)-positive tumors 1 and 3 and normal kidney cells using a primer set located within the helix–loop–helix coding domain of TFEB. To be able to correct for variations in TFEB expression in the kidney, renal cortex RNAs from 15 unrelated individuals were collected and used independently for real-time RT–PCR. By doing so, we measured a ~30-fold (tumor 1) and ~60-fold (tumor 3) increase in expression of TFEB transcripts in the tumors compared with the normal renal cortex samples (Fig. 3), whereas the Alpha gene expression levels did not change significantly (not shown). Since the bHLH-LZ subfamily members TFE3, TFEC and MITF are considered to be dimerization partners of TFEB (9), we also determined their expression levels relative to that of TFEB in both tumor cells and normal renal cortex cells. Each of the four MITF/TFE subfamily members was expressed in the normal cells at approximately the same level with only a little variation (Fig. 3). However, in the tumor cells the TFEB transcripts were most abundant, representing 91% (tumor 1) to 96% (tumor 3) of the total amount of MITF/TFE transcripts present, compared to ~22% in normal renal cortex samples. The absolute expression levels of TFE3, TFEC and MITF were not significantly influenced by the high expression levels of AlphaTFEB (Fig. 3).



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  		Figure 3. Quantitative real-time RT–PCR of MITF/TFE transcripts in normal kidney samples and renal tumors 1 and 3, respectively, using subfamily member-specific primer sets within the helix–loop–helix domains of the TFEB, TFE3, TFEC and MITF genes. Data were obtained from duplo experiments, and reflected to levels obtained from normal renal cortex RNA obtained from 15 unrelated individuals.

 
With an in-frame ATG codon present in exon 2 of the TFEB gene, the AlphaTFEB fusion transcript most likely encodes a full-length TFEB protein. In order to establish whether such a protein is expressed in the tumor cells, we performed western blot analysis on proteins extracted from normal human kidney cells, and renal tumors 1 and 3, using affinity-purified rabbit polyclonal anti-TFEB-N antibody. Hardly any TFEB protein could be detected in the normal human kidney cells and hybrid cell line MT8. However, in both tumors 1 and 3 a strong ~65 kDa band was observed (Fig. 4A). Moreover, TFEB protein expressed by COS1 cells transfected with either wild-type TFEB- or AlphaTFEB cDNA comigrated with this 65 kDa band (Fig. 4A), strongly suggesting that the AlphaTFEB transcript indeed encodes full-length TFEB protein. Based on these results, we conclude that the high expression of AlphaTFEB fusion RNA in the tumor cells leads to a similarly high expression of TFEB protein in these cells.



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  		Figure 4. Expression of AlphaTFEB-derived TFEB protein. (A) Western blot analysis of TFEB in renal tumors 1 and 3, and transfected COS1 cells. The unaffected part of the tumor 1 nephrectomy was used as a reference sample (first lane). COS1 cells were transiently transfected with a construct containing full-length TFEB cDNA (pSG8/TFEB), AlphaTFEB fusion cDNA (pSG8/{alpha}TFEB), or with empty vector (pSG8). Equal amounts of total protein were loaded in each lane (~30 µg). Proteins were visualized by chemiluminescence. The protein sizes are marked in kiloDalton. (B) Steady-state immunofluorescence localization of wild-type TFEB- and AlphaTFEB-derived protein in pSG8/TFEB- (left panel) and pSG8/{alpha}TFEB-transfected (right panel) COS1 cells, respectively. FITC and DAPI staining images were merged to illustrate the predominant nuclear localization of TFEB for both constructs.

 
For efficient targeting to the nucleus, the TFEB protein is equipped with a nuclear localization signal, which is postulated to be located C-terminally of the helix–loop–helix domain in the TFEB protein. To determine whether the AlphaTFEB-encoded TFEB protein is targeted to the nucleus similar to wild-type TFEB, we analyzed TFEB- and AlphaTFEB-transfected COS1 cells by immunofluorescence, using the anti-TFEB-N antibody. In both transfections, positive cells showed similar, strong nuclear staining patterns (Fig. 4B). Occasionally, the transfected cells showed a weak cytoplasmic staining. Neither the pre-immune serum nor the anti-TFEB-N serum pre-incubated with synthetic peptide yielded a fluorescent signal, illustrating the specificity of the antibody (not shown). Thus, like wild-type TFEB, also the AlphaTFEB-encoded TFEB protein appears to be efficiently targeted to the nucleus.

Taken together, we conclude that in three independent RCCs carrying the recurrent t(6;11)(p21;q13) translocation, the TFEB gene on 6p21 is highly activated due to promoter substitution, resulting in a dramatic increase in the absolute and relative levels of TFEB transcript and nuclear protein in the tumor cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Recently, a novel subgroup of human RCC with specific clinical and histologic characteristics and a recurrent t(6;11)(p21;q13) has been identified (13–15). Usually, this translocation is observed in children or young adults, although a 42-year-old patient has been reported as well (13). Typically, this translocation is the sole cytogenetic anomaly present. However, an additional case of t(6;11)(p21;q13)-positive RCC has been described that exhibits a complex karyotype (17). We have identified the genes involved in the t(6;11)(p21;q13) translocation, i.e. the Alpha gene on chromosome 11 and the TFEB gene on chromosome 6. As a result of the translocation, these two genes are fused. Importantly, the breakpoints in the TFEB gene were located just upstream of exon 2 in all three cases studied. This exon contains an in-frame ATG codon preceded by a perfect Kozak consensus sequence. This suggests that the complete coding sequence of the TFEB gene is preserved in the AlphaTFEB fusion gene, whereas its regulatory elements are exchanged for those of the more active Alpha gene promoter. Although both the AlphaTFEB and TFEBAlpha fusion genes were found to be expressed in the tumor cells, we were able to demonstrate that the AlphaTFEB mRNA expression levels were 30- to 60-fold higher compared with those of the endogenous TFEB and/or TFEBAlpha fusion mRNAs. Furthermore, we found that also the TFEB protein levels were dramatically upregulated in the tumor cells. Additional transfection experiments revealed that the AlphaTFEB-encoded TFEB protein is efficiently targeted to the nucleus. Based on these results we conclude that the bHLH-LZ transcription factor TFEB may act as a novel oncoprotein that is strongly up-regulated by the t(6;11)(p21;q13) translocation through promoter substitution.

Similar promoter substitutions have previously been reported for other translocation-associated genes, including those encoding the zinc finger transcription factor BCL6 in B-cell non-Hodgkin's lymphomas (18,19) and the zinc finger transcriptional activator PLAG1 in pleiomorphic adenomas of the salivary gland (20) and lipoblastomas (21). Both zinc finger genes fuse to distinct translocation partners, whose regulatory control elements up-regulate the mRNA and subsequent protein expression levels.

TFEB and its close relatives TFE3, TFEC and MITF, comprise a subfamily of bHLH-LZ transcription factors. They share a common structural organization and are known to form stable DNA binding homo- and heterodimers with each other, but not with bHLH-LZ transcription factors of other subfamilies (9). In vitro binding studies revealed that all possible MITF/TFE homo- and heterodimers can be formed in complexes with the Ebox consensus sequence CA[C/T]GTG (9,22–24). Additional in vivo evidence has been obtained for the presence of MITF-TFE3 heterodimers in osteoclast cell lines (25). Studies in mice, including single and double mutants of the four MITF/TFE members, have indicated that homodimeric interactions are essential for MITF/TFE function (26). It could be hypothesized that the relative expression levels of the four MITF/TFE subfamily members in the various tissues may determine which dimers are formed and, subsequently, which and/or how downstream target genes are regulated. We found that, in human kidney, the four MITF/TFE subfamily members are expressed at approximately equal levels. Although the MITF/TFE expression ratios may vary among the different cell types present within the renal cortex, our data suggest that various MITF/TFE dimers can be formed in this tissue. We speculate that the relatively high expression level of TFEB in t(6;11)(p21;q13)-positive tumor cells may change the dimeric state of the MITF/TFE proteins, leading to deregulation of the expression of downstream target genes. With >90% of the MITF/TFE proteins being TFEB, TFEB homodimers may be predominant. We are currently investigating the occurrence and nature of MITF/TFE dimers in tumor cells and whether the anticipated changes do indeed lead to a transcriptional deregulation that may trigger renal tumorigenesis.

TFEB was originally isolated from a human B-cell cDNA library using a binding sequence from the adenovirus major late promoter (27). In vitro overexpressed TFEB was also found to stimulate promoter activity of the tyrosinase related protein-1 gene, which is specifically expressed in melanocytes and pigmented retina epithelia through binding to the Ebox-related Mbox motif (28). We found that the TFEB gene is ubiquitously expressed, but relatively high levels were observed in lung, kidney, placenta and spleen (not shown). Transgenic mice have been generated carrying a mutation in the TFEB gene (TfebFcr) (29), which resulted in embryonic lethality due to defects in placental vascularization. The placental labyrinthine cells in these mice failed to express vascular endothelial growth factor (VEGF), the embryonic vasculature was unable to invade the placenta and the embryos died as a result of hypoxia. These data indicate that TFEB may be involved in the regulation of vascularization through the VEGF gene. Since none of the other MITF/TFE mutant mice showed defects in placental vascularization, TFEB may act as a homodimer in this process (26). However, we did not find any changes in VEGF expression in the TFEB overexpressing RCC tumor 3 (not shown), suggesting that down-regulation of VEGF in the TfebFcr mutant mouse placentas may be related to factors not present in these RCC cells.

Previously, we and others found that another member of the MITF/TFE subfamily of bHLH-LZ transcription factors, TFE3, is affected in a subgroup of papillary RCCs with Xp11-associated translocations or inversions (8,30–32). In papillary RCCs carrying the t(X;1)(p11;q21) translocation, the TFE3 gene is disrupted and fused to the PRCC gene on chromosome 1, leading to the formation of two fusion proteins (8). PRCC was found to interact with the mitotic checkpoint protein MAD2B, but this interaction is impaired in the PRCCTFE3 fusion protein, resulting in a dominant-negative mitotic checkpoint defect in PRCCTFE3 expressing cells (12). Furthermore, the PRCCTFE3 fusion protein, in which all domains of TFE3 necessary for transcriptional activation are retained, can bypass temperature-induced growth arrest in conditionally immortalized mouse renal proximal epithelial cells (11), and was shown to act as a three-fold better transactivator as compared to wild-type TFE3 (10). This increase in TFE3-specific transcriptional activity may lead to deregulation of downstream target genes. Since the MITF/TFE subfamily members appear to recognize Ebox sequences as homo- and/or heterodimers (9), these downstream target genes may be (partially) identical to those affected in TFEB-overexpressing t(6;11)(p21;q13)-positive RCCs. If so, this would indicate that there are parallels in the pathways that lead to tumor formation in both subgroups.

Interestingly, also the MITF and TFEC genes have been mapped to chromosomal regions that are frequently affected in RCCs, i.e. 3p13–14 and 7q31–32, respectively (4,5,33–35). It remains to be tested whether such aberrations may also lead to deregulation of these bHLH-LZ transcription factors and, subsequently, to RCC development. The MITF/TFE subfamily was found to be involved in the development of other tumor types as well. Fusions between the ASPL and TFE3 genes, for example, were observed in alveolar sarcomas of soft parts (36,37), and MITF has been found to play a role in melanoma cell growth and survival (38–40). Thus, the MITF/TFE subfamily of bHLH-LZ transcription factors seems to be involved in the development of distinct tumor types.

In conclusion, we found that through the RCC-associated t(6;11)(p21;q13) the Alpha gene on chromosome 11 is fused to the TFEB gene on chromosome 6, resulting in gene promoter substitution and a dramatic up-regulation of TFEB mRNA and protein expression levels. This up-regulation may lead to downstream target gene deregulation and, ultimately, RCC. In addition, we propose that the MITF/TFE subfamily of bHLH-LZ transcription factors may play a more general role in (renal) tumorigenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Tumor material, somatic cell hybrids, normal renal tissues, and antibodies
Three primary renal cell carcinomas displaying a t(6;11)(p21;q13) as the sole chromosomal abnormality were used in this study (Table 1). These tumors were derived from relatively young patients (14, 17 and 42 years), and were all classified as clear cell type composed of nests of polymorphous neoplastic cells with abundant clear cytoplasms, but with focal differentiation towards eosinophilic and granular (oncocytic-like) cells. A more detailed description of these cases will be published elsewhere. The generation of somatic cell hybrids through fusion of tumor 1 cells (RCCMT) with thymidine kinase-deficient Chinese hamster A3 cells was described previously (15). Hybrid clones MT1D [containing the der(6) chromosome], MT4C [containing the der(11) chromosome], and MT8 (containing both the normal chromosomes 6 and 11) were selected for this study. Renal tissues from normal kidneys were kindly provided by Dr E. Oosterwijk, Department of Urology, UMC Nijmegen, The Netherlands. A rabbit polyclonal TFEB antiserum (anti-TFEB-N) was raised against a synthetic peptide comprising 15 amino acids within the N-terminal moiety of human TFEB (HQKVREYLSETYGNK) coupled to keyhole limpet hemocyanin (Sigma Genosys, Cambridge, UK). The antibody was affinity-purified against covalently coupled synthetic peptide using AminoLink coupling (Pierce Biotechnology, Rockford, IL, USA).

Genomic PCR and Southern blot analysis
Genomic DNAs were isolated as previously described (8). The chromosome 11 breakpoint was mapped through PCR analysis using a series of STS markers flanking the breakpoint region. A 839 bp genomic PCR fragment that contains the breakpoint (obtained with primers AlphaRT-1f: 5' TAA CGC ATT TAC TAA ACG CAG ACG 3', and AlphaRT-1r: 5' TCT GTG TAG CAC CTG GGT CAG C 3') was subsequently labeled with [{alpha}-32P]dCTP by random priming and used as a probe on Southern blots, as previously described (8).

Vectorette PCR and DNA sequence analysis
A vectorette-PCR cloning procedure was used essentially as described before (41). Briefly, HinfI-digested MT4C DNA was ligated to a phosphorylated HinfI-Vectorette Top Strand Primer (VTSP-HinfI; 5' CCW GGC AAG GAG AGG ACG CTG TCT GTC GAA GGT AAG GAA CGG ACG AGA GAA GGG AGA G 3') and a phosphorylated Vectorette Bottom Strand Primer (VBSP; 5' CTC TCC CTT CTC GAA TCG TAA CCG TTC GTA CGA GAA TCG CTG TCC TCT CCT TG 3') o/n at 16°C. The resulting vectorette libraries were amplified using a Universal Primer (UVP; 5' CGA ATC GTA ACC GTT CGT ACG AGA ATC GCT 3'), and sequence specific primers AlphaVrt-der11 (5' AAC GCA TTT ACT AAA CGC AGA CGA AAA TGG AAA GA 3') and AlphaVrt-der11nest (5' ATT GGG AGT GGT AGG ATG AAA CAA TTT GGA GAA GA 3'). The latter was used in a hemi-nested PCR reaction. The PCR conditions were as follows: five cycles of 30 s at 94°C, 3 min at 70°C; five cycles of 30 s at 94°C, 3 min at 68°C; 25 cycles of 30 s at 94°C, 1 min at 66°C, 2 min at 70°C. The PCR products were separated on agarose gels, purified, subcloned into a pGemT vector (Promega, Leiden, The Netherlands), and subsequently sequenced using a Ready Reaction Dye Terminator Cycle sequencing kit (PE Applied Biosystems, Foster City, CA, USA) and an ABI 3700 automated sequencer (PE Applied Biosystems). The genomic breakpoint regions were amplified using AlphaRT-1f or AlphaRT-1r in combination with TFEBexon2r (5' AAC CCT ATG CGT GAC GCC ATG GTG G 3') or TFEBintron1f (5' CAC CTT GTG GCA GCA AAG GAG GGT GGA TAT 3'), respectively.

RNA isolation, northern blot analysis and RT–PCR
Total RNA from normal kidney and tumors 1 and 3 was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA from tumor 2 was extracted from paraffin-embedded tissue blocks. Therefore, ten 5 µm slides were deparaffinized, incubated with 200 µg/ml proteinase K for 1 h at 55°C, followed by an acidic phenol extraction and ethanol precipitation. Average size of RNA fragments obtained using this procedure was ~250 bp, and could only be used for non-quantitative RT–PCR. Northern blot analysis was performed as previously described (8). Reverse transcriptase (RT)–PCR was performed using 5 µg of total RNA and Superscript II (Life Technologies, Gaithersburg, MD, USA) according to the instructions of the manufacturer. The primers used were AlphaRT-1f, AlphaRT-1r, and TFEBexon2r (see above), AlphaRT-2f (5' TGA GAG AAA GGA CTA CAG AGC CC 3'), AlphaRT-2r (5' TCT TCG CCT TCC CGT ACT TCT GTC T 3'), TFEBexon1f (5' CGG ACA GAT TGA CCT TCA GAG CG 3'), TFEBexon2/3r (5' CTC CAG GTA GGA CTG CAC CTT CAA CAC CTC C 3'), and TFEBintron1r (5' AGA ATG ACC TGG GAC CGC ATC 3'). The TFEB-Alpha transcript could only be detected using TFEBintron1f as a forward primer, due to lack of TFEB intron 1 splicing.

Real-time quantitative PCR
Total RNA isolations and RT reactions (2.5 µg RNA in 50 µl) were performed as described above. Real-time RT–PCR was performed on a TaqMan ABI 7700 Sequence Detection System (PE Applied Biosystems) using heat-activated TaqDNA polymerase (Amplitaq Gold; PE Applied Biosystems). The reaction mixture included 200 nM of TET reporter-labeled TFE-specific TaqMan MGB probe (5' CGC TGG AAC AAG GG 3' for TFE3, TFEB and MITF, and 5' CGC TGG AAC AAA GG 3' for TFEC; PE Applied Biosystems), and 300 nM of both forward and reverse primers. The primers used were TFE3 forward (5' GGC ACT CTC ATC CCT AAG TCC AG 3') and reverse (5' GCT CCA GGG ATC GCT GC 3'), TFEB forward (5' CGC ATC AAG GAG TTG GGA AT 3') and reverse (5' CTC CAG GCG GCG AGA GT 3'), TFEC forward (5' CAC TCT TAT TCC AAA GTC TAA TGA TCC T 3') and reverse (5' TTG TAG CCA CTT GAT GTA CTC CAC T 3'), and MITF forward (5' TGA TTC CCA AGT CAA ATG ATC CA 3') and reverse (5' GCA ACT TTC GGA TAT AGT CCA CG 3'). For quantitative analysis of the data, TFEB CT -values were normalized to those of endogenous GAPDH (with use of standard TaqMan human GAPDH control reagents; PE Applied Biosystems) using the {Delta}{Delta}CT technique (42).

Western blot analysis
Proteins were extracted from tissues using Trizol (Invitrogen) according to the instructions of the manufacturer. Proteins from cell lines were extracted by sonification in lysis buffer (1% Tween-20, 1% SDS in PBS). Cell debris was removed by centrifugation. Equal amounts of protein were separated by SDS–PAGE (10%), and transferred to nitrocellulose membranes (Schleider & Schuell, Dassel, Germany). For immunodetection of TFEB, the affinity-purified rabbit polyclonal anti-TFEB-N antiserum was used in a 1 : 2500 dilution. Immunostaining was performed using chemoluminesce.

Transfection and immunofluorescence
Full-length cDNAs corresponding to the wild-type TFEB transcript, and the AlphaTFEB fusion transcript were cloned into the eukaryotic expression vector pSG8 (43). Subsequent transfection of these constructs into COS1 cells and immunofluorescence was performed as described before (10). For immunofluorescence analysis, the anti-TFEB-N antibody was used in a 1 : 300 dilution.


   ACKNOWLEDGEMENTS
 
The authors thank Professor S. Störkel for tumor material, advice and support, Dr E. Oosterwijk for normal renal tissues, and L. van der Logt and J. Kraan for technical assistance and advice. This work was supported by a grant from the Dutch Cancer Society (Koningin Wilhelmina Fonds).


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +31 243614017; Fax: +31 243540488; Email: r.kuiper{at}antrg.umcn.nl Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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