mutations : Identification of potential driver and passenger a factor HIF gene mutations and their effects on hypoxia inducible VHL

Mutations of the von Hippel-Lindau gene (VHL) are frequent in clear cell renal cell carcinomas (ccRCC). Nonsense and frameshift mutations abrogate the function of the VHL protein (pVHL), whereas missense mutations can have different effects. To identify those missense mutations with functional consequences, we sequenced VHL in 256 sporadic ccRCC and identified 187 different VHL mutations of which 65 were missense mutations. Location and destabilizing effects of VHL missense mutations were determined in silico . The majority of the thermodynamically destabilizing missense mutations were located in exon 1 in the core of pVHL, while protein surface mutations in exon 3 affected the interaction domains of elongin B and C. Their impact on pVHL's functionality was further investigated in vitro by stably re-introducing VHL missense mutations into a VHL null cell line and by monitoring the GFP signals after the transfection of a HIF α -GFP expression vector. pVHL's functionality ranged from no effect to complete HIF stabilization. Interestingly, Asn78Ser, Asp121Tyr, and Val130Phe selectively influenced HIF1 α and HIF2 α degradation. In sum, we obtained three different groups of missense mutations: one with severe destabilization of pVHL, a second without destabilizing effects on pVHL but relevance for the interaction with HIF α , elongin B, and elongin C, and a third with pVHL functions comparable to wild-type. We therefore conclude that the specific impact of missense mutations may help to distinguish between driver and passenger mutations and may explain responses of ccRCC patients to HIF targeted therapies. Multiple Comparison tests. A p-value of < 0.05 was considered significant.


Introduction
Renal cell carcinoma (RCC) is the most frequent malignant tumor arising from the kidney with approximately 210,000 new cases diagnosed per year worldwide (1). About 80% of the RCC belong to the clear cell subtype (ccRCC) which is commonly characterized by loss of one short arm of chromosome 3 and mutation of the von Hippel-Lindau (VHL) tumor suppressor gene on the second short arm of chromosome 3 at position 3p25. The high rate of VHL mutations suggests that the inactivation of the multiadaptor protein pVHL plays a critical part in ccRCC initiation (2).
The best investigated function of pVHL is its role as a substrate recognition component of an E3 ubiquitin protein ligase complex (3). Under normoxic conditions pVHL binds the hydroxylated HIFα subunits which leads to their ubiquitination and degradation (4). Under hypoxic conditions or absence of pVHL the stabilization of HIFα leads to the formation of a heterodimer with HIF1β which initiates enhanced transcription of HIF target genes. Both HIF1α and HIF2α show common but also distinct transcription patterns. HIF1α preferably drives the expression of genes important for apoptotic and glycolytic pathways, whereas HIF2α activates genes involved in cell proliferation and angiogenesis (5-7). Based on these results it was suggested that HIF2α is more oncogenic than HIF1α. This finding was supported by the observation that silencing of HIF2α in a human VHL-negative RCC cell line was sufficient to prevent tumor formation in mice and that HIF2α promotes c-myc activity (8)(9)(10)(11). However, recent evidence suggests that HIF1α is responsible for genomic instability which may favor the accumulation of additional genetic hits leading to carcinogenesis (12).
Over 800 VHL mutations were identified in both hereditary and sporadic ccRCC (13). More than 50% of these mutations are frameshift and nonsense mutations which are highly likely to cause loss of pVHL function (14,15). Due to of the large number of missense mutations distributed over the three exons of VHL, the consequences of such alterations on pVHL's integrity and HIFα stabilization are difficult to predict.
Several studies have been performed to classify VHL mutations identified in the hereditary VHL syndrome (reviewed in (16)). Nonsense and frameshift mutations generating VHL-null alleles are associated with ccRCC (Type 1 VHL disease), whereas Type 2 VHL disease is mainly characterized by missense mutations. This type is further subdivided into type 2A (with low risk of ccRCC), type 2B (with high risk of ccRCC) and type 2C which predisposes for pheochromocytoma. Missense mutations affecting pVHL's surface result in a higher risk for developing pheochromocytoma compared to substitutions altering the protein core (17).
Forman and co-workers used bioinformatic tools to determine the thermodynamic change of missense mutations and linked destabilizing mutations in the interface of HIFα and elongin B to a prevalence of ccRCC, while mutations interfering with the elongin C interface resulted in increased risk of pheochromocytoma (18).
Controversial data exist about the prognostic and predictive value of the VHL mutation type in sporadic ccRCC. Some groups found a correlation between "loss-of-function" mutations (nonsense, frameshift) and a worse prognosis for patients or higher HIF target-directed response rates, whereas other groups were not able to confirm these results (for review see (16)). A large study at the Dana-Faber Cancer Institute revealed an increased response in ccRCC patients with "loss-of-function" mutations (nonsense, frameshift, in frame) treated with antiangiogenic therapies (sunitinib, sorefanib, axitinib, or bevacizumab), blocking some of the downstream effects of pVHL (19). In contrast to nonsense, frameshift, and in frame mutations, the impact of missense mutations on the function of pVHL seems to be highly diversified ranging from imperceptable to complete functional loss (20-22).
The goal of our study was to functionally characterize missense mutations in sporadic ccRCC on pVHL and HIF using a combination of in silico and in vitro assays.

Tissue specimens
Two hundred and fifty-six formalin fixed, paraffin embedded (FFPE) ccRCC samples were histologically reviewed by one pathologist (H.M.). This study was approved by the local commission of ethics (ref. number StV 38-2005). Survival time was obtained for 123 patients (48%). The mean age of patients was 64 (31-88) and the mean follow-up of patients was 47 months (0-139). Tumors were graded according to tumor stage (pT) and the Fuhrman grading system and histologically classified according to the World Health Organization classification (23). The histological data are listed in supplementary Table S2.

VHL mutation analysis
For DNA extraction three tissue cylinders (diameter 0.6 mm) were punched from each paraffin block. DNA was extracted according to the QIAGEN EZ1 DNA Tissue protocol for automated purification of DNA from tissue (Qiagen, Hilden, Germany). PCR was performed as previously described (24) with slight modifications. Only one PCR step with 40 cycles was carried out using unlabelled primers. As mutations are rare in the 5' region of exon 1 the first 162 coding basepairs of VHL were excluded from sequence analysis (25). DNA sequencing was performed using the BigDye® Terminator v1.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA). The obtained sequences were compared with the NCBI sequence AF010238 using NCBIs Blast 2 Sequences. VHL point mutations were validated by a second separate PCR and sequencing analysis.

In silico analysis of VHL mutants
Two crystal structures of pVHL in complex with elongin B, elongin C, and the HIF peptides are available (26,27) and stored in the Piccolo database of protein interaction (PDB codes 1lm8 and 1lqb (28)). As input for in silico analyses pVHL was separated from the VCB
The distributions of nuclear differentiation grade (Fuhrman), tumor stage (pT) and VHL mutation type are summarized in supplementary table S2. Survival analysis revealed no significant differences neither between wild-type and mutated VHL nor between the different mutation types (Fig. 1B). The same was true when the mutations and the mutation types were subgrouped according to their exon locations.
The distribution and number of the identified mutations and mutation types within the three exons of VHL are shown in Fig. 2A-J. The number of VHL mutations was significantly increased in exon 1 compared to exon 2 and exon 3. The most frequent mutated amino acids (aa) were found at positions 65 (whole dataset and nonsense, respectively, Fig. 2A

In silico characterization of VHL missense mutations and protein structure
In a first step, we characterized the VHL missense mutations identified in our set of sporadic ccRCC using the Swiss PDB viewer 'Deep View v.4.0'. This program predicts whether the locations of missense mutations are pVHL surface-or core-related. With a threshold of accessibility of the aa of > 9% (defined as surface; (29)) we obtained the most surface mutations in exon 3 (n = 14), whereas the majority of the missense mutations in exons 1 (n = 25) and 2 (n = 16) were assigned to the core (Fig. 3A). Calculating the surface to core ratio in pVHL (aa 53-213) resulted in similar values for exon 1 (1.9) and 2 (1.7) but a higher value for exon 3 (5.6). This suggests an increased probability of having surface mutations in exon 3. Due to the crystallographic structure of the pVHL complex (pdb code 1lm8), 21 of 23 missense mutations located on the surface of pVHL could be assigned to interact with either HIF, elongin C, or elongin B (Supplementary Table S1, Fig. 3C, (18)). The remaining 2 missense mutations were not located in the domain of any known interaction partner. By using the software Crescendo which predicts the most essential regions and aa for proteinto-protein interactions, most of the missense mutations were also allocated to these conserved regions. Other binding partners (reviewed in (35)) listed in supplementary table S1 were found to be influenced by the surface missense mutations (Trp88Cys: aPKC, To predict changes of the pVHL structure due to missense mutations we used the program "Site Directed Mutator" (SDM). The algorithm calculates the thermodynamic change (ddG) caused by a mutation and output values with |ddG| > 2 are defined as "disease-associated".
By calculating the ddG values for all missense mutations we found 48% predicting a significant impact on the protein structure (ddG > 2) and the remaining 52% having "neutral" mutations (ddG < 2). A significant increased number of missense mutations changing the structure of pVHL was detected in exon 1 followed by exon 2 and 3 ( Fig. 3D). When we split surface-and core-related mutations according to their exon locations, we observed 'disease-associated' mutations primarily in the core of exon 1 and 2 and on the surface of exon 3 (Fig. 3F).
As expected, transduction of the negative control mutation Leu63fsX67 showed no expression in RCC4 and hTERT RPE-1 cells ( Fig. 4A and B). In both cell lines, a pVHL expression pattern was observed after transduction of wild-type pVHL and the neutral pVHL mutant Ser68Thr as previously described (36). In contrast, the pVHL mutants Gly93Glu, Trp117Arg, and Leu101Pro which had the highest destabilizing ddG values of 2.9, 4.3, and 6.1, respectively, showed much lower expression levels with bands between 20 and 30 kDa and additional abundant degradation products below 20 kDa which were not detected with the pVHL wild-type and the mutation Ser68Thr. The lower expression of the bands in these pVHL mutants were not due to decreased mRNA expression as verified by quantitative RT-PCR analysis (data not shown). The pVHL mutant Tyr112Asp (ddG = 2.6) was only minimally affected by the mutation underscored by the detection of a faint additional band below 20 kDa compared to pVHL wild-type. Interestingly, the pVHL mutant Pro86His (ddG = 2.3) expressed in the hTERT RPE-1 cell line showed an increased band intensity of fulllength pVHL (30 kDa) compared to the additional bands between 20 and 30 kDa present in the wild-type protein which reflects the predicted stabilizing effect of the mutation by SDM.
To further investigate the stability of mutant proteins, a cycloheximide assay was performed with two selected missense mutations. pVHL wild-type and pVHL mutant Ser68Thr exhibited similar band intensities after 1.5, 3, and 6h cycloheximide treatment whereas the pVHL mutant Leu101Pro was clearly less stable ( Fig. 4C and D). The bands between 20 and 30 kDa as well as the degradation product at 15 kDa were significantly decreased in intensity already after 1.5 h in the cell line hTERT RPE-1.

In vitro characterization of VHL missense mutations and protein function
To address the question whether the prediction of the stability of pVHL mutants influences its functionality, we set up a screening platform based on mouse embryonic fibroblasts negative for VHL and p53 (MEFs -/-). First, we transiently transfected hTERT RPE-1 and MEFs -/with an N-terminally HA-tagged VHL expressed from the pcDNA3.1 vector using Fugene6 with a vector DNA ratio 3:1, 3:2, and 6:1. In both cell lines, the expression pattern looked MEFs -/-that were stably transfected with a negative control vector, the three batches were able to decrease the GFP signal by 73%, 63%, and 75% respectively (Fig. 5E). To validate the system, we added DMOG to MEF -/-VHL wild-type cells after transfection with HIFα-GFP and measured the change of the FL-1 channel in gate 1 by FC 24h after transfection ( Fig. 5F). As a control pmaxGFP was transfected. After addition of DMOG the GFP signal increased in both the HIF1α-GFP and HIF2α-GFP transfected MEF -/-VHL wild-type cells 2.1-fold and 2.2 fold, respectively. DMOG treated and untreated MEF -/-VHL wild-type cells transfected with pmaxGFP showed no increased GFP signals. Next, we generated MEFs -/stably expressing the same set of VHL mutants (Fig. 5G)  As an additional validation we also analyzed the impact of VHL frameshift mutations on the ability to degrade the two HIFα isoforms. For each of the three exons we generated two frameshift mutations which were identified in our ccRCC patient set. All frameshift mutations except Glu204fsX44, which is located at the very end of exon 3, failed to destabilize HIF1α and HIF2α ( Fig. 6C-D). As expected the frameshift mutations in all 3 exons affected pVHL ability to degrade both HIFα isoforms in the same way (Fig. 6E).
Finally, we correlated the data obtained from pVHL stability prediction and functionality.
Spearman correlation coefficient for HIF1α and HIF2α were r = 0.5741, p = 0.0006 and r = 0.5494, p = 0.0011, respectively. A cut-off of 58% of the GFP signal for HIF1α and 54% for HIF2α resulted in 13 disease-associated missense mutations with a ddG > 2 for each of the HIF isoforms ( Figure 6A and B, boxes A). Eleven (34%) and 16 (50%) mutations were predicted to be "neutral" but were compromised in their ability to ubiquitinate HIF1α and HIF2α, respectively ( Figure 7A and B, boxes B). Only 8 (25%) and 3 (9%) missense mutations were identified with similar functionality on HIF1α and HIF2α destabilization, respectively, as pVHL wild-type ( Figure 7A and B, boxes C).

Discussion
In this study, we analyzed VHL missense mutations in sporadic ccRCC by determining their impact on pVHL's functionality and HIFα stability using in silico and in vitro assays. The frequencies of the different VHL mutation types as well as their distribution over the three exons were highly comparable to the results described in previous publications (2, 37).
Our in silico approach enabled us to locate missense mutations to the surface or the core of Therefore, loss of VHL/HIF interaction is more common in sporadic ccRCC compared to its hereditary form.
To resolve the question whether a missense mutation represents a driver mutation, which generates a growth advantage and thus potentially contributes to tumor formation, Carter and co-workers established a computational method termed Cancer-specific Highthroughput Annotation of somatic mutations (CHASM) (38). This approach is a powerful tool to analyze missense mutations in multiple genes especially when structures of their gene products are not available. Due to the fact that the crystal structure of the pVHL/elongin B/elongin C complex is known, we used an algorithm which calculates the structural change caused by a specific missense mutation in this complex. Assuming that loss of pVHL function increases HIF transcriptional activity leading to tumor formation and progression, we have considered defining mutations with high structural changes as driver mutations. By calculating the thermodynamic stability of pVHL we obtained 30 destabilizing driver ("disease-associated") and 32 (excl. 1 silent mutation) passenger ("neutral") mutations. Most of the driver missense mutations were located in exon 1 suggesting that the majority of pVHL missense mutations in exon 1 predispose to ccRCC by affecting whole protein stability rather than disrupting binding to interaction partners. Only 38% of missense mutations in exon 3 were predicted to destabilize the whole protein structure. However, previous publications reported that loss of elongin binding to pVHL also leads to instability and rapid degradation of the protein (39-41). As all passenger surface-specific missense mutations in exon 3 localized to either elongin C or B it cannot be excluded that at least some of these mutations exert compromising effects to the regulation of HIF. Interestingly, a recent publication showed that although the missense mutation Arg167Gln causes loss of pVHLelongin interaction, the HIFα ubiquitination was still functional (22). The authors suggested the formation of a remnant protein E3 ligase complex which is still partially capable of regulating HIF.
To analyse the impact of pVHL missense mutations on the thermodynamic stability and HIF regulation in more detail, we established an in vitro screening platform based on mouse embryonic fibroblasts (MEFs) negative for VHL and generated polyclonal stable cell lines expressing various VHL mutants. We used MEFs because higher transient transfection efficiencies are obtained in comparison to pVHL negative RCC4 or 786-O cell lines. We performed all experiments using polyclonal batches with similar levels of pVHL rather than individually picked clones to avoid any possible selection for a specific cellular background.
Our in vitro platforms verified the predicted thermodynamic changes of ddG > 2 for the 4 selected mutations which resulted in both pVHL degradation and HIF stabilization.
Additionally, the bioinformatic approach could also correctly predict the impact of different aa exchanges at the same position. For example, the missense mutation at aa Arg161Pro (ddG = -2.86) was completely impaired and could not degrade HIFα as expected due to structural loss of pVHL, whereas the passenger mutation Arg161Gln (ddG = -0.54) retained 56% functionality.
In the majority of the cases in our study, pVHL mutations affected both HIFα isoforms equally. Only three missense mutations seem to selectively influence HIF1α and HIF2α The majority of the identified pVHL missense mutations affected both HIFα isoforms.
Therefore it is tempting to speculate that additional factors on the activity (44), translational normoxic levels and allow the E3 ubiquitin ligase complex to interact with its substrate. It was suggested that different PHDs influence the levels of HIF1α or HIF2α depending on the cell type, the tissue, and the degree of oxygenation (48-51) which is independent of the VHL mutation status.
Our in silico tool was able to group VHL missense mutations. The used algorithm proved to be a good predictor of pVHL functionality in terms of thermodynamic stability changes. By combining the results from our in silico and in vitro analyses, we obtained three different groups of missense mutations which a) lead to a severe destabilization of pVHL; b) have no destabilizing effects on pVHL but affect the interaction with HIFα, elongin B, and elongin C; and c) have functionalities comparable to the wild-type protein. The first two groups represent driver mutations whereas the latter one consists of passenger mutations. It is to note that 11 and 16 of 32 missense mutations predicted in silico to be "neutral" were compromised in their ability to ubiquitinate HIF1α and HIF2α, respectively. The higher number of missense mutations leading to HIF2α stabilization suggests a more specific binding of pVHL and HIF2α. Taken together, these results imply a gradient effect of pVHL missense mutations on HIF regulation which may be caused by a combination of structural changes and alterations of the binding capability to interaction partners. Our systematic approach enabled us to group these VHL mutations according to their effects on HIF which may have important implications for tumor behavior or response to therapy. However, the translation of the HIF (de)stabilizing impact of a VHL missense mutation into clinical application needs further investigation.
Survival analysis of VHL wild-type, VHL mutation types, and subgrouping of VHL missense mutations into predicted destabilizing and neutral missense mutations, showed no differing effects on patient outcome. Our findings are in line with those described in a previous study (2) in which the impact of VHL mutation types on patient survival was also investigated.
Based on these results it is tempting to speculate that the analysis of VHL mutations in ccRCC patients may serve as predictive rather than as prognostic tool.

Literature
1 . w w w -d e p . i a r c . f r .

. B a n k s R E , T i r u k o n d a P , T a y l o r C , H o r n i g o l d N , A s t u t i D , C o h e n D , e t a l . G e n e t i c a n d e p i g e n e t i c a n a l y s i s o f v o n H i p p e l -L i n d a u
( V H L ) g e n e a l t e r a t i o n s a n d r e l a t i o n s h i p w i t h c l i n i c a l v a r i a b l e s i n s p o r a d i c r e n a l c a n c e r . C a n c e r R e s . 2 0 0 6 ; 6 6 : 2 0 0 0 -1 1 .         Figure 6C and 6D. F, correlation of HIF1α-GFP and HIF2α-GFP signals in MEFs -/-expressing the VHL missense mutations presented in Fig 6A and 6B.

. O h h M , P a r k C W , I v a n M , H o f f m a n M A , K i m T Y , H u a n g L E , e t a l . U b i q u i t i n a t i o n o
Open circles represent missense mutations registered in the UMD database as VHL disease causing. Bars represent the range in which the specific VHL disease type was detected.