VHL L169P Variant Does Not Alter Cellular Hypoxia Tension in Clear Cell Renal Cell Carcinoma

In the current era of tumor genome sequencing, single amino acid missense variants in the von Hippel–Lindau (VHL) tumor suppressor gene are frequently identified in clear cell renal carcinoma (ccRCC). Due to the incomplete knowledge of the structural architecture of VHL protein, the functional significance of many missense mutations cannot be assigned. L169P is one such missense mutation identified in the case of aggressive, metastatic ccRCC. Here, we characterized the biochemical activity, transcriptomic hypoxia signature and biological functions of the L169P variant. Lentiviral vector expressing either wildtype (WT) or L169P VHL were used to transduce two VHL-deficient human ccRCC cell lines, 786-O and RCC4. The stability of the VHL protein and the expression level of VHL, HIF1α and HIF2α were analyzed. The impact of restoring L169P or WT VHL on the hypoxia gene expression program in 786-O cells was assessed by mRNA sequencing (RNAseq) and computed hypoxic scores. The impact of restoring VHL expression on the growth of ccRCC models was assessed in cell cultures and in chorioallantoic membrane (CAM) xenografts. In the 786-O cells, the protein stability of L169P VHL was comparable to WT VHL. No obvious difference in the capability of degrading HIF1α and HIF2α was observed between WT and L169P VHL in the 786-O or RCC4 cells. The hypoxic scores were not significantly different in the 786-O cells expressing either wildtype or L169P VHL. From the cellular function perspective, both WT and L169P VHL slowed cell proliferation in vitro and in vivo. The L169P VHL variant is comparable to WT VHL in terms of protein stability, ability to degrade HIF1α factors and ability to regulate hypoxia gene expression, as well as in the suppression of ccRCC tumor cell growth. Taken together, our data indicate that the L169P VHL variant alone is unlikely to drive the oncogenesis of sporadic ccRCC.


Introduction
The VHL (von Hippel-Lindau) tumor suppressor gene is the most frequently mutated gene in clear cell renal cell carcinoma (ccRCC) followed by PBRM1, BAP1 and SETD2.Alterations in the VHL gene, such as missense mutations, deletion or epigenetic silencing, have been identified in all familial cases and about 85% of sporadic cases of ccRCC [1].Since the recognition of "VHL disease" in 1936, over 1600 mutations have been identified in this gene from ccRCC tumor tissues [2,3].Amongst the identified VHL mutations, over 900 are missense mutations with a single amino acid substitution [3].Most of these missense mutations were evaluated by computation models without comprehensive characterization in cellular or animal models [4].At this time, an atlas that accurately assigns a VHL genotype to its oncogenic phenotypes in ccRCC is lacking.VHL L169P is one such missense variant of unknown significance in exon 3, identified in a highly aggressive and metastatic case of ccRCC [5] and in four patients in the TCGA-KIRC and CPTAC-3 projects [6].
Three isoforms of hVHL have been identified, including the 213 aa (amino acids) isoform 1 (hVHL-v1), the 172 aa isoform 2 (hVHL-v2) that skips the second exon in hVHL-v1, as well as the third isoform (hVHL-v3) with 193 aa that has a distinct C-terminal exon from the other two isoforms.In addition, hVHL-v1 can be translated into two protein products due to an alternative start codon at amino acid 54, resulting in a 30 kD and a 19 kD protein [7].The protein structure of hVHL is composed of a β-domain in the N-terminal and an α domain in the C-terminal [8].The L169P substitution impacts the hVHL-v1 and v2 structure at the C-terminal α-domain.
VHL protein is known to be involved in two major cellular processes.First, it is well recognized as a component of an E3 ubiquitin ligase VCB-CR complex (Vhl/Elongin C/Elongin B-Cullin 2/RING finger protein), which is responsible for binding and degrading hypoxia-inducible factors (HIF1α and HIF2α) under normoxic condition [8][9][10].The N-terminal β-domain participates in substrate recognition and binding to HIFαs, while the α-domain interacts with elongin B and C [8,11,12].Therefore, mutations in this domain including L169P mainly affect the interface between pVHL and elongin C/B and are classified as type 2b VHL mutations [13][14][15].The second known function of VHL is involved in regulating cilia centrosome and microtubules to maintain cell polarity and chromosomal stability in cell division [16,17].
The VHL function in the first process, namely the regulation of the hypoxic response [18], is considered to be the main cause of ccRCC tumorigenesis.A wide range of VHL protein dysfunctions, ranging from the complete loss of VHL protein to the disabling of its enzymatic function, or just a reduced protein stability resulting in protein insufficiency, could contribute to oncogenesis attributed by dysregulated hypoxic responses.In this study, we undertook a comprehensive functional evaluation of the L169P VHL variant regarding its protein stability, cellular hypoxia tension and tumorigenic potential in two human ccRCC models.

Amino Acid L169P Substitution Does Not Alter VHL Protein Stability
Over the past 5 years, we collected fresh surgical tumor samples from 50 patients with RCC and generated 40 primary cell lines from these samples to study their biology [5].The most aggressive case amongst this cohort was a 59-year-old male who presented with a 10 cm, Fuhrman grade 4, primary tumor and bilateral lung metastases and who succumbed within one year of nephrectomy despite multiple interventions.The L169P VHL variant was identified in the tumor of this aggressive case of ccRCC by whole-exome sequencing (WES), revealing a homozygous in-frame T506C transition that resulted in the L169P amino acid substitution [5].Although this variant has previously been reported, its oncogenic and hypoxia regulation function was not closely examined [19].To study the oncogenic function of the L169P variant relative to WT VHL, we re-expressed the respective VHL gene in two common VHL-null human ccRCC cell lines, 786-O and RCC4.The coding sequence (CDS) of human VHL isoform 1 (designated as WT VHL) was cloned from normal human kidney tissue into a lentiviral expression vector, and the L169P was generated subsequently by the site-directed mutagenesis of the WT VHL construct.Sanger DNA sequencing verified the designated sequence for both constructs, as shown in Figure 1A.Upon lentiviral-mediated stable transduction, a comparable and robust expression of WT and L169P VHL mRNA (Figure 1B) and protein (pVHL) was achieved in the 786-O and RCC4 cells (Figure 1C,D).
Next, we evaluated the stability of L169P pVHL.The degradation of pVHL was assessed upon the addition of cycloheximide (CHX) [20], which halts the synthesis of new protein.The pVHL level was examined at 0, 3 and 6h post CHX treatment.As shown in Figure 1E, the decrement over time of WT and L169P pVHL was very comparable in the 786-O cells.This finding supports the unaltered protein stability of the L169P variant.Next, we evaluated the stability of L169P pVHL.The degradation of pVHL was assessed upon the addition of cycloheximide (CHX) [20], which halts the synthesis of new protein.The pVHL level was examined at 0, 3 and 6h post CHX treatment.As shown in Figure 1E, the decrement over time of WT and L169P pVHL was very comparable in the 786-O cells.This finding supports the unaltered protein stability of the L169P variant.

The Hypoxic Regulation of L169P VHL Is Comparable to WT VHL
We examined the hypoxic regulatory response of L169P VHL, as the hypoxia pathway is implicated in ccRCC oncogenesis.The protein level of HIF-1α and HIF-2α, the two major targets of VHL-mediated ubiquitin ligation and degradation, were examined in the parental VHL-null parental cells 786-O and RCC4 and their WT or L169P VHL-expressing derivative cells (Figure 2A,B).In the absence of VHL expression, a clear accumulation of HIF-1α and HIF-2α protein was observed (Figure 2A,B).Upon the reintroduction of WT or L169P VHL, the level of both targets was reduced to a similar extent (Figure 2A,B).These findings indicate that the L169P variant is capable of degrading both HIF-1α and HIF-2α as efficiently as its wildtype counterpart.

The Hypoxic Regulation of L169P VHL Is Comparable to WT VHL
We examined the hypoxic regulatory response of L169P VHL, as the hypoxia pathway is implicated in ccRCC oncogenesis.The protein level of HIF-1α and HIF-2α, the two major targets of VHL-mediated ubiquitin ligation and degradation, were examined in the parental VHL-null parental cells 786-O and RCC4 and their WT or L169P VHL-expressing derivative cells (Figure 2A,B).In the absence of VHL expression, a clear accumulation of HIF-1α and HIF-2α protein was observed (Figure 2A,B).Upon the reintroduction of WT or L169P VHL, the level of both targets was reduced to a similar extent (Figure 2A,B).These findings indicate that the L169P variant is capable of degrading both HIF-1α and HIF-2α as efficiently as its wildtype counterpart.Next, we evaluated the stability of L169P pVHL.The degradation of pVHL sessed upon the addition of cycloheximide (CHX) [20], which halts the synthesis protein.The pVHL level was examined at 0, 3 and 6h post CHX treatment.As sh Figure 1E, the decrement over time of WT and L169P pVHL was very comparabl 786-O cells.This finding supports the unaltered protein stability of the L169P var

The Hypoxic Regulation of L169P VHL Is Comparable to WT VHL
We examined the hypoxic regulatory response of L169P VHL, as the hypoxia p is implicated in ccRCC oncogenesis.The protein level of HIF-1α and HIF-2α, the tw targets of VHL-mediated ubiquitin ligation and degradation, were examined in the tal VHL-null parental cells 786-O and RCC4 and their WT or L169P VHL-expres rivative cells (Figure 2A,B).In the absence of VHL expression, a clear accumulation 1α and HIF-2α protein was observed (Figure 2A,B).Upon the reintroduction of L169P VHL, the level of both targets was reduced to a similar extent (Figure 2A,B findings indicate that the L169P variant is capable of degrading both HIF-1α and as efficiently as its wildtype counterpart.We examined the broader cellular hypoxia program further, beyond the regulation of alpha subunits of HIF.To assess possible alterations in cellular hypoxic tension, bulk mRNA sequencing was implemented to obtain the comprehensive genetic expression profiles of the 786-O, 786-O/VHL L169P and 786-O/VHL WT cells.Using the hypoxic signature panel developed by Ragnum [21], Buffa [22] and Winter [23], the hypoxia scores for each cell line were calculated and are shown in Figure 2C.The whole list of each gene panel and their scores are shown in Figure 3.A higher hypoxia score reflects a more hypoxic state of the cells.As shown in Figure 2C, both L169P and WT VHL manifested similar hypoxia tensions and were both less hypoxic compared to their parental cell lines.
signature panels from Ragnum et al. [21], Buffa et al. [22] and Winter et al. [23] on the parenta O cells as well as the derived cell lines that were either wildtype or L169 overexpressed.
We examined the broader cellular hypoxia program further, beyond the regul of alpha subunits of HIF.To assess possible alterations in cellular hypoxic tension, mRNA sequencing was implemented to obtain the comprehensive genetic expression files of the 786-O, 786-O/VHL L169P and 786-O/VHL WT cells.Using the hypoxic sign panel developed by Ragnum [21], Buffa [22] and Winter [23], the hypoxia scores for cell line were calculated and are shown in Figure 2C.The whole list of each gene p and their scores are shown in Figure 3.A higher hypoxia score reflects a more hy state of the cells.As shown in Figure 2C, both L169P and WT VHL manifested si hypoxia tensions and were both less hypoxic compared to their parental cell lines.
Figure 3. Gene list and heatmap from the hypoxic signature panels from Ragnum et al. [21], et al. [22] and Winter et al. [23].The darkness of the color indicates the relative expression lev each gene labeled on the bo om.The darker, the higher expression level of gene in that cell.

Restored Expression of WT or L169P VHL Slowed Cancer Cell Growth
Next, we evaluated the impact of L169P VHL on the tumorigenic phenotype.Th pression of either VHL L169P or WT VHL did not alter the cellular morphology in parison to the parental 786-O cells (Figure 4A).However, the restored expression of e VHL-L169P or WT VHL significantly suppressed the growth rate of the parental 7 cells (Figure 4B).Meanwhile, no distinguishable difference in the growth rate wa served between the two VHL-expressing cells (Figure 4B).We further assessed the in growth potential of these three tumor cell xenografts established on a duck CAM (c oallantoic membrane) model.We have previously reported that a rich CAM vascul effectively supports the growth and aggressive behaviors of numerous tumor xenog recapitulating their behaviors observed in mouse models [24,25].Corroborating t vitro results, the 786-O cells with a restored expression of either WT or L169P VHL e ited slower growth rates in vivo, resulting in a significantly lower terminal tumor siz weight compared to the parental 786-O xenografts (Figure 4C,D).However, the diffe in the tumor weight between the two VHL-expressing groups was insignificant (F 4D).Buffa et al. [22] and Winter et al. [23].The darkness of the color indicates the relative expression level for each gene labeled on the bottom.The darker, the higher expression level of gene in that cell.

Restored Expression of WT or L169P VHL Slowed Cancer Cell Growth
Next, we evaluated the impact of L169P VHL on the tumorigenic phenotype.The expression of either VHL L169P or WT VHL did not alter the cellular morphology in comparison to the parental 786-O cells (Figure 4A).However, the restored expression of either VHL-L169P or WT VHL significantly suppressed the growth rate of the parental 786-O cells (Figure 4B).Meanwhile, no distinguishable difference in the growth rate was observed between the two VHL-expressing cells (Figure 4B).We further assessed the in vivo growth potential of these three tumor cell xenografts established on a duck CAM (chorioallantoic membrane) model.We have previously reported that a rich CAM vasculature effectively supports the growth and aggressive behaviors of numerous tumor xenografts, recapitulating their behaviors observed in mouse models [24,25].Corroborating the in vitro results, the 786-O cells with a restored expression of either WT or L169P VHL exhibited slower growth rates in vivo, resulting in a significantly lower terminal tumor size and weight compared to the parental 786-O xenografts (Figure 4C,D).However, the difference in the tumor weight between the two VHL-expressing groups was insignificant (Figure 4D).
In this study, we analyzed the biochemical and tumorigenic activities of the L169P VHL variant, including protein stability, the degradation of alpha subunits of HIF, hypoxic tension and tumor-growth-suppressive ability.Overall, the L169P variant functions at a level that is comparable to WT VHL.In this study, we analyzed the biochemical and tumorigenic activities of the VHL variant, including protein stability, the degradation of alpha subunits of HIF, h tension and tumor-growth-suppressive ability.Overall, the L169P variant functio level that is comparable to WT VHL.

Discussion
The functional sequela of missense mutations is more difficult to predict com to nonsense or frameshift mutations that result in the complete loss of VHL prote crystal structure of VHL in the VCB complex has previously [8] been dete (h ps://www.rcsb.org(accessed on 1 June 2023)).Several computational algorithm been developed that can predict the stability and function of VHL missense variant licly accessible algorithms include I-mutant 2.0 [26], Site Directed Mutator (SDM) [2 Crescendo [28].Both I-mutant 2.0 and SDM calculate ddG to evaluate the thermod changes in an amino acid substitution [26,27].In these algorithms, a ddG value than 2 is considered "disease associated", while one less than 2 is considered "ne As shown in Table 1, the ddG values for L169P were −0.03 and −1.71, indicating it tral.In contrast, the CRESCENDO program assigns the L169P variant as disease ated.This program evaluates the impact of an amino acid change by interrogatin conserved the affected region is in the protein-protein interaction.The leucine 1 due, located in exon 3 of VHL, was postulated to interact with Elongin B due to i proximity to arginine 167, which was reported to have lower regulatory effects on [4].

Discussion
The functional sequela of missense mutations is more difficult to predict compared to nonsense or frameshift mutations that result in the complete loss of VHL protein.The crystal structure of VHL in the VCB complex has previously [8] been determined (https: //www.rcsb.org(accessed on 1 June 2023)).Several computational algorithms have been developed that can predict the stability and function of VHL missense variants.Publicly accessible algorithms include I-mutant 2.0 [26], Site Directed Mutator (SDM) [27], and Crescendo [28].Both I-mutant 2.0 and SDM calculate ddG to evaluate the thermodynamic changes in an amino acid substitution [26,27].In these algorithms, a ddG value greater than 2 is considered "disease associated", while one less than 2 is considered "neutral".As shown in Table 1, the ddG values for L169P were −0.03 and −1.71, indicating it is neutral.In contrast, the CRESCENDO program assigns the L169P variant as disease-associated.This program evaluates the impact of an amino acid change by interrogating how conserved the affected region is in the protein-protein interaction.The leucine 169 residue, located in exon 3 of VHL, was postulated to interact with Elongin B due to its close proximity to arginine 167, which was reported to have lower regulatory effects on HIF2α [4].Due to the equivocal designation of L169P VHL to "disease association" by in silico algorithms, we decided to pursue direct biochemical and functional experiments that reintroduced the VHL gene into two human VHL-null ccRCC models.In the 786-O cells, the protein stability of L169P and its capability to degrade HIFs were very comparable to WT pVHL (Figures 1D and 2A,B).Our findings regarding L169P VHL corroborates a prior study by Rechsteiner et al. [4] that used an HIFαs-GFP degradation reporter assay in MEF (mouse embryonic fibroblast) cells to assess the E3 ligase function of missense variants of VHL, identified in ccRCC tumors.The L169P variant was amongst the 29 variants studied, and it was reported to function as WT pVHL in terms of degrading HIF1α, while the degradation of HIF2α was slightly reduced [4].In the supplementary findings, they assigned the L169P variant as a passenger mutation, likely not pathogenic [4].
In this study, we pursued two additional assays besides those previously reported [4] to further clarify the potential oncogenic role of L169P VHL in hypoxia regulation and in tumor growth suppression.The first assay utilized mRNA sequencing and gene expression profiling to assess pVHL's functional impact on cellular hypoxia.Bhandari et al. [29,30] reported several hypoxia score algorithms based on sets of known hypoxia marker genes.In comparison to the parental VHL-null 786-O cells, both the WT or L169P VHL-expressing cells showed a lower score by three diffent sets of hypoxia signatures (Figures 2C and 3), indicating that the regulation of HIFα-governed hypoxia pathways were intact and were comparable under the WT or L169P VHL-controlled cells.The WT VHL gene is known to induce growth arrest by restoring cell entry into G0 or the quiescent state and to suppress tumor growth [31,32].The impact of L169P VHL on tumor growth suppression was again comparable to WT VHL in slowing the growth of VHL-null 786-O cells in cultures (Figure 4B) as well as in xenografts in a CAM model (Figure 4C,D).
In a recent study, we reported that spontaneous metastasis in ccRCC models requires the presence of and cross communication between two distinct tumor cell populations, one that does not express any pVHL (VHL-Neg) and one that expresses WT pVHL (VHL-Pos) [5].This cooperative metastasis model demonstrated that the VHL-Neg cells act as the metastatic driver, while the VHL-Pos cells play the accepting effector role [5].In this cooperative metastatic scenario, L169P functions the same as WT VHL-expressing cells to achieve distant metastasis to the lungs [5].
The current study serves to caution that the oncogenic function of missense VHL variants could be difficult to determine, especially for those variants involving a single amino acid substitution in exon 3 that do not obviously reduce VHL protein stability.As previously reported, 30-50% of this subtle class of variants are classified as passenger, nonpathogenic variants [4].The L169P variant belongs to this class of subtle missense variants that require thorough comprehensive biological function analyses, like those outlined here.
Many factors contribute to the difficulty in determining the oncogenic role of VHL variants in ccRCC, such as the complex multiple normal functions of VHL, its yet undetermined specific mechanism of oncogenesis, tumor heterogeneity and the presence of multiple oncogenic driver gene mutations in ccRCC.The association of VHL gene mutations with VHL syndrome and ccRCC has been known for over three decades [33].The precise oncogenic mechanism of VHL remains elusive.The sophisticated conditional knockout of the VHL gene in the proximal tubules of mice resulted in cystic dysplasia but not renal tumors [34].The deletion of VHL in conjunction with other oncogenic drivers such as Rb1 and Trp53 was able to generate renal tumors but unable to recapitulate the aggressive metastatic disease [35].In the context of other genetic backgrounds, the L169P variant could possibly synergize with other oncogenic gene mutation(s), such as BAP1 and p53 uncovered in the highly aggressive and metastatic case of ccRCC [5], to drive tumorigenesis.However, based on the multiple lines of evidence, such as protein stability, hypoxia regulation and tumor growth suppression, we conclude at this juncture that L169P VHL functions comparably to WT VHL.Thus, the L169P variant of VHL alone is unlikely to drive the oncogenesis of sporadic ccRCC.

The Cell Culture, Reagents and Generation of Genetic Engineered Cell Lines
The 786-O and RCC4 cell lines were kindly gifted from Dr. Brian M. Shuch at the University of California, Los Angeles (UCLA).Both cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum and 1x penicillin-streptomycin at 37 • C and in 5% CO 2 humidified cell incubators.The coding sequence (CDS) of wild-type human VHL was amplified by PCR with EcoRV restriction enzyme sites on both ends, where the forward primer was 5 -AAACGGATATCATGCCCCGGAGGGCG-3 and the reverse primer was 5 -CGATCGATATCATCTCCCATCCGTTGATGTGC-3 .The template was normal tissue cDNA from a healthy donor.The L169P variant of human VHL was amplified by bridging PCR in combination with primers for wildtype VHL.The first pair of primers were forward: 5 -AAACGGATATCATGCCCCGGAGGGCG-3 and reverse: 5 -CAGGCTTGACTGGGCTCCG-3 .The second pair of primers were forward: 5 -CGGAGCCCAGTCAAGCCTG-3 and reverse: 5 -CGATCGATATCATCTCCCATCCG TTGATGTGC-3 .The template was wildtype human VHL CDS, as mentioned above.The backbone for both VHL-wildtype and VHL-L169P overexpression plasmids was pLV-CMV-LoxP-DsRed-LoxP-eGFP (Cat#65726, Addgene, Watertown, MA, USA) without DsRed and eGFP sequences.
A cell proliferation assay was carried out with MTS reagent from Promega (Cat#G3580, Madison, WI, USA).On day 0, log phase cells were seeded in a flat-bottom 96-well plate at 1000 cells/well.The absorbance of each well in all groups was examined every 24 h by a Multiskan MK3 microplate reader (Thermofisher, Waltham, MA, USA) at 490 nm.
A chorioallantoic membrane (CAM) cancer cell xenograft model was established by implanting the cancer cells on top of the highly vascularized CAM, upon which the cancer cells invaded into the duck embryonic blood cells to obtain nourishment.The cancer cells were implanted on the embryo at day 15 upon the delivery of fertilized duck embryos and were allowed to grow until day 28.Tumors were harvested and weighted for statistical analysis [24,25,37].
For the VHL stability analysis, 786-O cells were cultured and seeded in a 6-well plate at 5 × 10 5 cells per well on day 0. On day 1, each well was transfected with either 3000 ng of wildtype VHL or L169P variant plasmid with 9 µL of Promega FuGENE transfection reagent (Cat#E2311, Promega, Madison, WI, USA) followed by 24 h of incubation.Upon harvesting, cycloheximide (CHX, 100 µg/mL, Cat#357420010, Acros Organics, Waltham, MA, USA) was added to each well, and RIPA lysis buffer was used to harvest the cells at 0, 3 and 6 h upon CHX addition.The harvested protein was subjected to Western blot analysis, as mentioned earlier.

Bioinformatic Analysis and Hypoxic Score Calculation
The mRNA from the 786-O parental cell line as well as the derived cells overexpressed with wildtype or L169P human VHL was extracted and sequenced for mRNA expression profiles at UCLA Technology Center for Genomics & Bioinformatics (TCGB) core.
Libraries were prepared with a KAPA Stranded mRNA-Seq kit.The workflow consisted of mRNA enrichment and fragmentation, first-strand cDNA synthesis using random priming followed by second-strand synthesis converting cDNA:RNA hybrid to doublestranded cDNA (dscDNA), and it incorporated dUTP into the second cDNA strand.cDNA generation was followed by end repairing to generate blunt ends, A-tailing, adaptor liga-tion and PCR amplification.Different adaptors were used for multiplexing samples in one lane.Sequencing was performed on an Illumina NovaSeq 6000 device for a PE 2 × 100 run.Data quality checking was carried out on an Illumina SAV device.Demultiplexing was performed with the Illumina Bcl2fastq v2.19.1.403software.
The Partek flow software (Partek ® Flow ® software, version 7.0 Copyright©; 2019 Partek Inc., St. Louis, MO, USA) was used for the data analysis.The alignment and generation of the raw read counts per gene were performed using STAR-2.7.9a [38,39] with human reference genome GRCh38 genome and were annotated with Ensembl 104 GTF.Normalization of transcript counts was performed using CPM (counts per million) normalization followed by adding 0.0001.The Partek GSA algorithm was used for differential gene expression, with p < 0.05 and FDR < 0.05, and 2Fold was used for cutoffs to obtain lists of significantly expressed genes from the sample comparisons.
The hypoxia scores of the three cell lines were calculated using the mRNA-based signatures developed by Ragnum [21], Buffa [22] and Winter [23].The raw read counts from the three cell lines were normalized with the TCGA database level 1 data using the DESeq2 package v1.38.3 accessed by 1 June 2023, and the hypoxia scores were calculated as described in Bhandari et al. [29].

Statistical Analysis
All experiments were repeated three times, and data are presented as means ± SEM.One-way ANOVA was used for the three group comparisons between the parental cell line, VHL-WTand VHL-L169P-overexpressed cells followed by planned post hoc comparisons.

Figure 1 .
Figure 1.Protein stability of L169P variant is comparable to WT pVHL.(A) Sequence alignment of the Sanger sequencing results from plasmids encoding wildtype VHL or c.506T>C point mutated VHL and the reference sequence, as made by the Vector NTI software v8.0.(B) qRT-PCR analysis of VHL in 786-O cells vector control, VHL/WT and VHL/L169P.(C,D) Western blots identifying VHL protein upon reintroduction into the parental cell line 786-O (C) and RCC4 (D).(E) A total of 100 µg/mL CHX was applied to evaluate the VHL degradation rate at time points of 0, 3 and 6 h after mRNA translation was blocked in 786-O cells.(**: p < 0.01, n.s.: no statistical significance).

Figure 2 .
Figure 2. L169P variant showed a similar capability to WT pVHL in decreasing hypoxic tension by degrading HIF-1A and HIF-2A.(A) Western blot analysis of HIF1A and HIF2A upon wildtype and L169P VHL overexpression in 786-O cells.(B) Western blot analysis of HIF1A and HIF2A upon wildtype and L169P VHL overexpression in RCC4 cells.(C) Hypoxic scores calculated by the gene

Figure 1 .
Figure 1.Protein stability of L169P variant is comparable to WT pVHL.(A) Sequence alignment of the Sanger sequencing results from plasmids encoding wildtype VHL or c.506T>C point mutated VHL and the reference sequence, as made by the Vector NTI software v8.0.(B) qRT-PCR analysis of VHL in 786-O cells vector control, VHL/WT and VHL/L169P.(C,D) Western blots identifying VHL protein upon reintroduction into the parental cell line 786-O (C) and RCC4 (D).(E) A total of 100 µg/mL CHX was applied to evaluate the VHL degradation rate at time points of 0, 3 and 6 h after mRNA translation was blocked in 786-O cells.(**: p < 0.01, n.s.: no statistical significance).

Figure 1 .
Figure 1.Protein stability of L169P variant is comparable to WT pVHL.(A) Sequence align the Sanger sequencing results from plasmids encoding wildtype VHL or c.506T>C point VHL and the reference sequence, as made by the Vector NTI software v8.0.(B) qRT-PCR an VHL in 786-O cells vector control, VHL/WT and VHL/L169P.(C,D) Western blots identify protein upon reintroduction into the parental cell line 786-O (C) and RCC4 (D).(E) A tot µg/mL CHX was applied to evaluate the VHL degradation rate at time points of 0, 3 and mRNA translation was blocked in 786-O cells.(**: p < 0.01, n.s.: no statistical significance).

Figure 2 .
Figure 2. L169P variant showed a similar capability to WT pVHL in decreasing hypoxic te degrading HIF-1A and HIF-2A.(A) Western blot analysis of HIF1A and HIF2A upon wild L169P VHL overexpression in 786-O cells.(B) Western blot analysis of HIF1A and HIF2 wildtype and L169P VHL overexpression in RCC4 cells.(C) Hypoxic scores calculated by

Figure 2 .
Figure 2. L169P variant showed a similar capability to WT pVHL in decreasing hypoxic tension by degrading HIF-1A and HIF-2A.(A) Western blot analysis of HIF1A and HIF2A upon wildtype and L169P VHL overexpression in 786-O cells.(B) Western blot analysis of HIF1A and HIF2A upon wildtype and L169P VHL overexpression in RCC4 cells.(C) Hypoxic scores calculated by the gene signature panels from Ragnum et al. [21], Buffa et al. [22] and Winter et al. [23] on the parental 786-O cells as well as the derived cell lines that were either wildtype or L169 overexpressed.

Figure 3 .
Figure 3. Gene list and heatmap from the hypoxic signature panels from Ragnum et al. [21],Buffa et al.[22] and Winter et al.[23].The darkness of the color indicates the relative expression level for each gene labeled on the bottom.The darker, the higher expression level of gene in that cell.

Figure 4 .
Figure 4. Overexpression of either wildtype or L169P VHL slowed cell growth both in vitr vivo.(A) Phase contrast images of the 786-O parental cells as well as the overexpressed c with either wildtype or L169P VHL in both dense and sparse cultured modes (×200 magn field).(B) Cell proliferation was analyzed by an MTS assay among the three cells for up days.(C) Gross images of the primary tumors formed in the duck CAM model from the th lines after 13 days post implant.(D) Statistical analysis of the tumor weight from (C). (*: p < p < 0.01).

Figure 4 .
Figure 4. Overexpression of either wildtype or L169P VHL slowed cell growth both in vitro and in vivo.(A) Phase contrast images of the 786-O parental cells as well as the overexpressed cell lines with either wildtype or L169P VHL in both dense and sparse cultured modes (×200 magnification field).(B) Cell proliferation was analyzed by an MTS assay among the three cells for up to three days.(C) Gross images of the primary tumors formed in the duck CAM model from the three cell lines after 13 days post implant.(D) Statistical analysis of the tumor weight from (C). (*: p < 0.05, **: p < 0.01).

Table 1 .
Human VHL in silico algorithm analysis results.* indicates algorithms were accessed and results were updated by 1 June 2023.