Quantitative proteomics to study a small molecule targeting the loss of von Hippel–Lindau in renal cell carcinomas

Inactivation of the tumor suppressor gene, von Hippel–Lindau (VHL), is known to play an important role in the development of sporadic clear cell renal cell carcinomas (ccRCCs). Even if available targeted therapies for metastatic RCCs (mRCCs) have helped to improve progression‐free survival rates, they have no durable clinical response. We have previously shown the feasibility of specifically targeting the loss of VHL with the identification of a small molecule, STF‐62247. Understanding its functionality is crucial for developing durable personalized therapeutic agents differing from those available targeting hypoxia inducible factor (HIF‐) pathways. By using SILAC proteomics, we identified 755 deregulated proteins in response to STF‐62247 that were further analyzed by ingenuity pathway analysis (IPA). Bioinformatics analyses predicted alterations in 37 signaling pathways in VHL‐null cells in response to treatment. Validation of some altered pathways shows that STF‐62247's selectivity is linked to an important inhibition of mTORC1 activation in VHL‐null cells leading to protein synthesis arrest, a mechanism differing from two allosteric inhibitors Rapamycin and Everolimus. Altogether, our study identified signaling cascades driving STF‐62247 response and brings further knowledge for this molecule that shows selectivity for the loss of VHL. The use of a global SILAC approach was successful in identifying novel affected signaling pathways that could be exploited for the development of new personalized therapeutic strategies to target VHL‐inactivated RCCs.

Kidney cancer is the ninth most diagnosed cancer in the world. 1,2 ccRCC, the most frequent kidney cancer, is associated with biallelic inactivation of the tumor suppressor gene VHL. 3,4 Multiregion exome sequencing studies indicated an elevated intratumor heterogeneity in sporadic ccRCCs and showed that VHL inactivation was the only truncal mutation present in all cases. 5 The most characterized function for VHL protein (pVHL) is as a recognition component of an E3 ubiquitin-ligase complex (VCB-Cul2 complex) that targets hypoxia inducible factors alpha (HIFa) for ubiquitination and consequent proteasomal degradation. [6][7][8][9] Comprehension of the VHL-HIF axis brought to light the development of targeted therapies for advanced ccRCC but is unfortunately of limited efficacy and carries significant toxicity. 9 Thus, treatment of advanced ccRCC remains an unresolved clinical challenge demonstrating the important need in developing novel therapeutic options.
We have previously shown the feasibility of specifically targeting VHL-deficient ccRCC by identifying a small molecule, STF-62247. [10][11][12][13] The HIF-independent selective response toward VHL-deficient cells was linked to a deregulation of autophagy, a highly conserved homeostatic process involved in the degradation and renewal of intracellular components. 14,15 The mammalian target of Rapamycin (mTOR) is an evolutionary conserved serine/threonine kinase that regulates cell division, growth and proliferation and its deregulation has been linked to metabolic diseases, cancer and diabetes. [16][17][18] Major intracellular and extracellular cues such as growth factors, stress, energy levels, oxygen and amino acids modulate mTORC1 signaling to assure anabolic processes such as protein, lipid and nucleotide synthesis and catabolic processes like autophagy. Upon activation, mTOR inhibits autophagy, phosphorylates and activates p70S6K and 4EBP1 driving protein synthesis, cell growth and cell proliferation. 16,19,20 Understanding the mechanism of action of STF-62247 and its targeted cytotoxicity is important for drug target identification and development of safer therapies. Thus, we hypothesized that using stable isotope labeling by amino acids in cell culture (SILAC) would build a global overview of proteome alterations in kidney cancer cells in response to STF-62247 treatment. SILAC is a powerful yet simple approach for incorporation of nonradioactive isotopecontaining amino acids into proteins for mass spectrometry (MS)-based quantitative proteomics. [21][22][23][24] IPA was used to analyze SILAC data and to predict deregulated pathways in response to STF-62247 in VHL-null cells. We show that a global SILAC proteomics approach coupled with bioinformatics analyses are successful methods for identifying important signaling pathways affected by a small bioactive compound. Validations of some of these pathways show that STF-62247 does not cause accumulation of reactive oxygen species (ROS) and that its selectivity is associated with an inhibition of mTOR phosphorylation in VHL-deficient cells leading to protein synthesis arrest.

Material and Methods
Mammalian cell culture and treatments RCC4 negative for VHL and its stably transfected counterpart RCC4-VHL (pcDNA3-VHL) were provided by Amato J. Giaccia (Stanford University, Stanford, CA). Authentication of VHL-expressing and negative (parental) cells was performed by short tandem repeat (STR) DNA profile at Genetica DNA Laboratories (Burlington, NC). Cells were cultured in Dulbecco's modified Eagle medium (DMEM) high glucose (HG) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1 mM sodium pyruvate. STF-62247 was provided by Michael Hay (University of Auckland, Auckland, New Zealand).

Metabolic labeling (SILAC)
RCC4 cells were cultured in DMEM-HG supplemented with 50 mg L-Lysine-2HCl and 50 mg L-Arginine-HCl (light) or with 50 mg 13 C 6 L-lysine-2HCl and 50 mg of 13 C 15 6 N L 4 -Arginine-HCl (heavy) (Cambridge Isotope Labeling, Tewksbury, MA). 3.5 3 10 5 cells were cultured in parallel in heavy or light media for 7 days and were lysed with the Mammalian-Protein Extraction Buffer (M-PER) lysis buffer. In-gel digestion of proteins was performed, offering better resolution and increasing the number and accuracy of proteins present in the samples. Briefly, proteins from both conditions were mixed in different ratio quantities and separated by SDS-PAGE on a 10% bis-acrylamide gel. The gel was stained with EZ Blue Gel Staining Reagent (Sigma-Aldrich Canada Co., ON, Canada) and bands were excised and digested with Trypsin for Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) analysis. Each lane was cut in 24 gel pieces, washed and destained with acetonitrile. Dithiothreitol (DTT) (10 mM) and iodoacetic acid (IAA) (25 mM) solutions were used for reduction and alkylation of protein bands, respectively. 20 ng/mL of Trypsin Gold (V5280, Promega, Madison, WI) was added to the gel pieces and incubated overnight at 378C. Extraction of peptides was achieved by adding 5% acetic acid in 50% acetonitrile. Heavy or light (INV)-labeled RCC4 cells were treated with 1.25 lM STF-62247 for 48 hr.

Mass spectrometry analysis
Protein digests were analyzed by gradient nanoLC-MS/MS using a Quadrupole-Orbitrap mass spectrometer (Q Exactive, Thermo Scientific, San Jose, CA) interfaced to a Proxeon Easy Nano-LC (Thermo Scientific). High-resolution chromatographic separation was achieved on an Easy C18 analytical column with dimensions of 100 mm by 75 lm i.d. using 3 lm diameter ReproSil-Pur particles. Peptide elution was achieved using an acetonitrile/water gradient system. Solvent A was 0.1% formic acid (Sigma-Aldrich) in water and solvent B was acetonitrile/water/formic acid (90/9.9/0.1). A linear acetonitrile gradient was applied to the C18 column from What's new? Mutations on the tumor suppressor gene VHL are observed in most RCC cases. We previously identified a small molecule, STF-62247 tha tis capable to kill VHL-inactivated tumor cells. Here they used SILAC proteomics, IPA, and bioinformatics to show that STF-62247 may arrest protein synthesis in VHL-negative cells, while sparing normal cells. This approach also identified novel signaling pathways that could provide new, personalized therapeutic targets for ccRCC. 5 to 30% solvent B in 60 min followed by 100% B for 10 min at a flow rate of 300 nL/min. Precursor ion spectra were collected at a resolution setting of 35,000 and an automatic gain control value of 1 3 10 6 . Peptide fragmentation was performed using high-energy collision-induced dissociation in the HCD cell and MS/MS spectra were collected in the orbitrap at a resolution of 17,500 and an AGC setting of 1 3 10 5 . Peptide precursors were selected using a repeat count of 2 and a dynamic exclusion period of 20 sec. Protein identification and quantification data were analyzed using Proteome Discoverer version 1.4 (Thermo Scientific) employing the Sequest scoring algorithm. FASTA databases were obtained from Uniprot for Homo Sapiens. Quantification of 2-plex SILAC data was performed by Proteome Discoverer Precursor Ions Quantifier node using a heavy labeled arginine (110.008 Da) and lysine (16.020 Da) with a minimum fold change of 2 to indicate a significant result. Scaffold Q1 (version Scaffold_4.3.4, Proteome Software, Inc., Portland, OR) was used to validate SILAC peptide and protein identifications and quantitation. Acquired intensities in the experiment were globally normalized across all acquisition runs. Individual quantitative samples were normalized within each acquisition run.

Microarray hybridization and analysis
Quality of total RNA samples was assessed using the Experion bioanalyzer system with RNA Stdsens chips and associated reagents (Bio-Rad, ON, Canada). One microgram of total RNA samples was amplified using the Amino Allyl Mes-sageAmp II aRNA amplification kit and subsequently labeled with AlexaFluor 555 or 647 (Thermo Scientific). Samples were compared in a dye swap experiment, with 2.5 lg of each labeled, fragmented aRNA (5 lg total per slide) hybridized to proprietary human cDNA microarray slides. These arrays consist of approximately 35,000 spots, representing roughly 17,000 different 50-mer oligonucleotides spotted in duplicate on Nexterion-E epoxy microarray slides (Schott). Hybridizations were performed in Ambion SlideHyb #2 buffer (Thermo Scientific) at 428C for 16 hr using the automated TECAN 4800 Hybridization station (TECAN). Analysis was done with Acuity 4.0 (Axon Instruments). The data was normalized using Lowess and analyzed by various statistical methods such as SOM (Self Organizing Maps), t test, PCA (principal component analysis) and volcano plot.

Bioinformatics and data analysis
Differentially expressed proteins were analyzed using IPA (Qiagen Ingenuity systems, Redwood City, CA). Functional networks, canonical pathways and molecular functions were generated based on information contained in the ingenuity pathways knowledge base. The database for annotation, visualization and integrated discovery (DAVID) was used as a web-based online bioinformatics resource for a functional interpretation of differentially expressed proteins identified by SILAC.

Measurement of reactive oxygen species (ROS)
Intracellular ROS production was assessed using 2 0 ,7 0 -dichlorofluorescein (H2DCFDA) probe (Life technologies, Thermo-Fisher). Cells were harvested and incubated with 10 lM H2DCFDA for 30 min at 378C. Fluorescence was measured using excitation at 485 nm and emission at 530 nm. Relative quantification was calculated between treated and untreated cells.

Transient transfection
Cells were transfected using validated siGENOME Human siRNA targeting EEF2 (SMARTpool) and siControl2 nontargeting pool from Dharmacon using Dharmafect reagent 1 for 72 hr. Cells were trypsinized for clonogenic assays and proteins were extracted for western blot.
Clonogenic assays. Five-hundred cells were plated in triplicate in 60 mm plates and treated 24 hr later with 1.25 lM STF-62247 and left at 378C for 8 days. Plates were stained with a solution of crystal violet and colony formation was quantified.

Protein synthesis measurement
First, protein synthesis was determined by Click-iT HPG AlexaFluor protein Synthesis Assay Kit (Thermo Fisher) and performed per manufacturer's instructions. Briefly, cells were platted in an eight-well chambered coverglass (LabTek) and treated with STF-62247. Click-iT HPG solution (in Lmethionine free media) was added for 30 min, fixed with 3.7% formaldehyde and permeabilized with 0.5% Triton X-100. Click-iT HPG detection was achieved by adding reaction cocktail for 30 min at RT and rinsed with the reaction buffer. DNA staining with HCS NuclearMask Blue Stain was incubated for 30 min. Images were acquired with an Olympus Fluoview FV1000 confocal microscope.
Second, a protocol for measuring the fractional rate of protein synthesis described by S.G. Lamarre's group 25 was adapted for mammalian cells. To establish the optimal incorporation time of ring-D 5 L-phenylalanine (Cambridge Isotope Laboratories, Inc., Tewksbury, MA) in cells, 300 lL of a 150 mM solution of phenylalanine and phenylalanine-D 5 (50:50) prepared in water was added to RCC4 and RCC4 VHL cells for 0, 15, 30, 45 and 60 min. One milliliter of media from each dish was kept and ice-cold perchloric acid was added (free-pool [FP] of amino acids). Cells were scrapped directly in 1 mL of 0.2 M ice cold perchloric acid and left on ice for 10 min. FP and cell fractions were centrifuged 15,000g, 5 min at 48C. Supernatants were discarded and protein pellets were resuspended in 0.2 M perchloric acid and centrifuged for 15,000g, 10 min. After repeating this step three times, the pellet is washed with acetone and centrifuged before being hydrolyzed in 6 mL 6 N hydrochloric acid (HCL) at 1108C overnight. Phenylalanine extraction and derivatization was done as per S.G. Lamarre et al. (2015). The determination of PHE specific enrichment was determined using an Agilent gas chromatograph (model 6890N) interfaced with a single quadrupole inert mass selective detector (MSD, model 5973).

SILAC experimental procedures and data compilation
Our optimized SILAC workflow consists of two main steps; (i) determination of isotope incorporation efficiency (Fig. 1a, left) and (ii) SILAC main experiment/treatment (Fig. 1a, right). First, VHL-deficient RCC4 cells were grown in parallel in light or heavy isotope containing-medium for five cell doublings and both protein populations were extracted and quantified (Fig. 1a, left). To ensure complete (>95%) incorporation efficacy and standard quantification, heavy and light samples were loaded separately on a polyacrylamide gel and different ratios of each were mixed (Heavy/Light; 1:1, 1:2, 1:5, 2:1, 5:1). A band with the same molecular mass was excised in each lane, trypsin digested and processed by LC-MS/MS to validate isotope incorporation ratios. Heavy isotope peptides were identified by a shift of 6 and 10 Da as expected by the presence of Lysine and Arginine (Fig. 1a, left). Secondly, cells in Heavy-medium were treated for 48 hr with STF-62247. Proteins from heavy-and light-untreated cells were separated on gel. "INVERT SILAC" (INV) was utilized as a control by treating cells in lightmedium with STF-62247. After staining, all protein bands from both gels (SILAC and INV) were processed by LC-MS/MS followed by bioinformatics analyses with IPA (Fig. 1a, right).
Nearly 3,000 proteins were identified in each STF-62247 and INV SILAC replicates (Fig. 1b). From this list, we noted 1,088 and 1,170 common proteins between STF-62247 and INV triplicates, respectively. The number of unique proteins for STF and INV experiments was 333 and 415, respectively (Fig. 1b). To reduce these numbers and eliminate off-target deregulated proteins not directly due to STF-62247 treatment, both lists were compared. Unlike STF-62247 SILACs, INV SILACs were performed after light-isotope containing media was treated with STF-62247. For this reason, only upregulated proteins identified as downregulated in INV-SILACs (or downregulated appearing upregulated in INV), were considered. A list of 755 identified proteins was obtained (Fig. 1b). A heat-map with respective significant quantitative values of all six replicates (STF-62247 and INVERT) was generated with a fold change cut-off of 0.25 and 1.25 with a p values of 0.05 (Fig. 1c). Our SILAC workflow was successful in identifying 755 proteins. Most importantly, differentially expressed proteins were validated by INV experiments, showing reproducibility of the method.

Validation of proteins identified by SILAC and classification of GO annotations using DAVID
A cDNA microarray (RCC4 vs. STF-62247-treated RCC4) was compared to the SILAC proteomic data and among 87 hits correlating between the two platforms, cathepsin D and p62 were the most upregulated (Figs. 1c, 2a, and 2b). SILAC experiments were performed in VHL-deficient cells, but protein expression was also assessed in cells that surmount STF-62247 treatment stably expressing VHL (RCC4 VHL). The top upregulated proteins identified by SILAC were TMEM59, Cathepsin D and p62 (Fig. 1c). Accordingly, western blots showed an increase in the uncleaved proform of Cathepsin D while its active cleaved form decreased (Fig. 2c). In contrast, no apparent decrease of the active form is seen in RCC4 VHL cells. TMEM59 was also validated with a clear increased expression in response to STF-62247 treatment while RCC4 VHL responded in an opposite manner, with a time-dependent decreased expression (Fig. 2c). No significant change in expression was quantified by SILAC and western blot analysis for UBA1.
We interrogated the DAVID to acquire gene ontology (GO) domains of quantified proteins identified by SILAC (Fig. 2d). Interestingly, many protein biological functions refer to protein localization, protein transport (vesicle-mediated transport, golgi vesicle transport or endosome transport), regulation of protein ubiquitination, protein translation or protein folding, all of which are directly related to dynamic processes such as autophagy and endocytosis. GO_Cell component results were also closely associated with the above-mentioned biological functions, including membrane-enclosed lumen, organelle membrane, endoplasmic reticulum, vesicle, golgi apparatus and transport vesicle (Fig. 2d). These results showed that expression of top upregulated proteins identified by SILAC proteomics corroborate at the mRNA level and were validated by western blot.
Deregulated signaling pathways in RCC4 cells predicted by ingenuity pathway analysis IPA was utilized to analyze SILAC data and link deregulated proteins to affected signaling pathways. Autophagy was of Molecular Cancer Biology interest to us as our previous study linked STF-62247 selectivity to this pathway. Interestingly, IPA generated a schematic view of up-(red) or down-(green) regulated proteins having direct roles in autophagy and linked them to predicted affected pathways; endoplasmic reticulum stress pathway, unfolded protein response, p70S6K, AMPK, mTOR and  Nuclear Factor, Erythoid 2 Like 2 (NRF2)-mediated oxidative stress response (Fig. 3a). These pathways were further confirmed by a histogram generated by IPA based on -log(p values) and represented by their interconnection (Figs. 3b and  3c). The z-scores serve as predictions in activation or inactivation of identified pathways represented in orange and in blue, respectively. Remodeling of epithelial junctions and NRF2-mediated oxidative stress-response were pathways predicted to be activated in response to STF-62247 (Figs. 3b and 3c). A major pathway directly affecting autophagy predicted to be inactivated in response to STF-62247 is mTOR and its downstream effectors, p70S6K, leading to protein synthesis (Fig. 3d). Because mTOR is known to be a major regulator of autophagy, this was of interest to be further validated. 11,12 IPA analyses were able to link protein expression quantified by SILAC to predicted affected pathways giving an idea on STF-62247's effect in ccRCC cells lacking VHL.

NRF2-mediated oxidative stress response is not triggered in response to STF-62247
To validate some predictions by IPA, dysregulated proteins in two above-mentioned pathways; Remodeling of Epithelial Adherens Junctions and NRF2-mediated oxidative stress were briefly explored. Activation z-scores were predicted by IPA for Rab5 and Rab7, two small GTPases. 26 Western blots showed an increased expression of these effectors in a timedependent manner, predominantly in RCC4 cells (Fig. 4a). We also explored components of NRF2-mediated oxidative stress pathway, predicted to be activated with STF-62247 treatment. However, no production of reactive oxygen species was measured using the H2DCFDA probe (Fig. 4b). Concomitantly, immunofluorescence by confocal microscopy did not show nuclear translocation of NRF2 in neither RCC4, nor RCC4 VHL cells (Fig. 4c). Furthermore, p62 is known to be a main transcriptional effector of NRF2 and its overexpression correlated between proteomic and transcriptomic data (Figs. 1c and 2a). 27 Despite the cytosolic localization of NRF2, p62 expression is markedly increased after STF-62247 treatment (Fig. 4d). These preliminary results show that p62 overexpression may not be due to an activation of NRF2mediated oxidative stress production, as predicted by IPA.

STF-62247 decreases mTORC1 activation differentially from Rapamycin and RAD001
Inactivation of mTOR signaling pathway leading to protein synthesis arrest was also predicted by IPA analysis of SILAC data (Fig. 3d). Thus, mTOR and some of its downstream effectors were investigated to validate IPA predictions and SILAC data. In cells lacking VHL, expression of P-mTOR (S2448) decreased in a time-dependent manner in response to treatment and is further confirmed by a decreased expression of its downstream effectors P-p70S6K (T389) and P-4EBP1 (T37/46) (Fig. 5a). Contrastingly, P-mTOR (S2448) levels in RCC4 VHL cells return to normal states at the 48 hr time point with a concurring expression pattern for P-p70S6K (T389) (Fig. 5a). Based on these results, mTORC1 would seem to be reactivated in cells expressing VHL coinciding with increased levels of P-4EBP1 (T37/46) (Fig. 5a). These results confirm IPA predictions and indicate that STF-62247 acts on mTORC1 phosphorylation and activation.
To compare the effect of STF-62247 with known allosteric mTOR inhibitors (Rapamycin and RAD001), western blot analysis and XTT assays were performed. Both inhibitors decreased mTOR phosphorylation in RCC4 cells with STF-62247 after 48 hr of treatment (Fig. 5b). Phosphorylation of mTOR downstream effector p70S6K (T389) is also decreased in both cell lines at 24 and 48 hr time points confirming mTOR downstream signaling inhibition. XTT assays showed a selectivity of STF-62247 for VHL-deficient RCC4 cells while none is observed when cells are treated with either RAD001 or Rapamycin alone (Fig. 5c). To monitor possible synergistic effects of mTOR inhibitors with STF-62247, increasing concentrations of either Rapamycin or RAD001 were combined with STF-62247 (Fig. 5d). When combined with STF-62247, both inhibitors abolished the specificity of the compound for VHL-deficient cells suggesting a different upstream signaling associated with STF-62247 (Fig. 5d). Unlike allosteric mTOR inhibitors that show a same inhibitory effect regardless of VHL status, these results suggest that STF-62247 sustains a decreased phosphorylation of mTOR and its downstream effectors in a selective manner, dependent on VHL status.

STF-62247 decreases protein synthesis in VHL-deficient cells
To further explore the implication of mTORC1 inhibition, we employed two approaches to measure its downstream effect on protein synthesis. Firstly, to measure global protein synthesis, Click-iT HPG AlexaFluor protein Synthesis Assay showed an important decrease in protein synthesis in VHLdeficient cells from 0 to 48 hr as assessed by fluorescence intensity (Fig. 6a). Cycloheximide (CHX), a protein synthesis inhibitor was used as a positive control and abolished Click-iT fluorescence. Concurrent with western blot results obtained for mTORC1 in VHL-proficient cells (Fig. 5), protein synthesis is shown to be unaffected, even slightly increased in RCC4 VHL treated cells with STF-62247 (Fig.  6a). Secondly, we optimized a quantitative protocol using labeled phenylalanine previously developed by Lamarre et al. The graph presented in Figure 6b shows individual experiments (N 5 1,2,3) in both RCC4 and RCC4 VHL from 0 to 48 hr. K s (% day 21 ) is representative of protein synthesis levels. These levels decreased significantly in RCC4-treated cells after 24 hr (Fig. 6b). At 48 hr, protein synthesis slightly increased in RCC4 albeit staying significantly lower compared to the untreated control. Again, protein synthesis seems to be increasing at 48 hr in VHL-containing cells in response to STF-62247 supporting the results obtained by the Click-it fluorescence (Fig. 6b) and reflecting mTORC1 phosphorylation states showed previously (Fig. 5a).    Figure 6c. Statistical analysis compared the relative levels of EEF2 for each siRNA treated cell lines relation their control using Student's t test. *p < 0.05. (e) Clonogenic assay of EEF2 silencing compared to the mock transfection (control) and nontargeting (NT) siRNA. Cells were plated 72 hr posttransfection and treated with 0-2.5 lM at STF-62247. Colony formation was quantified after 9 days incubation. Relative quantification was compared to cells transfected with the nontargeting sequence. Statistical analysis compared the relative levels of survival observed in siEEF2 cells to siControl cells using Student's t test (*p < 0.05, **p < 0.01, ***p < 0.001).
To explore the difference in protein synthesis levels observed between VHL-null and cells with a functional gene, we evaluated the effect of EEF2 inhibition on clonogenic ability. Knock-down of EEF2 was achieved by siRNA and compared to a nontargeting control (Figs. 6c and 6d). As previously reported, survival of RCC4 cells decreased in a concentration dependent manner while survival for RCC4 VHL remained unaffected. 11 Interestingly, knockdown expression of EEF2 in VHL-deficient cells caused a synergistic effect and further decreased survival of RCC4 cells in presence of 0-2.5 lM STF-62247. In contrast, cell survival was largely unaffected in VHL cells silenced for EEF2 (Fig.  6e). Taken together, SILAC quantification and IPA bioinformatics analyses helped us identify some of STF-62247 mechanisms of action in RCC4 -/-VHL. We have validated IPA predictions and showed that STF-62247 acts on phosphorylation of mTOR and its downstream effectors and significantly decreases protein synthesis, as measured by two methods.

Discussion
To date, available HIF-targeting therapies for the treatment of mRCC remain inefficient and patients too often live a progression of their metastatic disease. The previous identification of a small molecule compound STF-62247 has shown that targeting the loss of VHL could be an attractive new approach to target kidney cancer cells and spare normal tissue. The understanding of its mechanism of action is crucial for the development of novel treatments for mRCC. Our previous studies linked STF-62247 to a deregulation of autophagy and inhibition of key autophagy related genes (ATG) prevented VHL-null cells from death suggesting that this dynamic process was at least in part implicated in the cytotoxicity of this small molecule.
In our study, we used SILAC to paint a global picture of STF-62247 signaling in the cell and most importantly, to identify key pathways that could lead to a better understanding of the molecule's selective mechanism of action. We employed a global proteomic labeling approach rather than a targeted strategy to quantify and identify all proteins that were modulated in response to STF-62247. We identified 1,088 proteins that were similar between the biological triplicates. The INVERT SILAC was performed to eliminate possible off-target proteins. These steps made possible the identification of 755 proteins in response to STF-62247. Several upregulated proteins were involved in lysosomal processes such as Cathepsin D, p62, lysosomal hydrolases and v-ATPases pump subunits. In addition, we compared proteomics results with transcriptomic profiling performed by microarray in our laboratory. By investigating the 87 differentially expressed proteins that showed correlation at the mRNA level, Cathepsin D, p62 and lysosomal enzymes were present in both analyses. Western blot analysis for Cathepsin D in VHL-deficient cells indicated a clear increase in its proform while its active cleaved form was decreased supporting our ongoing findings suggesting that STF-62247 affects lysosomal integrity in these cells.
We used gene ontology to facilitate association of altered proteins quantified by SILAC and their implication in different biological processes, molecular functions and cell components. The functional annotation tool DAVID showed that most proteins had roles in trafficking functions linking autophagy and endocytosis. Both pathways are intimately linked and contribute to lysosomal integrity by trafficking of lysosomal hydrolases and v-ATPase subunits. 26,28,29 Although this tool identified GO annotations associated with SILAC data, it did not consider quantification values and this could explain some differences observed between the pathway analyses by DAVID and those identified by IPA. Nevertheless, bioinformatics analyses using DAVID and IPA lead us to investigate the NRF2 and the mTOR pathway that could be linked to autophagy and lysosomal processes.
By investigating components of predicted pathways, our results showed a decreased phosphorylation of mTOR (S2448) further confirmed by a decrease of direct mTOR effectors, P-p70S6K (T389) and P-4EBP1 (T37/46). Interestingly, inhibition of mTORC1 activation was specific to VHLnull cells, concurrent with protein synthesis arrest, measured by two different techniques. Contrastingly, phosphorylation states of mTOR and of its downstream effectors seem to return to levels comparable to the untreated control in VHLproficient cells concurrent with stable, even augmented protein synthesis. Moreover, small-interfering RNAs against EEF2 in VHL-deficient cells caused a synergistic effect and further decreased survival of RCC4 cells while remaining largely unaffected in VHL cells silenced for EEF2. Like us, previous studies have also linked VHL status to protein synthesis and have shown that the lack of VHL gene product sensitized RCC cells to the apoptotic effects of a protein synthesis inhibitor. 30 This could be further investigated as a potentially new therapeutic target to bypass development of resistance of RCC tumors to available targeted therapies.
The mechanism by which mTORC1 is inhibited by STF-62247 remains to be elucidated since many upstream sources may contribute to its altered signaling, i.e., amino acids, growth factors and even lipids. 16,[31][32][33] Inhibition of mTORC1 by STF-62247 would seem to be through a mechanism differing from both allosteric inhibitors Rapamycin and RAD001 as they abolish STF-62247 selectivity when combined and do not further decrease cell viability in VHL deficient cells. Lastly, one of the most upregulated protein in response to treatment was the signaling adaptor p62/SQSTM1. We have demonstrated that this over-expression was not due to NRF2-mediated oxidative stress pathway, since no translocation to the nucleus was observed. Recent studies have elucidated central roles of p62 for cell survival and proliferation through activation of mTORC1. [34][35][36][37][38][39] p62 has been shown to facilitate translocation of mTORC1 to lysosomes but importantly, acts as a scaffold, bringing components involved in the control of mTORC1 signaling to the correct cellular location. 40 Together, the selective inactivation of mTORC1 in VHL-deficient cells and the deregulated proteins and mRNA associated with lysosomal integrity could lead to hypothesize the possibility that mTORC1 may not be able to get reactivated at the lysosomes, especially if lysosomal membrane components are compromised in response to STF-62247 treatment. This however, remains to be investigated.
Our proteomic study identified signaling pathways implicated in STF-62247 response. Although further mechanistic investigations are needed to shed light on the inhibitory effects of the small molecule on mTORC1, we have validated data on the differences existing in protein synthesis progression and identified a synergistic effect of STF-62247 with knock-down of EEF2. We have succeeded in accomplishing a global proteomic study and have validated bioinformatics analysis and predictions of SILAC data. Although the target of this molecule remains to be identified, this research deepens our understanding of its mechanism of action.