HHS Public Access Author manuscript Author Manuscript
Clin Cancer Res. Author manuscript; available in PMC 2017 November 15. Published in final edited form as: Clin Cancer Res. 2016 November 15; 22(22): 5605–5616. doi:10.1158/1078-0432.CCR-15-1673.
Tyrosine Kinase Signaling in Clear Cell and Papillary Renal Cell Carcinoma Revealed by Mass Spectrometry-Based Phosphotyrosine Proteomics
Author Manuscript
Scott M. Haake1, Jiannong Li2, Yun Bai2, Fumi Kinose2, Bin Fang3, Eric Welsh4, Roy Zent5, Jasreman Dhillon6, Julio Pow-Sang7, Yian Ann Chen4, John Koomen8,9, W. Kimryn Rathmell1, Mayer Fishman7, and Eric B. Haura2 1Hematology
& Medical Oncology, Department of Medicine, Vanderbilt University Medical Center,
Nashville, TN 2Thoracic
Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL
3Proteomics,
H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL
4Biostatistics
and Bioinformatics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL
5Nephrology,
Department of Medicine, Department of Cancer Biology, Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN; Nephrology, Department of Medicine, Veterans Affairs Hospital, Tennessee Valley Healthcare System, Nashville, TN
Author Manuscript
6Pathology,
H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL
7Genitourinary 8Chemical
Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL
Biology and Molecular Medicine, H. Lee Moffitt Cancer Center & Research Institute,
Tampa, FL 9Molecular
Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL
Abstract Purpose—Targeted therapies in renal cell carcinoma (RCC) are limited by acquired resistance. Novel therapeutic targets are needed to combat resistance and, ideally, target the unique biology of RCC subtypes.
Author Manuscript
Experimental Design—Tyrosine kinases provide critical oncogenic signaling and their inhibition has significantly impacted cancer care. In order to describe a landscape of tyrosine kinase activity in RCC that could inform novel therapeutic strategies, we performed a mass spectrometry-based system-wide survey of tyrosine phosphorylation in 10 RCC cell lines as well
Address correspondence to: Scott M. Haake, MD, Division of Hematology and Medical Oncology, Department of Medicine, Vanderbilt University Medical Center, 2220 Pierce Ave, 777 PRB, Nashville, TN 37232. Phone: 615-879-9744; Fax: 615-343-2551; [email protected] or Eric B. Haura, MD, Department of Thoracic Oncology, Chemical Biology and Molecular Medicine Program, H. Lee Moffitt Cancer Center & Research Institute, MRC3East, Room3056F, 12902 Magnolia Drive, Tampa, FL 33612. Phone: 813-745-6827; Fax: 813-745-6817; [email protected]. There are no conflicts of interest to disclose by the authors.
Haake et al.
Page 2
Author Manuscript
as 15 clear cell and 15 papillary RCC human tumors. To prioritize identified tyrosine kinases for further analysis, a 63 tyrosine kinase inhibitor (TKI) drug screen was performed. Results—Among the cell lines, 28 unique tyrosine phosphosites were identified across 19 kinases and phosphatases including EGFR, MET, JAK2, and FAK in nearly all samples. Multiple FAK TKIs decreased cell viability by at least 50% and inhibited RCC cell line adhesion, invasion, and proliferation. Among the tumors, 49 unique tyrosine phosphosites were identified across 44 kinases and phosphatases. FAK pY576/7 was found in all tumors and many cell lines, while DDR1 pY792/6 was preferentially enriched in the papillary RCC tumors. Both tyrosine kinases are capable of transmitting signals from the extracellular matrix and emerged as novel RCC therapeutic targets. Conclusions—Tyrosine kinase profiling informs novel therapeutic strategies in RCC and highlights the unique biology amongst kidney cancer subtypes.
Author Manuscript
Keywords renal cell carcinoma; tyrosine kinase; mass spectrometry; FAK; DDR
Introduction Renal cell carcinoma (RCC) is among the most common cancers diagnosed in the United States. Metastatic RCC is relatively insensitive to traditional therapies like chemotherapy and radiation and is generally incurable (1). However, the emergence of active agents targeting vascular epithelial growth factor receptor (VEGFR) and mammalian target of rapamycin (mTOR) heralded a new era in the treatment of RCC. However, patient outcomes remain poor despite these contemporary therapies (2).
Author Manuscript Author Manuscript
There are multiple reasons that could explain the poor outcomes associated with the targeted treatment era. First, the responses to these targeted therapies are typically transient. The emergence of resistance is nearly inevitable resulting in poor 5-year survival (2). Second, the VEGFR-targeted therapies that have come to dominate the targeted treatment era in RCC were tailored for clear cell RCC, the most common histological subtype. These tumors are characterized by the near ubiquitous loss of function of the Von Hippel Lindau tumor suppressor, VHL (3). The result is inappropriate stabilization of HIF and a maladaptive hypoxic and angiogenic response, including markedly high levels of VEGF production (4). By targeting VEGF receptors, this class of tyrosine kinase inhibitors (TKIs) targets the core biology of clear cell RCC tumors. However, non-clear cell RCC tumors such as papillary RCC have functional VHL and thus classically do not exhibit the same angiogenic response. Predictably, their outcomes are inferior when treated with VEGFR therapies (5). Novel therapeutic targets could guide new drug development with a goal of delaying, treating, or preventing disease resistance. Furthermore, studying non-clear cell RCC offers the opportunity to tailor our treatment to their unique biology. Tyrosine kinases provide signal transduction that is critical for the growth and survival of several cancers (6). While several tyrosine kinases are overexpressed in RCC, including EGFR (7) and MET (8), few approved therapies target these epithelial drivers in RCC (9–
Clin Cancer Res. Author manuscript; available in PMC 2017 November 15.
Haake et al.
Page 3
Author Manuscript
11). In order to describe a broad landscape of tyrosine kinase activity in RCC that could inform novel therapeutic strategies, we performed a mass spectrometry (MS)-based systemwide survey of tyrosine phosphorylation in RCC cell lines as well as clear cell and papillary RCC tumors (Fig. 1). In order to prioritize emerging tyrosine kinase targets based on functional data, we concurrently performed a large TKI screen across the ten RCC cell lines. One potential target to emerge from this approach was focal adhesion kinase (FAK). FAK was phosphorylated at an activating site in cell lines and both clear cell and papillary tumors. FAK TKIs were active in the screen and were shown to inhibit cell adhesion, proliferation, and invasion. Additionally, the receptor tyrosine kinase DDR1, for which the only known ligand is collagen, was highly phosphorylated at multiple activating sites in the papillary RCC tumors relative to the clear cell RCC tumors. This receptor tyrosine kinase (RTK) may represent a novel mediator of stromal signaling and a therapeutic target in papillary RCC. The combination of systems level MS techniques to study tyrosine phosphorylations with integrated functional studies identified novel therapeutic targets that warrant further investigation.
Author Manuscript
Materials and Methods Full descriptions of all materials and methods can be found under Supplementary Methods and Materials. Cell lines
Author Manuscript
Cell lines A704 (HTB-45), A498 (HTB-44), ACHN (CRL-1611), Caki-1 (HTB-46), and Caki-2 (HTB-47) were purchased from American Type Culture Collection (ATCC). 786-O (CRL-1932), RXF393, UO31, SN12C, and TK10 (CRL-2396) were a gift from Dr. Javier Torres-Roca (Moffitt Cancer Center, Tampa, FL). All cell lines were cultured in RPMI1640 with 10% fetal bovine serum, maintained in a central repository at MCC, routinely tested for mycoplasma contamination, and have been authenticated with short-tandem repeat (STR) analysis (ATCC). Human tumor tissues
Author Manuscript
Tumor tissues were collected as part of the Total Cancer Care protocol (12) and approved by the University of South Florida Institutional Review Board (Tampa, FL). Patients gave informed consent before enrollment in the Total Cancer Care protocol. Tumor tissues were snap frozen following nephrectomy. All tissues contained more than 90% tumor cells when examined by light microscopy. Centralized pathology review was performed at time of TCC enrollment. So that phosphotyrosine (pY) peptide patterns could be compared between tumor types, clear cell and papillary RCC tumors were balanced for patient characteristics including age, gender, race, tumor size, stage, and nuclear grade (Supplementary Table 1). Type 1, type 2, and undefined papillary RCC were included in the analysis. Phosphopeptide immunoprecipitation, analysis, and data processing Immunoprecipitation and purification of pY peptides was performed using PhosphoScan pTyr100 (Cell Signaling Technology) according to the manufacturer’s recommendations and as described previously (13). Briefly, whole cell extracts were prepared from either 1) 2 ×
Clin Cancer Res. Author manuscript; available in PMC 2017 November 15.
Haake et al.
Page 4
Author Manuscript Author Manuscript
108 cells of each cell line or 2) 100 mg of tumor tissue using denaturing lysis buffer containing 9 M urea and 20 mM HEPES (pH 8.0) supplemented with phosphatase inhibitors (1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate) followed by sonication on ice. Extracted proteins (50 mg for each cell line) were then reduced with 4.5 mM DTT and alkylated with 10 mM iodoacetamide. Trypsin digestion was carried out at room temperature overnight and resulting tryptic peptides were then acidified with 1% trifluoroacetic acid and desalted with C18 Sep-Pak cartridges (Waters) according to the manufacturer’s procedure. Peptides were lyophilized and then dissolved in immunoaffinity purification (IAP) buffer containing 50 mM MOPS/NaOH (pH 7.2), 10 mM sodium phosphate, and 50 mM sodium chloride. The pY peptides were immunoprecipitated with immobilized pTyr100 antibody overnight at 4°C followed by three washes with IAP buffer and two washes with H2O. The pY peptides were eluted from beads twice with 0.15% trifluoroacetic acid and the volume was reduced to a final concentration of 20 μL via vacuum centrifugation. Before MS tumor analysis, 100 fmol of a multi-peptide standard (Pierce 88320) was spiked into each sample. Samples were analyzed by nano-liquid chromatography/tandem mass spectrometry (LC-MS/MS). Two technical replicates were performed for each sample. Aliquots of unused tumor pY immunoprecipitate was analyzed in a targeted quantification experiment (liquid chromatography-multiple reaction monitoring or LC-MRM) to validate the label free relative quantification data for select phosphopeptides. Further LC-MS/MS and LC-MRM details are described in Supplementary Materials and Methods. Generation of protein-protein interaction network for pY proteins
Author Manuscript
Drug screening and in vitro assays
Author Manuscript
The pY proteins corresponding to the clear cell RCC cell lines and the human tumors were input into Cytoscape (version 2.8.3; http://www.cytoscape.org; (14)). Protein-protein interactions (PPI) between nodes were imported using the PSICQUIC plug-in to search multiple databases (15). Network visualization was performed with Cytoscape.
Western blotting
Viability assays were performed in black-wall 384-well microtiter plates for the 10 RCC cell lines. Cells were seeded at a density of 1,000 cells/well. Drugs or DMSO were added after 24 hours and cells were incubated for another 72 hours. 63 tyrosine kinase inhibitors (Fig. 4a, Supplementary Fig. 1–2) were screened for each cell line at 0.5 μM and 2.5 μM (each in duplicate and averaged), a concentration that approximates or is slightly greater than predicted serum levels in humans and thus assures minimum false negatives. Viability was evaluated using the CellTiter-Glo assay (Promega) with addition of reagent after 72 hour drug incubation and luminescence was read on a SpectraMax M5 plate reader (Molecular Devices). Matrigel adhesion and invasion assays (16) as well as 3H-thymidine proliferation assays (17) were performed as described previously.
Western blotting was performed as described in our previous studies (13, 18). Primary antibodies used for our study were purchased from Cell Signaling Technology (EGFR pY1068, EGFR pY1197, MET pY1234/5, total MET), Santa Cruz (total EGFR, total FAK), BD Sciences (FAK pY397) and Sigma-Aldrich (β-actin). Clin Cancer Res. Author manuscript; available in PMC 2017 November 15.
Haake et al.
Page 5
Statistical Methods & Data Analysis
Author Manuscript Author Manuscript
Tumor samples were randomized prior to MS analysis to minimize potential batch effects. Internal controls within each sample (Thermo peptide standard) and external controls (MS analysis of control cell line before analysis of RCC tumors, after every 20 MS runs, and at conclusion of MS analysis of RCC tumors) were included to monitor potential batch effects (Supplementary Fig. 3 and 4). In order to maximize protein coverage, Mascot, Sequest, and Andromeda search engines were performed to identify phosphorylated proteins (see supplementary methods for details). To facilitate and streamline comparisons among samples, only proteins that were identified as phosphorylated in at least 5 samples (cell line or tumor) were considered for further analysis (Fig. 2 and 5). The data regarding individual phosphosites came from only the Andromeda search results (Fig. 3 and 6). Label free quantitative data was calculated using MaxQuant (version 1.2.2.5) using the Andromeda search results (Fig. 6) (19). Peptide ion signal intensities were log2 transformed to allow for the use of parametric statistical tests. A two-sample, two-tailed t-test was used to contrast phosphopeptide intensity in clear cell versus papillary tumors (Fig. 6a), phosphopeptide intensity in tumor samples calculated from extracted ion chromatograms (Fig. 6b), and output from multiple reaction monitoring experiments (Fig. 6c). To account for multiple hypothesis testing, a false discovery rate was estimated for phosphopeptide intensity in tumor samples and reported as a q-value with a cut-off of 0.05 (Fig. 6a). Protein and phosphosite functional annotation was performed with reference to databases at www.phosphosite.org and www.uniprot.org.
Author Manuscript
When contrasting the patient characteristics corresponding to human tumors (Supplementary Table 1), all continuous variables were described with the median and range values. Analyses of categorical data utilized a two-tail Fisher’s exact test. Difference of medians was evaluated with Mann-Whitney U test.
Results RCC cell lines and tumors display FAK phosphorylation but different patterns of receptor tyrosine kinase phosphorylation
Author Manuscript
In order to study RCC in an unbiased and systems level manner, we performed immunoprecipitation followed by LC-MS/MS to catalog pY peptides from 10 RCC cell lines and 30 tumors (Fig. 1). Overall, 198 proteins were identified and their function annotated (Fig. 2a, Supplementary Table 2). The proteins corresponded to several different classes including receptor tyrosine kinases (EGFR, MET), non-receptor tyrosine kinases (JAK2, FAK or FAK1 or PTK2), serine/threonine kinases (CDK1, MK01 or MAPK1), phosphatases (SHIP2 or INPPL1), cell adhesion proteins (ITB1 or ITGB1), cytoskeletal proteins (ABLM1, MYO1E), GTP-associated proteins (ARHG5 or ARHGEF5), and others. The cell lines and tumors had similarities with 102 shared tyrosine phosphorylated proteins (Fig. 2b). However, marked distinctions were also evident as 53 pY proteins were unique to cell lines and 22 unique to tumors. To further explore these patterns, the ~50 most commonly identified phosphorylated proteins were analyzed further. If the rate of phosphorylation for a protein was at least 50% higher for cell lines relative to tumors (or vice versa), it was considered “more common” in that subset (Fig. 2c and 2e). Proteins that were commonly
Clin Cancer Res. Author manuscript; available in PMC 2017 November 15.
Haake et al.
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Author Manuscript
phosphorylated but did not meet this cutoff were considered “common in tumors and cell lines” (Fig. 2d). The MAP kinase pathway was tyrosine phosphorylated in both tumors and cell lines as was PTK2 protein FAK1 or FAK), supporting their universal importance in RCC biology. However, multiple members of the PI3K pathway as well as EGFR were more common in the cell lines suggesting distinct signal transduction relative to tumors. By contrast, the collagen receptor DDR1 was phosphorylated in tumor samples only. Thus, while MAPK and FAK were phosphorylated in both cell lines and tumors, distinct patterns of activation emerged among some RTKs. Several kinases, including EGFR and MET, are phosphorylated in RCC cell lines
Author Manuscript
Rather than only examine whether a protein had any pY (Fig. 2), we also evaluated specific phosphosites in cell lines (Supplementary Table 3). Furthermore, our focus was narrowed to kinases and phosphatases given their key roles in regulating cancer signaling (Fig. 3a). EGFR pY1110, pY1172 and pY1197 was observed in multiple cell lines, consistent with receptor activation (20–22). Similarly, the presence of the MET activation site pY1234/5 in all cell lines implies the presence of ubiquitous MET activation. FAK pY576/7 was detected in nearly all the cell lines (Fig. 3a). This FAK phosphosite within the kinase domain of the enzyme is required for maximal enzymatic activity and thus suggests FAK activation (23). Thus, the identification of specific phosphosites was able to provide insight into the functional relevance of pY events.
Author Manuscript Author Manuscript
Western blotting was performed to further explore the pY patterns of the cell lines for EGFR, MET, and FAK (PTK2) (Fig. 3b). EGFR is known to be activated and phosphorylated in RCC, possibly related to VHL inactivation (24). EGFR pY1197 was weakly positive by western blot in several cell lines despite being identified in all cell lines via MS analysis (Fig. 3a and 3b). To further validate EGFR activation, western blot for EGFR pY1068 was performed. EGFR pY1068 is well known to correlate with EGFR activation (20, 21) and has an excellent performing antibody though, given the large size and double phosphorylation status of the corresponding tryptic peptide can be difficult to detect in MS analysis. The positivity of EGFR pY1068 on western blot supports the MS data that suggests EGFR activation. Thus, the weak EGFR pY1197 signal on western blot yet strong signal via MS may simply be due to increased sensitivity for MS techniques. MET is also known to be activated and phosphorylated in RCC and interest is increasing given the approval of cabozantinib, a TKI whose targets include MET (8, 11, 25). Western blot for MET pY1234/5 confirms that this phosphorylation event is common among the cell lines. Again, its detection in some cell lines via MS but not western blot suggests increased sensitivity of MS techniques. FAK (PTK2) was evaluated further as it was seen to be frequently phosphorylated in cell lines and tumors (Fig. 2d) and its function in RCC had not been well characterized in the literature. The performance of commercially available FAK pY576/7 antibodies was suboptimal and lacked reproducibility in our hands. However, the MS data (Fig. 2d and 3a) showed near-universal FAK activation in RCC and we verified its activation by testing phosphorylation of the canonical autophosphorylation and activation site, FAK pY397 (23). Overall, western blots validate the MS data that EGFR, MET, and FAK are phosphorylated at activating sites in RCC cell lines.
Clin Cancer Res. Author manuscript; available in PMC 2017 November 15.
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Page 7
TKI screen highlights activity of FAK tyrosine kinase inhibitors
Author Manuscript Author Manuscript
A functional screen utilizing 63 tyrosine kinase inhibitors (TKIs) was employed to assist in nominating functionally relevant signaling networks for further analysis (Supplementary Fig. 1–2). We focused our analysis on TKIs targeting tyrosine kinases observed to be phosphorylated in most cell lines, specifically EGFR, FAK, JAK2, and MET (Fig. 4a). Among the most active class of TKIs were those targeting FAK. Specifically, CEP-37440 at 2.5 µM reduced cell viability to
Clin Cancer Res. Author manuscript; available in PMC 2017 November 15. Published in final edited form as: Clin Cancer Res. 2016 November 15; 22(22): 5605–5616. doi:10.1158/1078-0432.CCR-15-1673.
Tyrosine Kinase Signaling in Clear Cell and Papillary Renal Cell Carcinoma Revealed by Mass Spectrometry-Based Phosphotyrosine Proteomics
Author Manuscript
Scott M. Haake1, Jiannong Li2, Yun Bai2, Fumi Kinose2, Bin Fang3, Eric Welsh4, Roy Zent5, Jasreman Dhillon6, Julio Pow-Sang7, Yian Ann Chen4, John Koomen8,9, W. Kimryn Rathmell1, Mayer Fishman7, and Eric B. Haura2 1Hematology
& Medical Oncology, Department of Medicine, Vanderbilt University Medical Center,
Nashville, TN 2Thoracic
Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL
3Proteomics,
H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL
4Biostatistics
and Bioinformatics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL
5Nephrology,
Department of Medicine, Department of Cancer Biology, Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN; Nephrology, Department of Medicine, Veterans Affairs Hospital, Tennessee Valley Healthcare System, Nashville, TN
Author Manuscript
6Pathology,
H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL
7Genitourinary 8Chemical
Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL
Biology and Molecular Medicine, H. Lee Moffitt Cancer Center & Research Institute,
Tampa, FL 9Molecular
Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL
Abstract Purpose—Targeted therapies in renal cell carcinoma (RCC) are limited by acquired resistance. Novel therapeutic targets are needed to combat resistance and, ideally, target the unique biology of RCC subtypes.
Author Manuscript
Experimental Design—Tyrosine kinases provide critical oncogenic signaling and their inhibition has significantly impacted cancer care. In order to describe a landscape of tyrosine kinase activity in RCC that could inform novel therapeutic strategies, we performed a mass spectrometry-based system-wide survey of tyrosine phosphorylation in 10 RCC cell lines as well
Address correspondence to: Scott M. Haake, MD, Division of Hematology and Medical Oncology, Department of Medicine, Vanderbilt University Medical Center, 2220 Pierce Ave, 777 PRB, Nashville, TN 37232. Phone: 615-879-9744; Fax: 615-343-2551; [email protected] or Eric B. Haura, MD, Department of Thoracic Oncology, Chemical Biology and Molecular Medicine Program, H. Lee Moffitt Cancer Center & Research Institute, MRC3East, Room3056F, 12902 Magnolia Drive, Tampa, FL 33612. Phone: 813-745-6827; Fax: 813-745-6817; [email protected]. There are no conflicts of interest to disclose by the authors.
Haake et al.
Page 2
Author Manuscript
as 15 clear cell and 15 papillary RCC human tumors. To prioritize identified tyrosine kinases for further analysis, a 63 tyrosine kinase inhibitor (TKI) drug screen was performed. Results—Among the cell lines, 28 unique tyrosine phosphosites were identified across 19 kinases and phosphatases including EGFR, MET, JAK2, and FAK in nearly all samples. Multiple FAK TKIs decreased cell viability by at least 50% and inhibited RCC cell line adhesion, invasion, and proliferation. Among the tumors, 49 unique tyrosine phosphosites were identified across 44 kinases and phosphatases. FAK pY576/7 was found in all tumors and many cell lines, while DDR1 pY792/6 was preferentially enriched in the papillary RCC tumors. Both tyrosine kinases are capable of transmitting signals from the extracellular matrix and emerged as novel RCC therapeutic targets. Conclusions—Tyrosine kinase profiling informs novel therapeutic strategies in RCC and highlights the unique biology amongst kidney cancer subtypes.
Author Manuscript
Keywords renal cell carcinoma; tyrosine kinase; mass spectrometry; FAK; DDR
Introduction Renal cell carcinoma (RCC) is among the most common cancers diagnosed in the United States. Metastatic RCC is relatively insensitive to traditional therapies like chemotherapy and radiation and is generally incurable (1). However, the emergence of active agents targeting vascular epithelial growth factor receptor (VEGFR) and mammalian target of rapamycin (mTOR) heralded a new era in the treatment of RCC. However, patient outcomes remain poor despite these contemporary therapies (2).
Author Manuscript Author Manuscript
There are multiple reasons that could explain the poor outcomes associated with the targeted treatment era. First, the responses to these targeted therapies are typically transient. The emergence of resistance is nearly inevitable resulting in poor 5-year survival (2). Second, the VEGFR-targeted therapies that have come to dominate the targeted treatment era in RCC were tailored for clear cell RCC, the most common histological subtype. These tumors are characterized by the near ubiquitous loss of function of the Von Hippel Lindau tumor suppressor, VHL (3). The result is inappropriate stabilization of HIF and a maladaptive hypoxic and angiogenic response, including markedly high levels of VEGF production (4). By targeting VEGF receptors, this class of tyrosine kinase inhibitors (TKIs) targets the core biology of clear cell RCC tumors. However, non-clear cell RCC tumors such as papillary RCC have functional VHL and thus classically do not exhibit the same angiogenic response. Predictably, their outcomes are inferior when treated with VEGFR therapies (5). Novel therapeutic targets could guide new drug development with a goal of delaying, treating, or preventing disease resistance. Furthermore, studying non-clear cell RCC offers the opportunity to tailor our treatment to their unique biology. Tyrosine kinases provide signal transduction that is critical for the growth and survival of several cancers (6). While several tyrosine kinases are overexpressed in RCC, including EGFR (7) and MET (8), few approved therapies target these epithelial drivers in RCC (9–
Clin Cancer Res. Author manuscript; available in PMC 2017 November 15.
Haake et al.
Page 3
Author Manuscript
11). In order to describe a broad landscape of tyrosine kinase activity in RCC that could inform novel therapeutic strategies, we performed a mass spectrometry (MS)-based systemwide survey of tyrosine phosphorylation in RCC cell lines as well as clear cell and papillary RCC tumors (Fig. 1). In order to prioritize emerging tyrosine kinase targets based on functional data, we concurrently performed a large TKI screen across the ten RCC cell lines. One potential target to emerge from this approach was focal adhesion kinase (FAK). FAK was phosphorylated at an activating site in cell lines and both clear cell and papillary tumors. FAK TKIs were active in the screen and were shown to inhibit cell adhesion, proliferation, and invasion. Additionally, the receptor tyrosine kinase DDR1, for which the only known ligand is collagen, was highly phosphorylated at multiple activating sites in the papillary RCC tumors relative to the clear cell RCC tumors. This receptor tyrosine kinase (RTK) may represent a novel mediator of stromal signaling and a therapeutic target in papillary RCC. The combination of systems level MS techniques to study tyrosine phosphorylations with integrated functional studies identified novel therapeutic targets that warrant further investigation.
Author Manuscript
Materials and Methods Full descriptions of all materials and methods can be found under Supplementary Methods and Materials. Cell lines
Author Manuscript
Cell lines A704 (HTB-45), A498 (HTB-44), ACHN (CRL-1611), Caki-1 (HTB-46), and Caki-2 (HTB-47) were purchased from American Type Culture Collection (ATCC). 786-O (CRL-1932), RXF393, UO31, SN12C, and TK10 (CRL-2396) were a gift from Dr. Javier Torres-Roca (Moffitt Cancer Center, Tampa, FL). All cell lines were cultured in RPMI1640 with 10% fetal bovine serum, maintained in a central repository at MCC, routinely tested for mycoplasma contamination, and have been authenticated with short-tandem repeat (STR) analysis (ATCC). Human tumor tissues
Author Manuscript
Tumor tissues were collected as part of the Total Cancer Care protocol (12) and approved by the University of South Florida Institutional Review Board (Tampa, FL). Patients gave informed consent before enrollment in the Total Cancer Care protocol. Tumor tissues were snap frozen following nephrectomy. All tissues contained more than 90% tumor cells when examined by light microscopy. Centralized pathology review was performed at time of TCC enrollment. So that phosphotyrosine (pY) peptide patterns could be compared between tumor types, clear cell and papillary RCC tumors were balanced for patient characteristics including age, gender, race, tumor size, stage, and nuclear grade (Supplementary Table 1). Type 1, type 2, and undefined papillary RCC were included in the analysis. Phosphopeptide immunoprecipitation, analysis, and data processing Immunoprecipitation and purification of pY peptides was performed using PhosphoScan pTyr100 (Cell Signaling Technology) according to the manufacturer’s recommendations and as described previously (13). Briefly, whole cell extracts were prepared from either 1) 2 ×
Clin Cancer Res. Author manuscript; available in PMC 2017 November 15.
Haake et al.
Page 4
Author Manuscript Author Manuscript
108 cells of each cell line or 2) 100 mg of tumor tissue using denaturing lysis buffer containing 9 M urea and 20 mM HEPES (pH 8.0) supplemented with phosphatase inhibitors (1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate) followed by sonication on ice. Extracted proteins (50 mg for each cell line) were then reduced with 4.5 mM DTT and alkylated with 10 mM iodoacetamide. Trypsin digestion was carried out at room temperature overnight and resulting tryptic peptides were then acidified with 1% trifluoroacetic acid and desalted with C18 Sep-Pak cartridges (Waters) according to the manufacturer’s procedure. Peptides were lyophilized and then dissolved in immunoaffinity purification (IAP) buffer containing 50 mM MOPS/NaOH (pH 7.2), 10 mM sodium phosphate, and 50 mM sodium chloride. The pY peptides were immunoprecipitated with immobilized pTyr100 antibody overnight at 4°C followed by three washes with IAP buffer and two washes with H2O. The pY peptides were eluted from beads twice with 0.15% trifluoroacetic acid and the volume was reduced to a final concentration of 20 μL via vacuum centrifugation. Before MS tumor analysis, 100 fmol of a multi-peptide standard (Pierce 88320) was spiked into each sample. Samples were analyzed by nano-liquid chromatography/tandem mass spectrometry (LC-MS/MS). Two technical replicates were performed for each sample. Aliquots of unused tumor pY immunoprecipitate was analyzed in a targeted quantification experiment (liquid chromatography-multiple reaction monitoring or LC-MRM) to validate the label free relative quantification data for select phosphopeptides. Further LC-MS/MS and LC-MRM details are described in Supplementary Materials and Methods. Generation of protein-protein interaction network for pY proteins
Author Manuscript
Drug screening and in vitro assays
Author Manuscript
The pY proteins corresponding to the clear cell RCC cell lines and the human tumors were input into Cytoscape (version 2.8.3; http://www.cytoscape.org; (14)). Protein-protein interactions (PPI) between nodes were imported using the PSICQUIC plug-in to search multiple databases (15). Network visualization was performed with Cytoscape.
Western blotting
Viability assays were performed in black-wall 384-well microtiter plates for the 10 RCC cell lines. Cells were seeded at a density of 1,000 cells/well. Drugs or DMSO were added after 24 hours and cells were incubated for another 72 hours. 63 tyrosine kinase inhibitors (Fig. 4a, Supplementary Fig. 1–2) were screened for each cell line at 0.5 μM and 2.5 μM (each in duplicate and averaged), a concentration that approximates or is slightly greater than predicted serum levels in humans and thus assures minimum false negatives. Viability was evaluated using the CellTiter-Glo assay (Promega) with addition of reagent after 72 hour drug incubation and luminescence was read on a SpectraMax M5 plate reader (Molecular Devices). Matrigel adhesion and invasion assays (16) as well as 3H-thymidine proliferation assays (17) were performed as described previously.
Western blotting was performed as described in our previous studies (13, 18). Primary antibodies used for our study were purchased from Cell Signaling Technology (EGFR pY1068, EGFR pY1197, MET pY1234/5, total MET), Santa Cruz (total EGFR, total FAK), BD Sciences (FAK pY397) and Sigma-Aldrich (β-actin). Clin Cancer Res. Author manuscript; available in PMC 2017 November 15.
Haake et al.
Page 5
Statistical Methods & Data Analysis
Author Manuscript Author Manuscript
Tumor samples were randomized prior to MS analysis to minimize potential batch effects. Internal controls within each sample (Thermo peptide standard) and external controls (MS analysis of control cell line before analysis of RCC tumors, after every 20 MS runs, and at conclusion of MS analysis of RCC tumors) were included to monitor potential batch effects (Supplementary Fig. 3 and 4). In order to maximize protein coverage, Mascot, Sequest, and Andromeda search engines were performed to identify phosphorylated proteins (see supplementary methods for details). To facilitate and streamline comparisons among samples, only proteins that were identified as phosphorylated in at least 5 samples (cell line or tumor) were considered for further analysis (Fig. 2 and 5). The data regarding individual phosphosites came from only the Andromeda search results (Fig. 3 and 6). Label free quantitative data was calculated using MaxQuant (version 1.2.2.5) using the Andromeda search results (Fig. 6) (19). Peptide ion signal intensities were log2 transformed to allow for the use of parametric statistical tests. A two-sample, two-tailed t-test was used to contrast phosphopeptide intensity in clear cell versus papillary tumors (Fig. 6a), phosphopeptide intensity in tumor samples calculated from extracted ion chromatograms (Fig. 6b), and output from multiple reaction monitoring experiments (Fig. 6c). To account for multiple hypothesis testing, a false discovery rate was estimated for phosphopeptide intensity in tumor samples and reported as a q-value with a cut-off of 0.05 (Fig. 6a). Protein and phosphosite functional annotation was performed with reference to databases at www.phosphosite.org and www.uniprot.org.
Author Manuscript
When contrasting the patient characteristics corresponding to human tumors (Supplementary Table 1), all continuous variables were described with the median and range values. Analyses of categorical data utilized a two-tail Fisher’s exact test. Difference of medians was evaluated with Mann-Whitney U test.
Results RCC cell lines and tumors display FAK phosphorylation but different patterns of receptor tyrosine kinase phosphorylation
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In order to study RCC in an unbiased and systems level manner, we performed immunoprecipitation followed by LC-MS/MS to catalog pY peptides from 10 RCC cell lines and 30 tumors (Fig. 1). Overall, 198 proteins were identified and their function annotated (Fig. 2a, Supplementary Table 2). The proteins corresponded to several different classes including receptor tyrosine kinases (EGFR, MET), non-receptor tyrosine kinases (JAK2, FAK or FAK1 or PTK2), serine/threonine kinases (CDK1, MK01 or MAPK1), phosphatases (SHIP2 or INPPL1), cell adhesion proteins (ITB1 or ITGB1), cytoskeletal proteins (ABLM1, MYO1E), GTP-associated proteins (ARHG5 or ARHGEF5), and others. The cell lines and tumors had similarities with 102 shared tyrosine phosphorylated proteins (Fig. 2b). However, marked distinctions were also evident as 53 pY proteins were unique to cell lines and 22 unique to tumors. To further explore these patterns, the ~50 most commonly identified phosphorylated proteins were analyzed further. If the rate of phosphorylation for a protein was at least 50% higher for cell lines relative to tumors (or vice versa), it was considered “more common” in that subset (Fig. 2c and 2e). Proteins that were commonly
Clin Cancer Res. Author manuscript; available in PMC 2017 November 15.
Haake et al.
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phosphorylated but did not meet this cutoff were considered “common in tumors and cell lines” (Fig. 2d). The MAP kinase pathway was tyrosine phosphorylated in both tumors and cell lines as was PTK2 protein FAK1 or FAK), supporting their universal importance in RCC biology. However, multiple members of the PI3K pathway as well as EGFR were more common in the cell lines suggesting distinct signal transduction relative to tumors. By contrast, the collagen receptor DDR1 was phosphorylated in tumor samples only. Thus, while MAPK and FAK were phosphorylated in both cell lines and tumors, distinct patterns of activation emerged among some RTKs. Several kinases, including EGFR and MET, are phosphorylated in RCC cell lines
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Rather than only examine whether a protein had any pY (Fig. 2), we also evaluated specific phosphosites in cell lines (Supplementary Table 3). Furthermore, our focus was narrowed to kinases and phosphatases given their key roles in regulating cancer signaling (Fig. 3a). EGFR pY1110, pY1172 and pY1197 was observed in multiple cell lines, consistent with receptor activation (20–22). Similarly, the presence of the MET activation site pY1234/5 in all cell lines implies the presence of ubiquitous MET activation. FAK pY576/7 was detected in nearly all the cell lines (Fig. 3a). This FAK phosphosite within the kinase domain of the enzyme is required for maximal enzymatic activity and thus suggests FAK activation (23). Thus, the identification of specific phosphosites was able to provide insight into the functional relevance of pY events.
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Western blotting was performed to further explore the pY patterns of the cell lines for EGFR, MET, and FAK (PTK2) (Fig. 3b). EGFR is known to be activated and phosphorylated in RCC, possibly related to VHL inactivation (24). EGFR pY1197 was weakly positive by western blot in several cell lines despite being identified in all cell lines via MS analysis (Fig. 3a and 3b). To further validate EGFR activation, western blot for EGFR pY1068 was performed. EGFR pY1068 is well known to correlate with EGFR activation (20, 21) and has an excellent performing antibody though, given the large size and double phosphorylation status of the corresponding tryptic peptide can be difficult to detect in MS analysis. The positivity of EGFR pY1068 on western blot supports the MS data that suggests EGFR activation. Thus, the weak EGFR pY1197 signal on western blot yet strong signal via MS may simply be due to increased sensitivity for MS techniques. MET is also known to be activated and phosphorylated in RCC and interest is increasing given the approval of cabozantinib, a TKI whose targets include MET (8, 11, 25). Western blot for MET pY1234/5 confirms that this phosphorylation event is common among the cell lines. Again, its detection in some cell lines via MS but not western blot suggests increased sensitivity of MS techniques. FAK (PTK2) was evaluated further as it was seen to be frequently phosphorylated in cell lines and tumors (Fig. 2d) and its function in RCC had not been well characterized in the literature. The performance of commercially available FAK pY576/7 antibodies was suboptimal and lacked reproducibility in our hands. However, the MS data (Fig. 2d and 3a) showed near-universal FAK activation in RCC and we verified its activation by testing phosphorylation of the canonical autophosphorylation and activation site, FAK pY397 (23). Overall, western blots validate the MS data that EGFR, MET, and FAK are phosphorylated at activating sites in RCC cell lines.
Clin Cancer Res. Author manuscript; available in PMC 2017 November 15.
Haake et al.
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TKI screen highlights activity of FAK tyrosine kinase inhibitors
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A functional screen utilizing 63 tyrosine kinase inhibitors (TKIs) was employed to assist in nominating functionally relevant signaling networks for further analysis (Supplementary Fig. 1–2). We focused our analysis on TKIs targeting tyrosine kinases observed to be phosphorylated in most cell lines, specifically EGFR, FAK, JAK2, and MET (Fig. 4a). Among the most active class of TKIs were those targeting FAK. Specifically, CEP-37440 at 2.5 µM reduced cell viability to
