Platelet-derived growth factor/vascular endothelial growth factor receptor inactivation by sunitinib results in Tsc1/Tsc2-dependent inhibition of TORC1

Abstract

Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptors are implicated in development and tumorigenesis and dual inhibitors like sunitinib are prescribed for cancer treatment. While mammalian VEGF and PDGF receptors are present in multiple isoforms and heterodimers, Drosophila encodes one ancestral PDGF/VEGF receptor, PVR. We identified PVR in an unbiased cell-based RNA interference (RNAi) screen of all Drosophila kinases and phosphatases for novel regulators of TORC1. PVR is essential to sustain target of rapamycin complex 1 (TORC1) and extracellular signal-regulated kinase (ERK) activity in cultured insect cells and for maximal stimulation by insulin. CG32406 (henceforth, PVRAP, for PVR adaptor protein), an Src homology 2 (SH2) domain-containing protein, binds PVR and is required for TORC1 activation. TORC1 activation by PVR involves Tsc1/Tsc2 and, in a cell-type-dependent manner, Lobe (ortholog of PRAS40). PVR is required for cell survival in vitro, and both PVR and TORC1 are necessary for hemocyte expansion in vivo. Constitutive PVR activation induces tumor-like structures that exhibit high TORC1 activity. Like its mammalian orthologs, PVR is inhibited by sunitinib, and sunitinib treatment phenocopies PVR loss in hemocytes. Sunitinib inhibits TORC1 in insect cells, and sunitinib-mediated TORC1 inhibition requires an intact Tsc1/Tsc2 complex. Sunitinib similarly inhibited TORC1 in human endothelial cells in a Tsc1/Tsc2-dependent manner. Our findings provide insight into the mechanism of action of PVR and may have implications for understanding sunitinib sensitivity and resistance in tumors.

Fig 1

PVR is required for TORC1 activation in insect cells. (A, C, and D) Western blot analyses were performed using the indicated cell types (below blots) grown in 10% serum and treated with the stated dsRNAs, and, where shown, transfected with HA-4E-BP (or an empty vector [EV]). UT, untreated; Ra, rapamycin; β-gal, β-galactosidase; Luc, luciferase. (B) Confocal images of Kc cells treated with the indicated dsRNAs and stained using a PVR polyclonal antibody. Magnification, ×650; red, propidium iodide; green, PVR.

Fig 2

PVR is required for maximal and sustained activation of TORC1 and ERK. Western blot analyses were performed using Kc cells treated with the indicated dsRNA for 2 days, starved for 2 to 3 days, and then stimulated with conditioned medium (A) or with insulin (1 μM) (B and C). R, rapamycin. Short and long indicate the length of exposure time.

Fig 3

Evaluation of the roles of Drk and Ras85D in PVR-mediated activation of ERK and TORC1. Western blot analyses were performed using Kc cells treated with the indicated dsRNA for 3 days (A, B, and C) or transfected with the indicated plasmids and then treated with dsRNA against either luciferase (−) or PVR (+) (D).

Fig 4

Restoration of TORC1 activity in Kc cells depleted of PVR by simultaneous inactivation of both the Tsc1/Tsc2 complex and Lobe. Western blotting was performed using Kc cells treated with the indicated dsRNAs (A to C) or after transfection with the empty vector (EV) or myristoylated-HA-Akt (D).

Fig 5

Tsc1/Tsc2-dependent regulation of TORC1 by Drk, CG32406, and other adaptor proteins. Western blot analysis was performed using Kc cells treated with the indicated dsRNA. Ra, rapamycin.

Fig 6

CG32406 protein interacts with PVR. (A) Domain prediction of CG32406 protein. SH2, Src homology 2; CC, coiled coil. (B) Western blot analysis of immunoprecipitated proteins. Kc cells harboring a CuSO₄-inducible form of HA-CG32406 were treated with Luc or PVR dsRNA in the absence (−) or presence (+) of CuSO₄. IP, immunoprecipitation; IB, immunoblot.

Fig 7

PVR is required for cell number expansion in vitro and in vivo, and TORC1 is implicated in this process. (A) Quantitation of the number of Kc cells treated with the indicated dsRNA. β, β-Galactosidase. (B) Blood cell numbers in larvae expressing PVR DN (DN), PVR RNAi, TOR RNAi, or Scylla/Charybdis using the hemocyte-specific driver He-Gal4. Wt, wild type (driver alone); DN, UAS-PVR DN; PVR RNAi, UAS-PVR RNAi; Scylla/Charybdis, UAS-Scylla/UAS-Charybdis. Data are means ± standard errors of the means (for each genotype, n ≥ 4). **, P < 0.01; ***, P < 0.001. (C, D, and E) TUNEL assay of cells treated as indicated. (C) Kc cells were treated with Luc, PVR, or Diap1 dsRNA followed by vehicle (DMSO) or the pan-caspase inhibitor Z-VAD. Maroon, Luc dsRNA plus vehicle; orange, PVR dsRNA plus vehicle; lime green, PVR dsRNA plus Z-VAD; dark green, Diap1 dsRNA plus vehicle; blue, Diap1 dsRNA plus Z-VAD. Results normalized to Luc, in relative units (RU), are shown in the bar graph. (D) Bar graph representation of apoptosis induction (in RU) in Kc cells treated with the indicated dsRNAs. (E) TUNEL assay of Kc or S2 cells treated with vehicle (Ve) or 25 nM rapamycin (Ra) for 48 h. Max, maximum; FITC-A, fluorescein-12-dUTP.

Fig 8

Constitutively active PVR induces hemocyte expansion and tumor-like structures with active TORC1. (A) Blood cell numbers in larvae expressing UAS-PVR (PVR) or UAS-λ-PVR (λ-PVR) using the hemocyte-specific driver He-Gal4. Data are means plus standard errors of the means (for each genotype, n ≥ 4). **, P < 0.01. (B) Differential interference contrast (DIC) confocal images of imaginal wing discs from larvae expressing UAS-PVR (PVR) or UAS-λ-PVR (λ-PVR) using the imaginal wing disc-specific driver MS1096-Gal4. Control, driver alone. (C) Confocal images of imaginal wing discs with the larvae described in panel B stained with P-4E-BP or propidium iodide (PI).

Fig 9

Alignment of the intracellular kinase domain of PVR and its successor RTKs or other RTKs known to be inhibited by sunitinib. *, mutations in KIT protein leading to sunitinib resistance. The conservation spectrum is represented from dark blue to white for identical to different. Red box, DFG motif.

Fig 10

Evaluation of PVR inhibition by sunitinib. (A) PVR model of transition from inactive to active state. (B and C) Structure of KIT (B) or VEGFR2 (C) bound to sunitinib. A conserved residue (depicted in stick form) within the JMR (shown in magenta) prevents activation of VEGFR2 and KIT. (D) Model structure of PVR bound to sunitinib. The A-loop (blue) adopts a DFG-out inactive conformation, forming the binding site for sunitinib (black stick). (E) A structure-based sequence alignment of the JMR (highlighted in magenta) and the A-loop (highlighted in blue). The DFG motif F residue and the hydrophobic JMR residue positions are indicated with an asterisk above the sequences. The conserved hydrophobic residue in the JMR competes with the F residues in the DGF motif to keep the receptor in the inactive conformation.

Fig 11

Sunitinib inhibits PVR and phenocopies PVR loss. (A and B) Western blot analyses of inputs or PVR immunoprecipitates (IP) were performed using Kc cells treated with the indicated dsRNAs for 3 days and then exposed to sunitinib for 6 h (A). Arrow, PVR. Western blot analyses were performed using Kc or S2 cells treated with the indicated concentrations of sunitinib overnight (B). (C) Cell number analyses of the indicated cells treated with vehicle (Ve), sunitinib (Su; 10 μM) or rapamycin (Ra; 25 nM). (D) TUNEL assay of Kc and S2 cells treated with vehicle or sunitinib for 48 h. (E) Hemocyte numbers from wandering larvae fed with vehicle or sunitinib (70 μM)-supplemented food starting at L1. Data are means ± standard errors of the means (n ≥ 4). **, P < 0.01.

Fig 12

Mutant PVR fails to confer resistance to sunitinib. Western blot analyses were performed of immunoprecipitated PVR from Kc cells transfected with the indicated plasmids and treated with PVR dsRNA for 2 days to deplete endogenous PVR followed by exposure to sunitinib for the indicated number of hours.

Fig 13

Constitutive PVR dimerization is sufficient to confer resistance to sunitinib in cells in culture as well as in hemocytes in vivo. Western blot analyses were performed of immunoprecipitated PVR from Kc cells transfected with the indicated plasmids, treated with either Luc or PVR dsRNA, and exposed to sunitinib (10 μM) for the indicated amounts of time. (B) Hemocyte numbers from wondering larvae expressing UAS-λ-PVR in blood cells using the He-Gal4 driver grown on food supplemented with either vehicle (Ve) or sunitinib (Su) starting from L1. Ve, vehicle; Su, sunitinib. Data are means ± standard errors of the means (n ≥ 4).

Fig 14

Tsc1/Tsc2-dependent inhibition of TORC1 by sunitinib in Drosophila cells. Western blot analysis was performed using the stated cell types pretreated with the indicated dsRNAs for 2 days and then treated with sunitinib for 24 h. UT, untreated; Ra, rapamycin; β-gal, β-galactosidase.

Fig 15

Sunitinib-mediated inhibition of TORC1 in primary human endothelial cells requires the TSC1/TSC2 complex. (A and C) Western blot analyses were performed using HUVEC or HDMEC treated with the indicated concentrations of sunitinib (A) or pretreated with the indicated siRNAs for 2 days and then exposed to sunitinib (10 μM) overnight (C). Ra or R, rapamycin. (B) Immunofluorescence and immunohistochemistry images of orthotopic renal cancer tumor grafts treated with vehicle (Ve) or sunitinib (Su) for 3 days.