A kinase inhibitor screen reveals protein kinase C-dependent endocytic recycling of ErbB2 in breast cancer cells

Abstract

ErbB2 overexpression drives oncogenesis in 20-30% cases of breast cancer. Oncogenic potential of ErbB2 is linked to inefficient endocytic traffic into lysosomes and preferential recycling. However, regulation of ErbB2 recycling is incompletely understood. We used a high-content immunofluorescence imaging-based kinase inhibitor screen on SKBR-3 breast cancer cells to identify kinases whose inhibition alters the clearance of cell surface ErbB2 induced by Hsp90 inhibitor 17-AAG. Less ErbB2 clearance was observed with broad-spectrum PKC inhibitor Ro 31-8220. A similar effect was observed with Go 6976, a selective inhibitor of classical Ca(2+)-dependent PKCs (α, β1, βII, and γ). PKC activation by PMA promoted surface ErbB2 clearance but without degradation, and ErbB2 was observed to move into a juxtanuclear compartment where it colocalized with PKC-α and PKC-δ together with the endocytic recycling regulator Arf6. PKC-α knockdown impaired the juxtanuclear localization of ErbB2. ErbB2 transit to the recycling compartment was also impaired upon PKC-δ knockdown. PMA-induced Erk phosphorylation was reduced by ErbB2 inhibitor lapatinib, as well as by knockdown of PKC-δ but not that of PKC-α. Our results suggest that activation of PKC-α and -δ mediates a novel positive feedback loop by promoting ErbB2 entry into the endocytic recycling compartment, consistent with reported positive roles for these PKCs in ErbB2-mediated tumorigenesis. As the endocytic recycling compartment/pericentrion has emerged as a PKC-dependent signaling hub for G-protein-coupled receptors, our findings raise the possibility that oncogenesis by ErbB2 involves previously unexplored PKC-dependent endosomal signaling.

Keywords: Breast Cancer; Confocal Microscopy; Endocytic Traffic; Endocytosis; ErbB2; High-Content Fluorescence Microscopy; Kinase Inhibitors; Protein Kinase C (PKC); Receptor Tyrosine Kinase; Small Molecule Screening.

FIGURE 1.

Development of an automated high-content fluorescence imaging-based assay for the measurement of surface levels of ERBB2. A–C, SKBR-3 cells were seeded in duplicate in the inner wells of 96-well plates (B, schematic at the top), labeled with Alexa Fluor® 488-conjugated anti-ErbB2 for 1 h at 4 °C, treated with DMSO (vehicle control), or increasing concentrations (5–500 nm) of 17-AAG for 8 h and fixed in 4% paraformaldehyde. The plates were scanned in a Cellomics^TM Arrayscan VTI fluorescent microscope imager (A, B) and the Cellomics^TM compartmental analysis software was used to quantify the fluorescence intensity of ErbB2 at the plasma membrane (C). The Y-axis represents the mean fluorescence intensity ± S.D. The presented data are from a single experiment representative of three. D and E, SKBR-3 cells were seeded in 6-well plates for 72 h and treated with DMSO (vehicle control) or increasing concentrations (5–500 nm) of 17-AAG for 8 h. In D, cells were trypsinized, washed in FACS buffer, and live cells stained with Alexa Fluor® 488-conjugated anti-ErbB2 for 1 h on ice. Cell surface levels of ErbB2 were quantified using flow cytometry and analyzed using BD Cellquest™ software. X-axis, mean fluorescence intensity; Y-axis, cell counts. In E, Triton X-100 lysates were prepared, and 25-μg aliquots of lysate protein resolved by SDS-PAGE followed by immunoblotting with anti-ErbB2 and anti-CHIP (loading control) antibodies.

FIGURE 2.

Small molecule kinase inhibitor library screen identifies protein kinase C as a potential regulator of ErbB2 down-regulation from the cell surface. A, SKBR-3 cells seeded and labeled with Alexa Fluor® 488-conjuagated anti-ErbB2 as in Fig. 1. Pre-warmed medium containing various inhibitors (1 μm final), with or without 17-AAG (100 nm final) was added, and cells were incubated at 37 °C for 8 h. Cell surface ErbB2 levels were computed and are presented as mean fluorescence intensity of duplicates. Inhibitors that reduced the 17-AAG-induced clearance of ErbB2 (i.e. more ErbB2 remaining at the cell surface) by >20% are indicated in red: Fasudil (no. 25), Ro-31–8220 (no. 28), and Genistein (no. 41). B, list of kinase inhibitor library (specificity per vendor is shown). C and D, to validate the selected inhibitors, SKBR-3 cells were treated with DMSO (vehicle control) or 1 μm final concentrations of Fasudil (C) or Ro 31–8220 (D), with or without 100 nm 17-AAG for 8 h, and cell surface ErbB2 levels analyzed by FACS-staining with Alexa Fluor® 488-conjugated-anti-ErbB2 antibody. E. SKBR-3 cells were seeded in 6-well plates and live cells stained with Alexa Fluor 488® conjugated anti-ErbB2 antibody for 1 h at 4 °C. Stained cells were pre-treated without or with Ro 31–8220 (1 μm) at 37 °C for 1 h followed by addition of DMSO (vehicle) or 17-AAG (25 nm) for 8 h. Cells were paraformaldehyde-fixed followed by imaging under a confocal microscope.

FIGURE 3.

Role of PKC-α in ErbB2 clearance from the cell surface. A, SKBR-3 cells were pre-treated with DMSO, Ro 31–8220 (1 μm), Go 6976 (1 μm), or PMA (100 nm) for 1 h, and incubations continued without or with 17-AAG (25 nm) for 8 h. Cells were trypsinized, stained with Alexa Fluor® 488-anti-ErbB2 antibody, and analyzed by FACS to quantify cell surface ErbB2 levels. The bars represent mean fluorescence intensity values ± S.D. Triton X-100 lysates were prepared from parallel cultures treated as those used for FACS and 25-μg aliquots of lysate protein resolved by SDS-PAGE followed by immunoblotting with anti-ErbB2, anti-PKC-α, and anti-β-actin (loading control) antibodies. The presented data are from a single experiment representative of three. B, SKBR-3 cells were transiently co-transfected with expression constructs encoding wild-type or constitutively active (CA) PKC-α together with dsRed plasmid. Live cells were stained with Alexa Fluor® 488 conjugated-anti-ErbB2, and cell surface levels of ErbB2 on ds-Red-negative (light bars) and ds-Red-positive (dark bars) cells were quantified using FACS. The mean fluorescence intensity values ± S.D. are presented; asterisks indicate statistically significant differences (p < 0.005). The presented data are from a single experiment representative of three.

FIGURE 4.

Requirement of ErbB2 kinase activity for PMA-induced down-regulation of cell surface ErbB2. SKBR-3 cells were cultured in low serum (0.5% FBS) medium without (A) or with (B) Lapatinib (100 nm) for 24h followed by further incubation in the absence (DMSO) or the presence of PMA (25 nm) for 8 h. Live cells were stained with Alexa Fluor® 488-conjugated anti-ErbB2 antibody and cell surface ErbB2 levels quantified using FACS.

FIGURE 5.

PKC-α-regulates surface ErbB2 translocation into a juxtanuclear compartment. A, live SKBR-3 cells on coverslips were stained with Alexa Fluor® 488-conjugated anti-ErbB2 antibody in cold, followed by treatment with DMSO (vehicle control) or PMA (100 nm) at 37 °C for 1 h. The cells were paraformaldehyde-fixed, permeabilized with saponin (0.2% in PBS), stained for PKC-α (red) and imaged under a confocal microscope. B, SKBR-3 cells were transfected with a non-targeting siRNA (control) or a PKC-α-specific siRNA (a smartpool or siRNA no. 2) for 48 h. Live cells were stained with Alexa Fluor® 488-conjugated anti-ErbB2 antibody in cold and treated with PMA (100 nm) for 1 h at 37 °C. The cells were stained for PKC-α (red) and imaged as in A. C, SKBR-3 cells were transfected with a non-targeting siRNA (control) or a PKC-α-specific siRNA smartpool for 48 h as in B, and 40-μg aliquots of Triton X-100 lysate protein subjected to Western blotting with anti-PKC-α and β-actin (loading control) antibodies.

FIGURE 6.

ErbB2 in the juxtanuclear compartment co-localizes with PKC-α and ERC marker Arf6. A and B, SKBR-3 cells were transiently transfected to co-express GFP-tagged wild-type PKC-α, ds-Red-tagged WT Arf6 (A) or Arf6-T27N mutant (B). The cells were stained with Alexa Fluor® 647-conjugated anti-ErbB2 antibody, treated with DMSO (vehicle) or PMA (100 nm) for 4 h, paraformaldehyde-fixed and permeabilized as in Fig. 5, and imaged under a confocal microscope. C, SKBR-3 cells were transiently transfected to co-express GFP-tagged wild-type PKC-α, and ds-Red-tagged WT Arf6 or Arf6-T27N mutant as in A and B, and 40-μg aliquots of Triton X-100 lysate protein were used for Western blotting with anti-Arf6 and β-actin (loading control) antibodies. D, SKBR-3 cells transiently transfected and stained as in A were treated with PMA (100 nm) for 4 h, paraformaldehyde fixed, stained with an anti-Arf6 antibody and imaged under a confocal microscope.

FIGURE 7.

ErbB2 kinase activity but not PKC-α are required for PMA-induced ERK phosphorylation. A, SKBR-3 cells seeded in 6-well plates were treated with DMSO (vehicle) or the indicated concentrations of PMA for 8 h. 25-μg aliquots of Triton X-100 lysate proteins were subjected to immunoblotting for p-ERK 1/2, total ERK 1/2, and β-actin (loading control). B, SKBR-3 cells were transfected with a non-targeting siRNA (control) or PKC-α-specific siRNAs (a smartpool or siRNA no. 2) for 48 h, treated without or with Lapatinib (1 μm) for 1 h, and further incubated in the presence of DMSO (vehicle control) or PMA (100 nm) for 1 h. Western blotting with anti-ErbB2-pY-1248, anti-ErbB2, anti-PKC-α, anti-p-ERK 1/2, anti-ERK 1/2, and anti-β-actin (loading control) antibodies was carried out as in A.

FIGURE 8.

PKC-δ co-localizes with ErbB2 in the Arf6-positive juxtanuclear compartment and is required for PMA-induced Erk phosphorylation. A and B, SKBR-3 cells were transiently transfected to co-express GFP-tagged wild-type PKC-δ and ds-Red-tagged WT Arf6 (A) or Arf6-T27N mutant (B). The cells were stained with Alexa Fluor® 647-conjugated anti-ErbB2 antibody and treated with DMSO (vehicle) or PMA (100 nm) for 1 h. The cells were paraformaldehyde-fixed and permeabilized as in Fig. 5, and imaged under a confocal microscope. C, SKBR-3 cells were transfected with a non-targeting control siRNA or a PKC-δ-specific siRNA smartpool for 48 h. Live cells were stained with Alexa Fluor® 488-conjugated anti-ErbB2 antibody in cold and treated with PMA (100 nm) for 1 h at 37 °C. The cells were stained for PKC-δ (red) and imaged as in A. D, SKBR-3 cells were transfected with a non-targeting control siRNA or a PKC-δ-specific siRNA smartpool for 48 h, treated without or with Lapatinib (1 μm) for 1 h, and further incubated in the presence of DMSO (vehicle control) or PMA (100 nm) for 1 h. Western blotting with anti-ErbB2-pY-1248, anti-ErbB2, anti-PKC-δ, anti-p-ERK 1/2, anti-ERK 1/2, and β-actin (loading control) antibodies was carried out as in Fig. 7.