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Involvement of de novo synthesized palmitate and mitochondrial EGFR in EGF induced mitochondrial fusion of cancer cells

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Pages 2415-2430 | Received 19 May 2014, Accepted 22 May 2014, Published online: 24 Jun 2014

Abstract

Increased expressions of fatty acid synthase (FASN) and epidermal growth factor receptor (EGFR) are common in cancer cells. De novo synthesis of palmitate by FASN is critical for the survival of cancer cells via mechanisms independent of its role as an energy substrate. Besides the plasma membrane and the nucleus, EGFR can also localize at the mitochondria; however, signals that can activate mitochondrial EGFR (mtEGFR) and the functions of mtEGFR of cancer cells remain unknown. The present study characterizes mtEGFR in the mitochondria of cancer cells (prostate and breast) and reveals that mtEGFR can promote mitochondrial fusion through increasing the protein levels of fusion proteins PHB2 and OPA1. Activation of plasma membranous EGFR (pmEGFR) stimulates the de novo synthesis of palmitate through activation of FASN and ATP-citrate lyase (ACLy). In vitro kinase assay with isolated mitochondria shows that palmitate can activate mtEGFR. Inhibition of FASN blocks the mtEGFR phosphorylation and palmitoylation induced by EGF. Mutational studies show that the cysteine 797 is important for mtEGFR activation and palmitoylation. Inhibition of FASN can block EGF induced mitochondrial fusion and increased the sensitivity of prostate cancer cells to EGFR tyrosine kinase inhibitor. In conclusion, these results suggest that mtEGFR can be activated by pmEGFR through de novo synthesized palmitate to promote mitochondrial fusion and survival of cancer cells. This mechanism may serve as a novel target to improve EGFR-based cancer therapy.

Introduction

It is common that cancer cells express elevated levels of fatty acid synthase (FASN)Citation1-Citation6 and epidermal growth factor receptor (EGFR).Citation7 Homodimeric FASN catalyzes the de novo synthesis of fatty acids with palmitate as the predominant product.Citation8 Tumor-associated de novo synthesized fatty acids provide tumor cells with growth and survival advantage independent of their roles as energy substrates.Citation9 EGFR is an oncogenic receptor tyrosine kinase involved in promoting tumorigenesis of a majority of tumors of epithelial origin.Citation10

The fact that cancer cells rely on de novo synthesized fatty acids for survival suggests that the activity of FASN is critically integrated into the basic survival mechanisms of cancer. Oncogenic signaling pathways, such as mitogen activated protein kinase (MAPK) pathway and phosphoinositol-3 kinase (PI-3K)/protein kinase B (Akt) pathways, participate in the upregulation of FASN in cancer cells.Citation11-Citation13 Increased de novo synthesis of fatty acids can provide cancer cells with the immediate need of fatty acids for membrane production and lipid rafts creation.Citation14 Long chain fatty acids including palmitate are ligands of transcription factors such as the peroxisome proliferator-activated receptors (PPAR).Citation15 Elevated de novo palmitate synthesis is associated with palmitoylation of Wnt1 and activation of Wnt1/β-catenin pathway.Citation16 Accumulating evidence indicates that FASN activity is closely associated with the survival functions of receptor tyrosine kinases of the EGFR family, which include EGFR/HER1, HER2, HER3, and HER4. Overexpression of FASN activates EGFR and HER2 signaling in breast epithelial cells.Citation17 Inhibition of FASN activity concomitantly suppresses EGFR signaling (Vazquez-Martin et al., 2008). HER2 can activate FASN through physical interaction and phosphorylation.Citation18

EGFR was primarily found in the plasma membrane, where it is activated by its extracellular ligands such as EGF. Besides the plasma membrane, EGFR can also exist in the nucleusCitation19,Citation20 and in the mitochondrion.Citation21-Citation23 In the nucleus, EGFR participates in transcriptional regulation.Citation24 The function of mitochondrial EGFR (mtEGFR) of cancer cells is largely unknown. Previous studies have shown that EGFR can localize to the inner mitochondrial membrane,Citation25 and activation of membranous EGFR increased the content of EGFR in the mitochondria.Citation21,Citation23 The localization of EGFR to the mitochondria is regulated by the activity of Src,Citation21,Citation22,Citation26 which is likely independent of the endocytosis of plasma membranous EGFR (pmEGFR).Citation23 Signals that can activate the mtEGFR and the role of mtEGFR activation in survival of cancer cells remain to be investigated.

Besides performing metabolic reactions, mitochondria also undergo fission/fusion changes, namely the mitochondrial dynamics, which plays critical roles in regulating cell metabolism, survival, and proliferation,Citation27 Fusion serves to unify the mitochondrial compartment, whereas fission generates morphologically and functionally distinct mitochondria. Mitochondrial fission often occurs early in the apoptotic eventCitation28 and the autophagic process.Citation29 Fusion of mitochondria is associated with increased cell survival.Citation30 The molecular mechanisms that govern the fission/fusion dynamics have been partially illustrated. A number of dynamin-related GTPases, such as Drp1, Mfn1, Mfn2, and OPA1, play key roles in regulating mitochondrial dynamics. Translocation of Drp1 from cytosol to mitochondria promotes mitochondria fission, Mfn1 and Mfn2 are involved in the fusion of outer mitochondria membrane, and OPA1 is needed for the fusion of inner mitochondria membrane.Citation31 Signaling factors found to regulate the mitochondria dynamics include PKA,Citation32 CaMKIα,Citation33 and activities of ubiquitin and SUMO ligases.Citation34 Mitochondrial fusion could be increased by peroxisome proliferator-activated receptor-γ coactivator 1β,Citation15 which could be activated by intracellular fatty acids,Citation35 suggesting signals initiated by fatty acids are involved in regulating the mitochondria dynamics.

In this study, we report that EGFR exists in the mitochondria of cancer cells of prostate and breast. The pmEGFR co-localizes and interacts with FASN independent of EGFR’s tyrosine kinase activity; pmEGFR activation by EGF induces de novo palmitate synthesis; de novo synthesized palmitate activates mtEGFR via inducing palmitoylation of mtEGFR and the activated mtEGFR promotes mitochondrial fusion and cell survival via increasing the levels of mitochondrial prohibitin 2 (PHB2) and OPA1.

Results

Cancer tissues and cell lines of prostate, breast, contain mitochondrial EGFR

To determine the cancer relevance of mtEGFR, we performed immunofluorescent co-staining of EGFR with a mitochondrial specific protein, the MTCO2 (mitochondrial Cytochrome c oxidase subunit II) on tissue arrays of 2 types of cancer, prostate (n = 63) and breast (n = 41), and their corresponding cancer cell lines, PC3 (prostate) and MDA-MB-231 (breast). We found that all the EGFR positive cancer tissues () and cell lines () contain mtEGFR. The existence of mtEGFR in PC3 and MDA-MB-231 cells was also determined by western blot analysis of mitochondria purified by ultra-centrifugation.Citation36 To determine the purity of isolated mitochondrial fraction, we measured the levels of marker proteins for plasma membrane (glucose transporter 1), endoplasmic reticulum (ER) (calreticulin), cytosol (tubulin) and Golgi apparatus (syntaxin 6) in the mitochondrial fraction. As shown in , mtEGFR was found in the purified mitochondrial fraction of both PC3 and MDA-MB-231 cells. We also performed immunofluorescent co-staining of EGFR and MTCO2 on smears of the isolated mitochondria. The results show that EGFR exists in the purified mitochondria (). To further determine the localization of mtEGFR within the mitochondria, we performed protease (thrombin) protection assay with intact mitochondria and mitoplast (outer membrane deprived mitochondria). PHB2 was used as a positive control of inner membranous protein. As shown in , intact mitochondria prevented mtEGFR from Thrombin treatment but mitoplast failed to protect mtEGFR, suggesting that mtEGFR is localized in the inner membrane of mitochondrion. To further validate the localization of mtEGFR at the inner membrane of mitochondria, we prepared inner and outer membrane protein fractions of mitochondria and performed western blot analysis for mtEGFR, MTCO2 (an inner membrane protein), and MAO (monoamine oxidase, an outer membrane protein). Consistent with the results shown in and with the findings of other groups,Citation22,Citation25 mtEGFR in PC3 and MDA-MB-231 cells also exists at the mitochondrial inner membrane (). Together, these data suggest that cancer cells of prostate and breast harbor mtEGFR, and mtEGFR may play roles in regulating mitochondria based cellular events of cancer cells.

Figure 1. EGFR localization in the mitochondria of cancer tissues and cell lines. (A) Immuno fluorescent co-staining of EGFR (green) and MTCO2 (for mitochondria) (red) in prostate and breast cancer tissues. (Arrows indicate EGFR localized in the mitochondria, bar = 10 μm) (B) Immuno fluorescent co-staining of EGFR (green) and MTCO2 (for mitochondria) (red) in prostate (PC3) and breast (MDA-MB-231) cancer cell lines. (Arrows indicate EGFR localized in the mitochondria, bar = 30 μm) (C) Western blot analysis of mitochondrial protein samples prepared from PC3 and MDA-MB-231 cells for EGFR, and marker proteins for plasma membrane (Glut1) enopalsmic reticulum (Calreticulin) Golgi apparatus (Syntaxin 6) cytoplasm (Tubulin) and mitochondria (PHB2) (D) Immunoflourescent co-staining of EGFR and MTCO2 on smears of isolated mitochondria from PC3 and MD-MB-231 cells. Colocalization of EGFR with mitochondria is in orange/yellow color in the merged confocal images (arrows) (E) Proteolysis coupled western blot analysis for EGFR and PHB2 in protein samples from intact mitochondria and mitoplast of PC3 cells. Intact mitochondria or mitoplast (outer membrane deprived) were treated with a protease, thrombin, at 37 °C for 15 min. PHB2 serves as positive control for mitochondrial inner membrane. (F) Western blot analysis of mitochondrial inner membrane and outer membrane for EGFR, MTCO2 (an inner membrane marker) and Monoamine oxidase (MAO, an outer membrane marker).

Figure 1. EGFR localization in the mitochondria of cancer tissues and cell lines. (A) Immuno fluorescent co-staining of EGFR (green) and MTCO2 (for mitochondria) (red) in prostate and breast cancer tissues. (Arrows indicate EGFR localized in the mitochondria, bar = 10 μm) (B) Immuno fluorescent co-staining of EGFR (green) and MTCO2 (for mitochondria) (red) in prostate (PC3) and breast (MDA-MB-231) cancer cell lines. (Arrows indicate EGFR localized in the mitochondria, bar = 30 μm) (C) Western blot analysis of mitochondrial protein samples prepared from PC3 and MDA-MB-231 cells for EGFR, and marker proteins for plasma membrane (Glut1) enopalsmic reticulum (Calreticulin) Golgi apparatus (Syntaxin 6) cytoplasm (Tubulin) and mitochondria (PHB2) (D) Immunoflourescent co-staining of EGFR and MTCO2 on smears of isolated mitochondria from PC3 and MD-MB-231 cells. Colocalization of EGFR with mitochondria is in orange/yellow color in the merged confocal images (arrows) (E) Proteolysis coupled western blot analysis for EGFR and PHB2 in protein samples from intact mitochondria and mitoplast of PC3 cells. Intact mitochondria or mitoplast (outer membrane deprived) were treated with a protease, thrombin, at 37 °C for 15 min. PHB2 serves as positive control for mitochondrial inner membrane. (F) Western blot analysis of mitochondrial inner membrane and outer membrane for EGFR, MTCO2 (an inner membrane marker) and Monoamine oxidase (MAO, an outer membrane marker).

Activation of mtEGFR promotes mitochondrial fusion

Mitochondria are dynamic intracellular organelles that constantly undergo fusion and fission processes. Activation of fusion process leads to the formation of tubular, elongated and inter-connected mitochondria. It was reported that a tyrosine kinase inhibitor caused mitochondrial fission in Hela cells.Citation37 Similarly, we found that inhibition of EGFR tyrosine kinase activity by AEE788Citation38 also resulted in mitochondrial fission and activation of pmEGFR by EGF promoted fusion of mitochondria in PC3 cells (). As shown in , EGF significantly increased highly fused, interconnected and elongated mitochondria (Type I and II) and decreased rod like mitochondria (Type III) and round shaped or fragmented mitochondria whereas EGFR kinase inhibitor (AEE788) decreased fused mitochondria (type I and II) and increased type III and fragmented mitochondria (Mitochondrial classification and quantification was performed according to the method used by othersCitation39). To explore the role of mtEGFR activation in regulating mitochondrial dynamics, we created plasmids expressing the kinase wild type (WT-EGFR) and the kinase dead (KD-EGFR) EGFR that can only localize to the inner membrane of mitochondria by replacing the membrane translocation signal sequence (the first 24 amino acids) of EGFR with the mitochondrial localization signal of cytochrome c oxidase subunit I. For detection purpose, we also fused a Flag tag to the C-terminus of the mitochondrial specific EGFRs. We named these EGFRs as mito-WT–EGFR and mito-KD-EGFR (serves as a dominant negative mutant for the endogenous mtEGFR) respectively. We then transfected these mitochondrial specific EGFRs into PC3 cells and determined their effects on mitochondrial dynamics. At the 24 h after transfection, we co-stained mitochondria with MTCO2 antibody and the mitochondrial specific EGFRs with Flag antibody for confocal imaging. We observed that, as compared with the control cells, the mito-WT-EGFR expressing cells exhibited less fragmented type of mitochondria and more fused mitochondria; the mitochondria of cells expressing the dominant negative mito-KD-EGFR were more fissed (). Together, these data suggest that the tyrosine kinase activation of mtEGFR can promote mitochondrial fusion.

Figure 2. mtEGFR kinase activation promotes mitochondrial fusion in PC3 cells. (A) Representative images of mitochondria of PC3 cells treated with DMSO, EGF (20 ng/ml) AEE788 (a small molecular EGFR inhibitor at 6 μM) or AEE788+EGF for 24 h. Mitochondria were stained with Mitotracker Red and images were taken with a confocal microscope. (B) Quantification of mitochondrial dynamics in PC3 cells treated as shown in (A) Mitochondria are classified into type I, II, III, and fragmented in the order of more fused to more fissed forms as described in “Materials and Methods”. Data are means +/− SD of triplicates. Asterisk indicates the statistical significance between treated group and DMSO group (P < 0.05) (C) Representative confocal images of mitochondria of PC3 cells transfected with empty vector, mito-WT-EGFR, or mito-KD-EGFR for 24 h. Nuclei were stained with DAPI (blue) Mitochondria (red) were stained with MTCO2 and ectopic mito-EGFRs (green) were stained with anti-Flag antibody. (D) Quantification of mitochondrial dynamics in PC3 cells transfected with mito-EGFRs as shown in (C) Quantification was performed the same way as described in (B) Data are means +/− SD of triplicates. Asterisk indicates the statistical significance between treated group and DMSO (P < 0.05).

Figure 2. mtEGFR kinase activation promotes mitochondrial fusion in PC3 cells. (A) Representative images of mitochondria of PC3 cells treated with DMSO, EGF (20 ng/ml) AEE788 (a small molecular EGFR inhibitor at 6 μM) or AEE788+EGF for 24 h. Mitochondria were stained with Mitotracker Red and images were taken with a confocal microscope. (B) Quantification of mitochondrial dynamics in PC3 cells treated as shown in (A) Mitochondria are classified into type I, II, III, and fragmented in the order of more fused to more fissed forms as described in “Materials and Methods”. Data are means +/− SD of triplicates. Asterisk indicates the statistical significance between treated group and DMSO group (P < 0.05) (C) Representative confocal images of mitochondria of PC3 cells transfected with empty vector, mito-WT-EGFR, or mito-KD-EGFR for 24 h. Nuclei were stained with DAPI (blue) Mitochondria (red) were stained with MTCO2 and ectopic mito-EGFRs (green) were stained with anti-Flag antibody. (D) Quantification of mitochondrial dynamics in PC3 cells transfected with mito-EGFRs as shown in (C) Quantification was performed the same way as described in (B) Data are means +/− SD of triplicates. Asterisk indicates the statistical significance between treated group and DMSO (P < 0.05).

mtEGFR interacts with PHB2 and increases OPA1

To investigate the mechanism by which mtEGFR regulates mitochondrial dynamics, we probed whether EGFR interacts with any mitochondrial proteins that are involved in the regulation of mitochondrial dynamics. Through proteomic approach, we found that a mitochondrial specific protein, prohibitin 2 (PHB2), was co-precipitated with EGFR independent of EGFR’s tyrosine kinase activity (Fig. S1). We co-transfected myc-tagged PHB2 and flag-tagged wild type and functional domain mutated EGFRs into HE293 cells. The EGFR-PHB2 interaction was then validated and characterized by immnoprecipitation coupled western blot analysis. We found that the EGFR-PHB2 interaction was indeed independent of the tyrosine kinase activity of EGFR and the transmembrane and the intracellular domain of EGFR are critical for the interaction (). OPA1 is a GTPase involved in the fusion process of mitochondria. Stabilization of OPA1 is required for promoting mitochondrial fusion, and PHB2 facilitates mitochondrial fusion by preventing OPA1 from cleavage by mitochondrial proteases such as the mAAA proteasesCitation40 or by the ATP-independent metalloprotease OMA1.Citation41 We found that the EGFR tyrosine kinase inhibitor AEE788 also significantly reduced the levels of EGF induced OPA1 and PHB2 in PC3 cells without changing the levels of other mitochondrial fusion related proteins such as Mfn1 and Mfn2 (), suggesting that PHB2 and OPA1 are also involved in EGFR regulated mitochondrial dynamics. Interestingly, the EGFR monoclonal antibody inhibitor, C225 that inhibits pmEGFR’s kinase activity, did not reduce OPA1 level (), suggesting that the tyrosine kinase activity of pmEGFR is not directly involved in AEE788 caused downregulation of OPA1. Because PHB2 is a mitochondrial protein, EGFR exists in mitochondria, EGFR interacts with PHB2, and AEE788 downregulates OPA1 and induces fission, we tested the tyrosine kinase activity of mtEGFR in regulating the levels of PHB2 and OPA1. We transfected the mito-WT-EGFR and the mito-KD-EGFR into PC3 cells, and determine their effects on PHB2 and OPA1. As shown in , the mito-WT-EGFR increased the levels of PHB2 and OPA1, the mito-KD-EGFR decreased them, suggesting that EGFR within the mitochondria upregulates PHB2 and OPA1 protein levels dependent of its tyrosine kinase activity. It is known that PHB2 protects OPA1 from proteolysis.Citation42 mtEGFR may protect OPA1 via PHB2, which is supported by the data that the AEE788 induced downregulation of OPA1 was prevented by overexpression of PHB2-myc in PC3 cells (). Together, these data suggest that mtEGFR can increase OPA1 by interacting with (independent of mtEGFR’s tyrosine kinase activity) and increasing PHB2 (dependent of mtEGFR’s tyrosine kinase activity).

Figure 3. mtEGFR regulates protein levels of mitochondrial profusion proteins, PHB2, and OPA1. (A) Western blot analysis of immunoprecipitated EGFR-flag mutants from HEK293T cells co-transfected with the indicated EGFR-flag mutants and PHB2-myc. Deletion of the entire intracellular domain or the transmembrane domain, abolished the EGFR-PHB2 interaction. WT, wild type EGFR; KD, kinase dead EGFR (R718M mutation); ∆intra, deletion of 684–1210AA; ∆extra, deletion of 1–644AA; ∆TM, deletion of 645–671AA; ∆JM, deletion of 669–684AA. (B) Western blot analysis of protein samples for PHB2, OPA 1, Mfn1, Mfn2, and Tubulin of PC3 cells treated with EGF (20 ng/ml) in the presence or absence of AEE788 (5 μM) for 30 h. (C) AEE788 but not C225 downregulated OPA1. PC3 cells were treated with vehicle, EGFR monoclonal antibody (inhibits the kinase activity of pmEGFR) or AEE 788 (inhibits both the plasma membrane and non-plasma membrane EGFRs) for 24 h and isolated proteins were analyzed for OPA1. GAPDH serves as loading control. (D) mtEGFR increases levels of the endogenous PHB2 and OPA1 dependent of mtEGFR’s tyrosine kinase activity. HEK 293T cells were transfected with Mito-WT-EGFR or Mito-KD-EGFR for 24 h and protein samples were analyzed by western blot for the indicated proteins. (E) PHB2 inhibited AEE788 induced downregulation of OPA1. PC3 cells were transfected with empty vector or PHB2-myc for 24 h followed by AEE788 for another 24 h. Protein samples were analyzed on western blot for OPA 1, myc (PHB2), and Tubulin.

Figure 3. mtEGFR regulates protein levels of mitochondrial profusion proteins, PHB2, and OPA1. (A) Western blot analysis of immunoprecipitated EGFR-flag mutants from HEK293T cells co-transfected with the indicated EGFR-flag mutants and PHB2-myc. Deletion of the entire intracellular domain or the transmembrane domain, abolished the EGFR-PHB2 interaction. WT, wild type EGFR; KD, kinase dead EGFR (R718M mutation); ∆intra, deletion of 684–1210AA; ∆extra, deletion of 1–644AA; ∆TM, deletion of 645–671AA; ∆JM, deletion of 669–684AA. (B) Western blot analysis of protein samples for PHB2, OPA 1, Mfn1, Mfn2, and Tubulin of PC3 cells treated with EGF (20 ng/ml) in the presence or absence of AEE788 (5 μM) for 30 h. (C) AEE788 but not C225 downregulated OPA1. PC3 cells were treated with vehicle, EGFR monoclonal antibody (inhibits the kinase activity of pmEGFR) or AEE 788 (inhibits both the plasma membrane and non-plasma membrane EGFRs) for 24 h and isolated proteins were analyzed for OPA1. GAPDH serves as loading control. (D) mtEGFR increases levels of the endogenous PHB2 and OPA1 dependent of mtEGFR’s tyrosine kinase activity. HEK 293T cells were transfected with Mito-WT-EGFR or Mito-KD-EGFR for 24 h and protein samples were analyzed by western blot for the indicated proteins. (E) PHB2 inhibited AEE788 induced downregulation of OPA1. PC3 cells were transfected with empty vector or PHB2-myc for 24 h followed by AEE788 for another 24 h. Protein samples were analyzed on western blot for OPA 1, myc (PHB2), and Tubulin.

EGFR interacts with FASN independent of the kinase activity of EGFR

In looking for upstream signals that may regulate the activity of mtEGFR, we focused on de novo synthesized palmitate because our proteomic analysis had identified that FASN interacted with EGFR independent of EGFR’s tyrosine kinase activity (Fig. S1). Immunoprecipitations of EGFR from PC3 cells treated with EGF with/without AEE788 show that FASN interacts with EGFR regardless of AEE788 (). To further characterize the EGFR-FASN interaction, we created a panel of mutated/truncated forms of EGFR with a Flag tag at their C-termini. We determined the interaction between these mutated EGFRs and FASN in HEK293T cells using co-immunoprecipitation coupled western blot analysis. We confirmed that EGFR interacts with FASN independent of EGFR’s tyrosine kinase activity, and found that deletion of the transmembrane domain (645–671AA) or the intracellular domain (684-1210AA) of EGFR demolished the interaction (), suggesting that membranous localization of EGFR and the intracellular domain of EGFR, but not the tyrosine kinase activity of EGFR, are critical for its interaction with FASN. Because both EGFR and FASN are frequently overexpressed in cancer cells, we determined their location relationship in cancer cells of prostate and breast using immunofluorescent co-staining based confocal imaging. It was found that FASN co-localized with pmEGFR at the plasma membrane of cells of cancer tissues and cell lines (). These data suggest that pmEGFR may regulate the function of FASN of cancer cells.

Figure 4. EGFR interacts with FASN independent of EGFR kinase activity. (A) Co-immunoprecipitation of endogenous EGFR (by C225) and endogenous FASN from PC3 cells treated with EGF+/−AEE788 for 30 min. Left panel shows western blot analysis of immunoprecipitated samples and right panel shows the analysis of whole cell lysates (WCL) (B) Co-immunoprecipitation of mutated flag tagged EGFRs (arrows) (using anti-flag antibodies) with exogenous FASN transfected into HEK293T cells. 1, WT EGFR; 2, deletion of 57–68AA (amino acids); 3, deletion of 69–184AA; 4, deletion of 185–337AA; 5, deletion of 338–360AA; 6, deletion of 361–481AA; 7, deletion of 482–496AA; 8, deletion of 497–598AA; 9, deletion of 599–644AA; 10, deletion of 1–644AA; 11, deletion of 645–671AA; 12, deletion of 669–684AA; 13, R817M; 14, deletion of 684–1210AA. (C) Immunofluorescent co-staining of EGFR (green) and FASN (red) in prostate and breast cancer tissues (arrows indicated colocalized EGFR-FASN, bar = 100 μm) (D) Immunofluorescent co-staining of EGFR (green) and FASN (red) in prostate (PC3) and breast (MDA-MB-231) cancer cell lines (arrows indicated colocalized EGFR-FASN, bar = 30 μm).

Figure 4. EGFR interacts with FASN independent of EGFR kinase activity. (A) Co-immunoprecipitation of endogenous EGFR (by C225) and endogenous FASN from PC3 cells treated with EGF+/−AEE788 for 30 min. Left panel shows western blot analysis of immunoprecipitated samples and right panel shows the analysis of whole cell lysates (WCL) (B) Co-immunoprecipitation of mutated flag tagged EGFRs (arrows) (using anti-flag antibodies) with exogenous FASN transfected into HEK293T cells. 1, WT EGFR; 2, deletion of 57–68AA (amino acids); 3, deletion of 69–184AA; 4, deletion of 185–337AA; 5, deletion of 338–360AA; 6, deletion of 361–481AA; 7, deletion of 482–496AA; 8, deletion of 497–598AA; 9, deletion of 599–644AA; 10, deletion of 1–644AA; 11, deletion of 645–671AA; 12, deletion of 669–684AA; 13, R817M; 14, deletion of 684–1210AA. (C) Immunofluorescent co-staining of EGFR (green) and FASN (red) in prostate and breast cancer tissues (arrows indicated colocalized EGFR-FASN, bar = 100 μm) (D) Immunofluorescent co-staining of EGFR (green) and FASN (red) in prostate (PC3) and breast (MDA-MB-231) cancer cell lines (arrows indicated colocalized EGFR-FASN, bar = 30 μm).

pmEGFR activation by EGF increased de novo palmitate synthesis

To determine the functional significance of the pmEGFR-FASN interaction, we measured the activity of de novo palmitate synthesis in EGFR positive PC3 cells treated with EGF in presence/absence of AEE788. 14C-acetate was used as a substrate to trace the de novo synthesized palmitate. Palmitate levels were measured by thin layer chromatography (TLC) using 14C-palmitate as a standard (Fig. S2).Citation43 We found that EGF treatment for 3 h significantly increased the levels of de novo synthesized palmitate, which was inhibited by AEE788 (). Similarly, EGF treatment also promoted the de novo palmitate synthesis of MCF-7 cells transfected with wild type EGFR but not with the kinase dead EGFR (Fig. S3). Given the facts that the function of FASN depends on the formation of FASN homodimer and EGFR physically interacts with FASN, we speculated that EGF induced dimerization of pmEGFR might promote dimerization of FASN. To test this possibility, we treated PC3 cells with EGF in the presence/absence of AEE788 and conducted western blot analysis on proteins isolated under reducing and non-reducing conditions. As shown in , EGF caused a molecular weight shift of FASN from 270kDa to about 540kDa regardless of AEE788, suggesting that EGF induced dimerization of FASN is independent of EGFR’s tyrosine kinase activity. ATP citrate lyase (ACLy) is another critical enzyme regulating de novo palmitate synthesis. Phosphorylation at S454 activates ACLy, which results in the breakdown of citrate into oxaloacetate and acetyl CoA, and the latter is a substrate for palmitate synthesis.Citation44 We found that activation of pmEGFR by EGF lead to activation of ACLy, which was blocked by AEE788 (). Because EGF increased palmitate synthesis without increasing the protein levels of FASN ( and ) and AEE788 decreased palmitate synthesis without affecting EGF induced FASN dimerization, we tested the possibility that pmEGFR activates de novo fatty acid synthesis through phosphorylation of tyrosine in FASN. To do this, we immunoprecipitated FASN and performed western blot analysis for phosphorylated tyrosine (pTyr) residues in FASN using anti-pTyr antibody. It was found that EGF treatment increased the levels of phosphorylated tyrosine of FASN, which was blocked by EGFR kinase inhibitor (AEE788) (). These data suggest that EGF increased de novo palmitate synthesis requires the tyrosine kinase activity of EGFR, although the tyrosine kinase activity of EGFR is neither needed for EGFR-FASN interaction nor for EGF induced dimerization of FASN.

Figure 5. EGF induces de novo synthesis of palmitate. (A) Quantification of de novo synthesized 14C palmitate in PC3 cells treated with EGF (20 nM) in the presence or absence of AEE788 (5 μM) for 3 h. Error bars indicate the mean +/− SD of triplicate samples. Asterisk indicates the statistical significance between treated group and DMSO (P < 0.05) (B) Western blot analysis of monomeric and dimeric FASN. Protein samples of PC3 cells treated with EGF +/− AEE788 for 15 min were prepared under reducing or non-reducing conditions for analysis. (C) Western blot analysis of protein samples for pACLY, ACLY of PC3 cells treated with EGFR+/−AEE788 for 1 h. (D) FASN was immunoprecipitated from PC3 cells treated with EGF+/−AEE788 for 30 min and subjected western blot analysis for phosphorylated tyrosine (pTyr) and FASN. The right panel shows the western blot analysis of whole cell lysates (WCL) for the indicated proteins.

Figure 5. EGF induces de novo synthesis of palmitate. (A) Quantification of de novo synthesized 14C palmitate in PC3 cells treated with EGF (20 nM) in the presence or absence of AEE788 (5 μM) for 3 h. Error bars indicate the mean +/− SD of triplicate samples. Asterisk indicates the statistical significance between treated group and DMSO (P < 0.05) (B) Western blot analysis of monomeric and dimeric FASN. Protein samples of PC3 cells treated with EGF +/− AEE788 for 15 min were prepared under reducing or non-reducing conditions for analysis. (C) Western blot analysis of protein samples for pACLY, ACLY of PC3 cells treated with EGFR+/−AEE788 for 1 h. (D) FASN was immunoprecipitated from PC3 cells treated with EGF+/−AEE788 for 30 min and subjected western blot analysis for phosphorylated tyrosine (pTyr) and FASN. The right panel shows the western blot analysis of whole cell lysates (WCL) for the indicated proteins.

De novo synthesized palmitate activated mtEGFR via palmitoylation

It has been reported that de novo synthesized fatty acids act as ligands for PPAR,Citation15 activates Wnt1 signaling pathway through palmitoylation.Citation16 Overexpression of FASN increases the oncogenic activity of ErbB members.Citation17 We hypothesized that EGF induced de novo synthesized palimitate may activate mtEGFR. To test this hypothesis, we treated PC3 cells with EGF in the presence or absence of FASN inhibitor, cerulenin, and measured the phosphorylation levels of mtEGFR in isolated mitochondria using western blot analysis. We also treated cells with AEE788 as a positive control for the inhibition of mtEGFR activity. We observed that EGF treatment increased phosphorylation of mtEGFR, which could be inhibited by both AEE788 and cerulenin (), suggesting that de novo synthesized palmitate mediates the EGF induced mtEGFR activation. This possibility was further supported by the immunocytochemistry data showing that the phosphorylation of mtEGFR by EGF was inhibited by cerulenin (). To further probe the mechanism by which de novo synthesized palmitate activates mtEGFR, we performed the following experiments. First, we tested whether palmitate can activate mtEGFR in vitro by treating isolated mitochondria with palmitate in a kinase reaction buffer and measured the levels of phosphorylated mtEGFR by western blot analysis. Indeed, palmitate potently increased the mitochondrial pEGFR levels in a reaction of 15 min (). These results suggest that de novo synthesized palmitate can serve as a signal molecule from pmEGFR to activate mtEGFR. Furthermore, we determined whether mtEGFR can be palmityolated in response to pmEGFR activation. We treated PC3 cells with EGF without/with AEE788 or cerulenin and immunoprecipitated mtEGFR from isolated mitochondria, and measured palmityolation level of the mtEGFR using the Acyl-Biotin exchange assay.Citation45 It was found that in deed EGF increased the level of palmitoylated mtEGFR, which was inhibited by both AEE788 and cerulenin (). Knowing that mtEGFR can be palmitoylated, we analyzed the amino acid sequence of EGFR for possible palmitoylation sites using the CSS-Palm 3.0 program (http://csspalm.biocuckoo.org/). The program predicted cysteine residues of 781, 797, 1058, and 1146 as highly possible sites for palmitoylation. We then mutated each of these cysteines to glycine in the mito-WT-EGFR and determined their effects on mito-WT-EGFR activation using HEK293T cells. We found that C781G and C797G significantly reduced the phosphorylation of mtEGFR, and mutations on cysteine 1058 and 1146 had no effect on the phosphorylation status of mito-WT-EGFR (). Therefore, we further determined the role of C797 on the palmitoylation of mito-WT-EGFR. As shown in , C797G mutation in the mito-WT-EGFR significantly reduced the palmitoylated level of mito-WT-EGFR transfected into HEK293T cells (). These data suggest that induction of palmitoylation of mtEGFR might be a mechanism by which de novo synthesized palmitate activates mtEGFR.

Figure 6. De novo Synthesized Palmitate activates mtEGFR via palmitoylation. (A) Western blot analysis for mtEGFR activation in mitochondrial protein samples of PC3 cells treated with EGF (20 nM) +/− AEE788 (5 μM) or Cerulenin (cells were pretreated at 5 μg/ml for 3 h before EGF+/−AEE788 treatments) for 30 min. MTCO2 was used as a loading control for mitochondria. Na+/K+ ATPase serves as plasma membrane marker. (B) Immunofluorescent co-staining of mitochondria (using antibody against MTCO2, red) and pEGFR (pY1173, green) in PC3 cells treated with EGF+/−cerulenin. Co-localized signals of pEGFR and mitochondria are in yellow color. Nucleus was stained by DAPI (blue) (C) Western blot analysis of mitochondrial samples for pEGFR and EGFR. Purified mitochondria were treated with ethanol or palmitate at 200 uM for 15 min in a kinase buffer at 37 °C. (D) Detection of Palmitoylated mtEGFR using Acyl-Biotin exchange assay as described in the “Materials and Methods” section. mtEGFR was immunoprecipitated from purified mitochondria of PC3 cells that were treated with 20 nM of EGF +/− AEE788 (5 μM) for 30 min. (E) Detection of palmitoylated endogenous mtEGFR immunoprecipitated from PC3 cells treated with 20 nM EGF +/− Cerulenin (cells were pretreated with cerulenin for 12 h) at 5 μg/ml for 30 min. (F) Western blot analysis of cysteine-to-glycine mutated mito-EGFR-flag transfected into HEK293T cells for their kinase activity (pEGFR) (G) Detection of palmitoylated mito-WT-EGFR and its C797G mutant transfected into HEK 293T cells. HAM, hydroxylamine treatment for detection of palmitoylation (see “Methods and Methods” for details).

Figure 6. De novo Synthesized Palmitate activates mtEGFR via palmitoylation. (A) Western blot analysis for mtEGFR activation in mitochondrial protein samples of PC3 cells treated with EGF (20 nM) +/− AEE788 (5 μM) or Cerulenin (cells were pretreated at 5 μg/ml for 3 h before EGF+/−AEE788 treatments) for 30 min. MTCO2 was used as a loading control for mitochondria. Na+/K+ ATPase serves as plasma membrane marker. (B) Immunofluorescent co-staining of mitochondria (using antibody against MTCO2, red) and pEGFR (pY1173, green) in PC3 cells treated with EGF+/−cerulenin. Co-localized signals of pEGFR and mitochondria are in yellow color. Nucleus was stained by DAPI (blue) (C) Western blot analysis of mitochondrial samples for pEGFR and EGFR. Purified mitochondria were treated with ethanol or palmitate at 200 uM for 15 min in a kinase buffer at 37 °C. (D) Detection of Palmitoylated mtEGFR using Acyl-Biotin exchange assay as described in the “Materials and Methods” section. mtEGFR was immunoprecipitated from purified mitochondria of PC3 cells that were treated with 20 nM of EGF +/− AEE788 (5 μM) for 30 min. (E) Detection of palmitoylated endogenous mtEGFR immunoprecipitated from PC3 cells treated with 20 nM EGF +/− Cerulenin (cells were pretreated with cerulenin for 12 h) at 5 μg/ml for 30 min. (F) Western blot analysis of cysteine-to-glycine mutated mito-EGFR-flag transfected into HEK293T cells for their kinase activity (pEGFR) (G) Detection of palmitoylated mito-WT-EGFR and its C797G mutant transfected into HEK 293T cells. HAM, hydroxylamine treatment for detection of palmitoylation (see “Methods and Methods” for details).

pmEGFR activates mtEGFR via de novo synthesized fatty acids to promote mitochondrial fusion and cell survival

Supported by the data that activation of pmEGFR enhances FASN activity (), de novo synthesized palmitate can activate mtEGFR (), and activation of mtEGFR promotes mitochondrial fusion (), we hypothesized that activation of pmEGFR may promote mitochondrial fusion via de novo synthesized palmitate induced activation of mtEGFR. To test this, we treated EGFR positive PC3 cells with EGF in the presence/absence of cerulenin, and determined the mitochondrial changes using confocal fluorescent image analysis. EGF treatment promoted mitochondrial fusion, which was inhibited by cerulenin and induced mitochondrial fission (). Supportively, cerulenin treatment decreased the levels of OPA1 and PHB2 without changing the levels of other mitochondrial fusion promoting proteins such as Mfn1 and Mfn2 () and caused recruitment of DRP1 to the mitochondria (). Considering de novo fatty acid synthesis participates in EGFR’s prosurvival functions, we thought to test the effect of inhibition of FASN on the sensitivity of prostate cancer cells to EGFR tyrosine kinase inhibitor. Inhibition of FASN by cerulenin increased the sensitivity of PC3 cells to AEE788 (). These data suggest that FASN is involved in mtEGFR activation by pmEGFR to promote mitochondrial fusion through increasing PHB2 and OPA1, and co-targeting EGFR and FASN may enhance the effects of EGFR tyrosine kinase inhibitors.

Figure 7. Inhibition of FASN leads to mitochondrial fission and increases the sensitivity of PC3 cells to the growth inhibitory effect of AEE788. (A) Representative confocal images of PC3 cells treated with 20 nM of EGF +/− cerulenin (5 μg/ml) for 24 h. Mitochondria were stained with MTCO2 (red) (B) Quantification of mitochondrial dynamics in PC3 shown in (A) Mitochondria are classified into type I, II, III, and fragmented in the order of more fused to more fissed forms as described in the Materials and Methods. Y-axis represents the percentage of cells in each group containing different types of mitochondria from experiments. Data are means +/− SD of triplicates. Asterisk indicates the statistical significance between treated group and DMSO group (P < 0.05) (C) Western blot analysis of OPA1, Mfn1, Mfn2, and PHB2 in PC3 cells treated with EGF+/−cerulenin. Actin was used as a loading control. (D) Cerulenin treatment caused DRP1 recruitment to the mitochondria. PC3 cells were treated with DMSO or Cerulenin (5 μg/ml) for 24 h and immuno stained for MTCO2 and DRP1. Inset shows the enlarged portion of the cell and yellow staining (white arrow heads) indicates DRP1 recruited to the mitochondria. Nuclei were stained with DAPI (bar = 30 μm) (E) MTS assay of PC3 cells treated with increasing dose of AEE788 with/without a constant dose of cerulenin (5 μg/ml) for 24 h. Asterisk marks indicate statistical significance between the indicated groups.

Figure 7. Inhibition of FASN leads to mitochondrial fission and increases the sensitivity of PC3 cells to the growth inhibitory effect of AEE788. (A) Representative confocal images of PC3 cells treated with 20 nM of EGF +/− cerulenin (5 μg/ml) for 24 h. Mitochondria were stained with MTCO2 (red) (B) Quantification of mitochondrial dynamics in PC3 shown in (A) Mitochondria are classified into type I, II, III, and fragmented in the order of more fused to more fissed forms as described in the Materials and Methods. Y-axis represents the percentage of cells in each group containing different types of mitochondria from experiments. Data are means +/− SD of triplicates. Asterisk indicates the statistical significance between treated group and DMSO group (P < 0.05) (C) Western blot analysis of OPA1, Mfn1, Mfn2, and PHB2 in PC3 cells treated with EGF+/−cerulenin. Actin was used as a loading control. (D) Cerulenin treatment caused DRP1 recruitment to the mitochondria. PC3 cells were treated with DMSO or Cerulenin (5 μg/ml) for 24 h and immuno stained for MTCO2 and DRP1. Inset shows the enlarged portion of the cell and yellow staining (white arrow heads) indicates DRP1 recruited to the mitochondria. Nuclei were stained with DAPI (bar = 30 μm) (E) MTS assay of PC3 cells treated with increasing dose of AEE788 with/without a constant dose of cerulenin (5 μg/ml) for 24 h. Asterisk marks indicate statistical significance between the indicated groups.

Discussion

One of the hallmarks of cancer is that cancer cells depend on altered metabolism for survival and growth.Citation46 Enhanced aerobic glycolysis and elevated de novo fatty acid synthesis are common in cancer cells.Citation47 Cellular events regulated by oncogenes are a major component of the survival machinery of cancer cells. Emerging evidence indicates that de novo fatty acid synthesis is connected with oncogenic pathways. The relationship between de novo fatty acid synthesis and members of the EGFR family is especially close and bi-directional.Citation17,Citation18 Our finding, pmEGFR signaling promotes mitochondrial fusion by interacting with/activating FASN to elevate the levels of de novo synthesized palmitate that in turn activates mtEGFR to promote mitochondrial fusion, further supports that FASN or de novo fatty acid synthesis is a part of EGFR’s oncogenic machinery.

Our understanding of the canonical EGFR functions is primarily from studies of EGFR in the plasma membrane, where it receives extracellular signals by the binding of its extracellular ligands. Recent years have witnessed expansions of understanding of EGFR and HER2 functions from the plasma membrane to the nucleusCitation19 and to the mitochondria.Citation21-Citation23,Citation25,Citation48 Our data reveal that cancer tissues contain mtEGFR and that mtEGFR play important roles in cancer cells. One of the questions that need to be addressed regarding the non-plasma membranous intracellular EGFRs is that what intracellular signal(s) is (are) involved in regulating their tyrosine kinase activity. Activation of mtEGFR by de novo synthesized palmitate suggests that de novo synthesized palmitate is a signal molecule that can activate non-plasma membranous EGFRs. Protein palmitoylation is one of the powerful posttranslational protein modifications that regulate protein functions.Citation49,Citation50 Our data indicate that induction of palmitoylation of mtEGFR might be a major mechanism by which de novo synthesized palmitate activates mtEGFR, however, deeper molecular mechanisms behind this event warrant further investigations. It is also possible that activation of FASN may also affect the activity of the plasma membranous EGFR.

The fission and fusion processes of mitochondria are tightly controlled by yet to be better understood mechanisms involving both cytoplasmic and mitochondrial signals.Citation27 Our data that activation of mtEGFR enhances mitochondrial fusion suggest that promoting mitochondrial fusion is a part of the prosurvival funciton of EGFR. Importantly, this function of mtEGFR involves both of its kinase dependent and kinase independent functions, i.e., activation of the kinase activity of mtEGFR inhibits the cleavage of OPA1 and mtEGFR interacts with PHB2 independent of its kinase activity. Because the endogenous pre-existing PHB2 was oppositely regulated by our mitochondrial specific mito-WT-EGFR and mito-KD-EGFR (), it is most likely that mtEGFR stabilizes PHB2 protein within the mitochondria. Further studies are needed to identify the mechanism whereby mtEGFR stabilizes PHB2.

As a cytoplasmic protein, FASN was found to participate in molecular events associated with the plasma membrane. FASN can accumulate at membrane lipid rafts where it interacts with caveolin-1 to promote the survival of prostate cancer cells.Citation51 The increase in FASN activity by pmEGFR activation was not associated with upregulation of the protein levels of FASN (), which indicates that pmEGFR dimerization induced FASN’s dimerization () and consequential phosphorylation on FASN () are likely the mechanisms by which pmEGFR activates FASN. In this mechanism, it is worth of notice that the EGFR-FASN interaction and the EGF induced dimerization of FASN are independent of EGFR’s tyrosine kinase activity. Previously, we have reported that EGFR interacts and stabilizes the sodium/glucose co-transporter 1 (SGLT1) independent of EGFR’s tyrosine kinase activity.Citation52,Citation53 It is intriguing that the proteins found to interact with EGFR independent of EGFR’s tyrosine kinase activity all own prosurvival functions, SGLT1 for glucose uptake, PHB2 for mitochondrial integrity, FASN for de novo fatty acids synthesis. Considering the facts that only about 10–20% of patients of solid cancers respond to treatment of EGFR tyrosine kinase inhibitorsCitation54-Citation56 and EGFR is overexpressed in the majority of cancers of epithelial origin,Citation57,Citation58 i.e., there is a majority of patients with EGFR positive cancers do not respond well to the inhibition of EGFR’s tyrosine kinase, we argue that a possibility exists, which is that EGFR can promote cancer progression via mechanisms that are independent of its tyrosine kinase activity.

This study not only unveiled a mechanism by which de novo synthesized fatty acids participate in EGFR’s signaling, but also identified a role of mtEGFR in regulating mitochondrial dynamics. The activation of intracellular EGFR by de novo synthesized palmitate may contribute to the development of resistance of cancer cells to EGFR tyrosine kinase inhibitors due to a possibility that the constant high levels of de novo synthesized palmitate in cancer cells may offset the effects of tyrosine kinase inhibitors during the intervals of drug administration at the clinic. Co-targeting EGFR and FASN may have a therapeutic application in treating cancers that are resistant to EGFR tyrosine kinase inhibitors.

Materials and Methods

Cell culture and materials

Prostate cancer cell line PC3 and breast cancer cell line MDA-MB-231 were from American Type of Cell Culture. Cells were cultured in DMEM (Invitrogen) supplemented with 5.5 mM glucose, 1% penicillin/streptomycin mixture and 10% fetal bovine serum at 37 °C in a cell culture incubator with 5% CO2. Human embryonic kidney cells (HEK 293T) were cultured in DMEM supplemented with 25 mM glucose. EGF, fatty acids and FASN inhibitor (Cerulenin, cat # C2389) were obtained from Sigma Aldrich. AEE788 (Cat# S1486) was purchased from Selleck Bio. For activity assay of fatty acid synthase, 14C labeled 2-acetate and 14C-palmitate were purchased from MP Biomedicals LLC. Antibodies for FASN (cat # sc-55580), EGFR (Cat # sc-03) β actin (Cat # sc-1616), Na+/K+ ATPase (Cat # sc-21712), Monoamine oxidase (MAO) (Cat # sc-50333), calreticulin (Cat# sc-7431), Tubulin (Cat # sc-5286) and Glut1 (Cat # sc-7903) were purchased from Santa Cruz Biotechnology. EGFR monoclonal antibody, C225, was from EMD Millipore. Antibody against EGFR for immunostaining (Cat #4267), was obtained from Cell Signaling. OPA 1 (Cat # NBP1-71656) antibody was from Novus Biologicals (CO, USA). PHB2 (Cat # AB10198) antibody was from Millipore. MTCO2 (Cat # Ab3298) was from Abcam (MA, USA). Anti-flag (M2) antibody was from Sigma Aldrich. Anti-pEGFR (Y1137) (Cat # 44794G) antibody was from Invitrogen.

Plasmid constructions

Plasmids expressing wild type EGFR and kinase dead EGFR (R817M) were cloned by PCR and point mutagenesis and inserted into the pDNA3.1 vector. EGFR and PHB2 were amplified by PCR from cDNA isolated from HEK293T cell. The PCR products with a Flag tag sequence (DYKDDDDK) fused at their 3′ were introduced into mammalian expression vector pRK5 (Clontech). Truncated mutations on EGFR were performed by two-rounds of overlapping PCR. EGFR 5′ forward primer is TATCTCGAGA TGCGACCCTC CGGGACGGC, common EGFR 3′ reverse primer is ACTATCTAGA TGCTCCAATA AATTCACTGC; the primer for L1 domain deletion(57–68Aa) is TGTTGCTGAG AAAGTCACTG CTATTGAACA TCCTCTGGAG; the primer for deletion of fragment between the L domain and the furin-like cyctein rich (69–184AA) is TGGATCACAC TTTTGGCAGC TGCCGACTAT GTCCCGCCAC TGGATGCT; the primer for deletion of the furin-like cyctein rich domain (185–337Aa) is TCACCAATAC CTATTCCGTT CAGGTGGTTC TGGAAGTCCA TC; the primer for deletion of the fragment between furin-like cyctein rich and the L2 domain (338–360 AA) is TCGCCACTGA TGGAGGTGCA GTTACACACT TTGCGGCAAG GCCCT; the primer for deletion of the L2 domain (361–481AA) is GTTTTCTGAC CGGAGGTCCC TTTGAAGTGT TTAATATTCG; the primer for deletion of the fragment between the L2 domain and the furin-like cysteine rich region (482–496AA) is TGGCAGACCT GGC- CTGTGGCCTT GCAGCTGTTA AACAGTTTTT TCCAGTTTAT TGTA; the primer for deletion of the furin-like cyctein rich region 2 (497–598 AA) is ACCAGGGTGTTGTTTTCTCCCATGACTTCACCTCTGTTGCTTATAAT; the primer for deletion of the fragment between the furin–like cysteine rich region 2 and the transmembrane domain (599–644 AA) is TGCGTCAAGA CCTGCCCGGC AGGAATCCCG TCCATCGCCA CTGGGA; the primer for deletion of the transmembrane domain is AAGGCTGTCC AACGAATGGG CCTAAGCACA TCGTTCGGAA GCGCAC; the primer for deletion of juxtamembrane domain is TGGCCCTGGG GATCGGCCTC TTCATGGAGA GGGAGCTTGT G- GAGCCTCTT; the extracellular deletion (1–642 Aa) was directly amplified with a forward primer TATCTCGAGA TCCCGTCCAT CGCCACTGGG A and reverse primer ACTATCTAGA TGCTCCAATA AATTCA- CTGC; the intracellular domain deletion (684–1210 AA) was directly achieved using a forward primer TATCTCGAG ATGCGACCCT CCGGGACGGC and a reverse primer ACTATCACTG CAGCAGCCTC CGCAGCGTGC GCTT. In order to specifically introduce EGFR into mitochondria, the DNA sequence of EGFR membrane signal peptide (1–24 AA) was replaced with the DNA sequence of mitochondria signal peptide, ACGCGTCGAC ATGTCCGTCC TGACGCCGCT GCTGCTGCGG GGCTTGACAG GCTCGGCCCG GCGGCTCCCA GTGCCGCGCG CCAAGATCCA TTC- GTTGCTGGAG GAAAAGAAAG TTTGC. Specific cysteine mutations on mito EGFR constructs were created using stratagene Quickchange site directed mutagenesis kit purchased from Agilent technologies.

Transfections and Co-immunoprecipitation coupled western blot analysis

Plasmid transfection, protein extraction, co-immunoprecipitation, and western blot analysis were performed as described previously.Citation52 Detection of FASN homodimer was performed according to a protocol published by others.Citation59 Briefly, we treated PC3 cells with vehicle alone or EGF (10 ng/ml) in the presence or absence of AEE788 (5 μM) for 15 min and cells were lysed on ice in RIPA buffer followed by centrifugation to remove the cell debris. The cell lysates were mixed with 2× non-reducing sample buffer (lamellae sample buffer [Biorad] without dithiothreitol) at 1:1 ratio and loaded (without boiling) on to the 6% SDS PAGE. For control (Monomeric FASN), protein sample was prepared in reducing sample buffer (lamellae sample buffer with dithiothreitol) and boiled for 5 min. Western blotting was performed to identify the FASN dimers using FASN antibody.

Measurement of FASN phosphorylation

Serum starved PC3 cells were treated with vehicle alone, EGF (20 ng/ml) for different time intervals of 30 min in the presence or absence of AEE788 (5 µM). FASN was immunoprecipitated with anti-FASN antibody. Tyrosine phosphorylation of FASN was determined using anti-phosphotyrosine antibody (Cat # 05-1050) (Millipore, MA) by western blot analysis.

Mitochondrial extraction and in vitro phosphorylation assay

Mitochondria were isolated using ultra-centrifugation according the protocol used by others.Citation36 To obtain pure mitochondrial pellets and separate from mitochondrial associated membranes, the mitochondrial pellets were centrifuged on 30% percoll medium at 94 000 g. for 30 min. Lower band was collected and further centrifuged at 3000 rpm to pellet pure mitochondria. Isolated mitochondria was then suspended in 20ul of kinase buffer (25 mM HEPES at PH 7.5, 100 mM NaCl, 5 mM MnCl2, 0.5 mM Na3VO4, 5 mM β glycerophosphate and 20 μM ATP) and treated with ethanol or 200 μM palmitate for 15 min at 37 °C. The reaction was stopped by adding 20 μl of lamellae sample buffer. The samples were subjected to western blot analysis for total and phosphorylated EGFR. The inner membranes and outer membranes of mitochondria were separated according a protocol used by others.Citation60

Protease treatment

Purified mitochondria were suspended in 60 μl of buffer A (50 mM HEPES-KOH pH 7.2, 0.75% bovine serum albumin, 0.5 M sorbitol, 80 mM KCl, 2.5 mM magnesium acetate, 1 mM potassium phosphate, 0.5 mM MnCl2). To prepare mitoplasts, mitochondria were suspended in 60 μl of hypotonic (swelling) solution (20 mM HEPES pH7.2, 0.3% BSA, 50 mM sorbitol, 8 mM KCl, 1mM magnesium acetate, 0.8 mM potassium phosphate and 0.2 mM MnCl2) and incubated on ice 10 min. 0.5 units of protease, Thrombin, was added to 25 μl of mitochondria either suspended in buffer A or swelling solution and incubated at 37 C for 15 min. Protease activity was stopped by adding 25 μl of reducing protein sample buffer and boiling for 5 min.

Acyl-Biotin Exchange Method for Palmitoylation analysis of Mitochondrial EGFR

The Acyl-Biotin exchange palmitoylation assay was performed according the protocol used by others.Citation45 Mitochondrial EGFR was immuno precipitated from purified mitochondrial protein samples using EGFR monoclonal antibody, C225. Acyl-Biotin exchange method was adopted to study the palmitoylation of EGFR as described elsewhere. Immuno precipitated EGFR was treated with 50mM NEM in RIPA buffer at 4 °C for 2 h on a shaker. Excess NEM was removed by washing EGFR with RIPA buffer 3 times. EGFR was treated with hydroxylamine buffer (1M Hydroxylamine, 50 mM tris, 150 mM Nacl, 5 mM EDTA, 0.2% TX100, pH 7.4) at room temperature for 2 h on a shaker. For mock, EGFR was treated with hydroxylamine buffer without hydroxyl amine. EGFR was then treated with 4uM HPDP-biotin in 50 mM tris, 150 mM Nacl, 5 mM EDTA, 0.2% TX100, pH 6.2 for 2 h followed by 3 washes to remove excess biotin. Sixty microliters (60 μl) of non-reducing protein sample buffer was added and samples were boiled for 2 min to elute EGFR from beads. 20% of sample was loaded on to SDS PAGE gel. Membrane was blocked with 5% BSA for overnight followed by incubation with streptavidin conjugated with HRP at 1:30 000 for 60 min at room temperature. Biotin-streptavidin HRP complex was visualized by exposing the membrane to ECL and then to X-ray film.

Immunocytochemistry and Immunohistochemistry

For immunocytochemistry (ICC), cells, grown on coverslips, were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature and permeabilized with 0.2% tritonX100 in PBST followed by 1 h blocking with 2.5% normal donkey serum. The cells were incubated with optimized concentrations of primary antibodies in 2.5% NDS for 16 h at 4 °C. Cells were washed with PBST 3 times and incubated in dark with alexa fluor secondary antibodies (Invitrogen) at RT for 1 h followed by 3 washes with PBST. The coverslips were mounted on microscopic slides using ultracruz mounting medium (Santa Cruz Biotechnology) with DAPI. For immunofluorescent staining, formalin fixed, paraffin embedded human cancer tissue samples were used. Briefly, after the tissue sections were treated with xylene and ethanol to remove paraffin, tissues were blocked with 2.5% normal donkey serum for 1 h followed by incubation with primary antibodies at optimized dilution for 16 h at 4 °C. Tissues sections were then washed with PBS 3 times and incubated with AlexaFluor conjugated secondary antibodies (Invitrogen, CA) at 1:300 dilution for one hour at room temperature. The samples were again washed with PBS 3 times and mounted on to microscopic slides using mounting medium. The images were taken using a confocal microscope.

For live cell mitochondrial imaging, cells were grown in 10mm dishes with glass bottom. After treating the cells with respective agents, cells were incubated with Mitotracker red (Invitrogen) at 1:6000 dilution for 5 min at 37 °C. The cells were then washed with fresh medium and live mitochondrial images were taken by a confocal microscope for further analyses. Mitochondrial classification and quantification was performed according to the method used by the others.Citation39 Cells with long tubular interconnecting mitochondria were counted as type I, cells with elongated mitochondria are counted as type II, cells with rod shaped mitochondria are counted as type III and cells with punctuated and round shaped mitochondria were counted as cells with fragmented mitochondria. We counted number of cells for each type of mitochondria in each group and calculated as percentage of cells of each type. For each group, we counted a total of 30–75 cells.

FASN activity assay

De novo synthesized palmitate was measured according a published protocol.Citation43 Briefly, cells were cultured in serum free medium containing 1uCi/ml 14C labeled acetate with/without addition of EGF (20 ng/ml), AEE788 (5 μM) or AEE788/EGF for 3 h. Cells were washed and collected into PBS followed by total lipid extraction using Folsch reagent (chloroform and methanol mixture at 2:1). Samples were centrifuged and the lower phase of the samples was collected into new tubes and air-dried. The dried lipid pellet was resuspended in 50 μl of Folsch reagent. The radioactive lipid samples were then separated on thin layer chromatography. Hexane, diethyl ether, acetic acid and methanol mixture (90:20:2:3) was used to develop the TLC chromatogram. The radiochromatogram was then read by an AR2000 TLC plate reader (BioScan technologies) to measure the de novo synthesized fatty acids. 14C labeled palmitate was used as standard run in parallel to identify the peak of palmitate on TLC plate.

Cell viability assay

Cell viability assay was performed using a MTS assay kit (Cat # G3582, Promega) following manufacturer instructions.

Statistics

Student 2-tailed t test was used to compare the values (mean ± SD) of triplicate control and experimental groups of 3 independent experiments. P < 0.05 is considered as significant difference.

Abbreviations:
Drp1 =

dynamin-related protein 1

Mfn1 =

mitofusin-1

Mfn2 =

mitofusin-2

OPA1 =

optic atrophy 1

Supplemental material

Additional material

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Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgment

Z.W. is supported in part by grants from the American Cancer Society (RSG-09-206-01), the Department of Defense Prostate Cancer Research Program (W91ZSQ8334N607), and a startup fund from the University of Houston. We thank Dr Stefan Andersson and Selvaraj Muthusamy for help in measuring fatty acids using thin layer chromatography.

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