VEGFR-1 Regulates EGF-R to Promote Proliferation in Colon Cancer Cells

International Journal of Molecular Sciences

Article VEGFR-1 Regulates EGF-R to Promote Proliferation in Colon Cancer Cells

Hikaru Nagano 1, Chisato Tomida 2, Naoko Yamagishi 3 and Shigetada Teshima-Kondo 1,*

1 Department of Medical Nutrition, Osaka Prefecture University, 3-7-30 Habikino, Habikino, Osaka 583-8555, Japan; [email protected] 2 Department of Food and Nutrition, Faculty of Home Economics, Tokyo Kasei University, 1-18-1 Kaga, Itabashi, Tokyo 173-8602, Japan; [email protected] 3 Department of Anatomy and Cell Biology, School of Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-8509, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-72-950-2111

Received: 23 October 2019; Accepted: 6 November 2019; Published: 9 November 2019

Abstract: The relationship between epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) pathways in tumor growth is well established. EGF induces VEGF production in cancer cells, and the paracrine VEGF activates vascular endothelial cells to promote tumor angiogenesis and thus supports tumor cell growth in an angiogenesis-dependent manner. In this study, we found angiogenesis-independent novel crosstalk between the VEGF and the EGF pathways in the regulation of colon cancer cell proliferation. Stimulation of colon cancer cells with VEGF-A and placental growth factor (PlGF) activated VEGF receptor-1 (VEGFR-1) and increased proliferation activity in an autocrine EGF/EGF receptor (EGF-R)-dependent manner. Mechanistically, VEGFR-1 interacted with and stabilized EGF-R, leading to increased EGF-R protein levels and prolonged its expression on cell surface plasma membrane. In contrast, VEGFR-1 blockade by a neutralizing antibody and an antagonistic peptide of VEGFR-1 suppressed the complex formation of VEGFR-1 and EGF-R and decreased EGF-R expression via a lysosome-dependent pathway, resulting in the suppression of proliferation activity. Our results indicated that VEGFR-1 regulated EGF-R expression to promote proliferation activity in a cell-autonomous-dependent manner.

Keywords: vascular endothelial growth factor receptor-1 (VEGFR-1); vascular endothelial growth factor (VEGF); epidermal growth factor receptor (EGF-R); proliferation; colon cancer cells

1. Introduction Tumor angiogenesis, the growth of new capillary blood vessels, is a hallmark of cancer and is essential for tumor growth and progression [1–3]. Vascular endothelial growth factor (VEGF) is a critical factor for tumor angiogenesis in numerous solid malignancies, and tumor cells overexpress and secrete VEGF. Paracrine VEGF acts on vascular endothelial cells and induces their proliferation, diﬀerentiation and migration, resulting in angiogenesis and providing oxygen and nutrients to the tumor [2,3]. The importance of VEGF-induced angiogenesis in tumor growth is strongly supported by studies showing that blockade of VEGF and its receptors results in decreased angiogenesis and subsequent abrogation of cancer growth [2–4]. The VEGF family of ligands includes VEGF-A, -B, -C, -D and placental growth factor (PlGF) [5,6]. PlGF and VEGF-B interact with vascular endothelial growth factor receptor-1 (VEGFR-1), whereas VEGF-A is able to bind to VEGFR-1 and VEGFR-2 [5,6]. In endothelial cells, VEGFR-2 is known to translate the full range of VEGF-A responses, i.e., regulating endothelial survival, proliferation, migration and formation of the vascular tube [5,6]. In contrast, VEGFR-1 binds VEGF-A with higher

Int. J. Mol. Sci. 2019, 20, 5608; doi:10.3390/ijms20225608 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 5608 2 of 17 aﬃnity than dose VEGFR-2, however, VEGFR-1 has a 10-fold lower kinase activity than VEGFR-2 in endothelial cells. Therefore, VEGFR-1 is known to function as a negative regulator of VEGF-A signaling in endothelial cells [5,6]. VEGFR-1 was initially believed to be expressed by only endothelial cells; however, recent studies, including from us, have demonstrated that VEGFR-1 is also expressed on a variety of tumor cells, including colon, breast, gastric, osteosarcoma, pancreatic, melanoma, prostate and ovarian cancers [7–18]. Although the biological function of VEGFR-1 on cancer cells is not fully understood, the concomitant expression of VEGF ligands and VEGFR-1 by tumor cells suggests that an autocrine VEGF/VEGFR-1 signaling loop exists [7–9]. This autocrine pathway is thus considered to be an angiogenesis-independent function of VEGF. In fact, VEGF/VEGFR-1 autocrine signaling regulates the proliferation and growth of several types of cancer cells, including breast, osteosarcoma, melanoma, ovarian and skin cancers [11,13,15–17]. For example, VEGF-A and PlGF stimulated cell proliferation through VEGFR-1 in breast cancer cells [11]. In osteosarcoma cells, autocrine VEGF-A signaling induced constitutive activation of VEGFR-1, resulting in increased proliferation and survival activities [13]. The angiogenesis-independent cell proliferation eﬀect in vivo has been well demonstrated in a K5-SOS conditional knockout mouse model [17]. When VEGFR-1 was genetically depleted in epidermal cancer cells but not in vascular endothelial cells in the mice, cancer cell proliferation was decreased, resulting in the suppression of skin tumor development [17]. However, the precise mechanism by which VEGFR-1 elicits cell proliferation is not yet fully understood. Here we show that VEGFR-1 interacted with and stabilized epidermal growth factor receptor (EGF-R), leading to an increased EGF-R-dependent proliferative activity of colon cancer cells. Thus, we revealed novel crosstalk between the VEGF and the EGF pathways in colon cancer cells.

2. Results

2.1. Effect of VEGFR-1 Activation on Cell Proliferation in Colon Cancer Cells There are several reports showing that human colon cancer HCT116 cells express VEGFR-1 [10,12,18]. Here, we confirmed that the VEGFR-1 was expressed on cell surface and functional in HCT116 cells. Flow cytometry analysis showed that VEGFR-1 was expressed on cell surface of HCT116 cells (Figure S1A). Stimulation of cells with VEGF-A and PlGF increased VEGFR-1 phosphorylation levels (Figure1A). We also examined the expression of VEGFR-2 and found that HCT116 cells weakly expressed VEGFR-2 (Figure S1A,B). We then examined the effect of VEGFR-1 activation on the proliferation activity of HCT116 cells using a modified thymidine analogue EdU (5-ethynyl-2’-deoxyuridine) incorporation assay. The result shown in Figure1B clearly indicated that VEGF-A and PlGF treatment significantly increased the number of EdU-positive proliferating cells compared with bovine serum albumin (BSA) control treatment. We also examined whether VEGFR-2 was involved in the VEGF-A-stimulated proliferation activity using a VEGFR-2 specific inhibitor (ZM323881) [19]. Treatment of cells with ZM323881 did not affect both basal and VEGF-A-stimulated proliferation (Figure S1C). These results indicate that VEGF-A-induced proliferation was mediated by VEGFR-1, but not by VEGFR-2. In colon cancer cells, autocrine EGF signaling is a well-known critical pathway that activates proliferation. In addition, it has been reported that crosstalk between EGF and VEGF-A signaling exists in tumor growth [20–22]. Thus, we hypothesized that an autocrine EGF/EGF-R pathway may be involved in the VEGFR-1 induced increase in cell proliferation activity. To address this hypothesis, autocrine EGF-R loop was blocked using neutralizing antibodies against EGF ligand (anti-EGF Ab) and against EGF-R (anti-EGF-R Ab) under VEGFR-1 activating conditions. Inhibition of EGF or EGF-R completely attenuated the proliferation activity induced by VEGF-A and PlGF stimulation (Figure1C). These results indicated that an increase in proliferation activity induced by VEGFR-1 activation was mediated by autocrine EGF/EGF-R pathway. Int. J. Mol. Sci. 2019, 20, 5608 3 of 17 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 17

FigureFigure 1. Vascular 1. Vascular endothelial endothelial growth growth factor factor receptor-1(VEGFR-1) (VEGFR-1) activation activation results results in increased in increased proliferationproliferation activity activity that that depend dependss on on autocrine epidermal epidermal growth growth factor factor (EGF) (EGF)/EGF/EGF receptor receptor (EGF-R) (EGF- R) pathway.pathway. ((AA)) Activation Activation of VEGFR-1of VEGFR-1 by vascular by vascular endothelial endo growththelial factor-A growth (VEGF-A) factor-A and (VEGF-A) placental and growth factor (PlGF) stimulation. Cells were treated with VEGF-A, (PlGF) or control bovine serum placental growth factor (PlGF) stimulation. Cells were treated with VEGF-A, (PlGF) or control bovine albumin (BSA) for 5 min. Phosphorylated VEGFR-1 was detected by immunoprecipitation with an serum albumin (BSA) for 5 min. Phosphorylated VEGFR-1 was detected by immunoprecipitation with anti-phospho-tyrosine antibody and immunoblotting with an anti-VEGFR-1 antibody. The same an anti-phospho-tyrosinelysates (10% input) were antibody immunoblotted and immunoblotting with an anti-VEGFR-1 with an antibody anti-VEGFR-1 to normalize antibody. the amounts The same lysatesof each(10% sample. input) were The levels immunoblotted of β-actin are with shown an anti-VEGFR-1 as a loading control. antibody (B) to Quantification normalize the of EdUamounts of each(5-ethynyl-2’-deoxyuridine) sample. The levels of positiveβ-actin are cells shown under VEGFR-1as a loading activating control. conditions. (B) Quantification Data are indicated of EdU (5- ethynyl-2’-deoxyuridine)by means SD (n = 6–8). positive* p < 0.01, cells statistically under VEGFR-1 significant activating increase comparedconditions. with Data the are BSA-treated indicated by ± meanscontrol ± SD cells. (n = 6–8). (C) Quantification * p < 0.01, statistically of EdU positive significant cells underincrease EGF compared/EGF-R inhibiting with the conditions. BSA-treated Cells control cells.were (C) pretreatedQuantification with neutralizing of EdU positive antibodies cells against under EGF EGF/EGF-R (anti-EGF Ab)inhibiting and EGF-R conditions. (anti-EGF-R Cells Ab), were pretreatedor control with non-immune neutralizing IgG antibodies (control) for against 1 h, and thenEGF treated(anti-EGF with Ab) VEGF-A and orEGF-R PlGF for(anti-EGF-R 24 h. Data areAb), or indicated by means SD (n = 6–8). control non-immune IgG± (control) for 1 h, and then treated with VEGF-A or PlGF for 24 h. Data are 2.2.indicated Effect ofby VEGFR-1 means ± ActivationSD (n = 6–8). on EGF-R Expression As recent studies demonstrated that several growth factors, such as HGF and PDGF, regulate 2.2. Effect of VEGFR-1 Activation on EGF-R Expression EGF-R expression at the protein level and affect cell proliferation [23–25], we investigated whether VEGF-AAs recent and studies PlGF a ffdemonstratedected EGF-R protein that several expression grow levelsth factors, by immunoblot such as HGF analysis. and EGF-R PDGF, levels regulate EGF-Rwere expression rapidly up-regulated at the protein by VEGF-A level and PlGFaffect stimulation cell proliferation within 1 h, [23–25], and the increasewe investigated continued whether in a VEGF-Atime-dependent and PlGF manneraffected compared EGF-R protein with the expression BSA control treatmentlevels by (Figure immunoblot2A,B). We analysis. further examined EGF-R levels whether VEGFR-1 actually up-regulated EGF-R activation (phosphorylation) by immunoblot analysis were rapidly up-regulated by VEGF-A and PlGF stimulation within 1 h, and the increase continued with an anti-phospho-EGF-R antibody. In correlation with the elevation of EGF-R protein levels, in a time-dependent manner compared with the BSA control treatment (Figure 2A,B). We further VEGF-A and PlGF stimulation increased and prolonged EGF-R phosphorylated levels (Figure2C,D). examinedTo examinewhether whetherVEGFR-1 the increasedactually EGF-Rup-regul wasated expressed EGF-R on cellactivation surface plasma (phosphorylation) membrane to by immunoblotreceive a continuous analysis with extracellular an anti-phospho-EGF-R EGF proliferation signal, antibody. we performed In correlation immunofluorescence with the elevation staining of EGF- R proteinusing anlevels, anti-EGF-R VEGF-A antibody and recognizingPlGF stimulation the extracellular increased domain and ofprolonged the receptor. EGF-R In agreement phosphorylated with levelsthe (Figure immunoblotting 2C,D). result (Figure2A), treatment with VEGF-A and PlGF significantly prolonged EGF-RTo examineexpression whether on the the cell increased surface comparedEGF-R was to expresse control BSAd on treatment cell surface (Figure plasma2E). membrane We determined to receive a continuousthe effect extracellular of VEGFR-1 activation EGF proliferation on EGF-R signal,mRNA we expression performed levels immunofluorescence by RT-qPCR analysis staining and found using an anti-EGF-Rthat the levelsantibody were recognizing not significantly the changedextracellular by VEGF-A domain and of PlGFthe receptor. stimulation In (Figure agreement2F). These with the immunoblottingobservations suggestresult (Figure that VEGFR-1 2A), treatment activation with increased VEGF-A EGF-R and protein PlGF stability. significantly prolonged EGF-R expression on the cell surface compared to control BSA treatment (Figure 2E). We determined the effect of VEGFR-1 activation on EGF-R mRNA expression levels by RT-qPCR analysis and found that the levels were not significantly changed by VEGF-A and PlGF stimulation (Figure 2F). These observations suggest that VEGFR-1 activation increased EGF-R protein stability.

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FigureFigure 2.2. VEGFR-1VEGFR-1 activation activation results results in inincreased increased EGF-R EGF-R expression expression levels. levels. (A–D) ( ACells–D) were Cells treated were treatedwith control with control BSA for BSA 18 for h, 18 or h, with or with VEGF-A VEGF-A or orPlGF PlGF for for the the indicated indicated times. EGF-R ((AA)) andand phosphorylatedphosphorylated EGF-R EGF-R ( C(C)) levels levels were were determined determined by by immunoblot immunoblot analysis. analysis. The The levels levels of ofβ β-actin-actin are are shownshown as as a a loading loading control. control. QuantificationQuantification ofof EGF-REGF-R levelslevels ((BB)) andand phosphorylatedphosphorylated EGF-REGF-R levelslevels ((DD)) normalizednormalized to toβ β-actin-actin from from three three independent independent experiments. experiments. * *p p< < 0.01,0.01, statisticallystatistically significantsignificantincrease increase comparedcompared with with the the BSA-treated BSA-treated control. control. (E) ( ImmunofluorescentE) Immunofluorescent staining staining with with cell surface cell surface EGF-R. EGF-R. Cells wereCells pre-treated were pre-treated with control with control BSA for BSA 4 h orfor with4 h or VEGF-A with VEGF-A and PlGF and for PlGF the indicatedfor the indicated times. Living times. cellsLiving were cells then were incubated then incubated with an anti-EGF-Rwith an anti-EGF-R antibody antibody conjugated conj withugated FITC with for FITC 30 min for at 30 4 degreesmin at 4 anddegrees fixed. and Nuclei fixed. were Nuclei stained were with stained 40,6-diamidino-2-phenylindole with 4′,6-diamidino-2-phenylindole (DAPI). Representative (DAPI). Representative fluorescent imagesfluorescent are shown. images Scaleare shown. bar = 10 Scaleµm. bar (F) Expression= 10 μm. ( levelsF) Expression of EGF-R levelsmRNA of wereEGF-R determined mRNA were by RT-qPCR analysis. Values were normalized for the amount of GAPDH mRNA (n = 5, means SD). determined by RT-qPCR analysis. Values were normalized for the amount of GAPDH mRNA± (n = 5, means ± SD). 2.3. Effect of VEGFR-1 Activation on EGF-R Stability 2.3. EffectTo address of VEGFR-1 whether Activation the stability on EGF-R of EGF-R Stability protein was increased by VEGFR-1 activation, we performed cycloheximide (a protein synthesis inhibitor) chase assay using an EGF-stimulated EGF-R To address whether the stability of EGF-R protein was increased by VEGFR-1 activation, we degradation model [26,27]. Cells were pretreated with VEGF-A, PlGF or BSA for 1 h, then treated with performed cycloheximide (a protein synthesis inhibitor) chase assay using an EGF-stimulated EGF- EGF and cycloheximide for the indicated times. The cycloheximide chase assay indicated that the R degradation model [26,27]. Cells were pretreated with VEGF-A, PlGF or BSA for 1 h, then treated BSA-treated cells showed a time-dependent decrease in EGF-R expression levels upon EGF stimulation with EGF and cycloheximide for the indicated times. The cycloheximide chase assay indicated that (Figure3A, lanes 6–9, and Figure3B). In contrast, VEGF-A and PlGF stimulation clearly increased the BSA-treated cells showed a time-dependent decrease in EGF-R expression levels upon EGF EGF-R stabilization (Figure3A, lanes 2–5 and 10–13, and Figure3B). stimulation (Figure 3A, lanes 6–9, and Figure 3B). In contrast, VEGF-A and PlGF stimulation clearly increased EGF-R stabilization (Figure 3A, lanes 2–5 and 10–13, and Figure 3B).

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FigureFigure 3. VEGFR-1 3. VEGFR-1 activation activation stabilizes stabilizes EGF-R. EGF-R. ( A) CellsCells were were pretreated pretreated with wi controlth control BSA, BSA, VEGF-A VEGF-A or PlGFor PlGF for for1 h, 1 then h, then incubated incubated with with EGF EGF plus cycloheximidecycloheximide for for the the indicated indicated times. times. EGF-R EGF-R protein protein levels were determined by immunoblot analysis. The levels of β-actin are shown as a loading control. levels were determined by immunoblot analysis. The levels of β-actin are shown as a loading control. (B) Quantification of EGF-R levels normalized to β-actin from three independent experiments. * p < 0.01, (B) Quantification of EGF-R levels normalized to β-actin from three independent experiments. * p < statistically significant increase compared with the BSA-treated control cells at the corresponding each 0.01,time statistically point. significant increase compared with the BSA-treated control cells at the corresponding each time point. 2.4. Effect of VEGFR-1 Activation on Interaction of VEGFR-1 and EGF-R 2.4. EffectA of recent VEGFR-1 report Activation demonstrated on Interaction that PDGF-R of VEGFR-1β prolongs and EGF-R EGF-R expression on cell surface by the β formationA recent ofreport a PDGF-R demonstrated/EGF-R heterodimer that PDGF-R [25].β Thus,prolongs we hypothesized EGF-R expression that EGF-R on stabilizationcell surface on by the cell surface may be induced by the interaction of VEGFR-1 with EGF-R. To test this hypothesis, formation of a PDGF-R β/EGF-R heterodimer [25]. Thus, we hypothesized that EGF-R stabilization immunoprecipitation in combination with immunoblotting analysis was performed (Figure4). on cell surface may be induced by the interaction of VEGFR-1 with EGF-R. To test this hypothesis, Immunoprecipitation results with an anti-EGF-R antibody showed that the basal interaction of immunoprecipitationVEGFR-1 with EGF-R in was combination observed in controlwith immunoblotting BSA treated cells (Figureanalysis4A). was Treatment performed with VEGF-A (Figure 4). Immunoprecipitationand PlGF increased theirresults interaction with an (Figure anti-EGF-R4A,B). Immunoblot antibody analysisshowed with that an the anti-phosphorylated basal interaction of VEGFR-1VEGFR-1 with antibody EGF-R was showed observed that phosphorylated in control BSA VEGFR-1 treated cells interacted (Figure with 4A). EGF-R Treatment (Figure with4A,C). VEGF- A andReciprocal PlGF immunoprecipitationincreased their interaction with an anti-VEGFR-1 (Figure 4A,B). antibody Immunoblot confirmed that analysis VEGFR-1 with activation an anti- phosphorylatedinduced the increase VEGFR-1 in VEGFR-1 antibody and showed EGF-R complexthat phosphorylated formation (Figure VEGFR-14D,E). interacted with EGF-R (Figure 4A,C). Reciprocal immunoprecipitation with an anti-VEGFR-1 antibody confirmed that VEGFR-1 activation induced the increase in VEGFR-1 and EGF-R complex formation (Figure 4D,E).

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FigureFigure 4. 4.VEGFR-1VEGFR-1 activation activation increases increases complexcomplex formation formation of of VEGFR-1 VEGFR-1 and and EGF-R. EGF-R. (A) Cells (A) Cells were were treatedtreated with with BSA, BSA, VEGF-A VEGF-A or or PlGF PlGF for for 30 min. 30 min. Cell lysatesCell lysates were immunoprecipitated were immunoprecipitated with an anti-EGF-R with an anti- EGF-Rantibody antibody (IP: EGF-R) (IP: andEGF-R) then immunoblottedand then immunoblotted for VEGFR-1 (Afor, upper VEGFR-1 panel) and(A, forupper phosphorylated panel) and for phosphorylatedVEGFR-1 (A, middle VEGFR-1 panel). (A In, middle parallel, panel). Western In blot parallel, was performed Western to controlblot was for performed EGF-R concentration to control for EGF-Rin the concentration immunoprecipitates in the (A ,immunoprecipitates lower panel). (B,C) Quantification (A, lower of panel). co-immunoprecipitated (B,C) Quantification VEGFR-1 of co- immunoprecipitatedlevels (B) and phosphorylated VEGFR-1 VEGFR-1levels (B levels) and ( Cphosphorylated) normalized to immunoprecipitated VEGFR-1 levels ( EGF-RC) normalized levels to from three independent experiments. * p < 0.01, statistically significant increase compared with the immunoprecipitated EGF-R levels from three independent experiments. * p < 0.01, statistically BSA-treated control. (D) Cells were treated with BSA, VEGF-A or PlGF for 30 min. Cell lysates were significant increase compared with the BSA-treated control. (D) Cells were treated with BSA, VEGF- immunoprecipitated with an anti-VEGFR-1 antibody (IP: VEGFR-1) and then immunoblotted for EGF-R A or(D ,PlGF upper for panel). 30 min. In parallel, Cell lysates Western were blot immunoprec was performedipitated to control with for an VEGFR-1 anti-VEGFR-1 concentration antibody in (IP: VEGFR-1)the immunoprecipitates and then immunoblotted (D, lower panel). for (EEGF-R) Quantification (D, upper of co-immunoprecipitatedpanel). In parallel, Western EGF-R levels blot was performednormalized to tocontrol immunoprecipitated for VEGFR-1 VEGFR-1concentration levels fromin the three immunoprecipitates independent experiments. (D, lower * p < panel).0.01, (E) Quantificationstatistically significant of co-immunoprecipitated increase compared withEGF-R the levels BSA-treated normalized control. to immunoprecipitated VEGFR-1 levels from three independent experiments. * p < 0.01, statistically significant increase compared with 2.5. Effect of VEGFR-1 Blockade on Cell Proliferation Activity the BSA-treated control. We then examined the effect of VEGFR-1 inhibition on cell proliferation activity using two different 2.5.VEGFR-1 Effect of VEGFR-1 inhibitors, Blockade a neutralizing on Cell anti-VEGFR-1 Proliferation antibody Activity (anti-R1 Ab) and an antagonistic peptide for VEGFR-1 (R1 antagonist) [28]. We confirmed that treatment with anti-R1 Ab and R1 antagonist significantlyWe then examined reduced VEGFR-1 the effect phosphorylation of VEGFR-1 uponinhibition VEGF-A on stimulation cell proliferation (Figure5 A).activity Compared using two differentwith control VEGFR-1 IgG inhibitors, treatment, treatmenta neutralizing with anti-VEGFR-1 anti-R1 Ab and antibody R1 antagonist (anti-R1 significantly Ab) and decreasedan antagonistic peptideproliferation for VEGFR-1 activity (Figure(R1 antagonist)5B). [28]. We confirmed that treatment with anti-R1 Ab and R1 antagonist significantly reduced VEGFR-1 phosphorylation upon VEGF-A stimulation (Figure 5A). Compared with control IgG treatment, treatment with anti-R1 Ab and R1 antagonist significantly decreased proliferation activity (Figure 5B).

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Figure 5. VEGFR-1Figure 5. VEGFR-1 blockade blockade decreases decreases proliferation proliferation activity. activity. (A) Inhibition (A of) VEGFR-1Inhibition by anti-VEGFR-1 of VEGFR-1 by anti- VEGFR-1 neutralizingneutralizing antibody antibody (anti-R1 (anti-R1 Ab) and VEGFR-1Ab) and antagonist VEGFR-1 (R1 antagonist). antagonist Cells (R1 were antagonist). treated with Cells were anti-R1 Ab, R1 antagonist or control IgG (control) in the presence of VEGF-A for 5 min. Phosphorylated treated with anti-R1 Ab, R1 antagonist or control IgG (control) in the presence of VEGF-A for 5 min. VEGFR-1 was detected as described in legend to Figure1. The same lysates (10% input) were Phosphorylatedimmunoblotted VEGFR-1 with was an anti-VEGFR-1 detected antibodyas described to normalize in legend the amounts to Figure of each sample. 1. The The same levels lysates (10% input) wereof βimmunoblotted-actin are shown as awith loading an control. anti-VEGFR-1 (B) Quantification antibody of EdU to positive normalize cells under the VEGFR-1 amounts of each sample. Theinhibiting levels conditions.of β-actin Cells are were shown treated as with a loading anti-R1 Ab, control. R1 antagonist (B) Quantification or control IgG for of 24 h.EdU Data positive cells are indicated by means SD (n = 6–8). * p < 0.01, statistically significant decrease compared with the ± under VEGFR-1control cells.inhibiting conditions. Cells were treated with anti-R1 Ab, R1 antagonist or control IgG for 24 h. Data are indicated by means ± SD (n = 6–8). * p < 0.01, statistically significant decrease 2.6. Effect of VEGFR-1 Blockade on EGF-R Expression compared with the control cells. We then examined the effect of VEGFR-1 inhibition on EGF-R expression levels. Treatment of cells with anti-R1 Ab and R1 antagonist rapidly reduced the EGF-R levels within 10 min (Figure6A,B). 2.6. Effect ofThe VEGFR-1 EGF-R levels Blockade were furtheron EGF-R decreased Expression after 60 min of VEGFR-1 blockade, and the decrease continued to 24 h (Figure6A,B). Knockdown of VEGFR-1 using a siRNA targeting VEGFR-1 mRNA We thenfor 24examined h (Figure6C) the also effect significantly of VEGFR-1 reduced EGF-R inhibiti expressionon on (FigureEGF-R6D). expression We further examined levels. Treatment of cells with anti-R1EGF-R expression Ab and R1 on theantagonist plasma membrane rapidly by redu immunofluorescenceced the EGF-R staining. levels within In agreement 10 min with (Figure 6A,B). The EGF-Rimmunoblotting levels were result further (Figure decreased6A), blockade after of VEGFR-1 60 min decreased of VEGFR-1 the cell surface blockade, EGF-R expression and the decrease continued comparedto 24 h (Figure to the control 6A,B). IgG-treated Knockdown conditions of VEGFR-1 (Figure6E). Weusing determined a siRNA the etargetingffect of VEGFR-1 VEGFR-1 mRNA inhibition on EGF-R mRNA expression levels by RT-qPCR analysis and found that the levels were not for 24 h (Figuresignificantly 6C) also changed significantly by VEGFR-1 blockadereduced (Figure EGF-R6F). expression (Figure 6D). We further examined EGF-R expression on the plasma membrane by immunofluorescence staining. In agreement with immunoblotting result (Figure 6A), blockade of VEGFR-1 decreased the cell surface EGF-R expression compared to the control IgG-treated conditions (Figure 6E). We determined the effect of VEGFR-1 inhibition on EGF-R mRNA expression levels by RT-qPCR analysis and found that the levels were not significantly changed by VEGFR-1 blockade (Figure 6F).

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FigureFigure 6. 6.VEGFR-1 VEGFR-1 blockade blockade decreases decreases EGF-R EGF-R expression. expression. ( A(A)) Cells Cells were were treated treated with with anti-R1 anti-R1 Ab Ab and and R1R1 antagonist antagonist forfor the the indicated indicated times, times, or or with with a a control control IgG IgG for for 24 24 h h (indicated (indicated by by control). control). EGF-R EGF-R proteinprotein levels levels were were determined determined by by immunoblot immunoblot analysis. analysis. The The levels levels of ofβ β-actin-actin are are shown shown as as a a loading loading control.control. ( B()B Quantification) Quantification of EGF-R of EGF-R levels normalizedlevels normalized to β-actin to from β-actin three independentfrom three experiments.independent * experiments.p < 0.01, statistically * p < 0.01, significant statistically decrease significant compared decrease withthe compared control cells.with (theC,D control) Cells werecells. transfected(C,D) Cells withwere the transfected indicated with siRNA the forindicated 24 h. Levels siRNA of for VEGFR-1 24 h. Levels protein of VEGFR-1 (C, left) and protein EGF-R (C, protein left) and (D EGF-R, left) wereprotein determined (D, left) were by immunoblot determined by analysis. immunoblot Quantification analysis. Quantification of VEGFR-1 levels of VEGFR-1 (C, right) levels and (C EGF-R, right) levelsand EGF-R (D, right) levels normalized (D, right) to normalizedβ-actin from to threeβ-actin independent from three experiments.independent experiments.* p < 0.01, statistically * p < 0.01, significantstatistically decrease significant compared decrease with the compared si-control-transfected with the cells. si-control-transfected (E) Immunofluorescent cells. staining (E) withImmunofluorescent cell surface EGF-R. staining After cellswith werecell surface treated withEGF- anti-R1R. After Ab, cells R1 were antagonist treated or with control anti-R1 IgG forAb, 1 R1 h, cellantagonist surface EGF-Ror control was IgG stained for 1 ash, describedcell surface in EGF-R legend was to Figure stained2E. as Scale described bar = in10 legendµm. (F )to Cells Figure were 2E. treatedScale bar with = anti-R110 μm. Ab,(F) R1Cells antagonist were treated or control with IgG anti-R1 for 12 Ab, h. Expression R1 antagonist levels or of controlEGF-R IgGmRNA for were12 h. determinedExpression bylevels RT-qPCR of EGF-R analysis. mRNA Values were weredetermined normalized by RT-qPCR for theamount analysis. of ValuesGAPDH weremRNA normalized (n = 5, meansfor the amountSD). of GAPDH mRNA (n = 5, means ± SD). ± 2.7. Effect of EGF-R Knockdown on Proliferation Activity. To confirm that EGF-R was essential for VEGFR-1-mediated proliferation activity, we examined the effect of EGF-R silencing on the proliferation. Transfection of cells with a siRNA targeting EGF-R mRNA significantly reduced EGF-R protein expression compared with control siRNA transfected cells (Figure 7A,B). Knockdown of EGF-R decreased basal proliferation activity to the same extent as VEGFR-1 blockade (Figure 7C). Furthermore, EGF-R silencing completely attenuated the VEGF-A- and PlGF-stimulated proliferation (Figure 7C).

Int. J. Mol. Sci. 2019, 20, 5608 9 of 17

2.7. Effect of EGF-R Knockdown on Proliferation Activity To confirm that EGF-R was essential for VEGFR-1-mediated proliferation activity, we examined the effect of EGF-R silencing on the proliferation. Transfection of cells with a siRNA targeting EGF-R mRNA significantly reduced EGF-R protein expression compared with control siRNA transfected cells (Figure7A,B). Knockdown of EGF-R decreased basal proliferation activity to the same extent as VEGFR-1 blockade (Figure7C). Furthermore, EGF-R silencing completely attenuated the VEGF-A- and PlGF-stimulatedInt. J. Mol. Sci. 2019, 20 proliferation, x FOR PEER REVIEW (Figure 7C). 9 of 17

Figure 7. EGF-R knockdown blocks VEGFR-1 activati activation-inducedon-induced proliferation activity. (A) Cells were transfected withwith thethe indicated siRNAsiRNA forfor 2424 h. EGF- EGF-RR protein levels were determineddetermined by immunoblot analysis. The The levels levels of β-actin-actin are shown as aa loadingloading control.control. ( B) QuantificationQuantification of EGF-REGF-R levelslevels normalized toto β-actin from three independentindependent experiments.experiments. ** p < 0.01,0.01, statistically statistically significant significant decrease compared with thethe si-control-treatedsi-control-treated cells.cells. ( (CC)) Quantification Quantification of EdU positive cells underunder EGF-REGF-R silencing conditions. Cells Cells were were transfected transfected with with the the indicated indicated siRNA siRNA for 24 h, then treated with VEGF-A, PlGF or BSA for the additionaladditional 24 h. For a control experiment, cells were treated with only anti-R1 Ab or control IgGIgG (indicated(indicated byby control)control) forfor 2424 h.h. DataData areare indicatedindicated byby meansmeans ± SD (n = 6–8).6–8). ± # pp << 0.01,0.01 ,statistically statistically significant significant decrease decrease compared compared with the control.control. * p < 0.01,0.01, statistically statistically significant significant increaseincrease comparedcompared withwith thethe control.control. 2.8. Effect of a Lysosomal and Proteasomal Inhibitor on EGF-R Expression 2.8. Effect of a Lysosomal and Proteasomal Inhibitor on EGF-R Expression The rapid decrease in EGF-R protein levels observed in Figure6A suggested that VEGFR-1 The rapid decrease in EGF-R protein levels observed in Figure 6A suggested that VEGFR-1 inhibition led to EGF-R degradation. Downregulation of EGF-R protein has been shown to occur inhibition led to EGF-R degradation. Downregulation of EGF-R protein has been shown to occur predominantly through lysosomal and/or proteasomal degradation pathways [26,27]. To determine predominantly through lysosomal and/or proteasomal degradation pathways [26,27]. To determine which degradation pathway was involved in the EGF-R downregulation under VEGFR-1 blocking which degradation pathway was involved in the EGF-R downregulation under VEGFR-1 blocking conditions, we used bafilomycin A (a lysosome inhibitor) and MG132 (a proteasome inhibitor). conditions, we used bafilomycin A (a lysosome inhibitor) and MG132 (a proteasome inhibitor). The The result indicated in Figure8A,B showed that the EGF-R reduction was completely inhibited by result indicated in Figure 8A and B showed that the EGF-R reduction was completely inhibited by bafilomycin A (lanes 4 and 5), but not by MG132 (lanes 6 and 7). These results indicated that the EGF-R bafilomycin A (lanes 4 and 5), but not by MG132 (lanes 6 and 7). These results indicated that the EGF- downregulation was mediated by a lysosomal degradation pathway. R downregulation was mediated by a lysosomal degradation pathway.

Int. J. Mol. Sci. 2019, 20, 5608 10 of 17 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 10 of 17

FigureFigure 8. EGF-R 8. EGF-R downregulation downregulation is is mediated mediated byby a a lysosomal lysosomal degradation degradation pathway pathway under under VEGFR-1 VEGFR-1 inhibitedinhibited conditions. conditions. (A ()A Cells) Cells were were pretreated pretreated withwith aa lysosomal lysosomal inhibitor inhibitor (bafilomycin (bafilomycin A) or A) a or a proteasomalproteasomal inhibitor inhibitor (MG132) (MG132) for for 1 1 h, then treatedtreated with with anti-R1 anti-R1 Ab Ab (lanes (lanes 4 and 4 and 6) and 6) and R1 antagonist R1 antagonist (lanes(lanes 5 and 5 and 7) 7)for for 1 1h. h. Without Without bafilomycin bafilomycin AA oror MG132, MG132, cells cells were were treated treated with with anti-R1 anti-R1 Ab alone Ab alone (lane 2), R1 antagonist alone (lane 3) or control IgG alone (lane 1, indicated by control) for 1 h. EGF-R (lane 2), R1 antagonist alone (lane 3) or control IgG alone (lane 1, indicated by control) for 1 h. EGF-R protein levels were determined by immunoblot analysis. The levels of β-actin are shown as a loading protein levels were determined by immunoblot analysis. The levels of β-actin are shown as a loading control. (B) Quantification of EGF-R levels normalized to β-actin from three independent experiments. control.* p < 0.01,(B) statisticallyQuantification significant of decreaseEGF-R comparedlevels normalized with the control. to β-actin from three independent experiments. * p < 0.01, statistically significant decrease compared with the control. 2.9. Effect of VEGFR-1 Blockade on Interaction of VEGFR-1 and EGF-R 2.9. EffectTo of examine VEGFR-1 whether Blockade VEGFR-1 on Interaction inhibition of VEGFR-1 decreased and the EGF-R interaction of VEGFR-1 and EGF-R, immunoprecipitationTo examine whether in combination VEGFR-1 withinhibition immunoblotting decreased was the performed. interaction Immunoprecipitation of VEGFR-1 and with EGF-R, anti-EGF-R antibody showed that the increased interaction of VEGFR-1 with EGF-R upon VEGF-A immunoprecipitation in combination with immunoblotting was performed. Immunoprecipitation stimulation was decreased by VEGFR-1 blockers (Figure9A,B). Reciprocal immunoprecipitation with with anti-EGF-R antibody showed that the increased interaction of VEGFR-1 with EGF-R upon anti-VEGFR-1 antibody confirmed the decrease in their interaction (Figure9C,D). These results suggest VEGF-Athat EGF-R stimulation destabilization was induceddecreased by VEGFR-1 by VEGFR-1 blockade wasblockers mediated (Figure by the dissociation 9A,B). ofReciprocal the immunoprecipitationVEGFR-1 and EGF-R with complex. anti-VEGFR-1 antibody confirmed the decrease in their interaction (Figure 9C,D). These results suggest that EGF-R destabilization induced by VEGFR-1 blockade was mediated by the dissociation of the VEGFR-1 and EGF-R complex.

Int. J. Mol. Sci. 2019, 20, 5608 11 of 17 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 11 of 17

FigureFigure 9. 9.VEGFR-1VEGFR-1 blockade blockade decreases decreases complex complex formation formation of VEGFR-1 of VEGFR-1 and EGF-R. and Cells EGF-R. were Cellsuntreated were oruntreated pretreated or with pretreated anti-R1 Ab, with R1 anti-R1 antagonist Ab, R1or control antagonist IgG (control) or control for IgG 3 min, (control) and then for stimulated 3 min, and with then VEGF-Astimulated for 2 with min. VEGF-A (A) Cell forlysates 2 min. were (A immunoprecipitated) Cell lysates were immunoprecipitatedwith an anti-EGF-R antibody with an (IP: anti-EGF-R EGF-R) andantibody then immunoblotted (IP: EGF-R) and for VEGFR-1 then immunoblotted (A, upper panel). for VEGFR-1In parallel,( WesternA, upper blot panel). was performed In parallel, to Westerncontrol forblot EGF-R was performedconcentration to control in the for immunoprecipitates EGF-R concentration (A in, lower the immunoprecipitates panel). (B) Quantification (A, lower of panel). co- immunoprecipitated(B) Quantification VEGFR-1 of co-immunoprecipitated levels normalized to VEGFR-1 immunoprecipitated levels normalized EGF-R from to immunoprecipitated three independent experiments.EGF-R from * p three < 0.01, independent statistically significant experiments. increase * p < compared0.01, statistically with the untreated significant cells. increase (C) Cell compared lysates werewith immunoprecipitated the untreated cells. with (C) Cellan anti-VEGFR-1 lysates were immunoprecipitatedantibody (IP: VEGFR-1) with and an then anti-VEGFR-1 immunoblotted antibody for EGF-R(IP: VEGFR-1) (C, upper andpanel). then In immunoblottedparallel, Western forblot EGF-R was performed (C, upper to panel).control for In parallel,VEGFR-1 Western concentration blot was in theperformed immunoprecipitates to control for(C, VEGFR-1lower panel). concentration (D) Quantification in the immunoprecipitates of co-immunoprecipitated (C, lower EGF-R panel). levels (D ) normalizedQuantification to immunoprecipitated of co-immunoprecipitated VEGFR-1 EGF-Rfrom three levels independent normalized experiments. to immunoprecipitated * p < 0.01, statistically VEGFR-1 significantfrom three increase independent compared experiments. with the untreated * p < 0.01, cells. statistically significant increase compared with the untreated cells. 3. Discussion 3. Discussion It has been demonstrated that there is angiogenesis-dependent crosstalk between the EGF and the VEGFIt has pathway been demonstrated in several types that of cancer there iscells angiogenesis-dependent [20]. Autocrine EGF signaling crosstalk consistently between induces the EGF VEGF-Aand the expression VEGF pathway and secretion in several by types colon, of cancergastric cellsand breast [20]. Autocrine cancer cells, EGF leading signaling to a consistently paracrine VEGF-A-inducedinduces VEGF-A tumor expression angiogenesis and secretion that supports by colon, tumor gastric growth and breastand progre cancerssion cells, [21,22,29]. leading toIn a contrast,paracrine inhibition VEGF-A-induced of EGF signaling tumor angiogenesis decreases VE thatGF-A supports production tumor growth and thus and inhibits progression angiogenesis [21,22,29 ]. andIn contrast,tumor growth inhibition [20,21,29]. of EGF signaling decreases VEGF-A production and thus inhibits angiogenesis andIn tumor the present growth [study,20,21,29 we]. elucidated novel angiogenesis-independent crosstalk between the VEGF Inand the the present EGF pathways study, we in elucidated colon cancer novel cell angiogenesis-independents. Activation of VEGFR-1 by crosstalk VEGF-A between and PlGF the stabilizedVEGF and EGF-R the EGF protein pathways and prolonged in colon cancerits expressi cells.on Activation and phosphorylation, of VEGFR-1 resulting by VEGF-A in increased and PlGF autocrinestabilized EGF/EGF-R-dependent EGF-R protein and prolonged proliferation its expression activity. In and contrast, phosphorylation, blockade of VEGFR-1 resulting led in increased to EGF- Rautocrine destabilization, EGF/EGF-R-dependent resulting in the suppression proliferation of activity. proliferation In contrast, activity. blockade Thus, ofour VEGFR-1 study suggests led to EGF-R that VEGFR-1destabilization, regulates resulting cancer incell the growth suppression via two of proliferationdifferent pathways: activity. an Thus, angiogenesis-independent our study suggests that autocrineVEGFR-1 pathway regulates and cancer an angiogen cell growthesis-dependent via two di paracrinefferent pathways: pathway. an angiogenesis-independent autocrineIn endothelial pathway cells, and anVEGFR-1 angiogenesis-dependent has been reported paracrine to be implicated pathway. as a negative regulator of VEGFR-2In endothelial signaling for cells, proliferation VEGFR-1 has[5,6]. been In contrast, reported in to several be implicated types of as cancer a negative cells, regulator VEGFR-1 of promotesVEGFR-2 proliferation signaling for signal proliferation [9–16]. [These5,6]. Infin contrast,dings suggest in several that typesinvolvement of cancer of cells, VEGFR-1 VEGFR-1 in mitogenicpromotes signal proliferation is distinctly signal different [9–16]. These between findings endothelial suggest cells that and involvement cancer cells. of VEGFR-1 in mitogenic signalIn cancer is distinctly cells, diit ffhaserent been between speculated endothelial that VEGFR-1 cells and signaling cancer cells. relies on abnormal link between unrelated receptors that function synergistically to enhance proliferation [30]. For example, disruption of VEGF-A and VEGFR-1 genes in epidermal tumor cells decreases their proliferation [17].

Int. J. Mol. Sci. 2019, 20, 5608 12 of 17

In cancer cells, it has been speculated that VEGFR-1 signaling relies on abnormal link between unrelated receptors that function synergistically to enhance proliferation [30]. For example, disruption of VEGF-A and VEGFR-1 genes in epidermal tumor cells decreases their proliferation [17]. Furthermore, VEGF-A disruption in an EGF-R-deficient background completely inhibits epidermal tumor growth, suggesting that there is an abnormal contribution of the VEGF-A and the EGF pathways in the tumor cells [17]. In this study, we provide the first demonstration that a direct interaction of VEGFR-1 and EGF-R contributed colon cancer cell proliferation. It is well established that the control of EGF-R trafficking and degradation is regulated mainly by its ligands [27,28]. Binding of EGF to EGF-R leads to internalization of EGF-R and subsequent degradation by lysosome and/or proteasome pathways [27,28]. In addition to EGF, it has recently been reported that HGF controls EGF-R degradation [23,24]. The HGF/cMet system accelerates EGF-R degradation by inhibiting the binding of SHIP2 (Src homology domain 2-containing inositol 50-phosphatase 2) to EGF-R, which is required to block the EGF-R degradation pathway [23]. In this study, we demonstrated novel growth factors that regulate EGF-R stabilization. The VEGF-A and PlGF/VEGFR-1 system stabilized EGF-R via the interaction of VEGFR-1 with EGF-R. In contrast, blockade of VEGFR-1 decreased their interaction and led to destabilization of EGF-R through a lysosome-dependent pathway. In addition to VEGFR-1, a recent report demonstrated that PDGF-Rβ formed a heterodimer with EGF-R and stabilizes EGF-R on the cell surface [25]. Thus, the stabilization of EGF-R is regulated by several growth factors and their receptors. Recently, the concept of molecular targeted therapy for the treatment of colorectal cancer has emerged. Two critical molecular targets for the treatment of colorectal cancer are EGF-R and VEGF-A, because these two molecules are often overexpressed and are associated with inferior outcomes [4,31]. Thus, anti-EGF-R monoclonal antibodies (cetuximab and panitumumab) and an anti-VEGF-A monoclonal antibody (bevacizumab) are mainly used as therapies for colorectal cancer patients [31]. Despite the initial clinical efficacy of the anti-EGF-R agents against colorectal cancer, acquired resistance develops during therapy [31]. The resistance is caused by the overexpression of VEGF-A, PlGF and VEGFR-1 and thus the activation of alternative VEGFR-1 signaling pathway [32]. In the case of anti-VEGF-A therapy, acquired resistance is also developed via alternative PlGF/VEGFR-1 pathway activation [9,33,34]. The therapeutic evidence suggests that VEGFR-1 targeting therapy provides a promising strategy for reducing the risk of the acquired resistance. In fact, VEGFR-1 targeted therapy has been recently found to block cancer growth by reducing the effects of VEGF-A and PlGF on cancer cells [9]. In addition to the direct anticancer effect, the VEGFR-1 blocking agents inhibit tumor angiogenesis by blocking the effects of VEGF-A and PlGF on vascular endothelial cells [9]. Intriguingly, in clinical studies, VEGFR-1 targeting therapies have been shown to be of potential benefit in patients with colorectal cancer [35–39]. For example, a VEGFR-1 blocking agent (aflibercept) that traps VEGF-A and PlGF [39,40] improved overall survival compared with a placebo in patients with metastatic colorectal cancer in a clinical study [36–39]. This benefit may be due to the inhibition of both colorectal cancer cells and vascular endothelial cells. Thus, the molecular elucidation of VEGFR-1 function in colon cancer cells will provide important insight into current therapies targeting VEGFR-1. Taken together, the present study provides the first evidence that VEGFR-1 plays, at least in part, an important role in cell proliferation through regulating EGF-R.

4. Materials and Methods

4.1. Reagents and Antibodies Cycloheximide, MG132, baﬁlomycin A and DMSO were from Wako Pure chemical (Osaka, Japan). A VEGFR-1 antagonistic peptide (anti-Flt1 peptide; GNQWFI) was from Alpha Diagnostic Int (San Antonio, TX, USA). A VEGFR-2 speciﬁc small molecule kinase inhibitor (ZM323881) [19] was obtained from Selleckchem (Houston, TX, USA). Recombinant human EGF was obtained from Int. J. Mol. Sci. 2019, 20, 5608 13 of 17

HIGETA SHOYU (Tokyo, Japan). Recombinant VEGF-A (293-VE), recombinant PlGF (264-PGB) and a neutralizing antibody against human EGF (MAB236) were purchased from R&D Systems (Minneapolis, MN, USA). A neutralizing antibody against human EGF-R (clone LA1) was obtained from Millipore (Burlington, MA, USA). A neutralizing antibody against human VEGFR-1 (Mab0702) was from Novus Biologicals (Oakville, Canada). For ﬂow cytometric analysis, PE-conjugated antibodies against human VEGFR-1 (clone 49560) and against human VEGFR-2 (clone 89106) were purchased from R&D Systems (Minneapolis, MN, USA). For immunoprecipitation analysis, a rabbit polyclonal anti-human VEGFR-1 antibody (C-17) and a mouse monoclonal anti-human EGF-R antibody (528) were from Santa Cruz Biotechnology (Dallas, TX, USA). A rabbit monoclonal anti-phospho-tyrosine antibody (P-Tyr-100) was from Cell Signaling Technology (Danvers, MA, USA). For Westernblot analysis, a rabbit monoclonal anti-human EGF-R antibody (D38B1), a rabbit monoclonal anti-human phospho-EGF-R antibody (D7A5) and a rabbit monoclonal anti-human VEGFR-2 antibody (D5B1) were from Cell Signaling Technology (Danvers, MA, USA). A rabbit monoclonal anti-human VEGFR-1 antibody (Y103) was purchased from Abcam (Cambridge, UK). A rabbit polyclonal anti-phospho-VEGFR-1 (Tyr1213) antibody was purchased from Merck (Darmstadt, Germany). A mouse monoclonal anti-human β-actin antibody (clone AC-74) was from Sigma Aldrich (St. Louis, MO, USA). For immunoﬂuorescence cell staining analysis, an anti-EGF-R antibody conjugated with VioBright FITC was from Miltenyi Biotech (Bergisch Gladbach, Germany).

4.2. Cell Culture and Treatment Human colon cancer cell line (HCT116) were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI1640 medium with 10% fetal bovine serum and antibiotics. For VEGFR-1 or EGF-R activation, 60% confluent cells were treated with control BSA (10 ng/mL), VEGF-A (2 ng/mL), PlGF (10 ng/mL) or EGF (10 ng/mL) for 0.5–48 h before RNA or protein lysates were harvested for further analyses. For VEGFR-1 inhibition, 70%–80% confluent cells were treated with the VEGFR-1 inhibitors; an anti-human VEGFR-1 antibody (2.5 µg/mL) and a VEGFR-1 antagonistic peptide (75 µM). For EGF-R inhibition, 70%–80% confluent cells were treated with the EGF-R inhibitors; an anti-EGF antibody (0.5 µg/mL) and an anti-EGF-R antibody (2.5 µg/mL), or non-immune control IgG (2.5 µg/mL) for 0.5–48 h before RNA or protein lysates were harvested for further analyses.

4.3. Cell Proliferation Assay For proliferation analysis, after serum starvation for 18 h, 50% confluent cells were incubated with the respective inhibitors or ligands for 24–48 h in 0.1% fetal bovine serum (FCS) containing medium. Then, cells were pulsed with 10 µM EdU for 2 h, then fixed with 4% paraformaldehyde. The EdU incorporation was detected using the Click-iT Plus EdU Alexa Fluor 488 Assay Kit, according to the manufacture’s protocol (Thermo Fisher Scientific, Waltham, MA, USA). EdU+ cells from eight to ten randomly chosen fields of at least three independent samples were counted.

4.4. Cycloheximide Chase Assay Cells were pre-treated with control (BSA) or VEGFR-1 ligands (VEGF-A and PlGF) for 1 h, then they were treated with EGF and cycloheximide (25 µg/mL) for the several times. Whole cell lysates were collected and analyzed Western blotting.

4.5. Western Blot Analysis Western immunoblotting was performed as described previously [18]. Total cell lysates were prepared using a lysis buffer containing 100 mM Tris-HCl (pH 6.8), 300 mM NaCl, 2 mM EDTA and 4% (v/v) SDS. Protein concentrations were determined with Protein assay BCA kit (Nakarai tesque, Kyoto, Japan). Western immunoblotting was performed using a rabbit monoclonal anti-human EGF-R antibody at a 1/5000 dilution, a rabbit monoclonal anti-human phospho-EGF-R antibody at a 1/1000 dilution, a rabbit monoclonal anti-human VEGFR-1 antibody at a 1/1000 dilution, a rabbit Int. J. Mol. Sci. 2019, 20, 5608 14 of 17 polyclonal anti-phospho-VEGFR-1 (Tyr1213) antibody at a 1/5000 dilution, a mouse monoclonal anti-human β-actin antibody at a 1/10,000 dilution. The membranes were developed using ECL Western blot detection reagents (GE Healthcare Life Sciences, Little Chalfont, UK). All immunoblots were performed in triplicate. Immunoblots were imaged using a ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA). Quantification was performed with Image J software (NIH, Bethsda, MD, USA). The median pixel intensity quantified for each band was normalized to the loading control. The experimental intensity values of experimental were divided with loading control.

4.6. Immunofluorescence Cell Staining Cells were grown on a glass chamber slide and either was pre-treated with BSA, VEGFR-1 ligands (VEGF-A and PlGF) or VEGFR-1 blockers (anti-VEGFR-1 antibody and VEGFR-1 antagonist) for 1–4 h. Then, cells were blocking with an FcR Blocking Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min at 4 degrees, and an anti-EGF-R antibody conjugated with VioBright FITC was added in the medium for 30 min at 4 degrees. Then, cells were fixed by the addition of 4% paraforlmaldehyde to the culture medium for 15 min. Nuclei were counter stained with 40,6-diamidino-2-phenylindole (DAPI). Fluorescence images were obtained using a BZ-X710 fluorescence microscope (Keyence Corporation, Osaka, Japan). Magnifications used were 40 for fluorescence microscopy. × 4.7. siRNA and Transfection

The siRNAs targeting human EGF-R Mrna (50-CCAUAAAUGCUACGAAUAU-30) and human VEGFR-1 mRNA (50-CAAUCAUAGAUGUCCAAAU-30) were obtained from Thermo Fisher Scientiﬁc (Waltham, MA, USA). Stelth RNAi negative control siRNA (medium GC content, Invitrogen) was used as a control siRNA, which has no homology to human gene products. RNA knockdown analysis was performed as described previously [18]. In brief, cells were transfected with siRNA using Lipofectamine RNAiMax reagent for 24 h (Thermo Fisher Scientiﬁc, Waltham, MA, USA), according to the manufacturer’s instructions. Then cell lysates were prepared for immunoblot analysis.

4.8. Immunoprecipitation Analysis Immunoprecipitation analysis was performed as described previously [18]. In brief, cells were harvested in ice-cold lysis buffer (50 mM HEPES, pH 7.0, 150 mM NaCl, 10 mM EDTA, 1.5 mM MgCl2, 1% Nonidet P-40, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 0.5% sodium deoxycholate, 5 mg/mL aprotinin, 5 mg/mL leupeptin, 20 mM sodium fluoride and 20 mM sodium pyrophosphate), homogenized through a 27-gauge needle ten times each, then centrifuged at 16,000 rpm for 20 min at 4 degrees. Cell lysates were incubated with a × rabbit monoclonal anti-phospho-tyrosine antibody (P-Tyr-100; Cell Signaling Technology) overnight at 4 degrees with constant gentle rocking for immunoprecipitation of tyrosine phosphorylated proteins, followed by the addition of protein G magnet Dynabeads (Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at 4 degrees. For VEGFR-1/EGF-R co-immunoprecipitation analysis, cells were crosslinked with dithiobis(succinimidyl propionate (DSP) (Thermo Fisher Scientific, Waltham, MA, USA) before cell lysis. Five hundred µg of each clarified lysate was incubated with a rabbit polyclonal anti-human VEGFR-1 antibody (C-17; Santa Cruz Biotechnology) or a mouse monoclonal anti-human EGF-R antibody (sc-120; Santa Cruz Biotechnology) overnight at 4 degrees with constant gentle rocking, followed by the addition of protein G magnet Dynabeads (Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at 4 degrees. Immunoprecipitates were washed, eluted with SDS-PAGE sample buffer, and subjected to 4%–20% polyacrylamide gradient gels for SDS-PAGE. Immunoblot analysis was performed as described above. To exclude the detection of IgG for immunoprecipitation, the TidyBlot HRP conjugated Western blot detection reagent (1:200, Bio-Rad) was used instead of the secondary antibody. Int. J. Mol. Sci. 2019, 20, 5608 15 of 17

4.9. Quantitative RT-PCR Quantitative RT-PCR was performed as described previously [18]. The levels of transcript for human EGF-R and human GAPDH were measured by quantitative RT-PCR using the following specific primer sets: EGF-R, 50-TTCCTCCCAGTGCCTGAA-30(forward) and 50-GGGTTCAGAGGCTGATTGTG-30(reverse); GAPDH, 50-GCTAGGGACGGCCTGAAG-30 (forward) and 50-GCCCAATACGACCAAATCC-30 (reverse). Amplification and quantification of the PCR products were performed using the Applied Biosystems 7500 System (Applied Biosystems; Thermo Fisher Scientific, Waltham, MA, USA). Standards were run in the same plate and the relative standard curve method was used to calculate the relative mRNA expression. RNA amounts were normalized against the GAPDH mRNA levels.

4.10. Flow Cytometric Analysis Cells were harvested and blocked with an FcR Blocking Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min at 4 degrees. Then, cells were stained with a PE-conjugated antibody against VEGFR-1 (clone 49560) or VEGFR-2 (clone 89106) for 30 min at room temperature. For negative control staining, cells were stained with a PE-conjugated isotype control IgG (R&D Systems, Minneapolis, USA). After cells were washed with Flow Cytometry Staining Buffer, flow cytometric analysis was performed using BD FACSVerse flow cytometer (BD Biosciences, Franklin Lakes, San Jose, CA, USA).

4.11. Statistical Analysis Results are expressed as means S.D. Statistical analyses of data were done using ANOVA and ± the Scheﬀé’s test. p value < 0.01 was considered signiﬁcant.

Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/20/22/5608/s1. Author Contributions: H.N., C.T. and N.Y. performed the experiments and analyzed the data; S.T.-K. conceived and designed the experiments; H.N. and S.T.-K. wrote the paper. All authors have read and approved the final manuscript. Funding: This study was supported in part by grants from Grants-in-Aid for Scientific Research (grant no. 19H04034 to STK) from JSPS and the Princess Takamatsu Cancer Research Fund (grant no. 14-24611 to STK). Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Abbreviations

VEGF vascular endothelial growth factor PlGF placental growth factor VEGFR-1 vascular endothelial growth factor receptor-1 EGF epidermal growth factor EGF-R epidermal growth factor receptor

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