PI3K/Akt and MAPK/ERK1/2 signaling pathways are involved in IGF-1-induced VEGF-C upregulation in breast cancer

Abstract

Objective

To investigate the signaling pathways involved in insulin-like growth factor-1 (IGF-1)-induced vascular endothelial growth factor C (VEGF-C) up-regulation and lymphatic metastasis in MDA-MB-231 breast cancer cells.

Methods

MDA-MB-231 breast cancer cells were exposed to IGF-1 with various concentrations. The expression level of VEGF-C was assessed by real-time PCR and Western blot. Akt and ERK1/2 phosphorylation was detected by Western blot. Signaling transduction inhibitors, LY294002 and PD98059, were used to block PI3K/Akt and MAPK/ERK1/2 signaling pathways, respectively.

Results

IGF-1 increased the level of VEGF-C expression in a dose-dependent manner in MDA-MB-231 breast cancer cells. In addition, phosphorylation of Akt and ERK1/2 was enhanced by IGF-1. Remarkably, inhibition of Akt phosphorylation by LY294002 completely blocked the effects on IGF-1-induced VEGF-C up-regulation. Inhibition of ERK1/2 phosphorylation by PD98059 reduced IGF-1-induced VEGF-C expression.

Conclusion

This study identified that PI3K/Akt and MAPK/ERK1/2 signaling pathways were involved in IGF-1-induced VEGF-C up-regulation and suggested their important roles in lymphatic metastasis in breast cancer.

Introduction

Lymph node metastasis is the hallmark of breast cancer progression and is considered one of the most important prognostic factors. Recent studies have suggested that lymphangiogenesis (formation of new lymphatic vessels) plays an important role in this process (Qian et al. 2006). However, the molecular events that lead to lymphangiogenesis are poorly understood. There has been accumulating evidence showing that vascular endothelial growth factor C (VEGF-C) is the central regulator of lymphangiogenesis. Increased expression of VEGF-C in primary tumors correlates with increased dissemination of tumor cells to regional lymph nodes in a variety of human carcinomas (Stacker et al. 2002; McColl et al. 2003). Cells over-expressing VEGF-C are implanted into transgenic mice-induced tumor-associated lymphangiogenesis in several orthotopic mouse models (Karpanen et al. 2006; Skobe et al. 2001). Anti-VEGF-C treatment inhibits VEGF-C-induced lymphatic hyperplasia and tumor cells delivery to draining lymph nodes (Hoshida et al. 2006).

Insulin-like growth factor-1 (IGF-1) is expressed in humans throughout the lifespan in multiple tissues. There is strong evidence that alteration of IGF-1 plays an important role in breast cancer (Samani et al. 2007). Pooled individual data analysis of 17 prospective studies found that circulating IGF-1 is positively associated with breast cancer risk (Endogenous Hormones Breast Cancer Collaborative Group et al. 2010). Immunohistochemical staining has revealed that the IGF-1 receptor is overexpressed in nearly half of all breast cancers (Pacher et al. 2007). Preclinical studies in cell culture and animal models suggest that dysregulation of the IGF-1 signaling pathway promotes the transformation, survival, growth and metastasis of breast cancer cells (Mester and Redeuilh 2008). However, the mechanisms by which IGF-1 mediates cancer metastasis are poorly understood.

In a recent study, IGF-1 was found to have the ability to induce and promote lymphangiogenesis as detected with LYVE-1 (Mester and Redeuilh 2008). In another study, stimulation of IGF-1 was found to promote the tumor growth and lymphatic metastasis through the induction of VEGF-C (Björndahl et al. 2005). Our previous study also showed that increased VEGF-C expression was closely related to lymphangiogenesis in breast cancer invasion and lymphatic metastasis (Gu et al. 2008). Therefore, in this study, we assessed the relationship of IGF-1 induction and VEGF-C expression and investigated the signaling pathways that involved in IGF-1-induced VEGF-C up-regulation and lymphatic metastasis in MDA-MB-231 breast cancer cells.

Materials and methods

Reagents and cells

Human breast cancer MDA-MB-231 cells were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). IGF-I was purchased from PeproTech EC LTD (London, UK). Signal transduction molecule inhibitor 2-(4-morpholinyl)-8-phenyl-chromone (LY294002) and 2′-amino-3′-methoxyflavone (PD98059) were purchased from Sigma Chemical Co. (Louis, MO). The inhibitors LY294002 and PD98059 were dissolved in dimethyl sulfoxide (DMSO). MDA-MB-231 cells were cultured in RPMI 1640 (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum medium (FBS) (Invitrogen, Carlsbad, CA, USA); 70–90% confluent MDA-MB-231 cells were harvested and grown on 12-well plates at a density of 1 × 10⁵ cells overnight to allow attachment. Cells were cultured in serum-free medium for a further 24 h before treating various doses of IGF-1 with or without the inhibitors, LY294002 and PD98059.

Real-time qRT-PCR

Real-time RT-PCR was performed to assess the mRNA level of VEGF-C in MDA-MB-231 breast cancer cells. Untreated cell served as a negative control. Total RNA was extracted from the cells using the RNeasy RNA Isolation Kit (Qiagen, Hilden, Germany) according to manufacturer instructions. One microgram of total RNA was reverse transcribed into cDNA with the high-capacity cDNA archive kit (Applied Biosystems, Foster City, USA). VEGF-C primers were designed on the basis of the nucleotide sequence of the human VEGF-C gene (GenBank Accession, NM_005429). The cDNA was amplified using the SYBR PrimeScript RT-PCR kit II (Takara, Dalian, China) and performed with the Rotor-Gene 3000 (Corbetty Research, Australia) according to manufacturer instructions. β-Actin was used as the internal control for RNA input and reverse transcription efficiency. Real-time PCR primers were listed as follows: VEGF-C forward primer: 5′-AGCTCGGATGTCCGGTTTC-3′; VEGF-C reverse primer: 5′-GCGTTCCCAACTTTGCAG-3′; β-actin forward primer: 5′-ATTGCCGACAGGATGCAGA-3′; β-actin reverse primer: 5′-GAGTACTTCGCTCAGGAGGA-3′. The PCR profile consisted of (1) 95°C for 5 min; (2) 40 cycles of 95°C for 15 s, 60°C for 15 s and 72°C for 20 s; (3) 72°C for 10 min. PCRs were done in triplicate for both the target gene and internal control. The cycle software was used for quantification of VEGF-C mRNA level relative to β-actin mRNA expression.

Western blot analysis

Western blot was performed to assess the protein level of VEGF-C and the expression and phosphorylation of signal transduction molecules Akt and ERK1/2. Untreated cells served as a negative control, and β-actin was used as controlled for equal loading as an internal loading control. Briefly, cells were harvested in the presence of protease inhibitor cocktail (Sigma-Aldrich, St Louis, USA) in RIPA lysis buffer(Beyotime, Jiangsu, China). Soluble proteins (40 μg) were subjected to SDS–PAGE and transferred electronically to PVDF membranes. Nonspecific binding was blocked with 5% nonfat milk in TBST [50 mM Tris (pH 7.5), 0.9% NaCl2, and 0.1% Tween-20] at room temperature (RT) for 1 h. Membranes were then incubated with anti-VEGF-C (1:1,000; sc-1881, Santa Cruz Biotechnology, CA, USA), anti-AKT (1:1,000; #9272; Cell Signaling Technology, Danvers, MA, USA), anti-pAKT (1:1,000; #9217 Cell Signaling Technology, Danvers, MA, USA), anti-ERK1/2 (1:1,000; #4695; Cell Signaling Technology, Danvers, MA, USA) and anti-pERK1/2 (1:1,000; #4376; Cell Signaling Technology, Danvers, MA, USA) at RT for 1 h. After 5 min washes with TBST, the membranes were incubated with Anti-horse/rabbit or anti-goat/rabbit immunoglobulin G (IgG)-horse radish peroxidase (HRP) (Dingguo Changsheng Biotech, Beijing, China) for 1 h. The membranes were washed four times for 5 min each with TBST. Bound antibody chemiluminescence was detected using chemiluminescence kits (Thermo Scientific, Germany). The optical density was determined using a scanning densitometer and analyzed using Quantity One software (Bio-Rad). β-Actin was used as the internal control.

Statistical analysis

Data were presented as mean ± standard deviations (SD) of at least three independent experiments with three or more replicates and analyzed by two-tailed Student’s t test, with P < 0.05 considered significant.

Results

IGF-1 significantly increased VEGF-C expression in MDA-MB-231 breast cancer cells

To determine the effects of IGF-1 on VEGF-C expression, MDA-MB-231 cells were incubated with various doses of IGF-1 (200, 400 and 600 ng/ml) for 24-h at 37°C. Cells without IGF-1 treatment served as the negative control. VEGF-C mRNA expression was assessed by real-time qRT-PCR, and protein expression was assessed by Western blot. IGF-1 increased VEGF-C mRNA levels in a dose-dependent manner (from 20 to 400 ng/ml) with maximal effect observed with 400 ng/ml IGF-1 (Fig. 1). This increase in the level of mRNA of VEGF-C was blocked when IGF-1 concentration was over 400 ng/ml, although still higher than that in control, P < 0.01. Consistently, IGF-1 increased the expression of VEGF-C in MDA-MB-231, P < 0.05 (Fig. 2). This effect was dose dependent and was attenuated when IGF-1 concentration was over 400 ng/ml. These data clearly demonstrated that IGF-1 stimulated VEGF-C expression both at mRNA and at protein levels in a dose-dependent manner.

Fig. 1.

IGF-1 increased VEGF-C mRNA expression measured by real-time RT-PCR in a dose-dependent manner in MDA-MB-231 cells. ^★ P < 0.05 versus control, ^# P < 0.01 versus control

Fig. 2.

IGF-1 increased VEGF-C protein level measured by Western blot in a dose-dependent manner in MDA-MB-231 cells. a Representative Western blot of the experiments. b Relative level of VEGF-C protein expression. Data are expressed as the ratio of band intensity for VEGF-C to β-actin ^★ P < 0.05 versus control, ^# P < 0.01 versus control

IGF-1 significantly promoted the phosphorylation of signal transduction molecules Akt and ERK1/2

Next, we tried to identify the signaling pathways that regulate IGF-1-induced VEGF-C up-regulation. We assessed Akt and ERK1/2 activity in MDA-MB-231 cells. Cells were incubated with IGF-1 (200, 400 and 600 ng/ml) for 24 h. Expression and phosphorylation levels of Akt and ERK1/2 were detected by Western blot. We found that although IGF-1 did not affect Akt and ERK1/2 expression, IGF-1 (200, 400 and 600 ng/ml) induction increased the phosphorylation levels of Akt and ERK1/2 in MDA-MB-231 breast cancer cells P < 0.01. Moreover, ERK1/2 phosphorylation was increased in a dose-dependent manner with increasing IGF-1 concentrations P > 0.05 (Fig. 3). In contrast, Akt phosphorylation was significantly up-regulated by IGF-1 (200 ng/ml), P < 0.01 but not increased further with increasing IGF-1 concentration. These data demonstrated that IGF-1 stimulated both PI3K/Akt and MAPK/ERK1/2 signal transduction pathways in MDA-MB-231 breast cancer cells. In addition, MDA-MB-231 breast cancer cells were pretreated with Akt inhibitor LY294002 (20 μM) or ERK1/2 inhibitor PD98059 (20 μM) for half an hour prior to IGF-1 (400 ng/ml) induction. Phosphorylation of AKT and ERK1/2 was assessed 48 h after IGF induction. IGF-1 completely blocked IGF-1-induced AKT or ERK1/2 phosphorylation in MDA-MB-231 breast cancer cells (Fig. 4). There were no significant effects of LY294002 and PD98059 on Akt and ERK1/2 protein expression, P > 0.05. These results suggested that IGF-1 activates both P13K/Akt and ERK1/2 pathways in MDA-MB-231 breast cancer cells and could be inhibited by Akt and ERK1/2 inhibitors.

Fig. 3.

IGF-1 increased phosphorylation of signal transduction molecules Akt and ERK1/2 measured by Western blotting analysis. a Representative Western blot of the experiments. b Quantification of three or four experiments from a. ^★ P < 0.05 versus control, ^# P < 0.01 versus control

Fig. 4.

Akt inhibitor LY294002 and ERK1/2 inhibitor PD98059 decreased phosphorylation of AKT and ERK in MDA-MB-231 cells. a Representative Western blot of the experiments. b Quantification of three or four experiments from a. ^★ P < 0.05 versus control, ^# P < 0.01 versus control

Akt inhibitor LY294002 decreased IGF-I-induced expression of VEGF-C

To determine whether the activated P13K/Akt and MAPK/ERK1/2 signaling pathways were involved in IGF-1-induced VEGF-C expression, MDA-MB-231 breast cancer cells were pretreated with Akt inhibitor LY294002 or ERK1/2 inhibitor PD98059 for half an hour prior before induced with 400 ng/ml IGF-1. VEGF-C expression was assessed 48-h after IGF induction by real-time qRT-PCR and Western blot. As previously observed, IGF-1 increased both VEGF-C mRNA and protein levels in MDA-MB-231 breast cancer cells, P < 0.05. Remarkably, Akt inhibitor LY294002 almost completely blocked this effect (Figs. 5, 6 P < 0.05). Similarly, ERK1/2 inhibitor PD98059 significantly reduced IGF-1-induced VEGF-C up-regulation. Interesting, without IGF-1 treatment, Akt inhibitor LY294002 significantly reduced VEGF-C expression. In contrast, ERK1/2 inhibitor PD98059 did not affect VEGF-C expression. These data suggested that both P13K/Akt and MAPK/ERK1/2 signaling pathways were involved in IGF-1-induced VEGF-C expression in MDA-MB-231 breast cancer cells. In addition, these data also suggested that P13K/Akt signaling pathway was also responsible for the baseline expression of VEGF-C (without IGF-1 induction).

Fig. 5.

LY294002 decreased VEGF-C mRNA expression measured by real-time RT-PCR in MDA-MB-231 cells. ^# P < 0.01 versus control, ^▲ P < 0.01 versus IGF-1 group

Fig. 6.

LY294002 significantly inhibited IGF-1-induced VEGF-C protein expression. a Representative Western blot of the experiments. b Quantification of three or four experiments from a. ^★ P < 0.05 versus control, ^# P < 0.01 versus control, ^● P < 0.05 versus IGF-1 group, ^▲ P < 0.01 versus IGF-1 group

Discussion

In the present study, we demonstrated that IGF-1 up-regulated VEGF-C expression through P13K/Akt and MAPK/ERK1/2 signaling pathways in MDA-MB-231 breast cancer cells. In addition, we recently reported that VEGF-C played an important role in lymphangiogenesis. Increased VEGF-C was significantly associated with lymph node metastasis, high TNM stage, and poor outcome in breast carcinoma patients¹². These data suggest that IGF-1-PI3K/Akt-VEGF-C and IGF-MAPK/ERK1/2-VEGF-C signaling pathways play important roles in lymphangiogenesis in breast cancer.

IGF-I signaling pathways and functions are mediated through the activities of a complex molecular network (Riedemann and Macaulay 2006). Under physiological conditions, the balance between the expression and activities of these molecules is tightly controlled. Changes in this delicate balance may trigger a cascade of molecular events that ultimately lead to malignancy (Samani et al. 2007). Accumulating evidences have demonstrated IGF-1, and its receptor IGF-1R provides a potent proliferative signaling system that stimulates growth in many different cell types and blocks apoptosis (Butt et al. 1999). Recently, IGF-1 was shown to induce lymphangiogenesis in vivo (Björndahl et al. 2005). IGF-1 signaling pathways appear to be much more complicated than previously recognized. VEGF-C is a crucial and potent promoter of lymphangiogenesis that functions under physiological and pathological conditions (McColl et al. 2003; Stacker et al. 2007). Over-expressing of VEGF-C in transgenic mice induces lymphatic vessel hyperplasia (Enholm et al. 2001). Increased lymph node metastases are correlated with increased expression of VEGF-D and VEGF-C/VEGFR3 by immunohistochemistry in invasive breast cancer (Giorgetti et al. 1993). Therefore, we hypothesized that IGF-1 might induce breast cancer lymphangiogenesis via up-regulation of VEGF-C levels. We found that IGF-1 increased VEGF-C expression in a dose-dependent manner, thereby promoting cancer lymphangiogenesis and lymphatic metastasis. Interestingly, this relationship was attenuated when the concentration of IGF-1 reached 400 ng/ml, indicating the maximal binding capacity of the receptor. It was previously reported that over dose of IGF-1 would lead to IGF-1R internalization. This could explain why the expression of VEGF-C decreased when the dosage of IGF-1 was over 400 ng/ml.

In many circumstances, upon IGF-1 binding, the intrinsic tyrosine kinase of the IGF-1R is activated which activates phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathway leading to activation of several downstream substrates (Enholm et al. 2001; Giorgetti et al. 1993; Vuori and Ruoslahti 1994; Hermanto et al. 2000). In this study, we identified two signaling pathways downstream of IGF-1, the P13K/Akt and MAPK/ERK1/2 signaling pathways, and were activated and contributed to IGF-1-induced VEGF-C up-regulation. IGF-1 induction leaded to increase in the Akt and ERK1/2 phosphorylation without affecting their protein levels. These effects could be almost completely blocked by AKT inhibitor LY294002 and reduced by ERK1/2 inhibitor PD98059. Interesting, in our model, in absence of IGF-1 induction, Akt inhibition reduced VEGF-C expression. In contrast, ERK1/2 inhibition has no effect on the baseline of VEGF-C expression. In conclusion, we identified both P13K/Akt and MAPK/ERK1/2 signaling pathways were involved in IGF-1-induced VEGF-C up-regulation and suggested their important roles in lymphatic metastasis in breast cancer. Further studies are required to investigate the detailed signaling events of these two signaling pathways in regulation of lymphangiogenesis in breast cancer.