- Email: [email protected]
LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2
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Research Report
LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2 Baohui Liua, Zhentao Guoa, Huimin Donga, Daofeng Tiana, Qiang Caia, Baowei Jia, Shenqi Zhanga, Liquan Wua, Junmin Wanga, Long Wanga, Xiaonan Zhua, Yue Liub, Qianxue Chena,n a
Renmin Hospital, Wuhan University, 238 Jiefang Street, Wuhan, Hubei 430060, China Shiyan Taihe Hospital, Hubei University of Medicine, 32 South Renmin Street, Shiyan, Hubei 442000, China
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art i cle i nfo
ab st rac t
Article history:
In our previous study, we have found that leucine-rich repeats and immunoglobulin-like
Accepted 12 March 2015
domains 1(LRIG1) can improve the chemosensitivity in U251 cells whereas the role of LRIG1 in multidrug resistance (MDR) remains unknown. Here, we reported that LRIG1 can reverse
Keywords:
MDR by inhibiting epidermal growth factor (EGF) receptor (EGFR) and secondary inhibiting
Leucine-rich repeats and
ATP-binding cassette, sub-family B member 1(ABCB1) and ATP-binding cassette, sub-
immunoglobulin-like domains 1
family G (WHITE), member 2 (ABCG2). Our data showed that the expression of LRIG1 was
(LRIG1)
significantly higher in O6-methylguanine DNA methyltransferase (MGMT) Promoter
Multidrug resistance (MDR)
Methylation positive glioblastoma tissues compared to MGMT Promoter Methylation
Epidermal growth factor receptor
negative glioblastoma tissues. In addition, we found that LRIG1 expression was signifi-
(EGFR)
cantly decreased in MDR cells U251/TMZ compared to U251cells. Our results demonstrated
ATP-binding cassette
that over-expression of LRIG1 can reverse the MDR. The expression of ABCB1 and ABCG2
Sub-family B member 1(ABCB1)
were markedly suppressed when LRIG1 was over-expressed, supporting the negative
ATP-binding cassette
relationship between LRIG1 level and ABCB1 and ABCG2 level in human specimen.
Sub-family G (WHITE)
Furthermore, we found that LRIG1 downregulated ABCB1 and ABCG2 through suppressing
Member 2 (ABCG2)
EGFR expression. In case of EGFR knockdown, the effect of LRIG1 on regulating MDR, ABCB1 and ABCG2 was partially compromised. Our results, for the first time, showed that LRIG1 can reverse MDR in glioblastoma, by negatively regulating EGFR and secondary suppressing the levels of ABCB1 and ABCG2. & 2015 Published by Elsevier B.V.
1.
Introduction
Glioblastoma multiforme (GBM) is the most common and lethal type of primary brain tumor (Kotliarova and Fine, 2012). Even treated aggressively by surgery, radiation, and chemotherapy, the survival of patients with GBM still remains
not more than 14 months (Nikaki et al., 2012). Since the introdution of temozolomide (TMZ), which has demonstrated promising activity against glioblastoma, as chemotherapeutic agent in clinic setting, chemotherapy has become more and more important. However, the clinical response to TMZ lasts only a few months and drug resistance subsequently
n
Corresponding author. E-mail address: [email protected] (Q. Chen).
http://dx.doi.org/10.1016/j.brainres.2015.03.023 0006-8993/& 2015 Published by Elsevier B.V.
Please cite this article as: Liu, B., et al., LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.03.023
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develops in most cases (Spinelli et al., 2012), especially multidrug resistance (MDR), which has become the major the problem needed to be solved as soon as possible. Unfortunately, the mechanism of MDR was still unclear. LRIG1 was an inhibitor of receptors tyrosine kinase, which had cDNA cloned and characterized in 1996 (Suzuki et al., 1996). The LRIG1 integral membrane protein has been demonstrated to regulate various oncogenic receptor tyrosine kinases, including EGFR, by cell-autonomous mechanisms (Yi et al., 2011). Previous studies have also showed that upregulation of LRIG1 suppresses malignant glioma cell growth by attenuating EGFR activity (Yi et al., 2011) and LRIG1 negatively regulates the oncogenic EGF receptor mutant EGFRvIII, which is the most common EGFR mutant observed in GBM (Stutz et al., 2008). Recently, the LRIG1 isoform has been linked to aggressive behaviors and drug resistance in esophageal carcinoma cell lines (Wu et al., 2012). Furthermore, our previous study identified LRIG1 as a possible candidate gene affecting the sensitivity of human glioblastoma to the TMZ (Liu et al., 2013). Recent studies found that octreotide enhances the sensitivity of the SKOV3/DDP ovarian cancer cell line to cisplatin chemotherapy in vitro by inhibiting EGFR (Shen et al., 2011), and more important, GW583340 and GW2974, two human EGFR and HER-2 inhibitors, reverse ABCG2- and ABCB1-mediated drug resistance (Sodani et al., 2012). Therefore, LRIG1 may reverse the MDR, but this still remains to be elucidated. We hypothesized that LRIG1 is involved in the MDR of GBM and that can reverse MDR through suppressing the expression of EGFR and then suppressing ABCB1 and ABCG2. To test this hypothesis, we manipulated expression of LRIG1 in MGMT Promoter Methylation positive and negative patient samples, and examined whether LRIG1 can reverse MDR in MDR cells as well as the effect of LRIG1 on EGFR, ABCB1 and ABCG2. LRIG1 attracted our attention because previous studies had shown that the expression of LRIG proteins played an important role in the pathogenesis of astrocytic tumors (Yi et al., 2009). In this study, we used U251 and U87 cells as research models, which are the most frequent human primary brain tumors and represent the most malignant stage of GBM progression.
2.
Results
2.1. LRIG1 protein and mRNA level were upregulated in MGMT Promoter Methylation positive glioblastoma and downregulated in MDR cell line U251/TMZ cells To explore the relationship of LRIG1 expression and glioblastoma MDR, we assessed LRIG1 mRNA and protein expression in a series of primary human glioblastoma by real-time PCR and western blot (Fig. 1A and B). As shown in Fig. 1A and B, all 17 MGMT Promoter Methylation positive primary human glioblastoma and 17 MGMT Promoter Methylation negative primary human glioblastoma showed LRIG1 mRNA and protein expression. In addition, we found that patients with MGMT Promoter Methylation positive had significantly higher LRIG1 expression compared to the patients with MGMT Promoter Methylation negative.
2.2.
LRIG1 knockdown abrogated MDR in U251/TMZ cells
To determine the role of LRIG1 in MDR development, we knocked down LRIG1 in U251 cells using siLRIG1. The downregulation of target protein was confirmed via western blot analysis (Fig. 2A). Article has reported that caspase 3 is activated in the apoptotic cell and has been widely used as readout for drug sensitivity (Yan et al., 2015). Our data showed that the expression of caspase 3 was increased (Fig. 2A) and the apoptosis rate of cells was significantly decreased when treated with TMZ (16 mg/ml) and etoposide (VP-16) (10 mg/ml) (Fig. 2E), suggesting that suppressing LRIG1 expression resulted in reversal of MDR. To solid this data, LRIG1 plasmid was also transfected into U251/TMZ cells which was established by us before (Liu et al., 2013). The upregulation of target protein was also confirmed via western blot analysis (Fig. 2B). In line with the results in U251 cells, our data showed that drug resistance to TMZ and VP-16 was abolished by upexpressed LRIG1 (Fig. 2B and F).
2.3.
LRIG1 inhibited ABCB1 and ABCG2
In order to explore the underlying mechanisms of how LRIG1 reversed MDR, LRIG1 was over expressed in U251/TMZ cells and the expression levels of ABCB1, LRP, ABCG2 and MRP1, which was related to MDR (Liu et al., 2013), was determined by western blot and real-time PCR. Our data showed that LRIG1 overexpression did not result in change in levels of LRP and MRP1, but remarkably decreased ABCB1 and ABCG2 expression by more than 50% at both mRNA and protein level (there was a significant difference between expression levels of ABCB1 and ABCG2 between control and LRIGI overexpressing groups (Po0.05)) (Fig. 3A and B). In addition, we found that LRIG1 expression was significantly negatively correlated with ABCB1 (Pearson, R2 ¼0.204, P ¼0.0037) and ABCG2 (Pearson, R2 ¼ 0.301, P¼ 0.0004) expression in human samples (Fig. 4C and D).
2.4.
LRIG1 suppressed EGFR level
Previous studies have pointed out that EGFR inhibition affected ABCG2 expression in EGFR-positive MDCK BCRP cells via the PI3K/Akt signaling pathway (Pick and Wiese, 2012) and ABCB1 expression in acute lymphoblastic leukemia cells through MAPK/ERK pathway (Tomiyasu et al., 2013). We have found that LRIG1 can reverse MDR and repress ABCG2 and ABCB1 expressions in U251/TMZ cells. Therefore, we conducted experiments to test whether LRIG1 regulate ABCG2 and ABCB1 by inhibiting EGFR activity and secondary inhibition of PI3K/Akt signaling pathway and MAPK/ERK pathway in human MDR cells. First, we test the effect of LRIG1 on EGFR, p-AKT and p-ERK expression level. As expected, forced overexpression of LRIG1 in U251/TMZ cells significantly inhibited EGFR and p-AKT and p-ERK protein expression (Fig. 4A and B). To solid this data, the expression levels of Q2 LRIG1 and EGFR in human glioblastoma were analyzed by western blot. The results also showed that LRIG1 expression was significantly negatively correlated with EGFR levels
Please cite this article as: Liu, B., et al., LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.03.023
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content induced by LRIG1 might not occur at the transcriptional level in the MDR cells. Next, we test whether LRIG1 downregulated ABCB1 and ABCG2 through inhibiting EGFR.
p=0.00578
10
p=0.00398
Relative LRIG1 protein level
Relative LRIG1 mRNA level
(Pearson, R2 ¼ 0.3299, P¼ 0.00027). However, we found that the mRNA level of EGFR was not changed in case of LRIG1 overexpression, indicating that the decrease in EGFR protein
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siCtrl siLRIG1
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GFP GFP-LRIG1
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siCtrl
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Fig. 1 – LRIG1 is upregulated in MGMT Promoter Methylation positive patient samples. (A) The expression level of LRIG1 mRNA on 17 MGMT promotor Methylation (MM) postive and 17 MGMT Promotor Methylation negative glioblastoma tissues. P-value was calculated via the mann-Whitney's test, O6-methylguanine DNA methyltransferase (MGMT). (B) The expression level of LRIG1 protein on 17 MGMT Promotor Methylation (MM) postive and 17 MGMT Promotor Methylation negative glioblastoma tissues. P-value was calculated via the mann-Whitney's test. (C) The protein expression level of LRIG1 in U251/ TMZ and U251 cells. nPo0.05 vs. U251 cells. (D) The protein expression level of LRIG1 in U251/TMZ and U251 cells.
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Fig. 2 – The effect of LRIG1 in MDR. (A) The protein expression of LRIG1 and caspase 3 when siLRIG1 and siCtrl were transfected into U251 cells. (B) The protein expression of LRIG1 and caspase 3 when plasmid of GFP and GFP-LRIG1 was transfected into U251/TMZ cells. (C) The mRNA expression of LRIG1 when siLRIG1 and siCtrl were transfected into U251 cells. (D) The mRNA expression of LRIG1 when plasmid of GFP and GFP-LRIG1 was transfected into U251/TMZ cells. (E) The apoptosis rate of U251 cells which were treated by TMZ (16 μg/ml) or VP-16(10 μg/ml), and indicated siRNA for 72 h. nPo0.05 vs. cells transfected with siCtrl. (F) The apoptosis rate of U251/TMZ cells which were treated by TMZ (16 μg/ml) or VP-16(10 μg/ml), and indicated plasmid for 72 h. nPo0.05 vs. cells transfected with GFP plasmid.
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2.5. EGFR knockdown can reverse MDR of U251/TMZ cells and inhibit ABCB1, ABCG2 expression
mRNA expression level
We knocked down EGFR in U251 cells and then knocked down LRIG1 by siRNA, our data showed that the effects of siLRIG1 on ABCB1 and ABCG2 expression were attenuated with EGFR knockdown (Fig. 6A). Collectively, our data showed that LRIG1 knockdown induced ABCB1 and ABCG2 downregulation via inhibition of EGFR.
ABCB1
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GAPDH
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Pearson Test R=-0.451 2
R = 0.2037
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ABCG2 protein expression level
ABCB1 protein expression level
It is well known that inhibition of EGFR can reverse ABCG2and ABCB1-mediated drug resistance in the KB-C2 cell line (Sodani et al., 2012), but whether inhibition of EGFR can
MRP1
LRIG1
P=0.0037˘0.05
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Pearson Test R=-0.-0.549 2
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R = 0.301 P=0.00038˘0.05
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LRIG1 protein expression level
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Flag Flag -Lrig1
LRIG1
140KD
EGFR
170KD
p-ERK1/2
42KD
p-AKT
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39KD
Flag Flag -Lrig1
EGFR protein expression level
Fig. 3 – LRIG1 over-expression induces ABCB1 and ABCG2 down-regulation. (A)The expression of LRIG1 and MDR related to gene when LRIG1 plasmid was transfected to U251/TMZ cells. Results are normalized relative to the amount of GAPDH mRNA and are plotted by the amount relative to Flag U251/TMZ cells sample. nPo0.05 vs. cells transfected with Flag plasmid. (B)The expression of LRIG1 and MDR related to protein when plasmid of Flag-LRIG1 was transfected into U251/TMZ cells. (C)LRIG1 and ABCB1 protein levels in glioblastoma and normal brain tissue were assessed using western blot. Correlation of the levels in 34 glioblastomas is shown. Statistical analysis was performed with Pearson's test. SE, standard error. GADPH, glyceraldehyde-3-phosphate dehydrogenase. (D) LRIG1 and ABCG2 protein levels in glioblastoma were assessed using western blot, correlation of the levels in 34 glioblastomas is shown. Statistical analysis was performed with Pearson's test.
EGFR mRNA expression level
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Pearson Test R=-0.-0.574 2
R = 0.3299
3
P=0.00019˘0.05
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LRIG1 protein expression level
Fig. 4 – LRIG1 over-expression induces EGFR down-regulation. (A) The expression of EGFR gene was detected when LRIG1 plasmid was transfected to U251/TMZ cells. Results are normalized relative to the amount of GAPDH mRNA and are plotted by the amount relative to Flag-U251/TMZ cells sample. (B) The protein expression level of LRIG1, EGFR, p-AKT and p-ERK1/2 when plasmid of Flag or Flag-LRIG1 was transfected into U251/TMZ cells. (C) LRIG1 and EGFR protein levels in glioblastoma and normal brain tissue were assessed using immunoblotting. Correlation of the levels in 34 glioblastomas tissues is shown. Statistical analysis was performed with Pearson's test. SE, standard error. GADPH, glyceraldehyde-3-phosphate dehydrogenase. Statistical analysis was performed with Pearson's test. Please cite this article as: Liu, B., et al., LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.03.023
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the MGMT Promoter Methylation negative patient samples, the methylation state of the MGMT gene determined whether tumor cells would be responsive to TMZ; If the promoter was methylated, TMZ was more effective (Hegi et al., 2005). Therefore, LRIG1 may act as MDR suppressor and lower the Q3 expression of LRIG1 may give an advantage to glioblastoma cells and contribute to the development of MDR. Cancer cells have the ability to become resistant to multiple drugs by many mechanisms, such as increased efflux of drug, enzymatic deactivation, decreased permeability, altered binding-sites and alternate metabolic pathways. Because efflux is a significant contributor for MDR in cancer cells, current research is aimed at blocking specific efflux mechanisms (Modok et al., 2006). Mounting studies have found that some certain proteins were related to efflux mechanisms (Jennings and Iyengar, 2001; Stupp et al., 2007). In present study, we found that knocked down LRIG1 in U251 cells, the drug sensitivity to TMZ and VP-16 was decreased, suggesting that down regulation of LRIG1 may be one of causes of MDR (Lu and Shervington, 2008). Consistent with these results, overexpression of LRIG1 in MDR cells U251/TMZ led to the loss of MDR in vitro (Fig. 2B and F). Although no publication has been reported on an effect of LRIG1 in MDR as yet, our results were in line with other's studies which suggested a role of LRIG1 in improving chemosensitivity in U251 cells (Qi et al., 2013). Accumulating evidences have shown that the development of multidrug resistance (MDR) is associated with overexpression of ATP-binding cassette (ABC) drug efflux transporters, which can reduce the intracellular concentrations of cytotoxic drugs and their metabolites (Alla et al., 2012; Greaves et al., 2012; Herraez et al., 2012). Moreover, MDR may be reversed if the expression of the proteins were downregulated (Michalak and Wesolowska, 2012; Minami et al., 2012; Tian et al., 2012). Our data showed that, when LRIG1 was upregulated in U251/TMZ cells, the expression of ABCB1 and ABCG2 was suppressed at both mRNA and protein levels, indicating that the effect of LRIG1 may occur in an ABC superfamily-dependent manner and LRIG1 reversed MDR
2.6. The effect of LRIG1 reversing MDR was attenuated in case of EGFR knockdown Our before mentioned results have shown that forced overexpression of LRIG1 reversed GBM cell MDR (Fig. 2B and F) and repressed ABCB1 and ABCG2 expressions (Fig. 3A and B). LRIG1 can repress EGFR (Fig. 4B) and EGFR can repress MDR, ABCB1, and ABCG2 (Fig. 5B and C), but whether LRIG1 regulated MDR, ABCB1, and ABCG2 through inhibiting EGFR was still unknown, so EGFR and LRIG1were knocked down by siRNA in U251 cells. Our results showed that the effect of siLRIG1 on MDR was attenuated when EGFR was knocked down (Fig. 6C and D). Collectively, our data showed that LRIG1 knockdown induced resistance to drugs in U251 cells through inhibition of EGFR and secondary suppression ABCB1 and ABCG2.
3.
Discussion
Our data showed that, the expression of LRIG1 was significantly lower in MDR cell line compared to glioblastoma cells. More important, we found that lower expression of LRIG1 in
1.5 ABCB1
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reverse ABCG2- and ABCB1-mediated drug resistance in glioma MDR cells remain unclear. In this study, EGFR siRNAs was used to knock down EGFR and the suppression of EGFR expreesion was confirmed through western blot analysis (Fig. 5A and B). Cells transfected with siRNA was treated by TMZ (16 mg/ml) for 72 h and apoptosis rate was assessed by Annexin-V-FITC assay. The results showed that the expression levels of ABCG2 and ABCB1 as well as the cells apoptosis rate were decreased (Fig. 5B and C). Similar findings were obtained in VP-16 treating cells. To confirm that EGFR siRNAs did not decrease downstream LRIG1 activity by inhibiting LRIG1 transcription, the mRNA expression of LRIG1 was detected by real time-PCR in U251 cells transfected with EGFR siRNA or control siRNA (Fig. 5D). As demonstrated by our results, EGFR knockdown did not have marked effect on LRIG1 mRNA level.
EGFR mRNA expression level
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Fig. 5 – EGFR knock down reversed MDR in U251/TMZ cells. (A) The mRNA expression of EGFR when siEGFR was transfected to U251/TMZ cells. nPo0.05 vs. siCtrl group. (B) The protein expression of EGFR, ABCB1 and ABCG2 when siEGFR was transfected to U251/TMZ cells. (C)The apoptosis rate of the cells treated by TMZ(16 g/ml) or VP-16(10 g/ml) compared with indicated siRNAs, nPo0.05 relative to U251/TMZ cells. (D) The expression of LRIG1 when siEGFR was transfected to U251/TMZ cells. Results are normalized relative to the amount of GAPDH mRNA and are plotted by the amount relative to siCtrl group, P40.05 vs. siCtrl group. Please cite this article as: Liu, B., et al., LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.03.023
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may by inhibiting ABCB1 and/or ABCG2. In consistent with the in vitro results, LRIG1 downregulation significantly correlated with ABCB1 and ABCG2 upexpression in human sample. These results also supported a previous report showing that Gefitinib, which is also Tyrosine Kinase Inhibitor, can inhibit ABCB1 and ABCG2 in glioma (Carcaboso et al., 2010). The mechanism by which LRIG1 regulates ABCB1 and ABCG2 was unclear, our data showed for the first time that LRIG1 regulates ABCB1 and ABCG2 through inhibiting EGFR. When we silenced the EGFR by siRNA, the expression levels of both ABCB1 and ABCG2 were decreased. More important, the effect of LRIG1 on regulating ABCB1 and ABCG2 was attenuated with EGFR knockdown. Taken together, our results suggested that LRIG1 regulated ABCB1 and ABCG2 through regulating EGFR expression. The mechanism by which EGFR inhibits ABCB1 and ABCG2 was unclear. However, it recently has been pointed out that EGFR inhibition affected ABCG2 expression in EGFR-positive MDCK BCRP cells via the PI3K/Akt signaling pathway (Pick and Wiese, 2012). In addition, MAPK/ERK pathway, which is regulated by EGFR (Kotliarova and Fine, 2012), has also been reported to up-regulate ABCB1 expression in acute lymphoblastic leukemia cell lines (Tomiyasu et al., 2013). Our data also showed that LRIG1 can inhibit the activity of PI3K/Akt signaling pathway and MAPK/ERK pathway in U251 cells. Taken together, these above findings highlighted the idea that knockdown EGFR might inhibit ABCB1 and ABCG2 via inhibiting PI3K/Akt signaling pathway and MAPK/ERK pathway. In summary, we have demonstrated that LRIG1 was a molecule that can enhance the chemosensitivity of glioblastoma to chemotherapeutics through inhibition of EGFR and secondary inhibition of ABCB1 and ABCG2. When cancer cells retain expression of LRIG1, they suppress ABCB1 and ABCG2
via suppressing EGFR. On the other hand, once cancer cells present with compromised expression of LRIG1, upregualted ABCB1 and ABCG2 can lead to chemo-resistance. Because numerous compounds and biological agents have been evaluated as EGFR inhibitors, it is highly possible that some of such agents will show promising effecacy in the treatment and prevention of cancer grows.
4.
Experimental procedures
4.1.
Reagents
Chemicals were purchased from Sigma-Aldrich (Wuhan, China). Flag-LRIG1 plasmids were saved by our lab.
4.2.
Specimens and patients
Specimens were collected at Renmin Hospital, Wuhan University from 34 patients with glioblastoma (primary tumors) with an average age of 36 years (range 23–71 years). Samples were harvested from the tumors at the time of surgery and subsequently snap frozen before stored at 80 1C. The present study was approved by Ethics Committee of the Faculty of Medicine of Renmin Hospital, Wuhan University (Appprove number: [2012]LKSZ(010)H), and all clinical investigation have been performed according to the principles of Declaration of Helsinki. Informed consent was obtained from the patients and/or guardians. All the tumors were located in the cerebral hemispheres. All the patients experienced neurosurgical operation(s) with the aim of gross radical tumor research between January 2010 and 2013. Pathological diagnoses of all specimens were panel reviewed by an experienced pathologist (HHa) according to WHO criteria (2000) (Gonzales, 2001).
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Fig. 6 – The effect of siLRIG1 was lost partly when EGFR was knocked down. (A) The expression of LRIG1, EGFR, ABCB1 and ABCG2 when cells were transfected for 72 h with indicated siRNAs, GAPDH was used as a loading control. (B) U251 cells transfected with LRIG1-targeting siRNA or scramble siRNA were assessed in cells apoptosis rate in the presence of siEGFR or siCtrl, P-value was for comparison to the siCtrl and from the Student's t test. In this experiment, siCtrl and siEGFR group, which were transfected siCtrl or siEGFR, were set first and then each group was divided into two subgroups: siCtrl and siLRIG1, which were transfected siCtrl or siLRIG1. The apoptosis index of siEGFR and siCtrl were calculated from apoptosis rate of the subgroups by (siLRIG1-siCtrl)/siCtrl. All the cells were treated by TMZ. Please cite this article as: Liu, B., et al., LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.03.023
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cytometer with Cell Quest software (Becton Dickinson, Mountain View, CA).
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MDR
Fig. 7 – A schematic representation of LRIG1 as a MDR Inhibitor. LRIG1 reversed MDR in glioblastoma, which involved negative inhibition of EGFR and secondary inhibition of ABCB1 and ABCG2.
4.3. Cell culture and establishment of a cell line with acquired resistance to TMZ. The U251 and U87 cells were purchased from The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. The cells were cultured in DMEM medium supplemented with 10% FBS in a humidified incubator at 37 1C/5% CO2. Basal cell culture was maintained in T-75 flasks (Sigma-Aldrich, USA). Cells were trypsinized at 80–90% confluency, seeded into either 6well plates or 96-well plates (Costar, USA). TMZ resistant cell line U251/TMZ was established as we previously described (Liu et al., 2013).
4.4.
7
Evaluation of apoptotic cells by Annexin-V-FITC
Sensitivity of U251 and U251/TMZ cells to TMZ (16 mg/ml) and VP-16 (10 mg/ml), was assessed by Annexin-V-FITC assay. Briefly, for Annexin-V-FITC assay, exponentially growing cells were plated into 6 well plates (2 105 cells/well). After 24 h, cells were treated by serial dilutions of the drugs and incubated for 96 h, then the cells were harvested and washed with phosphate-buffered saline. Cells were then resuspended in 100 μl of Annexin-V binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4) and incubated with 5 μl of Annexin-V-FITC for 15 min at room temperature before counterstained with propidium iodide (final concentration 1 μg/ml). After the incubation period, the cells were diluted with 190 μl of Annexin-V binding buffer. Cells were analyzed by flow cytometry using a Becton Dickinson FACS can flow
Cell transfections
To overexpress LRIG1, U251/TMZ and U251 cells (50–80% confluency) were transfected with LRIG1 plasmid (1 mg/well, 6-well plate) using FUGENE HD transfection reagent (Roche) according to the manufacturer's instructions. For knockdown experiments, siRNAs targeting human LRIG1 and EGFR, and control siRNA were purchased from Thermo Scientific. According to the manufacturer's instructions, 30–50% confluent cells were transfected with siRNAs at a final concentration of 40 nM using Lipofectamine 2000 transfection reagent (Invitrogen).
4.6.
RNA extraction and real time PCR
Total RNA was extracted using the Total RNA Isolation System from Promega according to the manufacturer's instructions. The Access real time PCR System supplied by Promega was utilized for reverse transcription. Based on the information of the LRIG1, EGFR, ABCB1, ABCG2, LRP, and GAPDH gene sequences, six pairs of gene-specific primers were designed the fowllowing: LRIG1 sense 50 -ttgctgatgttgtttcgctg-30 and antisense 50 -tgatggtctgtcacggtcg-30 , EGFR sense 50 -ggacgacgtggtggatgccg-30 and antisense 50 -ggcgcctgtggggtctgagc-30 , ABCB1 sense50 -aacaacgcattgccatagctcgtg-30 , and antisense50 -agtctgcattctggatggtggaca-30 LRP sense50 -aggagagcagctggaacaag-30 , and antisense 50 -cttgttccagctgctctcct-30 ; ABCG2sense50 -cacctattggcctcaggaa-30 ,and antisense50 -cctgcttggaaggctctatg-30 ; and GAPDH (internal control) sense 50 -gagtcaacggatttggtcgt-30 and antisense 50 -ttgattttggagggatctcg-30 . Quantitative PCR was carried out in a reaction mixture containing 2.5 μl of cDNA, 12.5 μl of SYBR Green mix, 2.5 μl of plus solution, 2 μl of primers(5pmol/ μl),5.5 μl of ddH2O, and 2.5 μl of cDNA. Furthermore, qPCR was performed on an ABI Prism 7500 Sequence Detection System (Applied Biosystems) using Sybr Green qPCR kit (Toyobo Biologics, Japan). The cycling conditions were as follows: a denaturation step at 95 1Cfor 2 min, followed by 40 cycles at 95 1C for 15 s, 57 1C for 15 s, 72 1C for 45 s, and a final extension step at 72 1C for 10 min. Results are normalized relative to the amount of GADPH mRNA and plotted by the amount relative to the reference sample
4.7.
Western blotting
Following treatments or transfections, total protein extracts were prepared by incubating cells in lysis buffer (20 mM Tris– HCl, pH 7.5, 150 mM NaCl, and 1% TritonX-100 5 mM EDTA) for 30 min at 0 1 C. Samples were normalized for protein content assay. 30 μg of lysates were separated on 12% polyacrylamide gels and electro-transferred onto PVDF membranes. Membranes were blocked in 5% BSA in TBS-Tween, incubated with primary antibody (Santa Cruz; 1: 200 for LRIG1, EGFR, ABCB1, ABCG2, MRP1, LRP, caspase 3, p-AKT, p-ERK and GAPDH), followed by incubation in secondary antibody (Santa Cruz; 1: 3000 for LRIG1 EGFR, ABCB1, ABCG2,MRP1,LRP, caspase 3, pAKT, p-ERK and GAPDH). The signals were visualized using
Please cite this article as: Liu, B., et al., LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.03.023
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BeyoECL Plus chemiluminescent substrate (Beyotime, Shanghai, China) and captured on chemiluminescence-sensitive film.
4.8.
Statistical analysis
Data were expressed as means7standard errors of the means. Statistical analysis was performed with (SigmaPlot, version 11.0 and spss 13.0). Differences between means were assessed with the student's t test or mann-Whitney's test. In multiple comparisons, one-way analysis of variance (ANOVA) was used. Pearson's test was used to detect the correction of two groups and compare quantitative values of expressions (Fig. 7).
Funding This work was supported by the National Natural Science Foundation of China (Nos. 30973072, 81372683), the Fundamental Research Funds for the Central Universities (No. 201130202020001), and the Fundamental Research Funds for the Central Universities (No. T2011302008).
r e f e r n c e s
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Michalak, K., Wesolowska, O., 2012. Polyphenols counteract tumor cell chemoresistance conferred by multidrug resistance proteins. Anticancer Agents Med. Chem. 12, 880–890. Minami, T., et al., 2012. HER2 as therapeutic target for overcoming ATP-binding cassette transporter-mediated chemoresistance in small cell lung cancer. Mol. Cancer Ther. 11, 830–841. Modok, S., Mellor, H.R., Callaghan, R., 2006. Modulation of multidrug resistance efflux pump activity to overcome chemoresistance in cancer. Curr. Opin. Pharmacol. 6, 350–354. Nikaki, A., Piperi, C., Papavassiliou, A.G., 2012. Role of microRNAs in gliomagenesis: targeting miRNAs in glioblastoma multiforme therapy. Expert Opin. Investig. Drugs 21, 1475–1488. Pick, A., Wiese, M., 2012. Tyrosine kinase inhibitors influence ABCG2 expression in EGFR-positive MDCK BCRP cells via the PI3K/Akt signaling pathway. Chem. Med. Chem. 7, 650–662. Qi, X.C., et al., 2013. LRIG1 dictates the chemo-sensitivity of temozolomide (TMZ) in U251 glioblastoma cells via downregulation of EGFR/topoisomerase-2/Bcl-2. Biochem. Biophys. Res. Commun. 437, 565–572. Shen, Y., et al., 2011. Octreotide enhances the sensitivity of the SKOV3/DDP ovarian cancer cell line to cisplatin chemotherapy in vitro. Exp. Ther. Med. 2, 1171–1176. Sodani, K., et al., 2012. GW583340 and GW2974, human EGFR and HER-2 inhibitors, reverse ABCG2- and ABCB1-mediated drug resistance. Biochem. Pharmacol. 83, 1613–1622. Spinelli, G.P., et al., 2012. Chemotherapy and target therapy in the management of adult high- grade gliomas. Curr. Cancer Drug Targets. 12, 1016–1031. Stupp, R., et al., 2007. Chemoradiotherapy in malignant glioma: standard of care and future directions. J. Clin. Oncol. 25, 4127–4136. Stutz, M.A., et al., 2008. LRIG1 negatively regulates the oncogenic EGF receptor mutant EGFRvIII. Oncogene 27, 5741–5752. Suzuki, Y., et al., 1996. cDNA cloning of a novel membrane glycoprotein that is expressed specifically in glial cells in the mouse brain. LIG-1, a protein with leucine-rich repeats and immunoglobulin-like domains. J. Biol Chem. 271, 22522–22527. Tian, C., et al., 2012. Common variants in ABCB1, ABCC2 and ABCG2 genes and clinical outcomes among women with advanced stage ovarian cancer treated with platinum and taxane-based chemotherapy: a gynecologic oncology group study. Gynecol. Oncol. 124, 575–581. Tomiyasu, H., et al., 2013. Regulations of ABCB1 and ABCG2 expression through MAPK pathways in acute lymphoblastic leukemia cell lines. Anticancer Res. 33, 5317–5323. Wu, X., et al., 2012. Expression of EGFR and LRIG proteins in esophageal carcinoma with emphasis on patient survival and cellular chemosensitivity. Acta Oncol. 51, 69–76. Yan, L.H., et al., 2015. Overexpression of CDX2 in gastric cancer cells promotes the development of multidrug resistance. Am. J. Cancer Res. 5, 321–332. Yi, W., et al., 2009. Expression of leucine-rich repeats and immunoglobulin-like domains (LRIG) proteins in human ependymoma relates to tumor location, WHO grade, and patient age. Clin. Neuropathol. 28, 21–27. Yi, W., et al., 2011. Paracrine regulation of growth factor signaling by shed leucine-rich repeats and immunoglobulin-like domains 1. Exp. Cell Res. 317, 504–512.
Please cite this article as: Liu, B., et al., LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.03.023
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Research Report
LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2 Baohui Liua, Zhentao Guoa, Huimin Donga, Daofeng Tiana, Qiang Caia, Baowei Jia, Shenqi Zhanga, Liquan Wua, Junmin Wanga, Long Wanga, Xiaonan Zhua, Yue Liub, Qianxue Chena,n a
Renmin Hospital, Wuhan University, 238 Jiefang Street, Wuhan, Hubei 430060, China Shiyan Taihe Hospital, Hubei University of Medicine, 32 South Renmin Street, Shiyan, Hubei 442000, China
b
art i cle i nfo
ab st rac t
Article history:
In our previous study, we have found that leucine-rich repeats and immunoglobulin-like
Accepted 12 March 2015
domains 1(LRIG1) can improve the chemosensitivity in U251 cells whereas the role of LRIG1 in multidrug resistance (MDR) remains unknown. Here, we reported that LRIG1 can reverse
Keywords:
MDR by inhibiting epidermal growth factor (EGF) receptor (EGFR) and secondary inhibiting
Leucine-rich repeats and
ATP-binding cassette, sub-family B member 1(ABCB1) and ATP-binding cassette, sub-
immunoglobulin-like domains 1
family G (WHITE), member 2 (ABCG2). Our data showed that the expression of LRIG1 was
(LRIG1)
significantly higher in O6-methylguanine DNA methyltransferase (MGMT) Promoter
Multidrug resistance (MDR)
Methylation positive glioblastoma tissues compared to MGMT Promoter Methylation
Epidermal growth factor receptor
negative glioblastoma tissues. In addition, we found that LRIG1 expression was signifi-
(EGFR)
cantly decreased in MDR cells U251/TMZ compared to U251cells. Our results demonstrated
ATP-binding cassette
that over-expression of LRIG1 can reverse the MDR. The expression of ABCB1 and ABCG2
Sub-family B member 1(ABCB1)
were markedly suppressed when LRIG1 was over-expressed, supporting the negative
ATP-binding cassette
relationship between LRIG1 level and ABCB1 and ABCG2 level in human specimen.
Sub-family G (WHITE)
Furthermore, we found that LRIG1 downregulated ABCB1 and ABCG2 through suppressing
Member 2 (ABCG2)
EGFR expression. In case of EGFR knockdown, the effect of LRIG1 on regulating MDR, ABCB1 and ABCG2 was partially compromised. Our results, for the first time, showed that LRIG1 can reverse MDR in glioblastoma, by negatively regulating EGFR and secondary suppressing the levels of ABCB1 and ABCG2. & 2015 Published by Elsevier B.V.
1.
Introduction
Glioblastoma multiforme (GBM) is the most common and lethal type of primary brain tumor (Kotliarova and Fine, 2012). Even treated aggressively by surgery, radiation, and chemotherapy, the survival of patients with GBM still remains
not more than 14 months (Nikaki et al., 2012). Since the introdution of temozolomide (TMZ), which has demonstrated promising activity against glioblastoma, as chemotherapeutic agent in clinic setting, chemotherapy has become more and more important. However, the clinical response to TMZ lasts only a few months and drug resistance subsequently
n
Corresponding author. E-mail address: [email protected] (Q. Chen).
http://dx.doi.org/10.1016/j.brainres.2015.03.023 0006-8993/& 2015 Published by Elsevier B.V.
Please cite this article as: Liu, B., et al., LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.03.023
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develops in most cases (Spinelli et al., 2012), especially multidrug resistance (MDR), which has become the major the problem needed to be solved as soon as possible. Unfortunately, the mechanism of MDR was still unclear. LRIG1 was an inhibitor of receptors tyrosine kinase, which had cDNA cloned and characterized in 1996 (Suzuki et al., 1996). The LRIG1 integral membrane protein has been demonstrated to regulate various oncogenic receptor tyrosine kinases, including EGFR, by cell-autonomous mechanisms (Yi et al., 2011). Previous studies have also showed that upregulation of LRIG1 suppresses malignant glioma cell growth by attenuating EGFR activity (Yi et al., 2011) and LRIG1 negatively regulates the oncogenic EGF receptor mutant EGFRvIII, which is the most common EGFR mutant observed in GBM (Stutz et al., 2008). Recently, the LRIG1 isoform has been linked to aggressive behaviors and drug resistance in esophageal carcinoma cell lines (Wu et al., 2012). Furthermore, our previous study identified LRIG1 as a possible candidate gene affecting the sensitivity of human glioblastoma to the TMZ (Liu et al., 2013). Recent studies found that octreotide enhances the sensitivity of the SKOV3/DDP ovarian cancer cell line to cisplatin chemotherapy in vitro by inhibiting EGFR (Shen et al., 2011), and more important, GW583340 and GW2974, two human EGFR and HER-2 inhibitors, reverse ABCG2- and ABCB1-mediated drug resistance (Sodani et al., 2012). Therefore, LRIG1 may reverse the MDR, but this still remains to be elucidated. We hypothesized that LRIG1 is involved in the MDR of GBM and that can reverse MDR through suppressing the expression of EGFR and then suppressing ABCB1 and ABCG2. To test this hypothesis, we manipulated expression of LRIG1 in MGMT Promoter Methylation positive and negative patient samples, and examined whether LRIG1 can reverse MDR in MDR cells as well as the effect of LRIG1 on EGFR, ABCB1 and ABCG2. LRIG1 attracted our attention because previous studies had shown that the expression of LRIG proteins played an important role in the pathogenesis of astrocytic tumors (Yi et al., 2009). In this study, we used U251 and U87 cells as research models, which are the most frequent human primary brain tumors and represent the most malignant stage of GBM progression.
2.
Results
2.1. LRIG1 protein and mRNA level were upregulated in MGMT Promoter Methylation positive glioblastoma and downregulated in MDR cell line U251/TMZ cells To explore the relationship of LRIG1 expression and glioblastoma MDR, we assessed LRIG1 mRNA and protein expression in a series of primary human glioblastoma by real-time PCR and western blot (Fig. 1A and B). As shown in Fig. 1A and B, all 17 MGMT Promoter Methylation positive primary human glioblastoma and 17 MGMT Promoter Methylation negative primary human glioblastoma showed LRIG1 mRNA and protein expression. In addition, we found that patients with MGMT Promoter Methylation positive had significantly higher LRIG1 expression compared to the patients with MGMT Promoter Methylation negative.
2.2.
LRIG1 knockdown abrogated MDR in U251/TMZ cells
To determine the role of LRIG1 in MDR development, we knocked down LRIG1 in U251 cells using siLRIG1. The downregulation of target protein was confirmed via western blot analysis (Fig. 2A). Article has reported that caspase 3 is activated in the apoptotic cell and has been widely used as readout for drug sensitivity (Yan et al., 2015). Our data showed that the expression of caspase 3 was increased (Fig. 2A) and the apoptosis rate of cells was significantly decreased when treated with TMZ (16 mg/ml) and etoposide (VP-16) (10 mg/ml) (Fig. 2E), suggesting that suppressing LRIG1 expression resulted in reversal of MDR. To solid this data, LRIG1 plasmid was also transfected into U251/TMZ cells which was established by us before (Liu et al., 2013). The upregulation of target protein was also confirmed via western blot analysis (Fig. 2B). In line with the results in U251 cells, our data showed that drug resistance to TMZ and VP-16 was abolished by upexpressed LRIG1 (Fig. 2B and F).
2.3.
LRIG1 inhibited ABCB1 and ABCG2
In order to explore the underlying mechanisms of how LRIG1 reversed MDR, LRIG1 was over expressed in U251/TMZ cells and the expression levels of ABCB1, LRP, ABCG2 and MRP1, which was related to MDR (Liu et al., 2013), was determined by western blot and real-time PCR. Our data showed that LRIG1 overexpression did not result in change in levels of LRP and MRP1, but remarkably decreased ABCB1 and ABCG2 expression by more than 50% at both mRNA and protein level (there was a significant difference between expression levels of ABCB1 and ABCG2 between control and LRIGI overexpressing groups (Po0.05)) (Fig. 3A and B). In addition, we found that LRIG1 expression was significantly negatively correlated with ABCB1 (Pearson, R2 ¼0.204, P ¼0.0037) and ABCG2 (Pearson, R2 ¼ 0.301, P¼ 0.0004) expression in human samples (Fig. 4C and D).
2.4.
LRIG1 suppressed EGFR level
Previous studies have pointed out that EGFR inhibition affected ABCG2 expression in EGFR-positive MDCK BCRP cells via the PI3K/Akt signaling pathway (Pick and Wiese, 2012) and ABCB1 expression in acute lymphoblastic leukemia cells through MAPK/ERK pathway (Tomiyasu et al., 2013). We have found that LRIG1 can reverse MDR and repress ABCG2 and ABCB1 expressions in U251/TMZ cells. Therefore, we conducted experiments to test whether LRIG1 regulate ABCG2 and ABCB1 by inhibiting EGFR activity and secondary inhibition of PI3K/Akt signaling pathway and MAPK/ERK pathway in human MDR cells. First, we test the effect of LRIG1 on EGFR, p-AKT and p-ERK expression level. As expected, forced overexpression of LRIG1 in U251/TMZ cells significantly inhibited EGFR and p-AKT and p-ERK protein expression (Fig. 4A and B). To solid this data, the expression levels of Q2 LRIG1 and EGFR in human glioblastoma were analyzed by western blot. The results also showed that LRIG1 expression was significantly negatively correlated with EGFR levels
Please cite this article as: Liu, B., et al., LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.03.023
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content induced by LRIG1 might not occur at the transcriptional level in the MDR cells. Next, we test whether LRIG1 downregulated ABCB1 and ABCG2 through inhibiting EGFR.
p=0.00578
10
p=0.00398
Relative LRIG1 protein level
Relative LRIG1 mRNA level
(Pearson, R2 ¼ 0.3299, P¼ 0.00027). However, we found that the mRNA level of EGFR was not changed in case of LRIG1 overexpression, indicating that the decrease in EGFR protein
5
0 MM
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0
MM
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GAPDH
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caspase3
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GAPDH
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LRIG1
155KD
GAPDH
siCtrl siLRIG1
39KD
GFP GFP-LRIG1
TMZ
0.7
17KD
TMZ
siCtrl
LRIG1 mRNA expression level
Fig. 1 – LRIG1 is upregulated in MGMT Promoter Methylation positive patient samples. (A) The expression level of LRIG1 mRNA on 17 MGMT promotor Methylation (MM) postive and 17 MGMT Promotor Methylation negative glioblastoma tissues. P-value was calculated via the mann-Whitney's test, O6-methylguanine DNA methyltransferase (MGMT). (B) The expression level of LRIG1 protein on 17 MGMT Promotor Methylation (MM) postive and 17 MGMT Promotor Methylation negative glioblastoma tissues. P-value was calculated via the mann-Whitney's test. (C) The protein expression level of LRIG1 in U251/ TMZ and U251 cells. nPo0.05 vs. U251 cells. (D) The protein expression level of LRIG1 in U251/TMZ and U251 cells.
1.5
6
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4
0.5
2
0
siCtr siLRIG1l
0
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0.2 0.3 0.2
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TMZ
VP-16
0
TMZ
VP-16
Fig. 2 – The effect of LRIG1 in MDR. (A) The protein expression of LRIG1 and caspase 3 when siLRIG1 and siCtrl were transfected into U251 cells. (B) The protein expression of LRIG1 and caspase 3 when plasmid of GFP and GFP-LRIG1 was transfected into U251/TMZ cells. (C) The mRNA expression of LRIG1 when siLRIG1 and siCtrl were transfected into U251 cells. (D) The mRNA expression of LRIG1 when plasmid of GFP and GFP-LRIG1 was transfected into U251/TMZ cells. (E) The apoptosis rate of U251 cells which were treated by TMZ (16 μg/ml) or VP-16(10 μg/ml), and indicated siRNA for 72 h. nPo0.05 vs. cells transfected with siCtrl. (F) The apoptosis rate of U251/TMZ cells which were treated by TMZ (16 μg/ml) or VP-16(10 μg/ml), and indicated plasmid for 72 h. nPo0.05 vs. cells transfected with GFP plasmid.
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2.5. EGFR knockdown can reverse MDR of U251/TMZ cells and inhibit ABCB1, ABCG2 expression
mRNA expression level
We knocked down EGFR in U251 cells and then knocked down LRIG1 by siRNA, our data showed that the effects of siLRIG1 on ABCB1 and ABCG2 expression were attenuated with EGFR knockdown (Fig. 6A). Collectively, our data showed that LRIG1 knockdown induced ABCB1 and ABCG2 downregulation via inhibition of EGFR.
ABCB1
130KD
ABCG2
85KD 190KD
LRP
72KD
LRIG1
140KD
GAPDH
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ABCB1 ABCG2
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LRP
5
Pearson Test R=-0.451 2
R = 0.2037
4
ABCG2 protein expression level
ABCB1 protein expression level
It is well known that inhibition of EGFR can reverse ABCG2and ABCB1-mediated drug resistance in the KB-C2 cell line (Sodani et al., 2012), but whether inhibition of EGFR can
MRP1
LRIG1
P=0.0037˘0.05
3 2 1 0
5
10
Pearson Test R=-0.-0.549 2
4
R = 0.301 P=0.00038˘0.05
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1
0
Flag Flag -Lrig1
LRIG1
140KD
EGFR
170KD
p-ERK1/2
42KD
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55KD
GAPDH
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Flag Flag -Lrig1
EGFR protein expression level
Fig. 3 – LRIG1 over-expression induces ABCB1 and ABCG2 down-regulation. (A)The expression of LRIG1 and MDR related to gene when LRIG1 plasmid was transfected to U251/TMZ cells. Results are normalized relative to the amount of GAPDH mRNA and are plotted by the amount relative to Flag U251/TMZ cells sample. nPo0.05 vs. cells transfected with Flag plasmid. (B)The expression of LRIG1 and MDR related to protein when plasmid of Flag-LRIG1 was transfected into U251/TMZ cells. (C)LRIG1 and ABCB1 protein levels in glioblastoma and normal brain tissue were assessed using western blot. Correlation of the levels in 34 glioblastomas is shown. Statistical analysis was performed with Pearson's test. SE, standard error. GADPH, glyceraldehyde-3-phosphate dehydrogenase. (D) LRIG1 and ABCG2 protein levels in glioblastoma were assessed using western blot, correlation of the levels in 34 glioblastomas is shown. Statistical analysis was performed with Pearson's test.
EGFR mRNA expression level
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Pearson Test R=-0.-0.574 2
R = 0.3299
3
P=0.00019˘0.05
2 1 0
0
5
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LRIG1 protein expression level
Fig. 4 – LRIG1 over-expression induces EGFR down-regulation. (A) The expression of EGFR gene was detected when LRIG1 plasmid was transfected to U251/TMZ cells. Results are normalized relative to the amount of GAPDH mRNA and are plotted by the amount relative to Flag-U251/TMZ cells sample. (B) The protein expression level of LRIG1, EGFR, p-AKT and p-ERK1/2 when plasmid of Flag or Flag-LRIG1 was transfected into U251/TMZ cells. (C) LRIG1 and EGFR protein levels in glioblastoma and normal brain tissue were assessed using immunoblotting. Correlation of the levels in 34 glioblastomas tissues is shown. Statistical analysis was performed with Pearson's test. SE, standard error. GADPH, glyceraldehyde-3-phosphate dehydrogenase. Statistical analysis was performed with Pearson's test. Please cite this article as: Liu, B., et al., LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.03.023
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the MGMT Promoter Methylation negative patient samples, the methylation state of the MGMT gene determined whether tumor cells would be responsive to TMZ; If the promoter was methylated, TMZ was more effective (Hegi et al., 2005). Therefore, LRIG1 may act as MDR suppressor and lower the Q3 expression of LRIG1 may give an advantage to glioblastoma cells and contribute to the development of MDR. Cancer cells have the ability to become resistant to multiple drugs by many mechanisms, such as increased efflux of drug, enzymatic deactivation, decreased permeability, altered binding-sites and alternate metabolic pathways. Because efflux is a significant contributor for MDR in cancer cells, current research is aimed at blocking specific efflux mechanisms (Modok et al., 2006). Mounting studies have found that some certain proteins were related to efflux mechanisms (Jennings and Iyengar, 2001; Stupp et al., 2007). In present study, we found that knocked down LRIG1 in U251 cells, the drug sensitivity to TMZ and VP-16 was decreased, suggesting that down regulation of LRIG1 may be one of causes of MDR (Lu and Shervington, 2008). Consistent with these results, overexpression of LRIG1 in MDR cells U251/TMZ led to the loss of MDR in vitro (Fig. 2B and F). Although no publication has been reported on an effect of LRIG1 in MDR as yet, our results were in line with other's studies which suggested a role of LRIG1 in improving chemosensitivity in U251 cells (Qi et al., 2013). Accumulating evidences have shown that the development of multidrug resistance (MDR) is associated with overexpression of ATP-binding cassette (ABC) drug efflux transporters, which can reduce the intracellular concentrations of cytotoxic drugs and their metabolites (Alla et al., 2012; Greaves et al., 2012; Herraez et al., 2012). Moreover, MDR may be reversed if the expression of the proteins were downregulated (Michalak and Wesolowska, 2012; Minami et al., 2012; Tian et al., 2012). Our data showed that, when LRIG1 was upregulated in U251/TMZ cells, the expression of ABCB1 and ABCG2 was suppressed at both mRNA and protein levels, indicating that the effect of LRIG1 may occur in an ABC superfamily-dependent manner and LRIG1 reversed MDR
2.6. The effect of LRIG1 reversing MDR was attenuated in case of EGFR knockdown Our before mentioned results have shown that forced overexpression of LRIG1 reversed GBM cell MDR (Fig. 2B and F) and repressed ABCB1 and ABCG2 expressions (Fig. 3A and B). LRIG1 can repress EGFR (Fig. 4B) and EGFR can repress MDR, ABCB1, and ABCG2 (Fig. 5B and C), but whether LRIG1 regulated MDR, ABCB1, and ABCG2 through inhibiting EGFR was still unknown, so EGFR and LRIG1were knocked down by siRNA in U251 cells. Our results showed that the effect of siLRIG1 on MDR was attenuated when EGFR was knocked down (Fig. 6C and D). Collectively, our data showed that LRIG1 knockdown induced resistance to drugs in U251 cells through inhibition of EGFR and secondary suppression ABCB1 and ABCG2.
3.
Discussion
Our data showed that, the expression of LRIG1 was significantly lower in MDR cell line compared to glioblastoma cells. More important, we found that lower expression of LRIG1 in
1.5 ABCB1
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reverse ABCG2- and ABCB1-mediated drug resistance in glioma MDR cells remain unclear. In this study, EGFR siRNAs was used to knock down EGFR and the suppression of EGFR expreesion was confirmed through western blot analysis (Fig. 5A and B). Cells transfected with siRNA was treated by TMZ (16 mg/ml) for 72 h and apoptosis rate was assessed by Annexin-V-FITC assay. The results showed that the expression levels of ABCG2 and ABCB1 as well as the cells apoptosis rate were decreased (Fig. 5B and C). Similar findings were obtained in VP-16 treating cells. To confirm that EGFR siRNAs did not decrease downstream LRIG1 activity by inhibiting LRIG1 transcription, the mRNA expression of LRIG1 was detected by real time-PCR in U251 cells transfected with EGFR siRNA or control siRNA (Fig. 5D). As demonstrated by our results, EGFR knockdown did not have marked effect on LRIG1 mRNA level.
EGFR mRNA expression level
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Fig. 5 – EGFR knock down reversed MDR in U251/TMZ cells. (A) The mRNA expression of EGFR when siEGFR was transfected to U251/TMZ cells. nPo0.05 vs. siCtrl group. (B) The protein expression of EGFR, ABCB1 and ABCG2 when siEGFR was transfected to U251/TMZ cells. (C)The apoptosis rate of the cells treated by TMZ(16 g/ml) or VP-16(10 g/ml) compared with indicated siRNAs, nPo0.05 relative to U251/TMZ cells. (D) The expression of LRIG1 when siEGFR was transfected to U251/TMZ cells. Results are normalized relative to the amount of GAPDH mRNA and are plotted by the amount relative to siCtrl group, P40.05 vs. siCtrl group. Please cite this article as: Liu, B., et al., LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.03.023
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may by inhibiting ABCB1 and/or ABCG2. In consistent with the in vitro results, LRIG1 downregulation significantly correlated with ABCB1 and ABCG2 upexpression in human sample. These results also supported a previous report showing that Gefitinib, which is also Tyrosine Kinase Inhibitor, can inhibit ABCB1 and ABCG2 in glioma (Carcaboso et al., 2010). The mechanism by which LRIG1 regulates ABCB1 and ABCG2 was unclear, our data showed for the first time that LRIG1 regulates ABCB1 and ABCG2 through inhibiting EGFR. When we silenced the EGFR by siRNA, the expression levels of both ABCB1 and ABCG2 were decreased. More important, the effect of LRIG1 on regulating ABCB1 and ABCG2 was attenuated with EGFR knockdown. Taken together, our results suggested that LRIG1 regulated ABCB1 and ABCG2 through regulating EGFR expression. The mechanism by which EGFR inhibits ABCB1 and ABCG2 was unclear. However, it recently has been pointed out that EGFR inhibition affected ABCG2 expression in EGFR-positive MDCK BCRP cells via the PI3K/Akt signaling pathway (Pick and Wiese, 2012). In addition, MAPK/ERK pathway, which is regulated by EGFR (Kotliarova and Fine, 2012), has also been reported to up-regulate ABCB1 expression in acute lymphoblastic leukemia cell lines (Tomiyasu et al., 2013). Our data also showed that LRIG1 can inhibit the activity of PI3K/Akt signaling pathway and MAPK/ERK pathway in U251 cells. Taken together, these above findings highlighted the idea that knockdown EGFR might inhibit ABCB1 and ABCG2 via inhibiting PI3K/Akt signaling pathway and MAPK/ERK pathway. In summary, we have demonstrated that LRIG1 was a molecule that can enhance the chemosensitivity of glioblastoma to chemotherapeutics through inhibition of EGFR and secondary inhibition of ABCB1 and ABCG2. When cancer cells retain expression of LRIG1, they suppress ABCB1 and ABCG2
via suppressing EGFR. On the other hand, once cancer cells present with compromised expression of LRIG1, upregualted ABCB1 and ABCG2 can lead to chemo-resistance. Because numerous compounds and biological agents have been evaluated as EGFR inhibitors, it is highly possible that some of such agents will show promising effecacy in the treatment and prevention of cancer grows.
4.
Experimental procedures
4.1.
Reagents
Chemicals were purchased from Sigma-Aldrich (Wuhan, China). Flag-LRIG1 plasmids were saved by our lab.
4.2.
Specimens and patients
Specimens were collected at Renmin Hospital, Wuhan University from 34 patients with glioblastoma (primary tumors) with an average age of 36 years (range 23–71 years). Samples were harvested from the tumors at the time of surgery and subsequently snap frozen before stored at 80 1C. The present study was approved by Ethics Committee of the Faculty of Medicine of Renmin Hospital, Wuhan University (Appprove number: [2012]LKSZ(010)H), and all clinical investigation have been performed according to the principles of Declaration of Helsinki. Informed consent was obtained from the patients and/or guardians. All the tumors were located in the cerebral hemispheres. All the patients experienced neurosurgical operation(s) with the aim of gross radical tumor research between January 2010 and 2013. Pathological diagnoses of all specimens were panel reviewed by an experienced pathologist (HHa) according to WHO criteria (2000) (Gonzales, 2001).
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Fig. 6 – The effect of siLRIG1 was lost partly when EGFR was knocked down. (A) The expression of LRIG1, EGFR, ABCB1 and ABCG2 when cells were transfected for 72 h with indicated siRNAs, GAPDH was used as a loading control. (B) U251 cells transfected with LRIG1-targeting siRNA or scramble siRNA were assessed in cells apoptosis rate in the presence of siEGFR or siCtrl, P-value was for comparison to the siCtrl and from the Student's t test. In this experiment, siCtrl and siEGFR group, which were transfected siCtrl or siEGFR, were set first and then each group was divided into two subgroups: siCtrl and siLRIG1, which were transfected siCtrl or siLRIG1. The apoptosis index of siEGFR and siCtrl were calculated from apoptosis rate of the subgroups by (siLRIG1-siCtrl)/siCtrl. All the cells were treated by TMZ. Please cite this article as: Liu, B., et al., LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.03.023
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cytometer with Cell Quest software (Becton Dickinson, Mountain View, CA).
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Fig. 7 – A schematic representation of LRIG1 as a MDR Inhibitor. LRIG1 reversed MDR in glioblastoma, which involved negative inhibition of EGFR and secondary inhibition of ABCB1 and ABCG2.
4.3. Cell culture and establishment of a cell line with acquired resistance to TMZ. The U251 and U87 cells were purchased from The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. The cells were cultured in DMEM medium supplemented with 10% FBS in a humidified incubator at 37 1C/5% CO2. Basal cell culture was maintained in T-75 flasks (Sigma-Aldrich, USA). Cells were trypsinized at 80–90% confluency, seeded into either 6well plates or 96-well plates (Costar, USA). TMZ resistant cell line U251/TMZ was established as we previously described (Liu et al., 2013).
4.4.
7
Evaluation of apoptotic cells by Annexin-V-FITC
Sensitivity of U251 and U251/TMZ cells to TMZ (16 mg/ml) and VP-16 (10 mg/ml), was assessed by Annexin-V-FITC assay. Briefly, for Annexin-V-FITC assay, exponentially growing cells were plated into 6 well plates (2 105 cells/well). After 24 h, cells were treated by serial dilutions of the drugs and incubated for 96 h, then the cells were harvested and washed with phosphate-buffered saline. Cells were then resuspended in 100 μl of Annexin-V binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4) and incubated with 5 μl of Annexin-V-FITC for 15 min at room temperature before counterstained with propidium iodide (final concentration 1 μg/ml). After the incubation period, the cells were diluted with 190 μl of Annexin-V binding buffer. Cells were analyzed by flow cytometry using a Becton Dickinson FACS can flow
Cell transfections
To overexpress LRIG1, U251/TMZ and U251 cells (50–80% confluency) were transfected with LRIG1 plasmid (1 mg/well, 6-well plate) using FUGENE HD transfection reagent (Roche) according to the manufacturer's instructions. For knockdown experiments, siRNAs targeting human LRIG1 and EGFR, and control siRNA were purchased from Thermo Scientific. According to the manufacturer's instructions, 30–50% confluent cells were transfected with siRNAs at a final concentration of 40 nM using Lipofectamine 2000 transfection reagent (Invitrogen).
4.6.
RNA extraction and real time PCR
Total RNA was extracted using the Total RNA Isolation System from Promega according to the manufacturer's instructions. The Access real time PCR System supplied by Promega was utilized for reverse transcription. Based on the information of the LRIG1, EGFR, ABCB1, ABCG2, LRP, and GAPDH gene sequences, six pairs of gene-specific primers were designed the fowllowing: LRIG1 sense 50 -ttgctgatgttgtttcgctg-30 and antisense 50 -tgatggtctgtcacggtcg-30 , EGFR sense 50 -ggacgacgtggtggatgccg-30 and antisense 50 -ggcgcctgtggggtctgagc-30 , ABCB1 sense50 -aacaacgcattgccatagctcgtg-30 , and antisense50 -agtctgcattctggatggtggaca-30 LRP sense50 -aggagagcagctggaacaag-30 , and antisense 50 -cttgttccagctgctctcct-30 ; ABCG2sense50 -cacctattggcctcaggaa-30 ,and antisense50 -cctgcttggaaggctctatg-30 ; and GAPDH (internal control) sense 50 -gagtcaacggatttggtcgt-30 and antisense 50 -ttgattttggagggatctcg-30 . Quantitative PCR was carried out in a reaction mixture containing 2.5 μl of cDNA, 12.5 μl of SYBR Green mix, 2.5 μl of plus solution, 2 μl of primers(5pmol/ μl),5.5 μl of ddH2O, and 2.5 μl of cDNA. Furthermore, qPCR was performed on an ABI Prism 7500 Sequence Detection System (Applied Biosystems) using Sybr Green qPCR kit (Toyobo Biologics, Japan). The cycling conditions were as follows: a denaturation step at 95 1Cfor 2 min, followed by 40 cycles at 95 1C for 15 s, 57 1C for 15 s, 72 1C for 45 s, and a final extension step at 72 1C for 10 min. Results are normalized relative to the amount of GADPH mRNA and plotted by the amount relative to the reference sample
4.7.
Western blotting
Following treatments or transfections, total protein extracts were prepared by incubating cells in lysis buffer (20 mM Tris– HCl, pH 7.5, 150 mM NaCl, and 1% TritonX-100 5 mM EDTA) for 30 min at 0 1 C. Samples were normalized for protein content assay. 30 μg of lysates were separated on 12% polyacrylamide gels and electro-transferred onto PVDF membranes. Membranes were blocked in 5% BSA in TBS-Tween, incubated with primary antibody (Santa Cruz; 1: 200 for LRIG1, EGFR, ABCB1, ABCG2, MRP1, LRP, caspase 3, p-AKT, p-ERK and GAPDH), followed by incubation in secondary antibody (Santa Cruz; 1: 3000 for LRIG1 EGFR, ABCB1, ABCG2,MRP1,LRP, caspase 3, pAKT, p-ERK and GAPDH). The signals were visualized using
Please cite this article as: Liu, B., et al., LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.03.023
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BeyoECL Plus chemiluminescent substrate (Beyotime, Shanghai, China) and captured on chemiluminescence-sensitive film.
4.8.
Statistical analysis
Data were expressed as means7standard errors of the means. Statistical analysis was performed with (SigmaPlot, version 11.0 and spss 13.0). Differences between means were assessed with the student's t test or mann-Whitney's test. In multiple comparisons, one-way analysis of variance (ANOVA) was used. Pearson's test was used to detect the correction of two groups and compare quantitative values of expressions (Fig. 7).
Funding This work was supported by the National Natural Science Foundation of China (Nos. 30973072, 81372683), the Fundamental Research Funds for the Central Universities (No. 201130202020001), and the Fundamental Research Funds for the Central Universities (No. T2011302008).
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Please cite this article as: Liu, B., et al., LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.03.023
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