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Crizotinib inhibits migration and expression of ID1 in MET-positive lung cancer cells: implications for MET targeting in oncology Emanuel Stutz1, Oliver Gautschi1,2, Martin F Fey1,3, Mathias Gugger4, Mario P Tschan1,3 & Sacha I Rothschild*1,5

ABSTRACT Aims: ID1 is an important component of the MET–SRC signaling pathway, which is a regulator of cell migration and invasion. We hypothesized that the ALK/MET inhibitor crizotinib inhibits migration via MET–SRC–ID1, rather than ALK. Materials & methods:  We used ALK fusion-positive and -negative lung cancer cell lines; crizotinib, PHA-665752, and saracatinib, and stable transfection with shMET. We performed western blotting for p-ALK, ALK, p-MET, MET, p-SRC, SRC and ID1, and quantitative real-time PCR for ID1. Results: Crizotinib decreased p-MET, p-SRC and ID1 levels in ALK- and or MET-positive cell lines and inhibited cell migration. Knockdown of MET was comparable with the effect of crizotinib. Conclusion: The effects of crizotinib on ID1 expression and cancer cell migration were associated with the presence of activated MET, rather than ALK fusion. Advances in our understanding of tumor biology are overturning the classification of tumors by histology in favor of grouping by molecular characteristics and key oncogenic drivers amenable to pharmacological modulation [1]. Several actionable oncogenic drivers have been identified in nonsmall-cell lung cancer (NSCLC), including EGFR, KRAS, MET, ALK, HER2, BRAF and ROS1, among others [2–5]. The EML4–ALK inversion was detected in 2007 [6]. Crizotinib, a dual ALK and MET tyrosine kinase inhibitor [7], had a high effect on ALK-positive tumors in Phase I, II and III clinical trials in NSCLC [8–11]. Based on these data, the US FDA granted approval of crizotinib for ALK-positive, pretreated lung adenocarcinoma in August 2011. Other groups showed that crizotinib has preclinical anti-tumor activity in MET amplification-positive lung cancer cells, but not in cells without MET amplification or with MET mutations [12]. In the clinics, crizotinib led to rapid and durable response in a NSCLC patient with de novo MET amplification in the absence of ALK rearrangement [13]. Recently, anti-tumor activity of crizotinib against ROS1 rearranged NSCLC has been described [14,15]. The anti-tumor activity of crizotinib may not be restricted to NSCLC. In a preclinical model of MET‑overexpressing ovarian cancer, crizotinib reduced tumor growth and metastasis [16]. Crizotinib also exhibited a marked anti-tumor effect in gastric cancer xenografts positive for MET amplification [17]. In esophagogastric adenocarcinoma, MET amplification defines an aggressive subgroup with sensitivity to crizotinb [18]. Antimetastatic effects of crizotinib have also been reported in preclinical breast, colorectal and lung cancer models, but without further investigation of the specific molecular mechanism [19]. The nonreceptor kinase SRC is part of the focal adhesion kinase complex, which mediates cell adhesion and migration [20]. SRC interacts with the MET receptor kinase [21,22], and in our

KEYWORDS

• ALK • crizotinib • ID1 • lung cancer • MET • migration

Department of Clinical Research, University of Bern, 3010 Bern, Switzerland Medical Oncology, Cantonal Hospital, 6000 Luzern, Switzerland 3 Department of Medical Oncology, Inselspital, Bern University Hospital, 3010 Bern, Switzerland 4 Institute of Pathology, University of Bern, 3010 Bern, Switzerland 5 Department of Medical Oncology, University Hospital Basel, 4031 Basel, Switzerland *Author for correspondence: Tel.: +41 612 655 074; Fax: +41 612 655 316; [email protected] 1 2

10.2217/FON.13.179 © 2014 Future Medicine Ltd

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RESEARCH ARTICLE  Stutz, Gautschi, Fey, Gugger, Tschan & Rothschild previous work, we identified ID1 as an important target of SRC and regulator of lung cancer cell migration and invasion [23]. ID1 belongs to the helix-loop-helix (HLH) protein superfamily and acts as a dominant-negative regulator of differentiation-specific transcription factors [24]. ID1 expression is elevated in lung adenocarcinoma compared with normal lung tissue, and is associated with poor prognosis [25]. More recently, we described a novel association between ID1 and miRNAs 29b and 381 [26,27]. In the present project, we tested the hypothesis that crizotinib inhibits migration via MET– SRC–ID1, rather than ALK. To the best of our knowledge, there is only one report about the effect of crizotinib on cell migration and invasion in NSCLC [19]. If positive, our study may contribute to the development of crizotinib and other MET inhibitors in the therapy of lung cancers, and other malignancies, which are driven by MET. Material & methods ●●Cell

lines & reagents

The A549 and H460 cell lines were obtained from the American Type Culture Collection (ATCC; VA, USA). H2228 was provided by John Minna (University of Texas Southwestern Medical Center, TX, USA). Cells were grown as previously described [26]. Saracatinib (AstraZeneca Pharmaceuticals, Macclesfield, UK), crizotinib (Selleck Chemicals, TX, USA) and PHA-665752 (Pfizer, CA, USA) were solubilized in dimethyl sulfoxide to obtain a 10 mmol/l stock solution, which was stored at -20°C and freshly diluted in cell culture medium for each experiment.

ALK variant 3b: 5´-TACCAGTGCTGTCTCAATTGCAGG-3´ (forward) and 5´-AGCTTGCTCAGCTTGTACTCAGGG-3´ (reverse). Primer sequences for ID1 and 18S RNA have been published previously [23]. All measurements were performed in triplicates. The method of the analysis was previously described [28]. Standard dilutions of cDNA and melting curve analyses were performed to confirm accuracy. All quantitative PCR reactions were carried out on a 7900HT Fast Real-Time PCR system (Applied Biosystems, Rotkreuz, Switzerland). ●●Western

blot analysis

Methods of cell lysis, protein quantification, transfer of separated protein onto polyvinylidene difluoride membranes and blocking were performed as previously described [26]. Membranes were incubated with antibodies against p-ALK (Tyr1604) 1:1000, ALK (C26G7) 1:1000, p-MET (Tyr1234/1235) 1:1000, p-SRC (Tyr416) 1:500 (all Cell Signaling Technology, MA, USA), MET (Santa Cruz Biotechnology, CA, USA) 1:500, SRC (Clone GD11; Millipore AG, Zug, Switzerland) 1:1000, and ID1 (BioCheck, Inc., CA, USA) 1:500. For loading control, β-actin antibody (Millipore AG) 1:10 4 was used. Horseradish peroxidase-linked anti-mouse (1:1000) and anti-rabbit (1:2500; Amersham ECL, GE Healthcare Bio-sciences, Uppsala, Sweden) secondary antibodies were used. Visualization was carried out by chemiluminescence (Amersham ECL). Results ●●EML4–ALK

●●Quantitative

real-time PCR

Total RNA was extracted using the RNeasy kit according to the manufacturer’s protocol (Qiagen, Hombrechtikon, Switzerland). Total RNA was reverse transcribed using random primers and M-MLV Reverse Transcriptase (Promega AG, Dübendorf, Switzerland). For mRNA analysis, RNA was reverse transcribed using the miScript Reverse Transcription Kit (Qiagen). Quantitative real-time PCR was performed using iQ Sybr Green Supermix (BioRad Laboratories AG, Reinach, Switzerland). The following primers were used: ALK variant 1: 5´-CCACACCTGGGA A AGGACCTAA AG-3´ (forward) and 5´-AGCTTGCTC AGCT TGTACTC AGGG-3´ (reverse);

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& MET expression in lung cancer cell lines

We first determined the basal expression levels of ALK and MET in our cell lines by western blotting. The detection of p-MET by western blot was indicative for the activation of MET. The KRAS-mutated A549 cell line did not harbor an EML4–ALK inversion, but contained activated MET. The H2228 cell line expressed the EML4–ALK variant 3b and activated MET, whereas the H460 cell line harbored neither EML4–ALK, nor activated MET (Figure 1). Expression of the EML4–ALK translocation in H2228 cells was confirmed by regular PCR (Supplementary Figure 1; see online at www.futuremedicine.com/doi/suppl/10.2217/ fon.13.179).

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Crizotinib inhibits migration & expression of ID1 in MET-positive lung cancer cells  ●●Crizotinib reduces p-MET levels in A549

A549

& H2228 cells

We then examined the effect of crizotinib on ALK, MET, SRC and ID1 protein levels in cell lines. Crizotinib decreased p-MET, p-SRC and ID1 levels in A549 and H2228, but not in H460 cells (Figure 2A). In H2228 cells, crizotinib also reduced ID1 mRNA levels in a dose-dependent way (Figure 2B). The p-MET expression size of A549 is different from the ones of the other cell lines, which was confirmed in repeated experiments. We incubated H2228 cells with the MET inhibitor PHA-665752 and with the SRC inhibitor saracatinib. PHA-665752 and saracatinib decreased the levels of p-MET, p-SRC and ID1 protein, but not p-ALK (Figure  2C). PHA-665752 and saracatinib both reduced ID1 mRNA expression in H2228 (Figure 2D).

H460

RESEARCH ARTICLE

H2228

p-ALK

90 kDa

ALK

90 kDa

p-MET

145 kDa

MET

145 kDa

p-SRC

60 kDa

SRC

60 kDa

ID1

17 kDa

β-actin

45 kDa

●●Crizotinib attenuates migration of

MET‑positive A549 cells

Previous studies showed that a decrease of ID1 protein expression by SRC inhibition reduces the migratory potential of lung cancer cells [26,29]. In addition, crizotinib reduces metastasis in a preclinical model of ovarian cancer via MET and SRC inhibition [16]. Given that crizotinib decreased p-SRC and ID1 levels in A549 cells, we investigated whether incubation with crizotinib would also impair A549 migration. To examine whether the antimigratory effect of crizotinib in A549 cells was due to MET inhibition, we specifically knocked down MET in A549 cells using lentiviral vectors expressing shRNA against c-MET (shMET). This resulted in decreased ID1 protein expression (Figure 3A) and migration (Figure 3B), compared with control cells (Shc in A549). Knockdown of MET was comparable with the effect of crizotinib (Figure  3B). Neither by incubating cells with physiological doses of crizotinib nor by stable knockdown of MET we could see an effect on cell survival. Conclusion & future perspective Tumor cell migration is one of the key factors leading to cancer metastasis. The availability of new drugs targeting migration and invasion would represent a major advance in oncology. Crizotinib as a dual inhibitor of ALK and MET has the potential to become one of the first drugs of this class of compounds. Crizotinib is presently only approved for the treatment of patients with advanced NSCLC harboring

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Figure 1. Basal expression of ALK and MET pathway proteins. Western blotting was performed for p-ALK, ALK, p-MET, MET, p-SRC, SRC and ID1 in A549, H460 and H2228 cell lines. H2228 cells expressed the EML4–ALK variant 3b with a protein length of 90 kDa. b-actin was used as a loading control.

EML4–ALK fusion, but case reports suggest that this drug may have clinical activity against MET-amplified tumors as well [30]. In the present study, we aimed to further delineate the in vitro effects of crizotinib, with regard to MET expression and function, in lung cancer cells. Our results indicate that crizotinib inhibited migration in cell lines with active MET, but not in cells with inactive MET. This functional effect appeared to be unrelated to the presence the of EML4–ALK fusion, suggesting that crizotinib activity on cell migration is mainly dependent on the presence of active MET. This finding is consistent with our hypothesis, and with previous preclinical studies by others, suggesting that MET is a valid molecular target for new anticancer therapies, and that crizotinib as a potent MET inhibitor is suitable for further development in lung cancers driven by MET [16–19]. Shojaei and colleagues demonstrated inhibitory potential of crizotinib on lung cancer migration by showing less metastases in a xenograft model upon

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RESEARCH ARTICLE  Stutz, Gautschi, Fey, Gugger, Tschan & Rothschild

p-ALK

90 kDa

ALK

90 kDa

p-MET

145 kDa

MET

145 kDa

p-SRC

60 kDa

SRC

60 kDa

ID1

17 kDa

β-actin

45 kDa

Relative ID1/18s RNA transcript level

Crizotinib 100 nM

H460 DMSO

Crizotinib 100 nM

A549 DMSO

Crizotinib 500 nM

DMSO

H2228

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0

DMSO

50

100

Crizotinib (nM)

H2228 PHA-665752

Saracatinib

p-ALK

90 kDa

ALK

90 kDa

p-MET

145 kDa

MET

145 kDa

p-SRC

60 kDa

SRC

60 kDa

ID1

17 kDa

β-actin

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Relative ID1/18s RNA transcript level

DMSO 100 nM 500 nM 100 nM 500 nM

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0

DMSO

100

500

PHA-665752 (nM)

100

500

Saracatinib (nM)

Figure 2. Effects of crizotinib, PHA-66752 and saracatinib in ALK-positive and -negative lung cancer cell lines. (A) Cells were incubated with crizotinib (100 or 500 nM). Western blotting was performed for p-ALK, ALK, p-MET, MET, p-SRC, SRC and ID1 in H2228 (ALK-positive), A549 and H460 (ALK-negative) cells. b-actin was used as a loading control. (B) H2228 cells were incubated with two different concentrations of crizotinib (50 and 100 nM). Quantitative real-time PCR for ID1 was performed. Results were normalized to 18sRNA and are shown as an n-fold decrease relative to the level in the control cells. (C) Incubation of H2228 cells with the MET inhibitor PHA-665752 (100 and 500 nM) and the SRC inhibitor saracatinib (100 and 500 nM). Western blotting was performed for p-ALK, ALK, p-MET, MET, p-SRC, SRC and ID1. b-actin was used as a loading control. (D) Incubation of H2228 cells with the MET inhibitor PHA-665752 (100 and 500 nM) and the SRC inhibitor saracatinib (100 and 500 nM). Quantitative real-time PCR for ID1 was performed. Results were normalized to 18sRNA and are shown as an n-fold decrease relative to the level in the control cells. DMSO: Dimethyl sulfoxide.

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Crizotinib inhibits migration & expression of ID1 in MET-positive lung cancer cells 

0h

A549 Shc

shMET 3120

MET

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ID1

17 kDa

β-actin

45 kDa

RESEARCH ARTICLE

12 h

A549 shc DMSO

A549 shc Crizotinib 100 nM

A549 shMET DMSO

Figure 3. Migratory potential of lung cancer cells is MET dependent. (A) A549 cells were transduced by lentiviral vectors expressing shRNA targeting c-MET (shMET) and a scrambled control vector (Shc), which were purchased from System Biosciences (CA, USA). Lentivirus production and transduction was performed as previously described [31]. Western blotting was performed to analyze total MET and ID1 in A549 cells. b-actin was used as the loading control. (B) Migration assay with A549 shMET cells and A549 Shc cells. Cells were seeded in a six-well plate (2.5 × 105 cells/well). After 24 h, monolayers were scratched with a 2‑ml pipette tip. Plates were washed three times with phosphate-buffered saline. Cells with a scrambled control vector were incubated with crizotinib in DMSO or with DMSO alone. Light microscopy images are shown immediately after scratching of the monolayer (0 h) and 12 h later. DMSO: Dimethyl sulfoxide.

treatment with crizotinib [19]. Our findings support their results and provide a potential underlying molecular mechanism. Tanizaki and coworkers showed marked anti-tumor activity in MET amplification-positive lung cancer cell lines but not in cells without MET amplification, including those with a MET mutation [12]. This seems to be due to the fact that levels of MET phosphorylation are significantly lower in cell lines with MET mutations than in those with MET amplification. These findings are supported by our results showing inhibition of migration upon crizotinib only in cell lines with MET phosphorylation. The second preliminary finding from our study is that crizotinib appears to be capable of modifying the expression of the tumor stem cell gene ID1. Given that not only crizotinib but also the MET inhibitor PHA-665752 and the SRC inhibitor saracatinib modified the expression of ID1, may link c-MET with the SRC-ID1 pathway in lung cancer cells with activated MET (Supplementary Figure 2). We previously described

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the role of ID1 in the SRC pathway, and more recently identified miRNAs targeting ID1 [26,27]. Further work is warranted to characterize the signaling pathway from MET via miRNAs to ID1, to better understand the manner in which MET inhibitors, especially crizotinib, impair cancer cell migration and metastasis, evaluate the effect of MET inhibition on EGFR-mutated cell lines, identify appropriate molecular markers for patient selection, and ultimately achieve maximal benefit with these new drugs in the clinic. Financial & competing interests disclosure The authors recieved grant support from the Swiss Cancer League (Grant KLS 02164‑02‑2008; to O Gautschi and M Gugger) and the Bernese Cancer League (to M Gugger, O Gautschi and MP Tschan). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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RESEARCH ARTICLE  Stutz, Gautschi, Fey, Gugger, Tschan & Rothschild EXECUTIVE SUMMARY EML4–ALK & MET expression in lung cancer cell lines ●●

Crizotinib reduces p-MET levels in A549 and H2228 cells. ūū The ALK inhibitor crizotinib decreased p-MET, p-SRC and ID1 levels in A549 (activated MET) and H2228 (EML4–ALK

translocation variant 3b and activated MET) lung cancer cell lines.

ūū The MET inhibitor PHA-665752 and the SRC inhibitor saracatinib decreased the levels of p-MET, p-SRC and ID1 in the

H2228 cell line.

●●

Crizotinib attenuates migration of MET-positive A549 cells. ūū Tumor cell migration is one of the key factors leading to cancer metastasis and the ID1 protein is critically involved in

the migratory potential of lung cancer cells.

ūū Crizotinib reduced ID1 expression and by that lung cancer cell migration, and the same effect was seen by a specific

knockdown of c-MET using lentiviral vectors.

●●

This study showed for the first time that crizotinib appears to be capable of modifying the expression of the tumor stem cell gene ID1 and proved the hypothesis that crizotinib might inhibit lung cancer cell migration.

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Crizotinib inhibits migration and expression of ID1 in MET-positive lung cancer cells: implications for MET targeting in oncology.

ID1 is an important component of the MET-SRC signaling pathway, which is a regulator of cell migration and invasion. We hypothesized that the ALK/MET ...
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