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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Rearrangements of the proto-oncogene RET are newly identified potential driver mutations in lung adenocarcinoma (LAD). However, the absence of cell lines harboring RET fusion genes has hampered the investigation of the biological relevance of RET and the development of RET-targeted therapy. Thus, we aimed to identify a RET fusion positive LAD cell line. Eleven LAD cell lines were screened for RET fusion transcripts by reverse transcription-polymerase chain reaction. The biological relevance of the CCDC6-RET gene products was assessed by cell growth, survival and phosphorylation of ERK1/2 and AKT with or without the suppression of RET expression using RNA interference. The efficacy of RET inhibitors was evaluated in vitro using a culture system and in an in vivo xenograft model. Expression of the CCDC6-RET fusion gene in LC-2/ad cells was demonstrated by the mRNA and protein levels, and the genomic break-point was confirmed by genomic DNA sequencing. Mutations in KRAS and EGFR were not observed in the LC-2/ad cells. CCDC6-RET was constitutively active, and the introduction of a siRNA targeting the RET 3' region decreased cell proliferation by downregulating RET and ERK1/2 phosphorylation. Moreover, treatment with RET-inhibitors, including vandetanib, reduced cell viability, which was accompanied by the downregulation of the AKT and ERK1/2 signaling pathways. Vandetanib exhibited anti-tumor effects in the xenograft model. Endogenously expressing CCDC6-RET contributed to cell growth. The inhibition of kinase activity could be an effective treatment strategy for LAD. LC-2/ad is a useful model for developing fusion RET-targeted therapy.

Lung cancer is the most common cause of cancer death worldwide.[1] The identification of oncogenic driver genes is to select the increasing number of small molecule inhibitors targeting these gene products.[2, 3] In particular, in lung adenocarcinoma (LAD), the most dominant histological subtype of lung cancer, the application of kinase inhibitors for cases with specific gene alterations has been successful, that is, gefitinib and erlotinib for EGFR mutation-positive cases and crizotinib for ALK fusion-positive cases.[4-7] Furthermore, accumulating evidence has demonstrated somatic mutations and rearrangements of potential oncogenes, including BRAF, ERBB2 and ROS1, in LAD.[8-10]

RET is one of the newest LAD driver genes.[11-15] RET gene is located on chromosome 10 and encodes a receptor tyrosine kinase,[16, 17] and the oncogenic potential of this gene product has been suggested in several tumors, including thyroid cancer.[18-20] Recently, five independent groups identified aberrant fusion genes, KIF5B-RET and CCDC6-RET in clinical samples of LAD.[11-15] Ectopically expressed RET fusion products afforded NIH3T3 cells with anchorage-independent growth and tumorigenicity in nude mice.[11, 14] Furthermore, KIF5B-RET-expressing H1299 cells exhibited growth factor-independent growth.[11] These findings strongly suggest the oncogenic activity of RET fusion products and also suggest the potential therapeutic efficacy of multi-kinase inhibitor targeting of RET using the abovementioned cells. However, LAD-derived cell lines harboring RET fusion genes had not been identified. Recently, Matsubara et al.[21] screened LAD cell lines that were sensitive to a RET inhibitor vandetanib and found a CCDC6-RET fusion gene-harboring cell line, LC-2/ad.

We have independently screened cell lines established from Japanese LAD samples by RT-PCR and found that LC-2/ad cells expressed the CCDC6-RET fusion gene product. We further examined whether LC-2/ad cells depend on RET fusion-mediated signaling. In addition, the antitumor effect of RET inhibitors in LC-2/ad cells was evaluated in vitro and in vivo.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Complete materials and methods were described in the supplementary information (Data S1. Materials and Methods).

Purchased materials

Cell lines were purchased from RIKEN Bio Resource Center, the Immuno-Biological Laboratories (Fujioka, Japan) and American Type Culture Collection. Procedures for western blotting was previously described.[22] Primary antibodies specific for RET and phospho-RET Tyr-905 were purchased from Epitomics (Burlingame, CA, USA) and Cell Signaling Technologies (Danvers, MA, USA), respectively. RET-targeting siRNA was purchased from Life Technologies (Carlsbad, CA, USA). Gefitinib, sunitinib malate and sorafenib were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), Sigma-Aldrich (St. Louis, MO, USA) and Toronto Research Chemicals (Toronto, ON, Canada), respectively. Vandetanib, AZD6244 and BEZ235 were purchased from Selleck (Houston, TX, USA).

Multiplex RT-PCR

Reported KIF5B/CCDC6-RET fusion variants were detected by multiplex RT-PCR according to the procedures described elsewhere.[11, 14]

Genomic DNA sequencing

LC-2/ad DNA was captured with custom hybridization probes targeting CCDC6 intron 1 and RET whole gene (Agilent) followed by parallel sequencing on the MiSeq system (Illumina).

Real-time RT-PCR

Procedures for real-time RT-PCR was previously described.[22] The PCR primers used in the present study are shown in Table S1.

In vivo studies

LC2/ad cells at 5.0 × 106 were subcutaneously inoculated to 8-week-old athymic nude mice (Clea Japan).[23] Vandetanib was administered once daily as a homogeneous suspension by oral gavage at a dosage of 50 mg/kg body weight.[24] The tumor volume was calculated as the product of a scaling factor (π/6) and the tumor length, width and height.[22] The study was approved by the Institutional Ethics Review Committee for animal experiments at the National Cancer Center.

Immunohistochemical analysis

The procedure for hematoxylin eosin staining and immunohistochemical (IHC) was previously described.[22, 25]

Microarray analysis

Background information of clinical samples was described in a previous report.[26] The study was approved by the Institutional Review Boards of the National Cancer Center. Total RNA was analyzed using Affymetrix (Santa Clara, CA, USA) U133Plus2.0 arrays. The data were processed by the MAS5 algorithm, and the mean expression level of a total of 54 675 probes was adjusted to 1000 for each sample.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Identification of the CCDC6-RET fusion gene in a Japanese LAD cell line

To identify RET fusion-derived mRNA expression in human LAD cell lines, all reported KIF5B-RET and CCDC6-RET gene products were screened by multiplex RT-PCR in 11 cell lines derived from Japanese patients. LC-2/ad cells were found to express CCDC6-RET mRNA at significantly higher levels, whereas the other cell lines did not exhibit any fusion gene products (Fig. 1a). The expressed fusion RET product was sequenced, and an in-frame fusion of CCDC6 exon 1 and RET exon 12, which was identical to the previously reported CCDC6-RET fusion products, was identified (Fig. 1b).[14] We then identified a breakpoint of chromosome 10 by retrieving genomic DNA fragments, including the entire RET gene and intron 1 of CCDC6, by target capture system followed by parallel sequencing. The identified break-point between CCDC6 intron 1 and RET exon 11 was confirmed by Sanger sequencing (Fig. 1b). Quantitative RT-PCR revealed that the expression of 3′ end of RET was increased comparable to that of CCDC6, whereas the transcript level of the 5′ end of RET was significantly lower (Fig. 1c). Consistent with the amount of transcript, western blotting using an antibody recognizing the C-terminus of RET isoform 2 detected a 60-kDa specific band equivalent to the estimated size of the fusion protein composed of 503 amino acids (GeneBank BAM36435), whereas no significant signal was detected that approximated the size of wild-type RET, 170-kDa (Fig. 1d).[11] Taken together, we concluded that LC-2/ad cells express CCDC6-RET fusion gene products. KRAS exon 2 and EGFR exon 19 and 21 were examined by Sanger sequencing, but no obvious mutation was confirmed (Fig. S1).

image

Figure 1. Identification of the CCDC6-RET fusion gene. (a) Detection of RET fusion transcripts in lung adenocarcinoma (LAD) cell lines by multiplex reverse transcription-polymerase chain reaction (RT-PCR). (b) Sanger sequencing around the fusion point of the cDNA (left) and the breakpoint of the genomic DNA (right) of CCDC6-RET in LC-2/ad cells. (c) 3′ region-specific expression of RET mRNA in LC-2/ad cells. The 5′ or 3′ region of RET and CCDC6 cDNA level was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. The data are shown as the mean ± standard deviation (SD) (n = 3). Asterisks indicate that mRNA expression were below the level of detection. (d) Specific expression of the CCDC6-RET fusion protein. Whole-cell lysates of LC2/ad and PC-9 cells and HEK293 cells transfected with wild-type RET (RET) or CCDC6-RET expression plasmids were subjected to western blot analysis to detect RET protein isoform 2. The LC-2/ad cells showed an approximately 60-kDa (red arrowhead) but not 170-kDa (blue arrowhead) band.

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CCDC6-RET-dependent ERK1/2 phosphorylation and the proliferation of LC-2/ad cells

We suppressed RET expression by RNAi to characterize the function of CCDC6-RET in LC-2/ad cells. For avoiding off-target siRNA effects, two different sequences of siRNA directed against the 3′ region of RET (siRET#1 and #2) and a nontargeting siRNA (siNC) were used. When compared to siNC, a significant reduction in mRNA expression was observed by quantitative RT-PCR detecting the 3′ end of the RET mRNA: 66.5% for siRET#1 and 94.2% for siRET#2 (Fig. S2). Western blot analyses also revealed significant decreases in the expression of CCDC6-RET protein (60-kDa) upon the introduction of siRET#1 and #2 compared to the control siNC in the LC-2/ad cells (Fig. 2a). To examine whether the downstream signaling pathway was altered by the introduction of siRNA, the phosphorylation of ERK1/2 and AKT was examined. The phospho-ERK1/2 signal was significantly decreased by the suppression of CCDC6-RET expression, whereas the decrease of AKT phosphorylation was marginal (Fig. 2a). The involvement of RET fusion in LC-2/ad cell proliferation was then examined. The number of live CCDC6-RET-suppressed cells decreased throughout the experiment, and the difference became significant at day 3 and thereafter (Fig. 2b). To address the growth suppression further, the cell cycle of the siRNA-treated cells were assessed by the DNA ploidy pattern. The LC-2/ad cells treated with siRET exhibited significant increases in the percent of cells arrested in the G1 phase relative to the cells treated with siNC (Fig. 2c). However, the apoptotic cells, as assessed by Annexin V positivity, was not significantly increased by the suppression of RET expression (Fig. 2d).

image

Figure 2. Suppression of CCDC-RET expression by siRNA in LC-2/ad cells. (a) Western blot analysis of siRET-treated LC-2/ad cells. The siRNA transfected cell lysates were applied to the western blotting. (b) Involvement of RET suppression in cell growth inhibition. LC-2/ad cells transfected with siRNAs were incubated for the indicated times. The data are shown as the mean ± standard deviation (SD) (n = 4). *P < 0.01 (Student's t-test). (c,d) The DNA ploidy (c) and Annexin V-positive population (d) of siRET-transfected LC-2/ad cells. After 72 h of siRNA transfection, the cells were subjected to DNA ploidy analysis and Annexin V staining. The data are shown as the mean ± SD (n = 4).

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RET-dependent transcriptome profile in LC-2/ad cell

To characterize the transcriptome profile, which is regulated by CCDC6-RET and its downstream signaling pathway, siRET#2 and siNC treated LC-2/ad cells were subjected to genome-wide expression profiling using Affymetrix U133Plus2.0 arrays. A total of 243 genes, evaluated with 285 probes were selected as those preferentially suppressed by less than half in siRET-treated cells.As well, 566 genes with 661 probes were expressed more than twice in siRET-treated cells (Table S2 and Fig. S3). The RET gene itself (probe ID = 211421_s_at) showed the highest fold-difference of 19.6 between siNC- and siRET#2-treated cells. Following RET, previously identified Gene Ontology-annotated Ras-MAPK downstream genes like DUSP6 was preferentially suppressed in the siRET-treated cells. In addition, cell cycle regulation-related genes like EREG, CDC6, MCM10, MAD2L1, CHEK1 and PLK4 were expressed <0.5-fold in siRET-treated cells (Table 1).

Table 1. Up- or downregulated genes associated with mitogen-activated protein kinase (MAPK) cascade or cell cycle
Gene symbolProbe set IDsiNC/siRETTumor/Non-tumor
Upregulated
RET 211421_s_at19.6319.52
205879_x_at3.765.03
215771_x_at2.374.72
DUSP6 208892_s_at4.455.22
208893_s_at4.176.34
208891_at4.173.56
EREG 1569583_at3.681.60
205767_at2.935.69
CDC6 203967_at2.424.82
203968_s_at1.955.32
MCM10 220651_s_at2.304.83
223570_at1.721.71
MAD2L1 203362_s_at2.285.91
1554768_a_at1.914.34
CHEK1 205394_at2.179.03
205393_s_at2.146.87
PLK4 204886_at2.074.38
204887_s_at1.564.08
Downregulated
MEF2C 209200_at0.210.46
209199_s_at0.260.65
GAB1 214987_at0.230.42
229114_at0.530.65
225998_at0.620.68
226002_at0.640.76
CDKN1C 216894_x_at0.260.41
213348_at0.320.23
213183_s_at0.350.30
219534_x_at0.420.27
213182_x_at0.440.21
PTEN 233314_at0.330.27
225363_at0.770.47
TIMP2 231579_s_at0.340.33
224560_at0.370.27
ID2 201566_x_at0.350.31
201565_s_at0.400.39
213931_at0.520.31
CCNL2 232274_at0.350.42
222999_s_at0.790.52
RPS6KA2 212912_at0.410.34
204906_at0.590.49

RET fusion gene screening of 300 consecutive surgically resected LAD samples identified one case of CCDC6-RET expressing LAD by RT-PCR and break-apart FISH (Tsuta et al., 2012, unpublished data). We checked the expression level of potential CCDC6-RET-driven genes identified above in the clinical sample. Among 285 preferentially expressed probes, 81 probes were also upregulated more than twofold in the CCDC6-RET positive LAD tissue compared to the surrounding non-cancerous tissue (Table 1 and Table S2).

RET inhibitor-induced cell cycle arrest and apoptosis in LC-2/ad cells

The phosphorylation status of the tyrosine 905 residue of RET isoforms 2 and 4 was high in the LC-2/ad cells, regardless of the presence or absence of serum in the culture medium, whereas the total amount of RET isoform 2 was not significantly altered. Similarly, the phosphorylation status of AKT and ERK1/2 was high under serum-starved conditions, and the enhanced phosphorylation of these molecules was slight with serum stimulation, suggesting that the fusion RET kinase was constitutively active and activated its downstream signaling pathways (Fig. 3a).

image

Figure 3. Effect of RET inhibitors on LC-2/ad cells. (a) Western blot analysis of inhibitor-treated cells. The cells were incubated under serum-starved conditions for 22 h and treated with 1 μM of inhibitor or dimethylsulfoxide (DMSO) for 2 h. Prior to cell lysis, the cells were treated with 10% fetal bovine serum (FBS) for 10 min. Whole-cell lysates were subjected to western blot analysis to detect the indicated proteins. G, gefitinib; So, sorafenib; Su, sunitinib; V, vandetanib. (b) Dose-dependent effect of vandetanib. Cells were treated with the indicated concentration of vandetanib for 12 h, and western blotting was used to detect the indicated proteins. (c) WST-8 assay with kinase inhibitors. Cells were treated with the indicated inhibitors for 72 h, and the viability was assessed using the WST-8 assay. The data are shown as the mean ± standard deviation (SD) (n = 6). (d) Effect of vandetanib for growth inhibition. Cells were treated with vandetanib and incubated for the indicated time. The data are shown as the mean ± SD (n = 3). *P < 0.01 (Student's t test). (e,f) DNA ploidy (e) and Annexin V-positive population (f) of the cells treated with vandetanib for 48 h. The data are shown as the mean ± SD (n = 4).

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Next, the effects of kinase inhibitors, which inhibit spectrum including RET were applied to evaluate their effects on the signaling pathways in the LC-2/ad cells. We treated the cells with RET inhibitors vandetanib, sunitinib and sorafenib at a final concentration of 10 μM, which was 10–30 times higher than the in vitro half maximal inhibitory concentration (IC50) for RET kinase activity of each compound. Gefitinib, another small molecule inhibitor targeting EGFR but not RET,[13] was also examined. All the inhibitors except gefitinib significantly suppressed the phosphorylation of RET, AKT and ERK1/2. Although vandetanib, sunitinib and sorafenib equivalently suppressed RET phosphorylation, vandetanib most significantly suppressed the phosphorylation of ERK1/2 (Fig. 3a). The inhibitory effect of vandetanib on RET, AKT and ERK1/2 phosphorylation exhibited concentration dependency (Fig. 3b). Gefitinib significantly suppressed EGFR phosphorylation while total EGFR protein level was not altered. Meanwhile, gefitinib did not alter the phosphorylation status of AKT and ERK1/2 (Fig. 3a). Meanwhile, vandetanib suppressed EGFR as well as AKT and ERK1/2 in EGFR-mutant PC-9 cells (Fig. S4).

We further examined the effect of the above inhibitors on the growth of the LC-2/ad cells using the WST-8 assay. Consistent with the effects of the inhibitors on the RET signaling pathway, vandetanib suppressed cell growth most significantly (IC50 = 0.32 μM), followed by sunitinib and sorafenib, whereas gefitinib only exhibited an apparent suppression at its highest dose (Fig. 3c). However, the effects of these inhibitors on KRAS-mutant A549 cells were much lower (Fig. S5). Gefitinib and vandetanib, both of which inhibit EGFR, suppressed EGFR-mutant PC-9 cells, whereas sunitinib and sorafenib had less effect (Fig. S5). Evaluating the number of live cells by trypan blue staining under the treatment of several doses of vandetanib suggested a dose-dependent suppression in the LC-2/ad cells. Furthermore, the number of cells treated with 0.5 and 1.0 μM vandetanib was apparently reduced to less than the starting amount, strongly suggesting that vandetanib induced both cell death and the suppression of cell proliferation (Fig. 3d). An assessment of the DNA ploidy revealed that vandetanib arrested the cell cycle in G1 phase in a dose-dependent manner (Fig. 3e), and an increased concentration of vandetanib induced an Annexin V-positive apoptotic cell population (Fig. 3f). The proapoptotic effect of vandetanib was confirmed by the detection of cleaved caspase-3 by western blotting (Fig. 3b). Meanwhile, 1.0 μM sunitinib and sorafenib induced cell cycle arrest but induction of apoptosis was marginal (Figs S6 and S7).

To further evaluate the contribution of Ras-ERK and AKT axes to cell survival, LC-2/ad cells were treated with MEK1/2 inhibitor AZD6244 or PI3K/mTOR inhibitor BEZ235. Cytotoxic effect of AKT-inhibiting BEZ235 was more than that of ERK-inhibiting AZD6244. However, both inhibitors did not completely reduce the cell survival even their maximal dose (Figs S8 and S9).

Anti-tumor effect of vandetanib in an LC-2/ad xenograft model

Subcutaneously transplanted LC-2/ad tumors exhibited typical adenocarcinoma morphology. These tumors were positive for SFTPA, Napsin A and carcinoembryonic antigen (CEA) but thyroid marker thyroglobulin negative using immunohistochemistry (IHC). Furthermore, using an antibody cross-reacting with both human and mouse RET protein, IHC revealed that RET was highly expressed specifically in the tumor cells but not in the interstitial cells (Fig. 4a). The overexpression of RET in these tumors was confirmed using quantitative RT-PCR and Western blotting. Similar to the results from cultured LC-2/ad cells, much more mRNA of the 3′ end of RET was detected than that of the 5′ end (Fig. 4b), and a specific band equivalent to the size of the CCDC6-RET fusion protein was detected (Fig. 4c). Vandetanib (50 mg/kg) was orally administrated to the mice harboring the LC-2/ad xenograft, and the daily administration of vandetanib significantly reduced the tumor size. Although the tumors were diminished at day 14 of the treatment, the body weight of the treated mice was not significantly reduced (Fig. 4d and Fig. S10). Sorafenib (30 mg/kg) and sunitinib (40 mg/kg) did not reduce the body weight, either (Fig. S10). Sorafenib reduced but not diminished the tumors at day14. Anti-tumor effect of sunitinib was not significant (Fig. S11).

image

Figure 4. Characterization of the LC-2/ad xenograft and anti-tumor effects of vandetanib. (a) Histological features of the xenograft. Hematoxylin and eosin staining and immunohistochemical staining with the indicated antibodies. Scale bars were 100 μm. Hematoxylin eosin (HE), RET, thyroglobulin (TG) and carcinoembryonic antigen (CEA) (×20); Napsin A and Sp-A (×40). (b) 3′ region-specific expression of RET mRNA in the xenograft. Total RNA extracted from tumors was subjected to real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis with the primer sets designed for the 5′ or 3′ region of the RET and CCDC6 cDNA. The data are shown as the mean ± standard deviation (SD) (n = 3). (c) Expression of the CCDC6-RET protein in mice xenografts. Whole-cell lysates of tumors were subjected to western blot analysis. (d) Anti-tumor effect of vandetanib in vivo. Vandetanib was administered once a day at a dosage of 50 mg/kg. The data are shown as the mean ± SD (n = 9). *< 0.01 (control vs sorafenib; Student's t test).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Previous reports suggest that the incidence of RET-fusion-positive cases in LAD is 1–2% and that these cases are concentrated in the EGFR mutation-, KRAS mutation-, and ALK-fusion-negative population.[10, 27] To identify cell lines expressing endogenous RET-fusion genes, we selected 11 cell lines that were derived from pathologically identified Japanese LAD cases. Among them, activating EGFR mutations have been reported in PC-3 and PC-9 cells.[28] However, the mutation status of known driver genes of other cell lines was not well investigated. The LC-2/ad cells were originally derived from pleural effusion of LAD in a patient who had received combined chemotherapy (endoxan, Adriamycin, Cisplatin and mitomycin C)[23]; the cancer was diagnosed by cytological examination of the patient's sputum and pleural effusion. The original report indicated that the LC-2/ad cells were positive for an adenocarcinoma marker, cytokeratin 18.[23] In addition, we detected surfactant protein, an aspartate proteinase, Napsin A, and CEA expression in the xenograft tumor (Fig. 4a). These findings support the origin of LC-2/ad as lung adenocarcinoma. The modal chromosome number described in the original report was 53–56, though an apparent translocation between the chromosomes was not reported, consistent with the fact that the inversion of chromosome 10 was not obvious in the conventional chromosome counts.

The Sanger sequencing in this study and the whole-transcriptome sequencing (Tsuchihara, 2012, unpublished data) revealed no driver mutations of KRAS, EGFR and known genes other than the CCDC6-RET fusion in the LC-2/ad cells, highly suggesting that the CCDC6-RET fusion protein plays pivotal roles in the proliferation of these cells. The autophosphorylation of CCDC6-RET was clearly observed in a serum-independent manner, accompanied with a constitutive elevation of ERK1/2 phosphorylation. The suppression of CCDC6-RET expression induced a decrease in ERK1/2 phosphorylation, accompanied with a decrease in the expression of the genes that regulate the cell cycle. As a result, the CCDC6-RET-suppressed cells exhibited significant growth retardation.

Recently, a Japanese group independently reported the CCDC6-RET fusion in LC2/ad cells.[21] However, the efficacy of RET inhibitors to the RET and downstream pathways and in vivo anti-tumor effects have been partially described.[21] Vandetanib, sorafenib and sunitinib suppress the activities of multiple kinases, including RET, and have been approved for several cancers.[29-31] In in vitro analyses, these compounds effectively suppressed the phosphorylation of CCDC6-RET and suppressed proliferation and induced death in LC-2/ad cells. It should be noted that the IC50 value for the growth suppression of these compounds was equivalent to the dose suggested in a previous study using culture cells expressing ectopic KIF5B-RET cDNA.[13] These effects were most likely dependent on RET inhibition. Sunitinib and sorafenib did not affect PC-9 and A549 cells, which have activating mutations of EGFR and KRAS, respectively. Vandetanib presumably suppressed the growth of PC-9 cells, as EGFR is included in its inhibitory spectrum. Meanwhile, gefitinib, which targets EGFR but not RET, did not significantly suppress the growth of LC-2/ad cells. Interestingly, gefitinib did not alter the phosphorylation of AKT and ERK1/2 in LC-2/ad cells albeit equivalently suppressing EGFR phosphorylation as vandetanib. Although precise molecular mechanisms should be further examined, LC-2/ad cells might not depend on EGFR for transducing downstream signaling.

Vandetanib exhibited apparent anti-tumor effects in the xenograft model in this study. Recently, efficacy of vandetanib on thyroid cancer cells harboring RET-fusion gene was also reported.[32] These findings strongly suggest that RET inhibition is a plausible therapeutic strategy for RET-fusion-positive tumors.

We noticed a discrepancy between the effects of RNA interference and inhibitor treatment on RET. Though RET suppression/inhibition equivalently reduced the level of phosphorylated RET and induced cell cycle arrest, obvious apoptosis was not found in the cells treated with siRNA. A possible explanation is that CCDC6-RET is mainly involved in the RAS-ERK pathway to regulate cell proliferation, whereas the anti-apoptotic signaling pathway mediated by AKT could be regulated by other signaling molecules inhibited by the multi-kinase inhibitors. A recent study using a Drosophila in vivo screening system suggested that the antitumor effects and toxicity of RET inhibitors were dependent on the profile of the “off-target” inhibition of multiple kinases in addition to the specific inhibition of RET.[33] Further investigation elucidating the molecules and signaling pathways relevant to the cytotoxic effect of vandetanib in LC-2/ad cells is anticipated.

Whether LC-2/ad-based models adequately represent clinical RET fusion-positive LAD cases is another challenging question. Takeuchi stated that clinically identified CCDC6-RET-positive LAD exhibited a histologically cribriform pattern.[14] Because the cribriform structure was presumably developed from normal alveolar architecture, this specific morphology was not observed in the subcutaneously transplanted LC-2/ad tumors. We assume that the comparison of the transcriptome profile between the LC-2/ad cells and clinically identified LAD tissue samples may provide clues. Approximately one-third of the genes suppressed by RNA interference directed at RET overlapped with the genes preferentially expressed in the clinical tumor sample. Because we have had only one example of paired data, it is difficult to estimate the similarity between the cell line and clinical samples. However, the above overlap appears promising, and we will continue to screen both cell lines and clinical samples to accumulate comprehensive data.

In this study, the screening of Japanese LAD cell lines was effective for the identification of RET fusion-positive cancer cells, representing a clinically rare subpopulation. LC-2/ad cells might be useful in the development of RET-targeted therapies, that is, new compound screening, clarifying the pharmacological mechanisms and investigating the mechanisms for acquired resistance.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

We thank Drs Hiroki Sasaki and Kazuhiko Aoyagi and the National Cancer Center Research Core Facility for the microarray analyses. The Core Facility was supported by National Cancer Center Research and Development Fund (23-A-7). This work was supported by National Cancer Center Research and Development Fund (23-A-8, 15, 24-A-1) and JSPS KAKENHI Grant number 24300345.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
cas12175-sup-0001-DataS1-FigS1-S11.docxWord document1640K

Data S1. Materials and methods.

Fig. S1. The absence of the known driver mutations.

Fig. S2. Suppression of RET mRNA in siRET-treated cells.

Fig. S3. RET-dependent transcriptome profile in LC-2/ad cells.

Fig. S4. Dose-dependent effect of vandetanib in PC-9 cells.

Fig. S5. WST-8 assay with various kinase inhibitors.

Fig. S6. Effect of sunitinib and sorafenib on G1 phase population of LC-2/ad cells.

Fig. S7. Effect of sunitinib and sorafenib on apoptosis of LC-2/ad cells.

Fig. S8. Dose-dependent effect of AZD6244 and BEZ235 in LC-2/ad cells.

Fig. S9. WST-8 assay of LC-2/ad cells treated with AZD6244 and BEZ235.

Fig. S10. Body weight of the vandetanib-, sunitinib-, sorafenib- and vehicle-treated mice.

Fig. S11. Effect of sunitinib and sorafenib in vivo.

cas12175-sup-0002-TableS1.docxWord document21KTable S1. Polymerase chain reaction primers.
cas12175-sup-0003-TableS2.xlsapplication/msexcel292KTable S2. Summary of the microarray data.

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