In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-014-9736-3

Establishment and characterization of a lung cancer cell line, SMC-L001, from a lung adenocarcinoma So-Jung Choi & Hyeseon Lee & Chungyoul Choe & Yong-Sung Shin & Jinseon Lee & Sung-Hwan Moon & Jhingook Kim

Received: 24 October 2013 / Accepted: 21 January 2014 / Editor: T. Okamoto # The Society for In Vitro Biology 2014

Abstract Lung cancer cell lines are a valuable tool for elucidating lung tumorigenesis and developing novel therapies. However, the majority of cell lines currently available were established from tumors in patients of Caucasian origin, limiting our ability to investigate how cancers in patients of different ethnicities differ from one another in terms of tumor biology and drug responses. In this study, we established a human non-small cell lung carcinoma cell line, SMC-L001, and characterized its genome and tumorigenic potential. SMC-L001 cells were isolated from a Korean lung adenocarcinoma patient (male, pStage IIb) and were propagated in culture. SMC-L001 cells were adherent. DNA fingerprinting analysis indicated that the SMC-L001 cell line originated from parental tumor tissue. Comparison of the genomic profile of the SMC-L001 cell line and the original tumor revealed an identical profile with 739 mutations in 46 cancer-related genes, including mutations in TP53 and KRAS. Furthermore, SMC-L001 cells were highly tumorigenic, as evidenced by the induction of solid tumors in immunodeficient mice. In summary, we established a new lung cancer cell line with point mutations in TP53 and KRAS from a Korean lung adenocarcinoma patient that will be useful for

S.17 μM with erlotinib (Fig. 4).

Table 2. DNA fingerprinting analysis of the SMC-L001 cell line and parental tumor tissue using 16 STR loci (15 short tandem repeat loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, Vwa, TPOX, D18S51, D5S818, FGA) and a segment of the X–Y homologous gene (amelogenin)) Sample

Tumor tissue

SMC-L001

D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 Vwa TPOX

13, 16 29, 32.2 8, 11 10, 12 15, 16 6, 7.3, 9 10, 11 9, 12 18, 24 14, 15 17, 18 8, 11

13, 16 29, 32.2 8, 11 10, 12 15, 16 6, 9 11 9, 12 18, 24 14, 15 17, 18 8, 11

D18S51 Amelogenin D5S818 FGA

12, 13 X, Y 11, 13 22, 24

13, 14 X, Y 11, 13 22, 24

Numbers indicate the number of short tandem repeats at STR loci on each chromosome

Conclusions. We established the SMC-L001 lung adenocarcinoma cell line from the lung tumor tissue of a Korean male lung cancer patient and confirmed that the cell line reflected the molecular characteristics of the original tumor. Ion torrent analysis showed that the SMC-L001 cell line and parental tumor tissues had point mutations in the tumor suppressor TP53 (R141H) and oncogene KRAS (G12V) genes. Mutations in EGFR, KRAS, and anaplastic lymphoma kinase (ALK) are mutually exclusive in patients with NSCLC (Choi et al. 2013). In agreement with this observation, the SMCL001 cell line did not show mutations in EGFR and ALK genes. Mutations in the TP53 gene are extremely frequent in SCLC (75%–100%), whereas in NSCLC, their frequency varies among histological types (Soussi and Beroud 2001; Olivier et al. 2002). To the best of our knowledge, this is the

CHOI ET AL. Table 3. Forty-six gene ion torrent cancer panel analysis for the SMC-L001 cell line and parental tumor tissue and adjacent normal tissue Sample

Chrom Gene Sym

Ploidy Ref Variant Var freq

Amino acid Exon REF_ID change number

Total Ref cov Var cov coverage

SMC-L001 Adjacent normal tissue Tumor tissue SMC-L001

Chr12 Chr12 Chr12 Chr17

KRAS KRAS KRAS TP53

Het Hom He Hom

C C C C

A A A T

65.36 0.31 50.92 99.8

p.G12V – p.G12V p.R141H

Exon2 Exon2 Exon2 Exon4

NM_033360 NM_033360 NM_033360 NM_001126115

3,805 1,301 2,019 2,023

1,317 1,294 990 4

2,487 4 1,028 2,019

TP53 TP53

Hom Het

C C

T T

0.25 – 34.91 p.R141H

Exon4 Exon4

NM_001126115 1,576 NM_001126115 2,028

1,572 1,320

4 708

Adjacent normal tissue Chr17 Tumor tissue Chr17

Mutations were validated by conventional Sanger sequencing using an ABI 3730 genetic analyzer (Macrogen, Seoul, Korea)

first report of an Asian cell line with point mutations in TP53 and KRAS. We believe that SMC-L001 cell line is of value in understanding the mechanisms underpinning the EGFR TKI therapy. EGFR TKI erlotinib is one of the most targeted drugs and important for lung cancer treatment. However, lung cancer patients who are KRAS mutation positive have a low response rate to erlotinib, which is estimated at 5% or less (Wistuba et al. 1999). Furthermore, erlotinib was highly effective to lung cancer patients harboring EGFR mutations. Consistent with these findings, SMC-L001 cell lines with EGFR wild type and KRAS mutation was resistant to EGFR TKI erlotinib.

Figure 3. Tumor formation ability of SMC-L001 primary lung cancer cells in nude mice. (a) Transplantation of cells at day 42. (b) Extract of tumor cells, (c) histological analysis of tumor tissue after H&E staining of the xenograft tumor and (d) the parental tumor tissue (magnification ×200).

KRAS is one of the most frequent mutations in lung cancer (Hingorani et al. 2005; Mitsudomi et al. 2013). Since KRAS mutations have been found frequently in NSCLC, KRAS mutations are found in 20–30% of NSCLC cases, predominately in adenocarcinomas (Sun et al. 2010). In NSCLC, the majority of KRAS mutations involves codons 12 or 13 (Pao et al. 2005). Many studies have observed lower efficacy of EGFR TKI therapy in KRAS-mutated NSCLC patients (Benesova et al. 2010; Suda et al. 2010). The reason for this is that the mutated KRAS proteins exhibit impaired ability to switch between active and inactive states of GTPase activity, resulting in constitutive activation of RAS signaling (Martin et al.

ESTABLISHMENT AND CHARACTERIZATION OF A LUNG CANCER CELL LINE

Viability (% of control)

125

inhibitor 17-AAG. However, this hypothesis remains to be tested in the future. In summary, we established the lung adenocarcinomaderived cell line SMC-L001. This cell line represents a model system for further studies of lung adenocarcinoma and the development and testing of molecular therapies targeted specifically to NSCLCs in Asian populations. This cell line was deposited to the Korean Cell Line Bank (KCLB, http:// cellbank.snu.ac.kr) and widely available to the scientific community through the KCLB.

100

75

50

25

cisplatin erlotinib PHA665752 17-AAG

0 -1.0

-0.5

0.0

0.5

1.0

1.5

Log[drug], uM IC50(uM)

cisplatin

erlotinib

PHA665752

17-AAG

SMC-L001

10

~17

5.12

3.2

Figure 4. Analysis of the chemosensitivity of SMC-L001 cells to cisplatin, erlotinib, PHA665752, and 17 AAG.

2013). Since KRAS is downstream of EGFR signaling pathway, constitutive activation of KRAS confer resistance to EGFR TKI therapy (Roberts and Stinchcombe 2013). The presence of EGFR mutations and KRAS mutations are mutually exclusive in the same tumor (Govindan et al. 2012). The development of therapeutics targeting mutated KRAS signaling is under intensive investigation. However, since mutant KRAS proteins entail loss of function, current approaches try either to inhibit the expression of mutated KRAS gene or to impede downstream effectors of mutant KRAS protein. We believe that the SMC-L001 lung cancer cell line with genome information as well as clinical information will be useful tools for the development of treatment for lung cancer with KRAS mutation. However, the mechanism by which KRAS mutation contributes to EGFR TKI remains to be elucidated in the future. Intriguingly, SMC-L001 cells were resistant to anti-tumor agent 17-AAG and BHA665752, with IC50 values of 3.2 and 5.12 μM, respectively. Given the observation that these drugs are effective in nanomolar range, the presence of TP53 mutation in SMC-L001 cells may be responsible for such resistance. In support of this idea, studies reported that gain of function mutations in TP53 are associated with drug resistance in several malignancies and cell lines (Bergh et al. 1995; Aas et al. 1996; Blandino et al. 1999; Brosh and Rotter 2009). Indeed, mutant p53 is known to interact with the molecular chaperon HSP90, leading to stabilization of p53 (Hinds et al. 1987; Blagosklonny et al. 1996; Sepehrnia et al. 1996; Whitesell et al. 1998). These findings led to the hypothesis that mutant p53 destabilization upon HSP90 with 17AAG attributes to the observed resistance to HSP90

Acknowledgments We thank Ga-Yun Kim for their technical assistance. This study was supported by a grant from the Seoul R&BD Program (SS100010) and the Converging Research Center Program funded by the Ministry of Science, ICT and Future Planning (Project no.2013K000278).

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