Cancer Res Treat.  2017 Oct;49(4):1065-1076. 10.4143/crt.2016.347.

Prevalence of Mutations in Discoidin Domain-Containing Receptor Tyrosine Kinase 2 (DDR2) in Squamous Cell Lung Cancers in Korean Patients

• Lee MS
• Jung EA
• An SB
• Kim YJ
• Oh DY
• Song JY
• Um SW
• Han J
• Choi YL

• KMID: 2394825
• DOI: http://doi.org/10.4143/crt.2016.347

Abstract

PURPOSE
The discoidin domain-containing receptor tyrosine kinase 2 (DDR2) is known to contain mutations in a small subset of patients with squamous cell carcinomas (SCC) of the lung. Studying the DDR2 mutations in patients with SCC of the lung would advance our understanding and guide the development of therapeutic strategies against lung cancer.
MATERIALS AND METHODS
We selected 100 samples through a preliminary genetic screen, including specimens from biopsies and surgical resection, and confirmed SCC by histologic examination. DDR2 mutations on exons 6, 15, 16, and 18 were analyzed by Sanger sequencing of formalin-fixed, paraffin-embedded tissue samples. The functional effects of novel DDR2 mutants were confirmed by in vitro assays.
RESULTS
We identified novel somatic mutations of DDR2 in two of the 100 SCC samples studied. One mutation was c.1745T>A (p.V582E) and the other was c.1784T>C (p.L595P), and both were on exon 15. Both patients were smokers and EGFR/KRAS/ALK-triple negative. The expression of the mutant DDR2 induced activation of DDR2 by the collagen ligand and caused enhanced cell growth and tumor progression. Moreover, dasatinib, a DDR2 inhibitor, showed potential efficacy against DDR2 L595P mutant-bearing cells.
CONCLUSION
Our results suggest that a mutation in DDR2 occurs naturally with a frequency of about 2% in Korean lung SCC patients. In addition, we showed that each of the novel DDR2 mutations were located in a kinase domain and induced an increase in cell proliferation rate.

Keyword

Discoidin domain receptor 2; Squamous cell carcinoma; Lung neoplasms; Somatic mutations

MeSH Terms

Biopsy
Carcinoma, Squamous Cell
Cell Proliferation
Collagen
Dasatinib
Epithelial Cells*
Exons
Humans
In Vitro Techniques
Lung Neoplasms*
Lung*
Phosphotransferases
Prevalence*
Protein-Tyrosine Kinases*
TYK2 Kinase*
Tyrosine*
Collagen
Dasatinib
Phosphotransferases
Protein-Tyrosine Kinases
TYK2 Kinase
Tyrosine

Figure

• 

  Fig. 1. Radiological and histological features and discoidin domain-containing receptor tyrosine kinase 2 (DDR2) mutation analysis of case 1. (A) Chest computed tomography (CT) in 2009 shows a 3 cm nonenhancing mass-like consolidation with a background of patchy fibrotic changes and scattered calcified and noncalcified sub-centimeter nodules in both lungs. (B) Chest CT in 2010 shows that the mass-like consolidation has increased in size to 6.2 cm. Numerous new cavitary and noncavitary nodules can be seen in both lungs. (C) Squamous cell carcinoma on hematoxylin and eosin section (×200). (D) Chromatogram of DDR2 shows a missense mutation, as depicted by the arrows, with a change in the amino acids from GTG (valine) to GAG (glutamic acid) on exon 15.

• 

  Fig. 2. Radiological and histological features and discoidin domain-containing receptor tyrosine kinase 2 (DDR2) mutation analysis of case 2. (A) Chest computed tomography scan showing a 5.3-cm necrotic cavitary mass. (B, C) Squamous cell carcinoma on hematoxylin and eosin section (×40 and ×200, respectively). (D) p63 staining showing nuclear staining of tumor cells. (E) Thyroid transcription factor 1 staining showing negative staining of tumor cells. (F) Chromatogram of DDR2 showing a missense mutation, as depicted by the arrows, with a change in the amino acids from CTA (leucine) to CCA (proline) on exon 15.

• 

  Fig. 3. Functional effects of discoidin domain-containing receptor tyrosine kinase 2 (DDR2) mutants observed through in vitro assays. (A) Schematic view shows a known mutation and novel mutations (red circles) within the kinase domain of DDR2. (B) Activation of DDR2 and Src molecules by the ectopic expression of DDR2 point mutants. NIH3T3 cells were stably expressed with the indicated DDR2 mutant constructs, stimulated with 10 μg/mL collagen, and then analyzed using western blot. WT, wild type. (C) In vitro transforming assay in soft agar. NIH3T3 cells expressing the indicated mutant protein were seeded in 6-well plates and cultured for 14 days in soft agar. The number of colonies formed per well, indicated as the mean±standard deviation, from the three wells in one representative experiment out of three. ***p < 0.001, scale bars=10 μm. (D) The effect of dasatinib in cells expressing DDR2 mutants. Dasatinib caused reduced proliferation and a decrease in phosphorylation of Src in cells expressing DDR2 V582E and L595P. In the left panel, the NIH3T3 cells expressing the DDR2 mutants were treated with the indicated doses of dasatinib for 72 hours, and cell viability was determined. ***p < 0.001. In the right panel, cells were treated with 0.1 μM dasatinib for 24 hours, followed by cell lysis and detection of the indicated protein using western blot. (E) In vitro colony-forming ability of cells expressing DDR2 mutants following dasatinib treatment. NIH3T3 cells expressing the DDR2 mutants were seeded in soft agar and cultured with or without dasatinib for 7 days. The colonies formed were stained with 0.001% crystal violet and the number of colonies was estimated. The images were obtained using a phase-contrast microscope at 40× magnification. The values shown represent the average of three independent experiments and error bars indicate standard deviations. *p < 0.05, ***p < 0.001.

Reference

References

1. Global Burden of Disease Cancer Collaboration, Fitzmaurice C, Dicker D, Pain A, Hamavid H, Moradi-Lakeh M, et al. The Global Burden of Cancer 2013. JAMA Oncol. 2015; 1:505–27.

2. Langer CJ, Besse B, Gualberto A, Brambilla E, Soria JC. The evolving role of histology in the management of advanced non-small-cell lung cancer. J Clin Oncol. 2010; 28:5311–20.

3. Hammerman PS, Sos ML, Ramos AH, Xu C, Dutt A, Zhou W, et al. Mutations in the DDR2 kinase gene identify a novel therapeutic target in squamous cell lung cancer. Cancer Discov. 2011; 1:78–89.

4. Belvedere O, Berri S, Chalkley R, Conway C, Barbone F, Pisa F, et al. A computational index derived from whole-genome copy number analysis is a novel tool for prognosis in early stage lung squamous cell carcinoma. Genomics. 2012; 99:18–24.

5. Kim Y, Hammerman PS, Kim J, Yoon JA, Lee Y, Sun JM, et al. Integrative and comparative genomic analysis of lung squamous cell carcinomas in East Asian patients. J Clin Oncol. 2014; 32:121–8.

6. Miyamae Y, Shimizu K, Mitani Y, Araki T, Kawai Y, Baba M, et al. Mutation detection of epidermal growth factor receptor and KRAS genes using the smart amplification process version 2 from formalin-fixed, paraffin-embedded lung cancer tissue. J Mol Diagn. 2010; 12:257–64.

7. Friboulet L, Li N, Katayama R, Lee CC, Gainor JF, Crystal AS, et al. The ALK inhibitor ceritinib overcomes crizotinib resistance in non-small cell lung cancer. Cancer Discov. 2014; 4:662–73.

8. Ding L, Getz G, Wheeler DA, Mardis ER, McLellan MD, Cibulskis K, et al. Somatic mutations affect key pathways in lung adenocarcinoma. Nature. 2008; 455:1069–75.

9. Tyner JW, Fletcher LB, Wang EQ, Yang WF, Rutenberg-Schoenberg ML, Beadling C, et al. MET receptor sequence variants R970C and T992I lack transforming capacity. Cancer Res. 2010; 70:6233–7.

10. Heist RS, Mino-Kenudson M, Sequist LV, Tammireddy S, Morrissey L, Christiani DC, et al. FGFR1 amplification in squamous cell carcinoma of the lung. J Thorac Oncol. 2012; 7:1775–80.

11. Weiss J, Sos ML, Seidel D, Peifer M, Zander T, Heuckmann JM, et al. Frequent and focal FGFR1 amplification associates with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. Sci Transl Med. 2010; 2:62ra93.

12. Ramsey MR, He L, Forster N, Ory B, Ellisen LW. Physical association of HDAC1 and HDAC2 with p63 mediates transcriptional repression and tumor maintenance in squamous cell carcinoma. Cancer Res. 2011; 71:4373–9.

13. Labrador JP, Azcoitia V, Tuckermann J, Lin C, Olaso E, Manes S, et al. The collagen receptor DDR2 regulates proliferation and its elimination leads to dwarfism. EMBO Rep. 2001; 2:446–52.

14. Kawai I, Hisaki T, Sugiura K, Naito K, Kano K. Discoidin domain receptor 2 (DDR2) regulates proliferation of endochondral cells in mice. Biochem Biophys Res Commun. 2012; 427:611–7.

15. Xu C, Buczkowski KA, Zhang Y, Asahina H, Beauchamp EM, Terai H, et al. NSCLC Driven by DDR2 Mutation Is Sensitive to Dasatinib and JQ1 Combination Therapy. Mol Cancer Ther. 2015; 14:2382–9.

16. Rammal H, Saby C, Magnien K, Van-Gulick L, Garnotel R, Buache E, et al. Discoidin Domain Receptors: Potential Actors and Targets in Cancer. Front Pharmacol. 2016; 7:55.

17. Hedberg ML, Goh G, Chiosea SI, Bauman JE, Freilino ML, Zeng Y, et al. Genetic landscape of metastatic and recurrent head and neck squamous cell carcinoma. J Clin Invest. 2016; 126:1606.

18. Iwai LK, Payne LS, Luczynski MT, Chang F, Xu H, Clinton RW, et al. Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants. Biochem J. 2013; 454:501–13.

19. Beauchamp EM, Woods BA, Dulak AM, Tan L, Xu C, Gray NS, et al. Acquired resistance to dasatinib in lung cancer cell lines conferred by DDR2 gatekeeper mutation and NF1 loss. Mol Cancer Ther. 2014; 13:475–82.

20. Terai H, Tan L, Beauchamp EM, Hatcher JM, Liu Q, Meyerson M, et al. Characterization of DDR2 Inhibitors for the Treatment of DDR2 Mutated Nonsmall Cell Lung Cancer. ACS Chem Biol. 2015; 10:2687–96.

21. Pitini V, Arrigo C, Di Mirto C, Mondello P, Altavilla G. Response to dasatinib in a patient with SQCC of the lung harboring a discoid-receptor-2 and synchronous chronic myelogenous leukemia. Lung Cancer. 2013; 82:171–2.

22. Rothschild SI. Targeted Therapies in Non-Small Cell Lung Cancer-Beyond EGFR and ALK. Cancers (Basel). 2015; 7:930–49.

23. Yashima H, Shimizu K, Araki T, Aomori T, Ohtaki Y, Nagashima T, et al. Assessment of DDR2, BRAF, EGFR and KRAS mutations as therapeutic targets in non-adenocarcinoma lung cancer patients. Mol Clin Oncol. 2014; 2:714–8.

24. Payne LS, Huang PH. Discoidin domain receptor 2 signaling networks and therapy in lung cancer. J Thorac Oncol. 2014; 9:900–4.

25. Sasaki H, Shitara M, Yokota K, Okuda K, Hikosaka Y, Moriyama S, et al. DDR2 polymorphisms and mRNA expression in lung cancers of Japanese patients. Oncol Lett. 2012; 4:33–7.

26. Miao L, Wang Y, Zhu S, Shi M, Li Y, Ding J, et al. Identification of novel driver mutations of the discoidin domain receptor 2 (DDR2) gene in squamous cell lung cancer of Chinese patients. BMC Cancer. 2014; 14:369.

27. Shin A, Oh CM, Kim BW, Woo H, Won YJ, Lee JS. Lung cancer epidemiology in Korea. Cancer Res Treat. 2017; 49:616–26.

28. Ichikawa O, Osawa M, Nishida N, Goshima N, Nomura N, Shimada I. Structural basis of the collagen-binding mode of discoidin domain receptor 2. EMBO J. 2007; 26:4168–76.

29. Terashima M, Togashi Y, Sato K, Mizuuchi H, Sakai K, Suda K, et al. Functional analyses of mutations in receptor tyrosine kinase genes in non-small cell lung cancer: double-edged sword of DDR2. Clin Cancer Res. 2016; 22:3663–71.