Expression of neurofibromin 1 in colorectal cancer and cetuximab resistance

Tak,Eunyoung; Kim,Minhee; Cho,Youngra; Choi,Sueun; Kim,Jihun; Han,Buhm; Kim,Hyung-Don; Jang,Chloe Soo‑Hyun; Kim,Jeong Eun; Hong,Yong Sang; Kim,Sun Young; Kim,Tae Won

doi:10.3892/or.2021.8226

Expression of neurofibromin 1 in colorectal cancer and cetuximab resistance

Authors:
- Eunyoung Tak
- Minhee Kim
- Youngra Cho
- Sueun Choi
- Jihun Kim
- Buhm Han
- Hyung-Don Kim
- Chloe Soo‑Hyun Jang
- Jeong Eun Kim
- Yong Sang Hong
- Sun Young Kim
- Tae Won Kim
View Affiliations

Affiliations: Department of Convergence Medicine, Asan Medical Center, University of Ulsan College of Medicine, Songpa, Seoul 05505, Republic of Korea, Asan Medical Institute of Convergence Science and Technology (AMIST), Asan Medical Center, University of Ulsan College of Medicine, Songpa, Seoul 05505, Republic of Korea, Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Songpa, Seoul 05505, Republic of Korea, Department of Biomedical Sciences, Seoul National University College of Medicine, Jongro, Seoul 03080, Republic of Korea, Department of Oncology, Asan Medical Center, University of Ulsan College of Medicine, Songpa, Seoul 05505, Republic of Korea
Published online on: November 12, 2021 https://doi.org/10.3892/or.2021.8226
Article Number: 15
Copyright: © Tak et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Neurofibromin 1 (NF1) is a tumor suppressor that has been previously reported to regulate RAS‑MAPK signaling. The present study investigated the possible relationship between NF1 expression and anti‑EGFR antibody (cetuximab) sensitivity in colorectal cancer cell lines. In addition, primary or metastatic colorectal cancer samples from patients treated with cetuximab were assessed for the association of cetuximab sensitivity. The quantities of the NF1 transcript, NF1‑related pathway enrichment and NF1 mutation profile were measured and investigated using RNA sequencing and targeted DNA sequencing. Based on growth inhibition and colony formation assay results, cell lines were designated to be cetuximab‑sensitive (NCI‑H508 and Caco2) or cetuximab‑resistant (KM12C and SM480). Western blotting revealed NF1 was highly expressed in cetuximab‑sensitive cell lines whilst there was little expression in their cetuximab‑resistant counterparts. Knocking down NF1 expression using small interfering RNA in the cetuximab‑sensitive cell lines enhanced the phosphorylation of MEK and ERK according to western blotting. NF1 knockdown also reduced apoptosis, as observed by the decreased number of apoptotic bodies by DAPI nuclear staining and reduced cleavage of caspase and poly‑(ADP ribose) polymerase. NF1 overexpression by transfection with GTPase‑activating protein‑related domain subunit rendered the cetuximab‑resistant cell lines, KM12C and SW480, more susceptible to cetuximab‑induced apoptosis. RNA sequencing of 111 RAS and BRAF^V600 wild‑type tumor samples collected from cetuximab‑treated patients with metastatic colorectal cancer revealed that the pre‑treatment NF1 expression levels were not associated with the cetuximab response. However, tumor samples obtained after cetuximab treatment displayed slightly lower NF1 transcript levels compared with those in the pre‑treatment samples, suggesting that exposure to the anti‑EGFR antibody may be associated with reduced NF1 expression levels. Next‑generation sequencing revealed that the frequency of inactivating mutations in NF1 were rare (1.8%) in patients with colorectal cancer and were not associated with the protein expression levels of NF1 except for in a small number of cases (0.5%), where the biallelic inactivation of NF1 was observed. To conclude, the present study showed that modification of NF1 expression can affect sensitivity to cetuximab in colorectal cancer cell lines, though a limitation exists in terms of its potential application as a biomarker for RAS and BRAF^V600 wild‑type tumors.

View Figures

View References

1	Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A and Bray F: Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 71:209–249. 2021. View Article : Google Scholar : PubMed/NCBI
2	Venook AP, Niedzwiecki D, Lenz HJ, Innocenti F, Fruth B, Meyerhardt JA, Schrag D, Greene C, O'Neil BH, Atkins JN, et al: Effect of first-line chemotherapy combined with cetuximab or bevacizumab on overall survival in patients with KRAS wild-type advanced or metastatic colorectal cancer: A randomized clinical trial. Jama. 317:2392–2401. 2017. View Article : Google Scholar : PubMed/NCBI
3	Kim SY and Kim TW: Current challenges in the implementation of precision oncology for the management of metastatic colorectal cancer. ESMO Open. 5:e0006342020. View Article : Google Scholar : PubMed/NCBI
4	Zhao B, Wang L, Qiu H, Zhang M, Sun L, Peng P, Yu Q and Yuan X: Mechanisms of resistance to anti-EGFR therapy in colorectal cancer. Oncotarget. 8:3980–4000. 2017. View Article : Google Scholar : PubMed/NCBI
5	Martinelli E, Ciardiello D, Martini G, Troiani T, Cardone C, Vitiello PP, Normanno N, Rachiglio AM, Maiello E, Latiano T, et al: Implementing anti-epidermal growth factor receptor (EGFR) therapy in metastatic colorectal cancer: Challenges and future perspectives. Ann Oncol. 31:30–40. 2020. View Article : Google Scholar : PubMed/NCBI
6	Li QH, Wang YZ, Tu J, Liu CW, Yuan YJ, Lin R, He WL, Cai SR, He YL and Ye JN: Anti-EGFR therapy in metastatic colorectal cancer: Mechanisms and potential regimens of drug resistance. Gastroenterol Rep (Oxf). 8:179–191. 2020. View Article : Google Scholar : PubMed/NCBI
7	Ratner N and Miller SJ: A RASopathy gene commonly mutated in cancer: The neurofibromatosis type 1 tumour suppressor. Nat Rev Cancer. 15:290–301. 2015. View Article : Google Scholar : PubMed/NCBI
8	de Bruin EC, Cowell C, Warne PH, Jiang M, Saunders RE, Melnick MA, Gettinger S, Walther Z, Wurtz A, Heynen GJ, et al: Reduced NF1 expression confers resistance to EGFR inhibition in lung cancer. Cancer Discov. 4:606–619. 2014. View Article : Google Scholar : PubMed/NCBI
9	Woolston A, Khan K, Spain G, Barber LJ, Griffiths B, Gonzalez-Exposito R, Hornsteiner L, Punta M, Patil Y, Newey A, et al: Genomic and transcriptomic determinants of therapy resistance and immune landscape evolution during Anti-EGFR treatment in colorectal cancer. Cancer Cell. 36:35–50.e39. 2019. View Article : Google Scholar : PubMed/NCBI
10	Cancer Genome Atlas Research Network, . Comprehensive genomic characterization of squamous cell lung cancers. Nature. 489:519–525. 2012. View Article : Google Scholar : PubMed/NCBI
11	Cancer Genome Atlas Network, . Comprehensive molecular portraits of human breast tumours. Nature. 490:61–70. 2012. View Article : Google Scholar : PubMed/NCBI
12	Krauthammer M, Kong Y, Bacchiocchi A, Evans P, Pornputtapong N, Wu C, McCusker JP, Ma S, Cheng E, Straub R, et al: Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas. Nat Genet. 47:996–1002. 2015. View Article : Google Scholar : PubMed/NCBI
13	Yap YS, McPherson JR, Ong CK, Rozen SG, Teh BT, Lee AS and Callen DF: The NF1 gene revisited -from bench to bedside. Oncotarget. 5:5873–5892. 2014. View Article : Google Scholar : PubMed/NCBI
14	Whittaker SR, Theurillat JP, Van Allen E, Wagle N, Hsiao J, Cowley GS, Schadendorf D, Root DE and Garraway LA: A genome-scale RNA interference screen implicates NF1 loss in resistance to RAF inhibition. Cancer Discov. 3:350–362. 2013. View Article : Google Scholar : PubMed/NCBI
15	Mendes-Pereira AM, Sims D, Dexter T, Fenwick K, Assiotis I, Kozarewa I, Mitsopoulos C, Hakas J, Zvelebil M, Lord CJ and Ashworth A: Genome-wide functional screen identifies a compendium of genes affecting sensitivity to tamoxifen. Proc Natl Acad Sci USA. 109:2730–2735. 2012. View Article : Google Scholar : PubMed/NCBI
16	Hölzel M, Huang S, Koster J, Ora I, Lakeman A, Caron H, Nijkamp W, Xie J, Callens T, Asgharzadeh S, et al: NF1 is a tumor suppressor in neuroblastoma that determines retinoic acid response and disease outcome. Cell. 142:218–229. 2010. View Article : Google Scholar : PubMed/NCBI
17	Berg KCG, Eide PW, Eilertsen IA, Johannessen B, Bruun J, Danielsen SA, Bjørnslett M, Meza-Zepeda LA, Eknæs M, Lind GE, et al: Multi-omics of 34 colorectal cancer cell lines-a resource for biomedical studies. Mol Cancer. 16:1162017. View Article : Google Scholar : PubMed/NCBI
18	Park HJ, Lee SJ, Sohn YB, Jin HS, Han JH, Kim YB, Yim H and Jeong SY: NF1 deficiency causes Bcl-xL upregulation in Schwann cells derived from neurofibromatosis type 1-associated malignant peripheral nerve sheath tumors. Int J Oncol. 42:657–666. 2013. View Article : Google Scholar : PubMed/NCBI
19	Schneider CA, Rasband WS and Eliceiri KW: NIH image to imageJ: 25 years of image analysis. Nat Methods. 9:671–675. 2012. View Article : Google Scholar : PubMed/NCBI
20	Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, Dancey J, Arbuck S, Gwyther S, Mooney M, et al: New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur J Cancer. 45:228–247. 2009. View Article : Google Scholar : PubMed/NCBI
21	Schuierer S, Carbone W, Knehr J, Petitjean V, Fernandez A, Sultan M and Roma G: A comprehensive assessment of RNA-seq protocols for degraded and low-quantity samples. BMC Genomics. 18:1–13. 2017. View Article : Google Scholar : PubMed/NCBI
22	Andrews S: FastQC: A quality control tool for high through-put sequence data. Babraham Institute. (Cambridge). 2010.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/November 11–2019
23	Krueger F: Trim galore: A wrapper tool around Cutadapt and FastQC to consistently apply quality and adapter trimming to FastQ files with some extra functionality for MspI-digested RRBS-type (Reduced Representation Bisufite-Seq) libraries. Babraham Institute. (Cambridge). 2015.https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/November 11–2019
24	Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M and Gingeras TR: STAR: Ultrafast universal RNA-seq aligner. Bioinformatics. 29:15–21. 2013. View Article : Google Scholar : PubMed/NCBI
25	Li B and Dewey CN: RSEM: Accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 12:3232011. View Article : Google Scholar : PubMed/NCBI
26	Love MI, Huber W and Anders S: Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15:5502014. View Article : Google Scholar : PubMed/NCBI
27	Yu G, Wang LG, Han Y and He QY: clusterProfiler: An R package for comparing biological themes among gene clusters. OMICS. 16:284–287. 2012. View Article : Google Scholar : PubMed/NCBI
28	Kanehisa M, Sato Y, Kawashima M, Furumichi M and Tanabe M: KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res. 44:D457–D462. 2015. View Article : Google Scholar : PubMed/NCBI
29	Kim JE, Chun SM, Hong YS, Kim KP, Kim SY, Kim J, Sung CO, Cho EJ, Kim TW and Jang SJ: Mutation burden and I index for detection of microsatellite instability in colorectal cancer by targeted next-generation sequencing. J Mol Diagn. 21:241–250. 2018. View Article : Google Scholar : PubMed/NCBI
30	Philpott C, Tovell H, Frayling IM, Cooper DN and Upadhyaya M: The NF1 somatic mutational landscape in sporadic human cancers. Hum Genomics. 11:132017. View Article : Google Scholar : PubMed/NCBI
31	Redig AJ, Capelletti M, Dahlberg SE, Sholl LM, Mach S, Fontes C, Shi Y, Chalasani P and Jänne PA: Clinical and molecular characteristics of NF1-mutant lung cancer. Clin Cancer Res. 22:3148–3156. 2016. View Article : Google Scholar : PubMed/NCBI
32	Hayashi T, Desmeules P, Smith RS, Drilon A, Somwar R and Ladanyi M: RASA1 and NF1 are preferentially co-mutated and define a distinct genetic subset of smoking-associated non-small cell lung carcinomas sensitive to MEK inhibition. Clin Cancer Res. 24:1436–1447. 2018. View Article : Google Scholar : PubMed/NCBI
33	Dischinger PS, Tovar EA, Essenburg CJ, Madaj ZB, Gardner EE, Callaghan ME, Turner AN, Challa AK, Kempston T, Eagleson B, et al: NF1 deficiency correlates with estrogen receptor signaling and diminished survival in breast cancer. NPJ Breast Cancer. 4:292018. View Article : Google Scholar : PubMed/NCBI
34	Pearson A, Proszek P, Pascual J, Fribbens C, Shamsher MK, Kingston B, O'Leary B, Herrera-Abreu MT, Cutts RJ, Garcia-Murillas I, et al: Inactivating NF1 mutations are enriched in advanced breast cancer and contribute to endocrine therapy resistance. Clin Cancer Res. 26:608–622. 2020. View Article : Google Scholar : PubMed/NCBI
35	Mei Z, Shao YW, Lin P, Cai X, Wang B, Ding Y, Ma X, Wu X, Xia Y, Zhu D, et al: SMAD4 and NF1 mutations as potential biomarkers for poor prognosis to cetuximab-based therapy in Chinese metastatic colorectal cancer patients. BMC Cancer. 18:4792018. View Article : Google Scholar : PubMed/NCBI
36	Cancer Genome Atlas Network, . Comprehensive molecular characterization of human colon and rectal cancer. Nature. 487:330–337. 2012. View Article : Google Scholar : PubMed/NCBI
37	Meng M, Zhong K, Jiang T, Liu Z, Kwan HY and Su T: The current understanding on the impact of KRAS on colorectal cancer. Biomed Pharmacother. 140:1117172021. View Article : Google Scholar : PubMed/NCBI
38	McFall T, Diedrich JK, Mengistu M, Littlechild SL, Paskvan KV, Sisk-Hackworth L, Moresco JJ, Shaw AS and Stites EC: A systems mechanism for KRAS mutant allele-specific responses to targeted therapy. Sci Signal. 12:eaaw82882019. View Article : Google Scholar : PubMed/NCBI
39	Georgiou A, Stewart A, Cunningham D, Banerji U and Whittaker SR: Inactivation of NF1 promotes resistance to EGFR inhibition in KRAS/NRAS/BRAF^V600 -wild-type colorectal cancer. Mol Cancer Res. 18:835–846. 2020. View Article : Google Scholar : PubMed/NCBI
40	Bray SM, Lee J, Kim ST, Hur JY, Ebert PJ, Calley JN, Wulur IH, Gopalappa T, Wong SS, Qian HR, et al: Genomic characterization of intrinsic and acquired resistance to cetuximab in colorectal cancer patients. Sci Rep. 9:153652019. View Article : Google Scholar : PubMed/NCBI
41	AACR Project GENIE: Powering precision medicine through an international consortium. Cancer Discov. 7:818–831. 2017. View Article : Google Scholar : PubMed/NCBI
42	Kandoth C, McLellan MD, Vandin F, Ye K, Niu B, Lu C, Xie M, Zhang Q, McMichael JF, Wyczalkowski MA, et al: Mutational landscape and significance across 12 major cancer types. Nature. 502:333–339. 2013. View Article : Google Scholar : PubMed/NCBI
43	Baeissa HM, Benstead-Hume G, Richardson CJ and Pearl FM: Mutational patterns in oncogenes and tumour suppressors. Biochem Soc Trans. 44:925–931. 2016. View Article : Google Scholar : PubMed/NCBI
44	Gutmann DH, Wu YL, Hedrick NM, Zhu Y, Guha A and Parada LF: Heterozygosity for the neurofibromatosis 1 (NF1) tumor suppressor results in abnormalities in cell attachment, spreading and motility in astrocytes. Hum Mol Genet. 10:3009–3016. 2001. View Article : Google Scholar : PubMed/NCBI
45	Brosseau JP, Liao CP, Wang Y, Ramani V, Vandergriff T, Lee M, Patel A and Ariizumi K: NF1 heterozygosity fosters de novo tumorigenesis but impairs malignant transformation. Nat Commun. 9:50142018. View Article : Google Scholar : PubMed/NCBI
46	Guo XE, Ngo B, Modrek AS and Lee WH: Targeting tumor suppressor networks for cancer therapeutics. Curr Drug Targets. 15:2–16. 2014. View Article : Google Scholar : PubMed/NCBI
47	Bai RY, Esposito D, Tam AJ, McCormick F, Riggins GJ, Clapp DW and Staedtke V: Feasibility of using NF1-GRD and AAV for gene replacement therapy in NF1-associated tumors. Gene Ther. 26:277–286. 2019. View Article : Google Scholar : PubMed/NCBI
48	Kong N, Tao W, Ling X, Wang J, Xiao Y, Shi S, Ji X, Shajii A, Gan ST, Kim NY, et al: Synthetic mRNA nanoparticle-mediated restoration of p53 tumor suppressor sensitizes p53-deficient cancers to mTOR inhibition. Sci Transl Med. 11:eaaw15652019. View Article : Google Scholar : PubMed/NCBI
49	El Sharkawi FZ, Ewais SM, Fahmy RH and Rashed LA: PTEN and TRAIL genes loaded zein nanoparticles as potential therapy for hepatocellular carcinoma. J Drug Target. 25:513–522. 2017. View Article : Google Scholar : PubMed/NCBI
50	Groelz D, Sobin L, Branton P, Compton C, Wyrich R and Rainen L: Non-formalin fixative versus formalin-fixed tissue: A comparison of histology and RNA quality. Exp Mol Pathol. 94:188–194. 2013. View Article : Google Scholar : PubMed/NCBI