Overexpression of miR-21-5p as a predictive marker for complete tumor regression to neoadjuvant chemoradiotherapy in rectal cancer patients

BMC Medical Genomics

Raphael Bessa Parmigiani³  Rodrigo Oliva Perez⁴  Anamaria Aranha Camargo⁴  Joaquim Gama-Rodrigues⁵  Pedro Alexandre Galante³  Paula Fontes Asprino³  Fernanda Christtanini Koyama³  Fabiana Bettoni³  Natália Mariana Felício¹  Bruna de Souza Quevedo⁴  Angelita Habr-Gama⁵  Camila Miranda Lopes-Ramos² 

[1] Hospital Alemão Oswaldo Cruz, São Paulo, Brazil;Fundação Antônio Prudente, São Paulo, Brazil;Centro de Oncologia Molecular, Hospital Sírio-Libanês, São Paulo, Brazil;Ludwig Institute for Cancer Research, São Paulo, Brazil;University of São Paulo School of Medicine, São Paulo, Brazil

关键词: Chemoradiotherapy;    SATB1;    miR-21-5p;    Rectal cancer;    Predictive biomarker;    miRNA;   

Others  :  1090116
DOI  :  10.1186/s12920-014-0068-7

 received in 2014-06-13, accepted in 2014-11-28,  发布年份 2014

【 摘 要 】

Background

Neoadjuvant chemoradiotherapy (nCRT) followed by radical surgery is the preferred treatment strategy for locally advanced rectal cancer. However, complete tumor regression is observed in a significant proportion of patients after nCRT, making them ideal candidates for alternative treatment strategies to this considerably morbid procedure. Identification of such patients based on clinical findings (complete clinical response - cCR) is difficult mainly because it relies on subjective clinical and imaging studies. Our goal was to identify biomarkers capable of predicting complete response to nCRT.

Methods

We analyzed miRNA expression profile using deep sequencing in rectal tumor biopsies prior to nCRT. Differential expression was investigated by EdgeR for a training (n = 27) and a validation (n = 16) set of patients to identify miRNAs associated with treatment response (complete vs. incomplete). In vitro experiments with two cancer cell lines were also performed in order to evaluate the possible role of miRNAs on response to nCRT.

Results

We found 4 miRNAs differentially expressed between complete and incomplete responders to nCRT. In addition, validation was performed using an independent group of patients and miR-21-5p was confirmed as being overexpressed in complete responders. Overall sensitivity and specificity of miR-21-5p expression in predicting complete response to nCRT was 78% and 86% respectively. Interestingly, in a subset of patients with cCR followed by early local recurrence, the expression level of miR-21-5p was considerably low, similarly to incomplete responders. We also found SATB1, a miR-21-5p target gene and known multidrug resistance gene, whose expression was inversely correlated with miR-21-5p expression. Finally, we performed functional experiments and showed that miR-21-5p and SATB1 may be directly involved with poor response to nCRT in rectal cancer patients.

Conclusions

This study suggests miR-21-5p as a promising predictive biomarker, which should aid in the selection of patients with cCR to nCRT that potentially could be spared from radical surgery.

【 授权许可】

   
2014 Lopes-Ramos et al.; licensee BioMed Central.

【 图 表 】

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【 参考文献 】

• [1]Kapiteijn E, Marijnen CA, Nagtegaal ID, Putter H, Steup WH, Wiggers T, Rutten HJ, Pahlman L, Glimelius B, van Krieken JH, Leer JW, van de Velde CJ: Preoperative radiotherapy combined with total mesorectal excision for resectable rectal cancer. N Engl J Med 2001, 345:638-646.
• [2]Minsky BD, Cohen AM, Kemeny N, Enker WE, Kelsen DP, Reichman B, Saltz L, Sigurdson ER, Frankel J: Combined modality therapy of rectal cancer: decreased acute toxicity with the preoperative approach. J Clin Oncol 1992, 10:1218-1224.
• [3]Habr-Gama A, Perez RO, Kiss DR, Rawet V, Scanavini A, Santinho PM, Nadalin W: Preoperative chemoradiation therapy for low rectal cancer. Impact on downstaging and sphincter-saving operations. Hepatogastroenterology 2004, 51:1703-1707.
• [4]Sanghera P, Wong DWY, McConkey CC, Geh JI, Hartley A: Chemoradiotherapy for rectal cancer: an updated analysis of factors affecting pathological response. Clin Oncol (R Coll Radiol) 2008, 20:176-183.
• [5]Habr-Gama A, Perez RO, Nadalin W, Sabbaga J, Ribeiro U, Sousa AH S e, Campos FG, Kiss DR, Gama-Rodrigues J: Operative versus nonoperative treatment for stage 0 distal rectal cancer following chemoradiation therapy: long-term results. Ann Surg 2004, 240:711-717.
• [6]Habr-Gama A, Perez RO, Wynn G, Marks J, Kessler H, Gama-Rodrigues J: Complete clinical response after neoadjuvant chemoradiation therapy for distal rectal cancer: characterization of clinical and endoscopic findings for standardization. Dis Colon Rectum 2010, 53:1692-1698.
• [7]Bartel DP, Lee R, Feinbaum R: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004, 116:281-297.
• [8]Croce CM: Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet 2009, 10:704-714.
• [9]Calin GA, Croce CM: MicroRNA signatures in human cancers. Nat Rev Cancer 2006, 6:857-866.
• [10]Shenouda SK, Alahari SK: MicroRNA function in cancer: oncogene or a tumor suppressor? Cancer Metastasis Rev 2009, 28:369-378.
• [11]Nakajima G, Hayashi K, Xi Y, Kudo K, Uchida K, Takasaki K, Yamamoto M, Ju J: Non-coding MicroRNAs hsa-let-7 g and hsa-miR-181b are associated with chemoresponse to S-1 in colon cancer. Cancer Genomics Proteomics 2006, 3:317-324.
• [12]Svoboda M, Izakovicova Holla L, Sefr R, Vrtkova I, Kocakova I, Tichy B, Dvorak J: Micro-RNAs miR125b and miR137 are frequently upregulated in response to capecitabine chemoradiotherapy of rectal cancer. Int J Oncol 2008, 33:541-547.
• [13]Hummel R, Hussey DJ, Haier J: MicroRNAs: predictors and modifiers of chemo- and radiotherapy in different tumour types. Eur J Cancer 2010, 46:298-311.
• [14]Dworak O, Keilholz L, Hoffmann A: Pathological features of rectal cancer after preoperative radiochemotherapy. Int J Colorectal Dis 1997, 12:19-23.
• [15]Kozomara A, Griffiths-Jones S: miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res 2011, 39:D152-D157.
• [16]Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ: miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res 2006, 34:D140-D144.
• [17]Robinson MD, McCarthy DJ, Smyth GK: edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 2010, 26:139-140.
• [18]Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn Y, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JYH, Zhang J: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 2004, 5:R80. BioMed Central Full Text
• [19]Benjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B 1995, 57:289-300.
• [20]Lewis BP, Burge CB, Bartel DP: Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005, 120:15-20.
• [21]Chang KH, Mestdagh P, Vandesompele J, Kerin MJ, Miller N: MicroRNA expression profiling to identify and validate reference genes for relative quantification in colorectal cancer. BMC Cancer 2010, 10:173. BioMed Central Full Text
• [22]Viprey VF, Corrias MV, Burchill SA: Identification of reference microRNAs and suitability of archived hemopoietic samples for robust microRNA expression profiling. Anal Biochem 2012, 421:566-572.
• [23]Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods San Diego Calif 2001, 25:402-408.
• [24]Han H-J, Russo J, Kohwi Y, Kohwi-Shigematsu T: SATB1 reprogrammes gene expression to promote breast tumour growth and metastasis. Nature 2008, 452:187-193.
• [25]Li Q-Q, Chen Z-Q, Xu J-D, Cao X-X, Chen Q, Liu X-P, Xu Z-D: Overexpression and involvement of special AT-rich sequence binding protein 1 in multidrug resistance in human breast carcinoma cells. Cancer Sci 2010, 101:80-86.
• [26]Meng W-J, Yan H, Zhou B, Zhang W, Kong X-H, Wang R, Zhan L, Li Y, Zhou Z-G, Sun X-F: Correlation of SATB1 overexpression with the progression of human rectal cancer. Int J Colorectal Dis 2012, 27:143-150.
• [27]Tomimaru Y, Eguchi H, Nagano H, Wada H, Tomokuni A, Kobayashi S, Marubashi S, Takeda Y, Tanemura M, Umeshita K, Doki Y, Mori M: MicroRNA-21 induces resistance to the anti-tumour effect of interferon-α/5-fluorouracil in hepatocellular carcinoma cells. Br J Cancer 2010, 103:1617-1626.
• [28]Deng J, Lei W, Fu J-C, Zhang L, Li J-H, Xiong J-P: Targeting miR-21 enhances the sensitivity of human colon cancer HT-29 cells to chemoradiotherapy in vitro. Biochem Biophys Res Commun 2014, 443:789-795.
• [29]Li Y, Zhao S, Zhen Y, Li Q, Teng L, Asai A, Kawamoto K: A miR-21 inhibitor enhances apoptosis and reduces G(2)-M accumulation induced by ionizing radiation in human glioblastoma U251 cells. Brain Tumor Pathol 2011, 28:209-214.
• [30]Valeri N, Gasparini P, Braconi C, Paone A, Lovat F, Fabbri M, Sumani KM, Alder H, Amadori D, Patel T, Nuovo GJ, Fishel R, Croce CM: MicroRNA-21 induces resistance to 5-fluorouracil by down-regulating human DNA MutS homolog 2 (hMSH2). Proc Natl Acad Sci U S A 2010, 107:21098-21103.
• [31]Feng Y-H, Wu C-L, Shiau A-L, Lee J-C, Chang J-G, Lu P-J, Tung C-L, Feng L-Y, Huang W-T, Tsao C-J: MicroRNA-21-mediated regulation of Sprouty2 protein expression enhances the cytotoxic effect of 5-fluorouracil and metformin in colon cancer cells. Int J Mol Med 2012, 29:920-926.
• [32]Beyer M, Thabet Y, Müller R-U, Sadlon T, Classen S, Lahl K, Basu S, Zhou X, Bailey-Bucktrout SL, Krebs W, Schönfeld EA, Böttcher J, Golovina T, Mayer CT, Hofmann A, Sommer D, Debey-Pascher S, Endl E, Limmer A, Hippen KL, Blazar BR, Balderas R, Quast T, Waha A, Mayer G, Famulok M, Knolle PA, Wickenhauser C, Kolanus W, Schermer B, et al.: Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation. Nat Immunol 2011, 12:898-907.
• [33]Yang S, Banerjee S, de Freitas A, Cui H, Xie N, Abraham E, Liu G: miR-21 regulates chronic hypoxia-induced pulmonary vascular remodeling. Am J Physiol Lung Cell Mol Physiol 2012, 302:L521-L529.
• [34]Maas M, Beets-Tan RGH, Lambregts DMJ, Lammering G, Nelemans PJ, Engelen SME, van Dam RM, Jansen RLH, Sosef M, Leijtens JWA, Hulsewé KWE, Buijsen J, Beets GL: Wait-and-see policy for clinical complete responders after chemoradiation for rectal cancer. J Clin Oncol 2011, 29:4633-4640.
• [35]Ghadimi BM, Grade M, Difilippantonio MJ, Varma S, Simon R, Montagna C, Füzesi L, Langer C, Becker H, Liersch T, Ried T: Effectiveness of gene expression profiling for response prediction of rectal adenocarcinomas to preoperative chemoradiotherapy. J Clin Oncol 2005, 23:1826-1838.
• [36]Watanabe T, Komuro Y, Kiyomatsu T, Kanazawa T, Kazama Y, Tanaka J, Tanaka T, Yamamoto Y, Shirane M, Muto T, Nagawa H: Prediction of sensitivity of rectal cancer cells in response to preoperative radiotherapy by DNA microarray analysis of gene expression profiles. Cancer Res 2006, 66:3370-3374.
• [37]Kim I-J, Lim S-B, Kang HC, Chang HJ, Ahn S-A, Park H-W, Jang S-G, Park J-H, Kim DY, Jung KH, Choi HS, Jeong S-Y, Sohn DK, Kim D-W, Park J-G: Microarray gene expression profiling for predicting complete response to preoperative chemoradiotherapy in patients with advanced rectal cancer. Dis Colon Rectum 2007, 50:1342-1353.
• [38]Rimkus C, Friederichs J, Boulesteix A-L, Theisen J, Mages J, Becker K, Nekarda H, Rosenberg R, Janssen K-P, Siewert JR: Microarray-based prediction of tumor response to neoadjuvant radiochemotherapy of patients with locally advanced rectal cancer. Clin Gastroenterol Hepatol 2008, 6:53-61.
• [39]Nishioka M, Shimada M, Kurita N, Iwata T, Morimoto S, Yoshikawa K, Higashijima J, Miyatani T: Gene expression profile can predict pathological response to preoperative chemoradiotherapy in rectal cancer. Cancer Genomics Proteomics 2011, 8:87-92.
• [40]Kheirelseid EAH, Miller N, Chang KH, Curran C, Hennessey E, Sheehan M, Newell J, Lemetre C, Balls G, Kerin MJ: miRNA expressions in rectal cancer as predictors of response to neoadjuvant chemoradiation therapy. Int J Colorectal Dis 2013, 28:247-260.
• [41]Della Vittoria Scarpati G, Falcetta F, Carlomagno C, Ubezio P, Marchini S, De Stefano A, Singh VK, D’Incalci M, De Placido S, Pepe S: A specific miRNA signature correlates with complete pathological response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer. Int J Radiat Oncol Biol Phys 2012, 83:1113-1119.
• [42]Svoboda M, Sana J, Fabian P, Kocakova I, Gombosova J, Nekvindova J, Radova L, Vyzula R, Slaby O: MicroRNA expression profile associated with response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer patients. Radiat Oncol 2012, 7:195. BioMed Central Full Text
• [43]Kalady MF, de Campos-Lobato LF, Stocchi L, Geisler DP, Dietz D, Lavery IC, Fazio VW: Predictive factors of pathologic complete response after neoadjuvant chemoradiation for rectal cancer. Ann Surg 2009, 250:582-589.
• [44]Yamamichi N, Shimomura R, Inada K, Sakurai K, Haraguchi T, Ozaki Y, Fujita S, Mizutani T, Furukawa C, Fujishiro M, Ichinose M, Shiogama K, Tsutsumi Y, Omata M, Iba H: Locked nucleic acid in situ hybridization analysis of miR-21 expression during colorectal cancer development. Clin Cancer Res 2009, 15:4009-4016.
• [45]Chan JA, Krichevsky AM, Kosik KS: MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res 2005, 65:6029-6033.
• [46]Ren J, Zhu D, Liu M, Sun Y, Tian L: Downregulation of miR-21 modulates Ras expression to promote apoptosis and suppress invasion of Laryngeal squamous cell carcinoma. Eur J Cancer 2010, 46:3409-3416.
• [47]Wu CW, Ng SSM, Dong YJ, Ng SC, Leung WW, Lee CW, Wong YN, Chan FKL, Yu J, Sung JJY: Detection of miR-92a and miR-21 in stool samples as potential screening biomarkers for colorectal cancer and polyps. Gut 2012, 61:739-745.
• [48]Toiyama Y, Takahashi M, Hur K, Nagasaka T, Tanaka K, Inoue Y, Kusunoki M, Boland CR, Goel A: Serum miR-21 as a Diagnostic and Prognostic Biomarker in Colorectal Cancer. J Natl Cancer Inst 2013, 105:849-859.
• [49]Xia X, Yang B, Zhai X, Liu X, Shen K, Wu Z, Cai J: Prognostic role of microRNA-21 in colorectal cancer: a meta-analysis. PLoS One 2013, 8:e80426.
• [50]Yu Y, Sarkar FH, Majumdar APN: Down-regulation of miR-21 induces differentiation of chemoresistant colon cancer cells and enhances susceptibility to therapeutic regimens. Transl Oncol 2013, 6:180-186.
• [51]Schetter AJ, Leung SY, Sohn JJ, Zanetti KA, Bowman ED, Yanaihara N, Yuen ST, Chan TL, Kwong DLW, Au GKH, Liu C-G, Calin GA, Croce CM, Harris CC: MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma. JAMA 2008, 299:425-436.
• [52]Drebber U, Lay M, Wedemeyer I, Vallböhmer D, Bollschweiler E, Brabender J, Mönig SP, Hölscher AH, Dienes HP, Odenthal M: Altered levels of the onco-microRNA 21 and the tumor-supressor microRNAs 143 and 145 in advanced rectal cancer indicate successful neoadjuvant chemoradiotherapy. Int J Oncol 2011, 39:409-415.
• [53]Sun F, Lu X, Li H, Peng Z, Wu K, Wang G, Tong Q: Special AT-rich sequence binding protein 1 regulates the multidrug resistance and invasion of human gastric cancer cells. Oncol Lett 2012, 4:156-162.
• [54]Han B, Luan L, Xu Z, Wu B: Expression and biological roles of SATB1 in human bladder cancer. Tumour Biol 2013, 34:2943-2949.