Decitabine impact on the endocytosis regulator RhoA, the folate carriers RFC1 and FOLR1, and the glucose transporter GLUT4 in human tumors

Stewart et al. Clinical Epigenetics 2014, 6:2 http://www.clinicalepigeneticsjournal.com/content/6/1/2 RESEARCH Open Access Decitabine impact on the endocytosis regulator RhoA, the folate carriers RFC1 and FOLR1, and the glucose transporter GLUT4 in human tumors David J Stewart1*, Maria I Nunez2, Jaroslav Jelinek3, David Hong2, Sanjay Gupta2, Jean-Pierre Issa3†, Ignacio I Wistuba2† and Razelle Kurzrock4† Abstract Background: In 31 solid tumor patients treated with the demethylating agent decitabine, we performed tumor biopsies before and after the first cycle of decitabine and used immunohistochemistry (IHC) to assess whether decitabine increased expression of various membrane transporters. Resistance to chemotherapy may arise due to promoter methylation/downregulation of expression of transporters required for drug uptake, and decitabine can reverse resistance in vitro. The endocytosis regulator RhoA, the folate carriers FOLR1 and RFC1, and the glucose transporter GLUT4 were assessed. Results: Pre-decitabine RhoA was higher in patients who had received their last therapy >3 months previously than in patients with more recent prior therapy (P = 0.02), and varied inversely with global DNA methylation as assessed by LINE1 methylation (r = −0.58, P = 0.006). Tumor RhoA scores increased with decitabine (P = 0.03), and RFC1 also increased in patients with pre-decitabine scores ≤150 (P = 0.004). Change in LINE1 methylation with decitabine did not correlate significantly with change in IHC scores for any transporter assessed. We also assessed methylation of the RFC1 gene (alias SLC19A1). SLC19A1 methylation correlated with tumor LINE1 methylation (r = 0.45, P = 0.02). There was a small (statistically insignificant) decrease in SLC19A1 methylation with decitabine, and there was a trend towards change in SLC19A1 methylation with decitabine correlating with change in LINE1 methylation (r = 0.47, P <0.15). While SLC19A1 methylation did not correlate with RFC1 scores, there was a trend towards an inverse correlation between change in SLC19A1 methylation and change in RFC1 expression (r = −0.45, P = 0.19). Conclusions: In conclusion, after decitabine administration, there was increased expression of some (but not other) transporters that may play a role in chemotherapy uptake. Larger patient numbers will be needed to define the extent to which this increased expression is associated with changes in DNA methylation. Keywords: Decitabine, RhoA, RFC1, FOLR1, GLUT4, LINE1 methylation, Promoter methylation Background Resistance to chemotherapy can arise from overexpression of resistance factors or from underexpression of factors required for drug efficacy [1,2]. Dose–response curve flattening at higher chemotherapy doses suggests that incurability of epithelial malignancies may be due primarily to underexpression of factors required for cytotoxicity [3]. If resistance were instead due to overexpression of resistance * Correspondence: † Equal contributors 1 Head, Division of Medical Oncology, The Ottawa Hospital/University of Ottawa, 501 Smyth Road, Ottawa, ON K1H 8 L6, Canada Full list of author information is available at the end of the article factors, one would expect a shoulder on a log-linear dose–response curve (with increasing efficacy at higher doses) instead of curve flattening at higher doses [1]. Examples of factors required for cytotoxicity that may be deficient in resistant cells include drug uptake transporters (for example, CTR1 for platinums and folate transporters for pemetrexed), drug activating enzymes (for example, deoxycytidine kinase for gemcitabine), obligate drug targets (for example, topoisomerase II for etoposide and doxorubicin) and pro-apoptotic molecules [2,4,5]. Deficiency of factors required for drug uptake and activation might directly potentiate resistance by reducing © 2014 Stewart et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Stewart et al. Clinical Epigenetics 2014, 6:2 http://www.clinicalepigeneticsjournal.com/content/6/1/2 the amount of active drug in a cell, but could also have a secondary effect, in that these transporters may also play a role in uptake and cellular metabolism of nutrients. Hence, deficiency in these factors could reduce the rate of tumor cell division, and quiescent cells are generally more resistant to chemotherapy than are actively dividing cells [1,2,4]. Decreased uptake of several agents [6] and downregulation of expression of various transporters including folate binding protein and the endocytosis regulator RhoA [7] has been described in cisplatin-resistant cells that also have a reduced cell growth rate. The mechanism by which transporters and other factors required for drug efficacy may be downregulated in cancer cells remains unclear. However, promoter hypermethylation is one mechanism by which gene expression may be downregulated, and hence DNA methylation could play a role in underexpression of factors required for drug efficacy [8-18]. Cancer cells often have abnormal hypermethylation and silencing of tumor suppressor genes [8-11] and of genes that support chemotherapy cytotoxicity [12,13]. Several genes may be hypermethylated in resistant cell lines [14,15] or tumors [18]. If DNA hypermethylation might play a role in resistance, then it follows that agents that reduce DNA methylation might sensitize cells to chemotherapy. The DNA demethylating agent decitabine reversed folate binding protein downregulation in cisplatin-resistant cells [7], augmented cellular uptake of methotrexate and carboplatin [7], and reversed resistance to various chemotherapy [15-17,19-24] or targeted agents [25] by upregulating expression of proapoptotic factors [19,20,25] and by other mechanisms [13]. DNA methylation also protected the anti-apoptotic factor survivin from repression by p53, and decitabine reversed this, permitting survivin repression by p53 [26]. Decitabine also partially reversed resistance to carboplatin in patients with advanced ovarian cancer [18,27,28]. The related DNA demethylating agent 5-azacytidine downregulated telomerase expression [29] and potentiated cisplatin [30-32], carboplatin [33], and docetaxel [31,32,34] preclinically by decreasing expression of pAKT [30,31] and other anti-apoptotic factors [31], by increasing expression of the tumor suppressor gene TMS1 [34] and various proapoptotic factors [31,33], and by other mechanisms [30]. Also, 5-Azacytidine potentiated irinotecan in p53-mutant cells by upregulating expression of its obligate target topoisomerase-I via mechanisms involving p16 demethylation and Sp1 upregulation [35]. Decitabine is active clinically in myelodysplasia and leukemia [36-38]. Low-dose administration days 1 to 5 +/− da (...truncated)