Streszczenie

Wstęp: Oporność na terapię oraz związany z nią profil ekspresji genów dyskryminują wyniki leczenia, a także wskazują podgrupy pacjentów z niekorzystnym rokowaniem. Celem badań była analiza profilu ekspresji genów w korelacji z profilem chemiooporności ex vivo na etopozyd u dzieci z ostrymi białaczkami.
Materiał/Metody: Profil oporności ex vivo oznaczono w teście cytotoksyczności MTT na komórkach białaczkowych pobranych od 56 pacjentów. Profil ekspresji genów opracowano na podstawie wyników hybrydyza­cji cRNA do macierzy oligonukleotydowych HGU133A 2.0 Chip. Do zidentyfikowanego zestawu sond przeprowadzono analizę korelacji, grupowanie hierarchiczne, przypisanie lokalizacji i funk­cji. Weryfikację danych o poziomie ekspresji dla 4 wybranych genów (MNDA, GH1, NUDT21, RHOG) przeprowadzono techniką QPCR w badanej oraz niezależnej grupie 54 pacjentów.
Wyniki: Za pomocą permutacyjnego testu korelacji Spearmana wyselekcjonowano zestaw 233 sond/209 genów. Ekspozycja blastów białaczkowych na etopozyd inicjuje złożoną odpowiedź komórko­wą, będącą odzwierciedleniem globalnych zmian ekspresji genów. Globalny test istotności różnic potwierdził (p<0,001) związek profilu ekspresji genów z opornością na etopozyd. Gen NUDT21 (Nudix, nucleoside diphosphate linked moiety X-type, motif 21) wykazywał najsilniejszą kore­lację z opornością na etopozyd (FDR<0,0001%).
Podsumowanie: Profilowanie ekspresji genów może pomóc w ocenie wrażliwości na leki stosowane w chemio­terapii dużymi dawkami. Oporność na etopozyd prawdopodobnie jest związana ze znaczną licz­bą biologicznie ważnych genów i jest konsekwencją współistnienia w komórce nowotworowej różnych mechanizmów.

Summary

Introduction: Drug resistance and the gene expression profiles might discriminate the therapy outcome, and in­dicate the subgroup of patients with poor prognosis. In this study we analyzed the gene expres­sion profile in correlation with the profile of ex vivo resistance to etoposide in children with acu­te leukemias.
Methods: The ex vivo drug resistance profile was determined by the MTT cytotoxicity assay performed on leukemic blasts of 56 patients. Gene expression profiles were obtained from the results of hybri­dization of cRNA to Human Genome U133A 2.0 ologonucleotide arrays. The following analy­ses were performed: correlation analysis, hierarchical clustering, the assignment of location and function. Verification of data for four selected genes (MNDA, GH1, NUDT21, RHOG) was per­formed by quantitative real time polymerase chain reaction in the studied population and in an independent group of 54 leukemic patients.
Results: Using the permutation Spearman correlation test, a set of 233 probes/209 genes was selected. The global test confirmed the significance of the correlation of gene expression profile and resistan­ce to etoposide (p<0.001). The NUDT21 (nudix, nucleoside diphosphate linked moiety X-type, motif 21) gene showed the strongest correlation with resistance to etoposide (FDR<0.0001%).
Conclusions: Profiling of transcriptome may help in assessing the sensitivity to drugs used in chemotherapy. Resistance to etoposide is possibly associated with a change of expression of a large number of biologically important genes that influence several cellular mechanisms.

Key words:ostra białaczka limfoblastyczna • ostra białaczka mieloblastyczna • lekooporność • etopozyd • VP-16 • mikromacierze • profil ekspresji genów • dzieci

Background

The most important obstacle in cancer chemotherapy is the development of resistance. Except for the overexpression of ATP-binding cassette transporters and the selection of mutated cells capable of avoiding pro-apoptotic signals, the knowledge of the resistance mechanisms to various cyto­statics is still unsatisfactory. Research on the scale of the whole genome/transcriptome is being used more frequen­tly to identify genes important for the cellular sensitivity to drugs [4,16,17,27,33,40]. This approach offers the possi­bility of creating a better characterization of the multige­ne nature as well as understanding the pathways involved in the cellular response to chemotherapeutic agents [20].

Etoposide (VePesid – VP16) is an alkaloid – a semisynthe­tic analogue of podophyllotoxin. As a cytostatic it is used in chemotherapy of malignant tumors such as Ewing’s sar­coma, glioblastoma, lung cancer, testicular cancer, lympho­ma, non-lymphocytic leukemia, osteosarcoma and erythema multiforme [5,20,25,31,39,45]. It is most commonly used in therapy combined with other drugs. Its application also includes a fixing treatment before transplantation of bone marrow or blood stem cells.

The cytostatic effect is a consequence of cell cycle arrest after a disruption of single- and double-stranded DNA [7]. It belongs to the phase-specific drugs, operating mainly in the interphase (later in the S and G2 phase as well). The primary target of the medicine is an enzyme that repairs damaged DNA – topoisomerase II. Topoisomerases regu­late DNA replication and transcription, and participate in chromosome segregation, cell cycle progression and RNA metabolism [32]. VP16 inhibits DNA synthesis after the creation of complexes of enzyme and Deoxyribonucleic acid [1,32]. Formation of such complexes induces the for­mation of breaks in double-stranded DNA and simulta­neously prevents bound topoisomerase II from repairing them [22,23]. Accumulation of DNA breaks prevents cell entry into the mitosis phase of the cell cycle, thus leading to programmed cell death [8,10]. Etoposide-induced DNA damage leads to activation of the p53-dependent path­way, which in turn is associated with changes in stimula­tion of tens of genes such as Waf-1/p21, PCNA, GPX and S100A2 [36,44].

For the purpose of the study an assumption was made that the ex vivo drug susceptibility profile combined with the gene expression profile may provide new insights into the choice of drugs used in high-dose therapy prior to trans­plantation of hematopoietic stem cells.

Patients and Methods

In vitro sensitivity test. Bone marrow samples were collec­ted from 56 pediatric patients (43 de novo ALL, 8 relapse ALL, 5 AML de novo) and analyzed (Table 1). Patients with relapsed ALL and AML were entered into the study in order to increase the size of the group resistant to etopo­side. At the same time, differences in expression between different subtypes of leukemia were not investigated (due to the very large disparities in the number of patients with ALL, relapsed ALL and AML). The profile of ex vivo resi­stance and sensitivity to etoposide (Bristol-Myers Squibb) was determined in a 4-daily cytotoxicity assay (MTT). The essence of the test is to reduce the yellow soluble bromi­de 3 [4.5-dimethyl-2-yl]-2.5-2,5-diphenyl tetrazolium to blue insoluble formazan. This reaction is characteristic for living cells. As a measure of resistance to etoposide the LC50 parameter was adopted (lethal concentration of the drug, in which 50% of the cells underwent apoptosis). Concentration used in the test was 0.048-50 mg/ml. Cells were classified as sensitive if the LC50 value was <=0.04 mg/ml, or as resistant when the LC50 was >=1.7 mg/ml.

Table 1. Characteristics of patient group analyzed with microarray and QPCR technique

Preparation of RNA. Samples (6-10×10⁶ isolated blasts) were homogenized in RLT (Qiagen) buffer. Cell lysates were stored at -80°C. Total RNA was extracted using Trizol (Invitrogen) and then digested with DNase I (Fermentas). RNA integrity assessment was based on the RIN coeffi­cient obtained after a capillary electrophoresis was perfor­med (Agilent 2100 Bioanalyzer).

Microarray analysis. The procedure for nucleic acid prepa­ration was carried out in accordance with the recommenda­tions of the manufacturer (Affymetrix), and based on sets of reagents for hybridization reactions appropriate for the selected type of microarray. The baseline in each test was 5 µg of total RNA as a template. Fragmented and labeled cRNA was subjected to hybridization for Human Genome U133A 2.0 microarrays HG-U133A 2.0 Chip (Affymetrix).

Quantitative real time polymerase chain reaction (QRT-PCR). In order to confirm the data derived from microarray technique, for 4 selected genes (MNDA, GH1, NUDT21, RHOG) a verification by real time PCR was conducted. The synthesis of cDNA library and quantitati­ve PCRs reactions were performed according to the manu­facturer’s reagents recommendation (Roche Diagnostics).

Reactions were carried out in a Mastercycler ep realplex thermocycler (Eppendorf). To analyze the relative expres­sion levels the REST^© (QIAGEN) program was used. The expression of selected genes was evaluated in a study gro­up and in an independent group of 54 patients.

Statistical analysis. Arrays were grouped according to the MTT assay resistance to etoposide profile. For each gene an expression median was defined. The median was calcu­lated in 4 analyzed groups (sensitive, moderately sensitive, moderately resistant, resistant). As a measure of changes in the level of gene expression, the median ratio between resistant and sensitive patients was adopted. Comparative analysis was performed on the basis of the algorithms im­plemented in the MAS 5.0 program (Affymetrix). For each gene, the correlation coefficient (Spearman test), the signi­ficance of the difference (Wilcoxon test) and the significan­ce adjusted for multiple comparisons (FDR, the Benjamin and Hochberg test) were calculated.

Results

Selection of relevant genes. Genes were ranked by fold change in expression level as up-regulation or down-regu­lation. Using the permuted Spearman correlation test, 233 probe sets significantly correlated with sensitivity to eto­poside (p<0.001) were selected. In the global test of signi­ficance of differences, the probability of obtaining such a number of genes is low (R=0.008), which confirms the si­gnificance of the observed difference.

For 150 sets of probes/134 genes an increase in expres­sion was observed (p<0.001, FDR<0.09, correlation coef­ficient from 0.418 to 0.625). The biggest changes in the le­vel of expression were observed for the following genes: RAB32 (member of RAS oncogene family), HPGDS (he­matopoietic prostaglandin D synthase), XCL1 (chemoki­ne (C motif) ligand 1), CSF3R (colony stimulating factor 3 receptor (granulocyte)), PTGS2 (prostaglandin-endope­roxide synthase 2 (prostaglandin G/H synthase and cyclo­oxygenase)) and CSTA (cystatin A (stefin A)). For 83 sets of probes/75 genes the expression silencing was observed (p<0.001, FDR <0.09, correlation coefficient from -0.418 to -0.611). The largest decrease in expression level was obse­rved for: SMARCA4 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, mem­ber 4), CELSR2 (cadherin, EGF LAG seven-pass G-type receptor 2 (flamingo homolog, Drosophila)), TCF3 (trans­cription factor 3 (E2A immunoglobulin enhancer binding factors E12/E47)), QRSL1 (glutaminyl-tRNA synthase (glu­tamine-hydrolyzing)-like 1), VASH2 (vasohibin 2), TPD52 (tumor protein D52), QRSL1 (glutaminyl-tRNA synthase (glutamine-hydrolyzing)-like 1) and PLXNB1 (plexin B1).

Hierarchical clustering. On the resulting set of discrimi­natory genes a cluster analysis was performed (Figure 1). The set of tested probe was used for hierarchical cluste­ring of both genes and patients. The results of this analy­sis, illustrated in the form of a dendrogram, are shown in Figure 1. On the basis of this analysis it was concluded that a specific set of genes shows a significant expression de­pendence on the value of chemoresistance of the samples.

Figure 1. Supervised hierarchical clustering discriminating etoposide resistant and sensitive ALL patients (genes grouped in rows, patients in the columns). Left side – shows the grouping according to the similarity of the expression, right side – contains a grouping of patients by increasing the LC50 values

Ontological analysis of molecular functions. Detailed ana­lysis of each functional class within a designated set of ge­nes revealed that the genes most strongly represented in the profile were those associated with nucleic acids (16.8%), receptor genes (13.2%) and genes participating in signal transduction (5.3%).

Diverse expression of genes was also observed for enzymes performing functions associated with the metabolism of proteins such as kinases, hydrolases and proteases. A de­crease in expression levels was observed for genes of RNA binding proteins and for genes involved in post-transcrip­tional mRNA splicing such as NUDT21 (nudix (nucleosi­de diphosphate linked moiety X)-type motif 21), FUSIP1 (FUS interacting protein (serine/arginine-rich) 1), SFRS3 (splicing factor, arginine/serine-rich 3). Stronger expres­sion silencing (by approximately 40%) was observed in the case of 4 helicases: BRIP1 (BRCA1 interacting protein C-terminal helicase 1), DHX9, 34 (DEAH (Asp-Glu-Ala-His) box polypeptide 9, 34) and SMARCA4 (SWI/SNF re­lated, matrix associated, actin dependent regulator of chro­matin, subfamily a, member 4). Topoisomerase II, a gene for the target protein for etoposide, also belongs to the ana­lyzed functional group. For the TOP2B gene (topoisomera­se (DNA) II beta 180 kDa) the analysis showed results in­dicating that the amount of transcripts in resistant cells is reduced by 10%. Changes in the transcription level show a strong correlation with the profile of resistance to VP16 (p=8.43 E-05, FDR=0.032).

Among the genes performing receptor functions, the big­gest changes were observed for the following receptor ge­nes: G protein-bound, cytokines and immunoglobulins.

The diverse expression within the class of genes perfor­ming regulatory functions focused mainly on genes of G proteins and their modulators. Overexpression was obse­rved for small GTPases such as RAP2B (RAP2B, mem­ber of RAS oncogene family), RAB31 (RAB31, member of RAS oncogene family), RHOG (ras homolog gene family, member G (rho G)), RAB27A (RAB27A, member of RAS oncogene family), RAB32 (RAB32, member of RAS on­cogene family) and their regulators: RGS10 (regulator of G-protein signaling 10), RIN3 (Ras and Rab interactor 3), HP (haptoglobin), REPS2 (RALBP1 associated Eps doma­in containing 2), ARAP1 (centaurin, delta 2). RAB32 and RIN3 belong to a group of genes for which the highest fold change in expression level (p<0.001, FDR<0.06) was ob­tained, which suggests a strong relationship with the pro­file of resistance to VP16.

The analysis of molecular functions in the resulting list of genes was narrowed down so as to select only classes whe­re at least a double overrepresentation of genes – in relation to the number of expected genes based on the frequency of a given group of genes on the used oligonucleotide micro­array – occurred. The results indicate that the most signi­ficant changes correlating with resistance to VP16 apply to genes performing functions associated with DNA me­tabolism and protein modifications (Table 2).

Table 2. Statement of functional classes for which it has been observed 2-fold genes overrepresentation (molecular function)

Analysis of biological processes. Classification of selected genes according to their participation in known biological processes showed that the largest group consisted of genes associated with signal transduction (23.7%), metabolism and protein modifications (20%), nucleic acids metabo­lism (17.9%), and immune processes (18.4%).

Among signal transducers, over a dozen genes were iden­tified, including genes involved in cell communication, si­gnal transduction from surface receptors and intracellular cascades. Overexpression was observed mainly for 9 ge­nes involved in cell adhesion, 8 genes involved in signal transduction of chemokines and cytokines, 7 genes asso­ciated with the path of signals involving G proteins and 5 genes of the JAK-STAT cascade.

Diverse changes in expression level were observed for ge­nes involved in regulating mRNA transcription, which ac­counted for the largest percentage of nucleic acid metaboli­zing genes. DNA metabolism genes including regulators of replication and damaged helix repair enzymes’ genes, were numerously represented in the profile. For repair genes such as CSNK1E (casein kinase 1, epsilon), BRIP1 (BRCA1 in­teracting protein C-terminal helicase 1), ATR (ataxia te­langiectasia and Rad3 related), and MSH3, 6 (mutS ho­mologue 3, 6 (E. coli)), a decline in the level of expression was observed.

Analysis of cellular pathways. The strongest association and the largest number of genes were obtained for a tra­il associated with inflammatory processes, which are as­sociated with chemokines and cytokines. The following genes belonging to this pathway were identified: ITGAM (integrin, alpha M (complement component receptor 3, alpha, also known as CD11b (P170), macrophage antigen alpha polypeptide)), STAT3 (signal transducer and ac­tivator of transcription 3 (acute-phase response factor)), IL8RB (interleukin 8 receptor, beta), FPR1 (formyl pepti­de receptor 1), FGR (Gardner-Rasheed feline sarcoma vi­ral (v-FGR) oncogene homolog), ALOX5AP (arachido­nate 5-lipoxygenase-activating protein), CISH (cytokine inducible SH2-containing protein), STAT4 (signal trans­ducer and activator of transcription 4), ITGB2 (integrin, beta 2 (antigen CD18 (p95), lymphocyte function-asso­ciated antigen 1, macrophage antigen 1 (mac-1) beta sub­unit)), IKBKB (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta).

Five overexpressed genes belonging to the integrin pathway were also identified, including the ITGB2 gene (integrin, beta 2 (antigen CD18 (p95), lymphocyte function-associa­ted antigen 1, macrophage antigen 1 (mac-1) beta subunit)). The increased activity of beta 2 integrin is associated with the reduction of drug-induced DNA damage reduction of caused by etoposide activity. Expression of this gene sho­wed a strong correlation with the profile of resistance at high significance (p=8.07 E-05, FDR=0.032, R=0.492), which confirms the significance of the observed change.

Discussion

Studies in cell lines indicate that the causes of cellular in­sensitivity to etoposide may be overexpression of MDR-1 and anti-apoptotic BCL-2 genes, as well as topoisomera­se IIa expression silencing [28]. It was shown that in cells resistant to etoposide an increase in drug efflux from cells and avoiding the apoptosis associated with BCL occurs. In cells insensitive to VP16, chromosomal instability and po­lyploidy induction were observed [9,28,29]. Another re­ason may be the changes in topoisomerase activity asso­ciated with changes in the phosphorylation of the enzyme (in a catalytic site) [3,9,10,12,26,34]. Changes in the cel­lular localization of the enzyme can also affect cellular re­sistance. Phosphorylation of the end of C terminus of the nuclear form of topoisomerase IIα plays a role in prote­in translocation into the cytoplasm [4]. The decline of the nuclear form affects the reduction of DNA damage un­der the influence of etoposide [1,30]. Cells with a resistant phenotype are also characterized by a decrease in intracel­lular calcium ion concentration [3]. Another cause of in­hibition of the therapeutic effect of etoposide is the reduc­tion of after drug-induced DNA damage, associated with β1 integrin pathway cell adhesion. Resistance associated with this pathway may be a consequence of the reduction in the amount of DNA strand breaks and increased cellular tolerance for these types of damage [15]. One of the me­chanisms of cell response to VP16 is hypoxia and the as­sociated variable expression of tens of genes, allowing the inhibition of p53-dependent apoptosis [37]. Changes in the level of expression of oncogenes from the RAS and SRC family have an indirect impact on the sensitivity of cells to VP16 [24,38].

In order to identify genetic mechanisms leading to the emer­gence of resistance in etoposide-induced leukemic blasts, expression arrays were used, which allowed us to define the changes in the level of gene expression. The study shows that the most significant changes of expression, correla­ting with resistance to VP16, apply to genes performing functions associated with DNA metabolism and protein modifications. Within the resistance profile a significant percentage consists of genes involved in the regulation of mRNA transcription and DNA metabolism genes, inclu­ding those controlling replication as well as those belon­ging to the double helix damage repair enzymes. Reduction of gene expression was observed for RNA-binding prote­ins, helicases, and genes participating in post-transcriptio­nal mRNA splicing. In the group of nucleic acid metabo­lizing genes we also observed changes in gene expression levels for the target protein for etoposide – topoisomera­se II. A strong correlation with the profile of resistance was shown by a decrease in TOP2B gene (topoisomera­se (DNA) II beta 180 kDa) expression. The consequence of lowering the level of topoisomerase II in cells is the re­duction of the amount of so-called fissile complexes (co­valent connections between enzyme and double-stranded DNA), which become susceptible to stabilization by eto­poside. A cell with a smaller pool of target molecules for cytostatic is less susceptible to its effects.

Silencing of expression was also observed for a group of genes involved in DNA strand repair, such as CSNK1E, BRIP1, ATR, MSH3 and MSH6. Cysteine kinase 1 is one of the enzymes that affect the activity of topoisomerase II. It is also responsible for the phosphorylation of the enzyme in a catalytic spot in a Ser1106 position [3,9,10]. This post­translational modification is required for full activation of topoisomerase. Reduction of expression of CSNK1E cau­ses a decrease in the amount of active enzyme and indirec­tly reduces the efficiency of VP16. Ten genes involved in protein phosphorylation and proteolysis were identified wi­thin the resistance profile, which suggests a significant ef­fect of such modification of proteins in leukemic cells’ de­fense mechanism against the cytostatic effect of etoposide.

Changes of expression in regulatory genes and their prote­in products from the BCL-2/BAX family are believed to be the cause of apoptosis abnormalities in cancer cells. Within the resistance profile to etoposide an increased expres­sion of BCL2A1 (BCL2-related protein A1, p=1.70 E-06, FDR=0.00541, R=0.583) was observed. Overexpression of BCL-2 is related to resistance to many chemotherapeutic agents acting through the induction of programmed de­ath of cancer cells. Silencing of BCL-2 significantly en­hances the sensitivity of breast cancer cells to etoposide and doxorubicin [35]. Research by Hong et al. [18] sho­wed a significant increase in expression of BCL-2 in leu­kemic line HL-60 cells with a multi-drug resistant pheno­type. Bednarek et al. [2] confirmed that the combination of etoposide with the inhibitor gene (oligonucleotide an­ti-BCL-2) significantly increases the therapeutic effect by reducing cell proliferation.

Several genes from the signal transduction pathway con­nected with integrins were identified within our analyzed profile. Increased activity of integrins is associated with the reduction in after drug-induced DNA strand damage induced by etoposide [19]. The role of overexpression of genes for integrins in resistance to cytostatics has been pre­viously demonstrated in studies on liver tumor by Zhang et al. [46]. It has also been proven that intracellular signa­ling pathways, associated with integrins, modulate Fas T lymphocyte-dependent apoptosis [11]. Overexpression of genes of this group in resistant leukemic blasts shows that they may play an important role in inhibition of the pro­grammed death pathway.

Microarray technology (expression, cDNA, miRNA) is increasingly being used in studies aimed at identification of new markers of resistance of tumor cells to etoposi­de [5,13,14,21,25,43,45]. Several genes from our profile in common with the analysis of resistance to etoposide in cell lines described by Györffy et al. [14] were identified. In compared profiles, large groups of genes were genes of kinases and endopeptidases, and genes related to the me­tabolism of nucleic acids. Maximum compliance, both in terms of the number of selected genes and their functions, were found for the analysis conducted by Györffy et al. [14], although those studies were carried out on cell lines.

Recent studies of the past few years, performed using mi­croarray technology, have led to the identification of new candidate genes responsible for cell resistance to etoposi­de. Re-expression of transcription factor TWIST2 resul­ted in increased sensitivity of lymphoblasts to this che­motherapeutic agent [41]. De Tayrac et al. [5] suggested PCDH9 and STARD13 as candidate tumor suppressor genes, participating in resistance to etoposide, probably through changes in ceramide signaling to the RhoA pa­thway. Lawson et al. [25] indicated 2 genes as key genes for resistance to etoposide in small cell lung cancer: the DNA repair enzyme DNA polymerase β (Pol β) and the neuroendocrine transcription factor NKX2.2. Inhibition of Pol β potentiates the cytotoxicity of etoposide, by pro­motion of DNA double strand breaks. Resistance to eto­poside in SCLC cell lines was related to higher levels of NKX2.2. Wong et al. [44] reported that changes in expres­sion of the TOP2B gene are associated with histological grading, microvascular invasion, early age of onset of the malignancy response to etoposide and survival in hepato­cellular carcinoma.

Despite the presence of the used HG-U133A 2.0 probe ar­rays for known genes linked to resistance (e.g., 93 probes for the ABC family of transporters, including MDR1 and MRP1), significant changes in their expression correlating with resistance to etoposide have not been found. Intensive studies of various mechanisms of resistance to therapy (in­cluding known multidrug resistance genes) prove their li­mited relevance, especially in the establishment of de novo drug resistance [6,42]. These studies stand in opposition to results presented by Iijima et al. [5], Walters et al. [42] and Wong et al. [44], which indicate a strong association of changes in expression level of ABC family genes with the lack of sensitivity of tumor cells to etoposide.

Studies on the scale of the whole genome or transcripto­me are a relatively objective approach to identifying ge­netic determinants of cell responses to drugs and their re­sistance to them. The identification of genes contributing to resistance to etoposide and determining the role of tho­se genes could help in characterizing patient responsive­ness and overcoming resistance in children with leukemia.

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The authors have no potential conflicts of interest to declare.

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