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MAPK7 and MAP2K4 as prognostic markers in osteosarcoma
Human Pathology (2012) 43, 994–1002
www.elsevier.com/locate/humpath
Original contribution
MAPK7 and MAP2K4 as prognostic markers in osteosarcoma☆ Francine Tesser-Gamba MSc a, b , Antonio Sergio Petrilli MD, PhD a , Maria Teresa de Seixas Alves MD, PhD a, c , Reynaldo Jesus Garcia Filho MD, PhD d , Yara Juliano PhD e , Sílvia Regina Caminada Toledo PhD a, b,⁎ a
Department of Pediatrics, Pediatric Oncology Institute (Grupo de Apoio ao Adolescente e à Criança com Câncer), Federal University of São Paulo, São Paulo, SP 04023-062, Brazil b Department of Morphology and Genetics, Federal University of São Paulo, São Paulo, SP 04023-062, Brazil c Department of Pathology, Federal University of São Paulo, São Paulo, SP 04023-062, Brazil d Department of Orthopedic Surgery and Traumatology, Federal University of São Paulo, São Paulo, SP 04023-062, Brazil e Department of Public Health, University of Santo Amaro, São Paulo, SP 04829-300, Brazil Received 15 April 2011; revised 10 August 2011; accepted 12 August 2011
Keywords: Osteosarcoma (OS); Gene expression; OS markers; MAPK7; MAP2K4
Summary Osteosarcoma is a class of cancer originating from the bone, affecting mainly children and young adults. Cytogenetic studies showed the presence of rearrangements and recurrent gains in specific chromosomal regions, indicating the possible involvement of genes located in these regions during the pathogenesis of osteosarcoma. These studies investigated expression of 10 genes located in the chromosomal region involved in abnormalities in osteosarcoma, 1p36, 17p, and chromosome 19. The purpose of this study was to investigate the expression profile of genes located in regions involved in chromosomal rearrangements in osteosarcoma. We used quantitative real-time polymerase chain reaction to investigate the expression of 10 genes located in 1p36.3 (MTHFR, ERRFI1, FGR, E2F2), 17p (MAPK7, MAP2K4), and chromosome 19 (BBC3, FOSB, JUND, and RRAS), in 70 samples taken from 30 patients (30 prechemotherapy, 30 postchemotherapy, and 10 metastases specimens) and 10 healthy bones as a control sample. The most interesting results showed a strong association between the expression levels of MAPK7 and MAP2K4 genes and clinical parameters of osteosarcoma. Overexpression of these genes was significantly associated to a poor response to treatment (P = .0001 and P = .0049, respectively), tumor progression, and worse overall survival (P = .0052 and P = .0085, respectively), suggesting that MAPK7 and MAP2K4 could play an important role in osteosarcoma tumorigenesis. Thus, these genes could be good markers in assessing response to treatment and development of osteosarcoma. © 2012 Elsevier Inc. All rights reserved.
☆ This study was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (São Paulo, Brazil), Grant nos. 07/53869-3 and 07/02693-2, and Grupo de Apoio ao Adolescente e à Criança com Câncer/Federal University of São Paulo (São Paulo, Brazil). ⁎ Corresponding author. Pediatrics Oncology Institute (Grupo de Apoio ao Adolescente e à Criança com Câncer), Federal University of São Paulo, Rua Botucatu, São Paulo, SP 04023-062, Brazil. E-mail addresses: [email protected] (F. Tesser-Gamba), [email protected] (A. S. Petrilli), [email protected] (M. T. de Seixas Alves), [email protected] (R. J. G. Filho), [email protected] (Y. Juliano), [email protected] (S. R. C. Toledo).
0046-8177/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2011.08.003
MAPK7 and MAP2K4 as prognostic markers in osteosarcoma
1. Introduction Osteosarcoma (OS) is the most common type of primary bone cancer. It can occur at any age, but most patients are aged between 12 and 25 years [1]. OS is classified as a malignant mesenchymal neoplasm in which the tumor directly produces defective osteoid (immature bone). Approximately 10% to 20% of patients with OS have the metastatic disease at diagnosis. The most frequent site of metastasis is the lung; however, a smaller percentage of patients have bone and soft tissue metastases. The presence of metastasis at diagnosis is a prognostic factor with strong impact in the overall survival of patients with OS [2]. In Brazil, 20% of patients present metastatic disease at diagnosis, a rate that is twice as high compared with that reported in developed countries [3]. The current treatment of OS involves an integrated strategy incorporating chemotherapy and surgery. Despite the dramatic advances in OS treatment, patient survival reached a plateau. Recent clinical trials that attempted to improve outcome through intensification of therapy or incorporation of new agents have not been widely successful. The exact cause of OS remains unknown. Therefore, increasing focus has been placed in achieving a greater understanding of the basic biology of OS to improve treatment [4]. Unlike other sarcomas, such as synovial sarcoma, alveolar rhabdomyosarcoma, and Ewing sarcoma, no specific translocations or genetic abnormalities have been identified in OS [5]. Nevertheless, nearly 70% of OS tumors display a multitude of cytogenetic abnormalities. These findings further highlight the complexity and the instability of the genetic makeup of OS tumors [5]. Cytogenetic studies showed the presence of rearrangements and recurrent gains in specific chromosomal regions, indicating the possible involvement of genes located in these regions in the pathogenesis of OS. Molecular cytogenetic studies use comparative genomic hybridization, as a method, identified frequent chromosomal gain involving 1p36.3, 17 and chromosome 19 [6-9]. For our study, we selected genes located in these 3 chromosomal regions that could function when genomic gain occurs. We have used quantitative polymerase chain reaction (PCR) for gene expression investigation, and we have compared these genes expression with healthy bone samples. The genes of this study were selected, considering the regions with more frequent genomic gain reported in previous work [10]. Our objectives were to validate the increase in the gene expression related to the genomic gain using the technique of quantitative PCR and to correlate these values with the clinical data of patients with OS. Among other gene, in the 1p36.3 region are, 5,10methylenetetrahydrofolate reductase (MTHFR), GardnerRasheed feline sarcoma viral oncogene homolog (FGR), E2F transcription factor 2 (E2F2), and ERBB-receptor feedback inhibitor 1 (ERRFI1). MTHFR codifies an enzyme that catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a cosubstrate for homocysteine remethylation to methionine. Genetic variation in this gene influences susceptibility to occlusive
995 vascular disease, neural tube defects, colon cancer, and acute leukemia. Mutations in this gene are associated with methylenetetrahydrofolate reductase deficiency [11]. The transcription factor E2F2 is a protein encoded by the E2F2 gene. The E2F family plays a crucial role in the control of the cell cycle and the action of tumor suppressor proteins, also being a target of the transforming proteins of small DNA tumor viruses [12]. ERBB receptor feedback inhibitor 1 is a protein encoded by the ERRFI1 gene. This gene codifies a cytoplasmic protein whose expression is up-regulated with cell growth, which is induced during cell stress and mediates cell signaling [13]. The FGR gene is a member of the Src family of protein tyrosine kinases. This gene codifies a protein localized in plasma membrane ruffles that functions as a negative regulator of cell migration and adhesion triggered by the β-2 integrin signal transduction pathway [14]. In the chromosome 17p region, 2 genes of the mitogenactivated protein kinases (MAPKs), MAPK7 and MAP2K4, are localized. MAPKs are serine/threonine-specific protein kinases that respond to extracellular stimuli and regulate various cellular activities such as gene expression, mitosis, differentiation, proliferation, and cell survival/apoptosis [15]. MAPKs are involved in the activation of most nonnuclear oncogenes. Mitogen-activated protein kinase 7 (MAPK7) is an enzyme encoded by the MAPK7 gene and is specifically activated by mitogen-activated protein kinase kinase 5 (MAP2K5/MEK5). It is involved in the downstream signaling processes of various receptor molecules including receptor type kinases and G protein–coupled receptors. In response to extracellular signals, this kinase translocates to the cell nucleus, where it regulates gene expression by phosphorylating and activating different transcription factors [16]. Dual-specificity mitogen-activated protein kinase 4 (MAP2K4) is an enzyme encoded by the MAP2K4 gene that also encodes a dual-specificity protein kinase that belongs to the Ser/Thr protein kinase family. This kinase is a direct activator of MAP kinases in response to various environmental stresses or mitogenic stimuli [17]. Finally, the genes BCL2 binding component 3 (BBC3), jun D proto-oncogene (JUND), related RAS viral oncogene homolog (RRAS), and FBJ murine OS viral oncogene homolog B (FOSB) are located in chromosome 19. The BBC3 and JUND genes are related to apoptosis and cell senescence. BBC3 is a Bcl-2 family member and is a critical mediator of p53-dependent and p53-independent apoptosis induced by a wide variety of stimuli, including genotoxic stress, deregulated oncogene expression toxins, altered redox status, growth factor/cytokine withdrawal, and infection. BBC3 transduces death signals primarily to the mitochondria, where it acts indirectly on the Bcl-2 family members Bax and/or Bak by relieving the inhibition imposed by antiapoptotic members. It directly binds and antagonizes all known antiapoptotic Bcl-2 family members to induce mitochondrial dysfunction and caspase activation [18]. JUND protein encoded by the intronless JUND gene is a member of the JUN family and a functional component of the activator protein 1 (AP1)
996 transcription factor complex. This protein has been proposed to protect cells from p53-dependent senescence and apoptosis [19]. RRAS gene is a member of the Ras family of small GTPases that are involved in cellular signal transduction, implicated in promoting cell adhesion and neurite outgrowth through integrin activation [20]. FOSB is a protein encoded by the FOSB gene that encodes leucine zipper proteins that can dimerize with proteins of the JUN family, thereby forming the transcription factor complex AP1 [21]. Thus, the objective of the current study was to examine the expression profile of 10 genes located in recurrent regions of chromosomal gains: 1p36.3 (MTHFR, FGR, E2F2, ERRFI1) 17p (MAPK7, MAP2K4), and chromosome 19 (BBC3, RRAS, JUND, FOSB) in a cohort of OS tumors. In addition, we analyzed the association of the expression profile of these genes with the OS clinicopathologic parameters.
2. Materials and methods 2.1. Samples All 70 flash-frozen OS samples from 30 patients (30 prechemotherapy specimens, 30 postchemotherapy specimens, and 10 metastasis specimens) used in this study were obtained from patients treated at the Pediatric Oncology Institute Grupo de Apoio ao Adolescente e a Criança com Câncer/Federal University of São Paulo. This was a retrospective study of samples collected sequentially between 2002 and 2008. Ten healthy bone samples were used as controls. These samples were obtained from healthy individuals without genetic and/or musculoskeletal diseases who underwent orthopedic surgery due to trauma (Supplementary Data 1 (on the journal's website at www.humanpathol.com)). Samples from each OS tumor and healthy bone were collected after informed consent was signed by patients/guardians according to the university's institutional review board (IRB/Federal University of São Paulo no. 0050). All patients were treated following the standard regimens—the Brazilian Osteosarcoma Treatment Group— comprising cisplatin, doxorubicin, and methotrexate. The team of orthopedists in collaboration with the pediatric oncology team determined the appropriate surgical procedure for each patient. Nonconventional endoprosthesis, resection of expendable bones, plaques, and bone graft fixation (autograft or bone bank) were used. Whenever possible, all pulmonary metastases were surgically removed after resection of the primary tumor.
2.2. RNA isolation and reverse transcription reaction Total RNA was extracted from tumor samples using TRIzol reagents (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA quality and quantity were determined by gel electrophoresis and spectrophotometry,
F. Tesser-Gamba et al. respectively. DNase treatment was used to remove genomic contamination using deoxyribonuclease I Amplification Grade (Invitrogen). Synthesis of complementary DNA (cDNA) was performed using 1 μg of total RNA according to the manufacturer's protocol of SuperScript III Kit (Invitrogen).
2.3. Primers The primers of the 10 genes investigated in this study and the endogenous gene b-actin (ACTB) were designed using Primer Express (3.0) Software from Applied Biosystems (Foster City, CA) taking care that the forward and reverse sequences were in different exons. We conducted in NCBI a Basic Local Alignment Search Tool (BLAST) search to confirm the total gene specificity of the nucleotide sequences chosen for primers. The sequences of primers are presented in Supplementary Data 2 (on the journal's website at www.humanpathol.com).
2.4. Real-time PCR Expression levels of FGR, E2F2, ERRFI1, MTHFR, MAP2K4, MAPK7, RRAS, FOSB, BBC3, and JUND were determined by quantitative real-time PCR. This analysis was performed in a thermocycler Applied Biosystems Prism 7500 Sequence Detection System (PE Applied Biosystems, Inc, Foster City, CA) using relative quantification. The expression of the housekeeping gene ACTB was used as an endogenous control in each assay performed. The reaction mixture combined 6 μL SYBR Green PCR Master Mix (PE Applied Biosystems) reaction mix, 3 μL sense/antisense primers, and 3 μL cDNA. The cycling conditions were as follow: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. Reactions were done in triplicate. For each sequence, a standard curve was constructed to determine the sensitivity and efficiency assays. For each sample, the cycle threshold (Ct) was determined (mean of the 3 reactions) for both the target gene and the endogenous control gene and was normalized for cDNA quantity. Subtracting the Ct of the endogenous control gene from the Ct of the target gene yields the ΔCt. The ΔCt of the control reference was then subtracted from the ΔCt of the tumor sample, yielding the ΔΔCt, and the relative quantification value was expressed as 2 −ΔΔCt [22].
2.5. Clinical parameters Clinical variables that were correlated with gene expression included age at diagnosis, sex, histologic OS subtype, site of primary tumor, presence of metastasis at diagnosis, and necrosis grade after chemotherapy (Huvos grade) [1]. The Huvos grading system was used to rate the level of tumor necrosis after preoperative chemotherapy: grade I, little or no effect of chemotherapy noted; grade II, partial response to chemotherapy, with between 50% and 90%
MAPK7 and MAP2K4 as prognostic markers in osteosarcoma
997
Fig. 1 A, Kruskal-Wallis test. Quantitative analysis of gene expression from prechemotherapy, postchemotherapy, and metastasis specimens compared with healthy bone samples for MAPK7 gene. B, Mann-Whitney test. Quantitative analysis of MAPK7 gene expression for metastatic and nonmetastatic patients. C, Mann-Whitney test. MAPK7 gene expression in postchemotherapy specimens for patients with good and bad response. D, Mann-Whitney test. MAPK7 gene expression in samples of patients in complete remission at the end of treatment. E, Overall survival curve using quantitative values of MAPK7 gene expression in prechemotherapy specimens using the median value of all samples of prechemotherapy specimens. F, Overall survival curve using quantitative values of MAPK7 gene expression in postchemotherapy specimens using the median value of all samples of postchemotherapy specimens. *P b .05.
998 necrosis; grade III, greater than 90% necrosis; and grade IV, no viable tumor cells are apparent [1]. Patients with nonmetastatic disease were considered to be in complete remission on the date of the primary tumor resection, and patients with metastatic tumors were considered to be in complete remission on the date of the pulmonary metastases resection. Overall survival was defined as the time from diagnosis until the date of either the last contact or death.
2.6. Statistical analysis Data analyses were performed using GraphPad Prism 4 Software (San Diego, CA). Continuous data (gene expression levels) were evaluated and compared using nonparametric tests: Mann-Whitney, Wilcoxon, Kruskal-Wallis, correlation coefficient, Spearman, and Friedman. Categorical data (age at diagnosis, sex, histologic OS subtype, primary site, presence of metastasis at diagnosis, and necrosis grade) were studied using χ 2 and Fisher exact test applying software VassarStats (Website for Statistical Computation; Poughkeepsie, NY, USA). The analysis for high expression and low expression of each group of samples (prechemotherapy, postchemotherapy, and metastasis specimens) used the median relative quantification value of the 11 samples of healthy bones as the normal reference. Statistical significance was set at P b .05 (Figs. 1 and 2).
3. Results A summary of the clinicopathologic characteristics is detailed in Supplementary Data 3 and 4 (on the journal's website at www.humanpathol.com). The clinicopathologic parameters analyzed were age at diagnosis, sex, presence of metastasis at diagnosis, Huvos grade [1], site of the primary tumor, use of methotrexate in treatment, and histologic type. We performed quantitative real-time PCR to quantify messenger RNA levels of MTHFR, E2F2, FGR, ERRFI1, MAPK7, MAP2K4, FOSB, JUND, BBC3, and RRAS genes in 30 prechemotherapy, 30 postchemotherapy, and 10 metastasis specimens from 30 patients with OS (Table 1). Results of all genes in all OS samples versus healthy samples, clinical parameters, and overall survival are described in Table 2.
Fig. 2 A, Kruskal-Wallis test. Quantitative analysis of gene expression from prechemotherapy, postchemotherapy, and metastasis specimens compared with healthy bone samples for MAP2K4 gene. Overall survival using quantitative values of MAP2K4 gene expression in prechemotherapy specimens using the median value of all samples of prechemotherapy specimens (B) and postchemotherapy specimens (C) using the median value of all postchemotherapy specimens. *P b .05.
F. Tesser-Gamba et al.
MAPK7 and MAP2K4 as prognostic markers in osteosarcoma Table 1
999
Number and percentage of OS samples with overexpression of the genes analyzed
Gene
Specimens
Total
Overexpression n
%
MTHFR
PreCH PostCH Metastasis PreCH PostCH Metastasis PreCH PostCH Metastasis PreCH PostCH Metastasis PreCH PostCH Metastasis PreCH PostCH Metastasis PreCH PostCH Metastasis PreCH PostCH Metastasis PreCH PostCH Metastasis PreCH PostCH Metastasis
30 30 10 30 30 10 30 30 10 30 30 10 30 30 10 30 30 10 30 30 10 30 30 10 30 30 10 30 30 10
21 14 7 25 24 10 26 14 8 5 3 2 26 16 7 25 11 4 12 12 7 11 18 7 17 18 4 8 17 6
70 47 70 83 80 100 86 46 80 16 30 20 86 53 70 83 37 40 40 40 70 37 60 70 57 60 40 27 57 60
ERRFI1
E2F2
FGR
MAPK7
MAP2K4
FOSB
JUND
BBC3
RRAS
Abbreviations: PreCH, prechemotherapy specimens; PostCH, postchemotherapy specimens.
4. Discussion In OS, some aspects regarding its pathogenesis are still unknown, and questions about the genetic determinants for its development are still unanswered. Moreover, any molecular marker that could be related to its development, evolution, and progression has not been described. Progress related to the improved survival of patients with OS is at a plateau, and the intensification of therapy or the addition of new therapeutic agents has been successful, particularly in the metastatic patients at diagnosis [4]. In this study, patients with OS presented distribution by sex, age, and clinical and pathologic examination similar to those described in the literature [3,23]. As expected, the number of affected men (70%) was higher than women (30%), and the peak of incidence was in the second decade of life (60%). The most frequent site of tumor was femur (53%), and the most frequent histologic type was osteoblastic (50%). Using Huvos classification, the necrosis grades I and II were observed in most metastatic patients (43%).
Previous studies using comparative genomic hybridization showed a gain of genomic DNA from specific chromosomal regions in OS samples [24]. The premise of our study was to determine if gains of specific chromosomal regions could be truly representing an increase of gene expression in OS. To contribute to the current knowledge of chromosomal abnormalities in OS, we investigated expression profiles of 10 genes located in the chromosomal gain regions, 1p36, 17p, and chromosome 19. MTHFR, ERRFI1, E2F2, FGR, FOSB, JUND, BBC3, and RRAS genes showed no significant correlation with the development of OS. From 10 genes investigated, 2 of them, MAPK7 and MAP2K4, located in 17p, were overexpressed in most OS samples analyzed (Table 1). In our study, we found a higher expression of MAP2K4 and MAPK7 genes in prechemotherapy OS specimens than in postchemotherapy specimens or healthy bone controls. Between the postchemotherapy groups, patients carrying specimens with a high expression of these genes were significantly associated with the presence of metastasis at diagnosis, poor Huvos grade,
1000 Table 2
F. Tesser-Gamba et al. Expression of all genes according to clinical parameters and overall survival
High expression in PreCH specimens vs PostCH specimens PostCH specimens vs PreCH specimens PreCH specimens vs metastasis specimens PreCH specimens vs healthy bone specimens Healthy bone vs PreCH specimens Healthy bone vs PostCH specimens Metastasis specimens vs healthy bone specimens Metastasis specimens vs patients who did not relapse PreCH specimens vs relapse patients PostCH specimens vs relapse patients PostCH specimens vs patients with tumor b12 cm Metastasis specimens vs patients with tumor N12 cm PostCH specimens vs use of MTX in OS treatment PreCH specimens vs poor Huvos grade (I and II) PostCH specimens vs poor Huvos grade (I and II) PreCH specimens vs complete remission Metastasis specimens vs complete remission PostCH specimens vs metastatic patients at diagnosis PostCH specimens vs nonmetastatic patients at diagnosis PostCH specimens vs patients without complete remission Comparison of moments PreCH vs PostCH vs metastasis vs healthy bone Overall survive PreCH specimens PostCH specimens
Genes located at 1p36
Genes located at 17p
MTHFR ERRFI1 E2F2 (P) (P) (P)
FGR (P)
.0276 ⁎
.0004 ⁎
Genes located at chromosome 19
MAPK7 MAP2K4 FOSB (P) (P) (P)
JUND (P)
BBC3 (P)
.0114 ⁎ b.0001 ⁎
.0262 ⁎
.0351⁎⁎ .0244 ⁎
.0003 ⁎ .0085 ⁎ .0092 ⁎ .0129 ⁎
.0083 ⁎ .0444 ⁎
RRAS (P)
.0253 ⁎
.0444 ⁎ .0210 ⁎
.0052 ⁎
.0458 ⁎ .0016 ⁎ .0399 ⁎
.0084 ⁎ .0329 ⁎
.0001 ⁎ .0049 ⁎ .0005 ⁎ .0317 ⁎ .0266 ⁎ .0239 ⁎
.0296 ⁎
.0446 ⁎ .0014 ⁎
.067
.0092 ⁎ .0054 ⁎ .0528
.0016 ⁎ .0013 ⁎
.0838
.328
.56
.078
.745 .0181 ⁎ .0052 ⁎ .0085 ⁎
Abbreviations: PreCH, prechemotherapy specimens; PostCH, postchemotherapy specimens; MTX, methotrexate. ⁎ P b .05.
and worse overall survival. Furthermore, the MAP2K4 overexpression in postchemotherapy specimens was also associated with patients without complete remission of disease at the end of treatment and in the last contact. MAPKs have been described in several hormonal systems, being related to proliferation and differentiation of various cell types. In bone cells, parathyroid hormone (PTH) and parathyroid hormone–related protein (PTHrP) show both upregulation and down-regulation of MAPKs during osteoblast
proliferation and differentiation, respectively. The molecular mechanisms that regulate PTH activity in the bone remain incompletely understood; however, it is believed that PTH stimulates the ERK (extracellular signal-regulated kinases) group of MAPKs by multiple mechanisms in cells with PTH/PTHrP receptor 1 (PTH1R) [25]. A previous study demonstrated a bidirectional effect of PTH and PTHrP on MAPK in osteoblasts, increasing phosphorylated ERK (pERK) in undifferentiated cells but
MAPK7 and MAP2K4 as prognostic markers in osteosarcoma decreasing pERK in differentiated cells. In proliferating osteoblastic cells, PTH and PTHrP up-regulate pERK, increase cyclin D1 expression, and promote cell proliferation. In contrast, in differentiated osteoblasts, PTHrP downregulates pERK, decreases cyclin D1 expression, and induces osteoblastic cell growth arrest, allowing more cellular differentiation and bone matrix formation [26]. A proposed mechanism in OS involves the tumoral secretion of bone-modulating compounds, such as PTHrP. In response to PTHrP, osteoclastic bone resorption is stimulated and growth factors are released from the surrounding extracellular bone matrix, further stimulating PTHrP secretion as well as stimulating tumor growth. This positive feedback loop culminates in further tumor growth and metastasis [27]. Previously, one of our studies also showed that overexpression of genes related to bone remodeling process in OS was significantly associated with poor overall survival [28]. In this way, the MAPK7 and MAP2K4 overexpression observed in the current study could be stimulated by PTH/PTHrP and, consequently, would increase the proliferation of undifferentiated OS cells. Therefore, strategies aimed at interrupting this vicious cycle may be useful in OS. In addition, we observed a significant association between MAPK7 overexpression in prechemotherapy specimens and the presence of metastasis at diagnosis. Recently, MAPK7 has also been involved in the control of cellular proliferation and cell death, as well as in tumorigenesis. Elegant in vivo studies using animals in which MAPK7 expression can be regulated have demonstrated that MAPK7 is important for sustaining tumor growth probably because of its supportive role in vasculogenesis and blood vessel homeostasis [29]. Our results demonstrated that MAPKs expression were strongly associated with important clinical parameters, including grade of necrosis. The necrosis grade still is the only one parameter to evaluate the response to treatment. We observed a significant association between MAPK overexpression in postchemotherapy specimens and a poor response to preoperative chemotherapy (grades I and II). Studies have shown that MAPKs expression may be an underlying mechanism contributing to the generation of chemoresistance in cancer [30]. The importance of MAPK activation in cellular response to cisplatin has gradually been appreciated in the last few years. Cisplatin is one of the most effective chemotherapeutic agents and is widely used in the treatment of a variety of pediatric and adult solid tumors including OS. This platinum compound is a DNA-damaging agent that forms cisplatin-DNA adducts and kills cells via several mechanisms, including induction of apoptosis. Resistance to cisplatin is multifactorial and leads to a poor response to chemotherapy and treatment failure. Failure to induce apoptosis is believed to be one of the major mechanisms underlying cisplatin resistance [31]. MAPKs control cell proliferation, differentiation, and cell death. Their role in response to cisplatin is complex, and the response varies with the type of cells as well as their
1001 proliferation and differentiation status [32]. It is believed that MAPK activities are regulated through reversible phosphorylation of both threonine and tyrosine residues by upstream kinases, as well as by the members of MAPK phosphatase (MKP) family, which can dephosphorylate both phosphothreonine and phosphotyrosine residues and inactivate MAPKs. Recent studies suggest that MKP-1, the founding member of the MKP family, plays an important role in the response of cancer cells to chemotherapy, especially to cisplatin [32]. Because cisplatin is one of the most important chemotherapeutic agents used in OS treatment, the MAPK overexpression observed in our study could be contributing to a worse treatment response of patients with OS and, consequently, low overall survival. Therefore, further investigation is necessary to study the role of MAPKs and MKPs in pediatric cancers including OS [31].
5. Conclusion Unfortunately, some groups of patients remain at high risk of eventual relapse and short-term survival. These patients may benefit from future investigations into innovative treatment approaches based on biological markers, such as antiangiogenesis factors or growth factor receptor modulation. However, our study is a pilot study and suggests that further investigations are needed. We suggest that abnormalities in the expression levels of genes involved in important cell signaling pathways, such as MAPKs pathway, may contribute to OS progression and aggressiveness.
Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.humpath.2011.08.003.
Acknowledgments We would like to thank Indhira Dias, MSc; Carolina Salinas, BSc; and Patrícia Pavoni, MSc.
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F. Tesser-Gamba et al. [19] Hernandez JM, Floyd DH, Weilbaecher KN, Green PL, Boris-Lawrie K. Multiple facets of junD gene expression are atypical among AP-1 family members. Oncogene 2008;27:4757-67. [20] Negishi M, Oinuma I, Katoh H. R-ras as a key player for signaling pathway of plexins. Mol Neurobiol 2005;32:217-22. [21] Limb JK, Yoon S, Lee KE, et al. Regulation of megakaryocytic differentiation of K562 cells by FosB, a member of the Fos family of AP-1 transcription factors. Cell Mol Life Sci 2009;66:1962-73. [22] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;4:402-8. [23] Campanacci M. High grade osteosarcomas. In Bone and soft tissue tumors: clinical features, imaging, pathology and treatment. p. 463-515. 2nd ed. Springer-Verlag; New York, NY. [24] Tarkkanen M, Elomaa I, Blomqvist C, et al. DNA sequence copy number increase at 8q: a potential new prognostic marker in high-grade osteosarcoma. Int J Cancer 1999;84:114-21. [25] Ferrari SL, Pierroz DD, Glatt V, et al. Bone response to intermittent parathyroid hormone is altered in mice null for arrestin2. Endocrinology 2005;146:1854-62. [26] Datta NS, Chen C, Berry JE, McCauley LK. PTHrP signaling targets cyclin D1 and induces osteoblastic cell growth arrest. J Bone Miner Res 2005;20:1051-64. [27] Lamoureux F, Trichet V, Chipoy C, Blanchard F, Gouin F, Redini F. Recent advances in the management of osteosarcoma and forthcoming therapeutic strategies. Expert Rev Anticancer Ther 2007;7:169-81. [28] Toledo SR, Oliveira ID, Okamoto OK, et al. Bone deposition, bone resorption, and osteosarcoma. J Orthop Res 2010;9:1142-8. [29] Hayashi M, Kim SW, Imanaka-Yoshida K, et al. Targeted deletion of BMK1/ERK5 in adult mice perturbs vascular integrity and leads to endothelial failure. J Clin Invest 2004;113:1138-48. [30] Haagenson KK, Wu GS. The role of MAP kinases and MAP kinase phosphatase-1 in resistance to breast cancer treatment. Cancer Metastasis Rev 2010;29:143-9. [31] Wang Z, Zhou JY, Kanakapalli D, Buck S, Wu GS, Ravindranath Y. High level of mitogen-activated protein kinase phosphatase-1 expression is associated with cisplatin resistance in osteosarcoma. Pediatr Blood Cancer 2008;51:754-9. [32] Wu GS. Role of mitogen-activated protein kinase phosphatases (MKPs) in cancer. Cancer Metastasis Rev 2007;26:579-85.
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Original contribution
MAPK7 and MAP2K4 as prognostic markers in osteosarcoma☆ Francine Tesser-Gamba MSc a, b , Antonio Sergio Petrilli MD, PhD a , Maria Teresa de Seixas Alves MD, PhD a, c , Reynaldo Jesus Garcia Filho MD, PhD d , Yara Juliano PhD e , Sílvia Regina Caminada Toledo PhD a, b,⁎ a
Department of Pediatrics, Pediatric Oncology Institute (Grupo de Apoio ao Adolescente e à Criança com Câncer), Federal University of São Paulo, São Paulo, SP 04023-062, Brazil b Department of Morphology and Genetics, Federal University of São Paulo, São Paulo, SP 04023-062, Brazil c Department of Pathology, Federal University of São Paulo, São Paulo, SP 04023-062, Brazil d Department of Orthopedic Surgery and Traumatology, Federal University of São Paulo, São Paulo, SP 04023-062, Brazil e Department of Public Health, University of Santo Amaro, São Paulo, SP 04829-300, Brazil Received 15 April 2011; revised 10 August 2011; accepted 12 August 2011
Keywords: Osteosarcoma (OS); Gene expression; OS markers; MAPK7; MAP2K4
Summary Osteosarcoma is a class of cancer originating from the bone, affecting mainly children and young adults. Cytogenetic studies showed the presence of rearrangements and recurrent gains in specific chromosomal regions, indicating the possible involvement of genes located in these regions during the pathogenesis of osteosarcoma. These studies investigated expression of 10 genes located in the chromosomal region involved in abnormalities in osteosarcoma, 1p36, 17p, and chromosome 19. The purpose of this study was to investigate the expression profile of genes located in regions involved in chromosomal rearrangements in osteosarcoma. We used quantitative real-time polymerase chain reaction to investigate the expression of 10 genes located in 1p36.3 (MTHFR, ERRFI1, FGR, E2F2), 17p (MAPK7, MAP2K4), and chromosome 19 (BBC3, FOSB, JUND, and RRAS), in 70 samples taken from 30 patients (30 prechemotherapy, 30 postchemotherapy, and 10 metastases specimens) and 10 healthy bones as a control sample. The most interesting results showed a strong association between the expression levels of MAPK7 and MAP2K4 genes and clinical parameters of osteosarcoma. Overexpression of these genes was significantly associated to a poor response to treatment (P = .0001 and P = .0049, respectively), tumor progression, and worse overall survival (P = .0052 and P = .0085, respectively), suggesting that MAPK7 and MAP2K4 could play an important role in osteosarcoma tumorigenesis. Thus, these genes could be good markers in assessing response to treatment and development of osteosarcoma. © 2012 Elsevier Inc. All rights reserved.
☆ This study was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (São Paulo, Brazil), Grant nos. 07/53869-3 and 07/02693-2, and Grupo de Apoio ao Adolescente e à Criança com Câncer/Federal University of São Paulo (São Paulo, Brazil). ⁎ Corresponding author. Pediatrics Oncology Institute (Grupo de Apoio ao Adolescente e à Criança com Câncer), Federal University of São Paulo, Rua Botucatu, São Paulo, SP 04023-062, Brazil. E-mail addresses: [email protected] (F. Tesser-Gamba), [email protected] (A. S. Petrilli), [email protected] (M. T. de Seixas Alves), [email protected] (R. J. G. Filho), [email protected] (Y. Juliano), [email protected] (S. R. C. Toledo).
0046-8177/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2011.08.003
MAPK7 and MAP2K4 as prognostic markers in osteosarcoma
1. Introduction Osteosarcoma (OS) is the most common type of primary bone cancer. It can occur at any age, but most patients are aged between 12 and 25 years [1]. OS is classified as a malignant mesenchymal neoplasm in which the tumor directly produces defective osteoid (immature bone). Approximately 10% to 20% of patients with OS have the metastatic disease at diagnosis. The most frequent site of metastasis is the lung; however, a smaller percentage of patients have bone and soft tissue metastases. The presence of metastasis at diagnosis is a prognostic factor with strong impact in the overall survival of patients with OS [2]. In Brazil, 20% of patients present metastatic disease at diagnosis, a rate that is twice as high compared with that reported in developed countries [3]. The current treatment of OS involves an integrated strategy incorporating chemotherapy and surgery. Despite the dramatic advances in OS treatment, patient survival reached a plateau. Recent clinical trials that attempted to improve outcome through intensification of therapy or incorporation of new agents have not been widely successful. The exact cause of OS remains unknown. Therefore, increasing focus has been placed in achieving a greater understanding of the basic biology of OS to improve treatment [4]. Unlike other sarcomas, such as synovial sarcoma, alveolar rhabdomyosarcoma, and Ewing sarcoma, no specific translocations or genetic abnormalities have been identified in OS [5]. Nevertheless, nearly 70% of OS tumors display a multitude of cytogenetic abnormalities. These findings further highlight the complexity and the instability of the genetic makeup of OS tumors [5]. Cytogenetic studies showed the presence of rearrangements and recurrent gains in specific chromosomal regions, indicating the possible involvement of genes located in these regions in the pathogenesis of OS. Molecular cytogenetic studies use comparative genomic hybridization, as a method, identified frequent chromosomal gain involving 1p36.3, 17 and chromosome 19 [6-9]. For our study, we selected genes located in these 3 chromosomal regions that could function when genomic gain occurs. We have used quantitative polymerase chain reaction (PCR) for gene expression investigation, and we have compared these genes expression with healthy bone samples. The genes of this study were selected, considering the regions with more frequent genomic gain reported in previous work [10]. Our objectives were to validate the increase in the gene expression related to the genomic gain using the technique of quantitative PCR and to correlate these values with the clinical data of patients with OS. Among other gene, in the 1p36.3 region are, 5,10methylenetetrahydrofolate reductase (MTHFR), GardnerRasheed feline sarcoma viral oncogene homolog (FGR), E2F transcription factor 2 (E2F2), and ERBB-receptor feedback inhibitor 1 (ERRFI1). MTHFR codifies an enzyme that catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a cosubstrate for homocysteine remethylation to methionine. Genetic variation in this gene influences susceptibility to occlusive
995 vascular disease, neural tube defects, colon cancer, and acute leukemia. Mutations in this gene are associated with methylenetetrahydrofolate reductase deficiency [11]. The transcription factor E2F2 is a protein encoded by the E2F2 gene. The E2F family plays a crucial role in the control of the cell cycle and the action of tumor suppressor proteins, also being a target of the transforming proteins of small DNA tumor viruses [12]. ERBB receptor feedback inhibitor 1 is a protein encoded by the ERRFI1 gene. This gene codifies a cytoplasmic protein whose expression is up-regulated with cell growth, which is induced during cell stress and mediates cell signaling [13]. The FGR gene is a member of the Src family of protein tyrosine kinases. This gene codifies a protein localized in plasma membrane ruffles that functions as a negative regulator of cell migration and adhesion triggered by the β-2 integrin signal transduction pathway [14]. In the chromosome 17p region, 2 genes of the mitogenactivated protein kinases (MAPKs), MAPK7 and MAP2K4, are localized. MAPKs are serine/threonine-specific protein kinases that respond to extracellular stimuli and regulate various cellular activities such as gene expression, mitosis, differentiation, proliferation, and cell survival/apoptosis [15]. MAPKs are involved in the activation of most nonnuclear oncogenes. Mitogen-activated protein kinase 7 (MAPK7) is an enzyme encoded by the MAPK7 gene and is specifically activated by mitogen-activated protein kinase kinase 5 (MAP2K5/MEK5). It is involved in the downstream signaling processes of various receptor molecules including receptor type kinases and G protein–coupled receptors. In response to extracellular signals, this kinase translocates to the cell nucleus, where it regulates gene expression by phosphorylating and activating different transcription factors [16]. Dual-specificity mitogen-activated protein kinase 4 (MAP2K4) is an enzyme encoded by the MAP2K4 gene that also encodes a dual-specificity protein kinase that belongs to the Ser/Thr protein kinase family. This kinase is a direct activator of MAP kinases in response to various environmental stresses or mitogenic stimuli [17]. Finally, the genes BCL2 binding component 3 (BBC3), jun D proto-oncogene (JUND), related RAS viral oncogene homolog (RRAS), and FBJ murine OS viral oncogene homolog B (FOSB) are located in chromosome 19. The BBC3 and JUND genes are related to apoptosis and cell senescence. BBC3 is a Bcl-2 family member and is a critical mediator of p53-dependent and p53-independent apoptosis induced by a wide variety of stimuli, including genotoxic stress, deregulated oncogene expression toxins, altered redox status, growth factor/cytokine withdrawal, and infection. BBC3 transduces death signals primarily to the mitochondria, where it acts indirectly on the Bcl-2 family members Bax and/or Bak by relieving the inhibition imposed by antiapoptotic members. It directly binds and antagonizes all known antiapoptotic Bcl-2 family members to induce mitochondrial dysfunction and caspase activation [18]. JUND protein encoded by the intronless JUND gene is a member of the JUN family and a functional component of the activator protein 1 (AP1)
996 transcription factor complex. This protein has been proposed to protect cells from p53-dependent senescence and apoptosis [19]. RRAS gene is a member of the Ras family of small GTPases that are involved in cellular signal transduction, implicated in promoting cell adhesion and neurite outgrowth through integrin activation [20]. FOSB is a protein encoded by the FOSB gene that encodes leucine zipper proteins that can dimerize with proteins of the JUN family, thereby forming the transcription factor complex AP1 [21]. Thus, the objective of the current study was to examine the expression profile of 10 genes located in recurrent regions of chromosomal gains: 1p36.3 (MTHFR, FGR, E2F2, ERRFI1) 17p (MAPK7, MAP2K4), and chromosome 19 (BBC3, RRAS, JUND, FOSB) in a cohort of OS tumors. In addition, we analyzed the association of the expression profile of these genes with the OS clinicopathologic parameters.
2. Materials and methods 2.1. Samples All 70 flash-frozen OS samples from 30 patients (30 prechemotherapy specimens, 30 postchemotherapy specimens, and 10 metastasis specimens) used in this study were obtained from patients treated at the Pediatric Oncology Institute Grupo de Apoio ao Adolescente e a Criança com Câncer/Federal University of São Paulo. This was a retrospective study of samples collected sequentially between 2002 and 2008. Ten healthy bone samples were used as controls. These samples were obtained from healthy individuals without genetic and/or musculoskeletal diseases who underwent orthopedic surgery due to trauma (Supplementary Data 1 (on the journal's website at www.humanpathol.com)). Samples from each OS tumor and healthy bone were collected after informed consent was signed by patients/guardians according to the university's institutional review board (IRB/Federal University of São Paulo no. 0050). All patients were treated following the standard regimens—the Brazilian Osteosarcoma Treatment Group— comprising cisplatin, doxorubicin, and methotrexate. The team of orthopedists in collaboration with the pediatric oncology team determined the appropriate surgical procedure for each patient. Nonconventional endoprosthesis, resection of expendable bones, plaques, and bone graft fixation (autograft or bone bank) were used. Whenever possible, all pulmonary metastases were surgically removed after resection of the primary tumor.
2.2. RNA isolation and reverse transcription reaction Total RNA was extracted from tumor samples using TRIzol reagents (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA quality and quantity were determined by gel electrophoresis and spectrophotometry,
F. Tesser-Gamba et al. respectively. DNase treatment was used to remove genomic contamination using deoxyribonuclease I Amplification Grade (Invitrogen). Synthesis of complementary DNA (cDNA) was performed using 1 μg of total RNA according to the manufacturer's protocol of SuperScript III Kit (Invitrogen).
2.3. Primers The primers of the 10 genes investigated in this study and the endogenous gene b-actin (ACTB) were designed using Primer Express (3.0) Software from Applied Biosystems (Foster City, CA) taking care that the forward and reverse sequences were in different exons. We conducted in NCBI a Basic Local Alignment Search Tool (BLAST) search to confirm the total gene specificity of the nucleotide sequences chosen for primers. The sequences of primers are presented in Supplementary Data 2 (on the journal's website at www.humanpathol.com).
2.4. Real-time PCR Expression levels of FGR, E2F2, ERRFI1, MTHFR, MAP2K4, MAPK7, RRAS, FOSB, BBC3, and JUND were determined by quantitative real-time PCR. This analysis was performed in a thermocycler Applied Biosystems Prism 7500 Sequence Detection System (PE Applied Biosystems, Inc, Foster City, CA) using relative quantification. The expression of the housekeeping gene ACTB was used as an endogenous control in each assay performed. The reaction mixture combined 6 μL SYBR Green PCR Master Mix (PE Applied Biosystems) reaction mix, 3 μL sense/antisense primers, and 3 μL cDNA. The cycling conditions were as follow: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. Reactions were done in triplicate. For each sequence, a standard curve was constructed to determine the sensitivity and efficiency assays. For each sample, the cycle threshold (Ct) was determined (mean of the 3 reactions) for both the target gene and the endogenous control gene and was normalized for cDNA quantity. Subtracting the Ct of the endogenous control gene from the Ct of the target gene yields the ΔCt. The ΔCt of the control reference was then subtracted from the ΔCt of the tumor sample, yielding the ΔΔCt, and the relative quantification value was expressed as 2 −ΔΔCt [22].
2.5. Clinical parameters Clinical variables that were correlated with gene expression included age at diagnosis, sex, histologic OS subtype, site of primary tumor, presence of metastasis at diagnosis, and necrosis grade after chemotherapy (Huvos grade) [1]. The Huvos grading system was used to rate the level of tumor necrosis after preoperative chemotherapy: grade I, little or no effect of chemotherapy noted; grade II, partial response to chemotherapy, with between 50% and 90%
MAPK7 and MAP2K4 as prognostic markers in osteosarcoma
997
Fig. 1 A, Kruskal-Wallis test. Quantitative analysis of gene expression from prechemotherapy, postchemotherapy, and metastasis specimens compared with healthy bone samples for MAPK7 gene. B, Mann-Whitney test. Quantitative analysis of MAPK7 gene expression for metastatic and nonmetastatic patients. C, Mann-Whitney test. MAPK7 gene expression in postchemotherapy specimens for patients with good and bad response. D, Mann-Whitney test. MAPK7 gene expression in samples of patients in complete remission at the end of treatment. E, Overall survival curve using quantitative values of MAPK7 gene expression in prechemotherapy specimens using the median value of all samples of prechemotherapy specimens. F, Overall survival curve using quantitative values of MAPK7 gene expression in postchemotherapy specimens using the median value of all samples of postchemotherapy specimens. *P b .05.
998 necrosis; grade III, greater than 90% necrosis; and grade IV, no viable tumor cells are apparent [1]. Patients with nonmetastatic disease were considered to be in complete remission on the date of the primary tumor resection, and patients with metastatic tumors were considered to be in complete remission on the date of the pulmonary metastases resection. Overall survival was defined as the time from diagnosis until the date of either the last contact or death.
2.6. Statistical analysis Data analyses were performed using GraphPad Prism 4 Software (San Diego, CA). Continuous data (gene expression levels) were evaluated and compared using nonparametric tests: Mann-Whitney, Wilcoxon, Kruskal-Wallis, correlation coefficient, Spearman, and Friedman. Categorical data (age at diagnosis, sex, histologic OS subtype, primary site, presence of metastasis at diagnosis, and necrosis grade) were studied using χ 2 and Fisher exact test applying software VassarStats (Website for Statistical Computation; Poughkeepsie, NY, USA). The analysis for high expression and low expression of each group of samples (prechemotherapy, postchemotherapy, and metastasis specimens) used the median relative quantification value of the 11 samples of healthy bones as the normal reference. Statistical significance was set at P b .05 (Figs. 1 and 2).
3. Results A summary of the clinicopathologic characteristics is detailed in Supplementary Data 3 and 4 (on the journal's website at www.humanpathol.com). The clinicopathologic parameters analyzed were age at diagnosis, sex, presence of metastasis at diagnosis, Huvos grade [1], site of the primary tumor, use of methotrexate in treatment, and histologic type. We performed quantitative real-time PCR to quantify messenger RNA levels of MTHFR, E2F2, FGR, ERRFI1, MAPK7, MAP2K4, FOSB, JUND, BBC3, and RRAS genes in 30 prechemotherapy, 30 postchemotherapy, and 10 metastasis specimens from 30 patients with OS (Table 1). Results of all genes in all OS samples versus healthy samples, clinical parameters, and overall survival are described in Table 2.
Fig. 2 A, Kruskal-Wallis test. Quantitative analysis of gene expression from prechemotherapy, postchemotherapy, and metastasis specimens compared with healthy bone samples for MAP2K4 gene. Overall survival using quantitative values of MAP2K4 gene expression in prechemotherapy specimens using the median value of all samples of prechemotherapy specimens (B) and postchemotherapy specimens (C) using the median value of all postchemotherapy specimens. *P b .05.
F. Tesser-Gamba et al.
MAPK7 and MAP2K4 as prognostic markers in osteosarcoma Table 1
999
Number and percentage of OS samples with overexpression of the genes analyzed
Gene
Specimens
Total
Overexpression n
%
MTHFR
PreCH PostCH Metastasis PreCH PostCH Metastasis PreCH PostCH Metastasis PreCH PostCH Metastasis PreCH PostCH Metastasis PreCH PostCH Metastasis PreCH PostCH Metastasis PreCH PostCH Metastasis PreCH PostCH Metastasis PreCH PostCH Metastasis
30 30 10 30 30 10 30 30 10 30 30 10 30 30 10 30 30 10 30 30 10 30 30 10 30 30 10 30 30 10
21 14 7 25 24 10 26 14 8 5 3 2 26 16 7 25 11 4 12 12 7 11 18 7 17 18 4 8 17 6
70 47 70 83 80 100 86 46 80 16 30 20 86 53 70 83 37 40 40 40 70 37 60 70 57 60 40 27 57 60
ERRFI1
E2F2
FGR
MAPK7
MAP2K4
FOSB
JUND
BBC3
RRAS
Abbreviations: PreCH, prechemotherapy specimens; PostCH, postchemotherapy specimens.
4. Discussion In OS, some aspects regarding its pathogenesis are still unknown, and questions about the genetic determinants for its development are still unanswered. Moreover, any molecular marker that could be related to its development, evolution, and progression has not been described. Progress related to the improved survival of patients with OS is at a plateau, and the intensification of therapy or the addition of new therapeutic agents has been successful, particularly in the metastatic patients at diagnosis [4]. In this study, patients with OS presented distribution by sex, age, and clinical and pathologic examination similar to those described in the literature [3,23]. As expected, the number of affected men (70%) was higher than women (30%), and the peak of incidence was in the second decade of life (60%). The most frequent site of tumor was femur (53%), and the most frequent histologic type was osteoblastic (50%). Using Huvos classification, the necrosis grades I and II were observed in most metastatic patients (43%).
Previous studies using comparative genomic hybridization showed a gain of genomic DNA from specific chromosomal regions in OS samples [24]. The premise of our study was to determine if gains of specific chromosomal regions could be truly representing an increase of gene expression in OS. To contribute to the current knowledge of chromosomal abnormalities in OS, we investigated expression profiles of 10 genes located in the chromosomal gain regions, 1p36, 17p, and chromosome 19. MTHFR, ERRFI1, E2F2, FGR, FOSB, JUND, BBC3, and RRAS genes showed no significant correlation with the development of OS. From 10 genes investigated, 2 of them, MAPK7 and MAP2K4, located in 17p, were overexpressed in most OS samples analyzed (Table 1). In our study, we found a higher expression of MAP2K4 and MAPK7 genes in prechemotherapy OS specimens than in postchemotherapy specimens or healthy bone controls. Between the postchemotherapy groups, patients carrying specimens with a high expression of these genes were significantly associated with the presence of metastasis at diagnosis, poor Huvos grade,
1000 Table 2
F. Tesser-Gamba et al. Expression of all genes according to clinical parameters and overall survival
High expression in PreCH specimens vs PostCH specimens PostCH specimens vs PreCH specimens PreCH specimens vs metastasis specimens PreCH specimens vs healthy bone specimens Healthy bone vs PreCH specimens Healthy bone vs PostCH specimens Metastasis specimens vs healthy bone specimens Metastasis specimens vs patients who did not relapse PreCH specimens vs relapse patients PostCH specimens vs relapse patients PostCH specimens vs patients with tumor b12 cm Metastasis specimens vs patients with tumor N12 cm PostCH specimens vs use of MTX in OS treatment PreCH specimens vs poor Huvos grade (I and II) PostCH specimens vs poor Huvos grade (I and II) PreCH specimens vs complete remission Metastasis specimens vs complete remission PostCH specimens vs metastatic patients at diagnosis PostCH specimens vs nonmetastatic patients at diagnosis PostCH specimens vs patients without complete remission Comparison of moments PreCH vs PostCH vs metastasis vs healthy bone Overall survive PreCH specimens PostCH specimens
Genes located at 1p36
Genes located at 17p
MTHFR ERRFI1 E2F2 (P) (P) (P)
FGR (P)
.0276 ⁎
.0004 ⁎
Genes located at chromosome 19
MAPK7 MAP2K4 FOSB (P) (P) (P)
JUND (P)
BBC3 (P)
.0114 ⁎ b.0001 ⁎
.0262 ⁎
.0351⁎⁎ .0244 ⁎
.0003 ⁎ .0085 ⁎ .0092 ⁎ .0129 ⁎
.0083 ⁎ .0444 ⁎
RRAS (P)
.0253 ⁎
.0444 ⁎ .0210 ⁎
.0052 ⁎
.0458 ⁎ .0016 ⁎ .0399 ⁎
.0084 ⁎ .0329 ⁎
.0001 ⁎ .0049 ⁎ .0005 ⁎ .0317 ⁎ .0266 ⁎ .0239 ⁎
.0296 ⁎
.0446 ⁎ .0014 ⁎
.067
.0092 ⁎ .0054 ⁎ .0528
.0016 ⁎ .0013 ⁎
.0838
.328
.56
.078
.745 .0181 ⁎ .0052 ⁎ .0085 ⁎
Abbreviations: PreCH, prechemotherapy specimens; PostCH, postchemotherapy specimens; MTX, methotrexate. ⁎ P b .05.
and worse overall survival. Furthermore, the MAP2K4 overexpression in postchemotherapy specimens was also associated with patients without complete remission of disease at the end of treatment and in the last contact. MAPKs have been described in several hormonal systems, being related to proliferation and differentiation of various cell types. In bone cells, parathyroid hormone (PTH) and parathyroid hormone–related protein (PTHrP) show both upregulation and down-regulation of MAPKs during osteoblast
proliferation and differentiation, respectively. The molecular mechanisms that regulate PTH activity in the bone remain incompletely understood; however, it is believed that PTH stimulates the ERK (extracellular signal-regulated kinases) group of MAPKs by multiple mechanisms in cells with PTH/PTHrP receptor 1 (PTH1R) [25]. A previous study demonstrated a bidirectional effect of PTH and PTHrP on MAPK in osteoblasts, increasing phosphorylated ERK (pERK) in undifferentiated cells but
MAPK7 and MAP2K4 as prognostic markers in osteosarcoma decreasing pERK in differentiated cells. In proliferating osteoblastic cells, PTH and PTHrP up-regulate pERK, increase cyclin D1 expression, and promote cell proliferation. In contrast, in differentiated osteoblasts, PTHrP downregulates pERK, decreases cyclin D1 expression, and induces osteoblastic cell growth arrest, allowing more cellular differentiation and bone matrix formation [26]. A proposed mechanism in OS involves the tumoral secretion of bone-modulating compounds, such as PTHrP. In response to PTHrP, osteoclastic bone resorption is stimulated and growth factors are released from the surrounding extracellular bone matrix, further stimulating PTHrP secretion as well as stimulating tumor growth. This positive feedback loop culminates in further tumor growth and metastasis [27]. Previously, one of our studies also showed that overexpression of genes related to bone remodeling process in OS was significantly associated with poor overall survival [28]. In this way, the MAPK7 and MAP2K4 overexpression observed in the current study could be stimulated by PTH/PTHrP and, consequently, would increase the proliferation of undifferentiated OS cells. Therefore, strategies aimed at interrupting this vicious cycle may be useful in OS. In addition, we observed a significant association between MAPK7 overexpression in prechemotherapy specimens and the presence of metastasis at diagnosis. Recently, MAPK7 has also been involved in the control of cellular proliferation and cell death, as well as in tumorigenesis. Elegant in vivo studies using animals in which MAPK7 expression can be regulated have demonstrated that MAPK7 is important for sustaining tumor growth probably because of its supportive role in vasculogenesis and blood vessel homeostasis [29]. Our results demonstrated that MAPKs expression were strongly associated with important clinical parameters, including grade of necrosis. The necrosis grade still is the only one parameter to evaluate the response to treatment. We observed a significant association between MAPK overexpression in postchemotherapy specimens and a poor response to preoperative chemotherapy (grades I and II). Studies have shown that MAPKs expression may be an underlying mechanism contributing to the generation of chemoresistance in cancer [30]. The importance of MAPK activation in cellular response to cisplatin has gradually been appreciated in the last few years. Cisplatin is one of the most effective chemotherapeutic agents and is widely used in the treatment of a variety of pediatric and adult solid tumors including OS. This platinum compound is a DNA-damaging agent that forms cisplatin-DNA adducts and kills cells via several mechanisms, including induction of apoptosis. Resistance to cisplatin is multifactorial and leads to a poor response to chemotherapy and treatment failure. Failure to induce apoptosis is believed to be one of the major mechanisms underlying cisplatin resistance [31]. MAPKs control cell proliferation, differentiation, and cell death. Their role in response to cisplatin is complex, and the response varies with the type of cells as well as their
1001 proliferation and differentiation status [32]. It is believed that MAPK activities are regulated through reversible phosphorylation of both threonine and tyrosine residues by upstream kinases, as well as by the members of MAPK phosphatase (MKP) family, which can dephosphorylate both phosphothreonine and phosphotyrosine residues and inactivate MAPKs. Recent studies suggest that MKP-1, the founding member of the MKP family, plays an important role in the response of cancer cells to chemotherapy, especially to cisplatin [32]. Because cisplatin is one of the most important chemotherapeutic agents used in OS treatment, the MAPK overexpression observed in our study could be contributing to a worse treatment response of patients with OS and, consequently, low overall survival. Therefore, further investigation is necessary to study the role of MAPKs and MKPs in pediatric cancers including OS [31].
5. Conclusion Unfortunately, some groups of patients remain at high risk of eventual relapse and short-term survival. These patients may benefit from future investigations into innovative treatment approaches based on biological markers, such as antiangiogenesis factors or growth factor receptor modulation. However, our study is a pilot study and suggests that further investigations are needed. We suggest that abnormalities in the expression levels of genes involved in important cell signaling pathways, such as MAPKs pathway, may contribute to OS progression and aggressiveness.
Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.humpath.2011.08.003.
Acknowledgments We would like to thank Indhira Dias, MSc; Carolina Salinas, BSc; and Patrícia Pavoni, MSc.
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