Correlation between ERK1 and STAT3 expression and chemoresistance in patients with conventional osteosarcoma

Osteosarcoma, the most common type of primary bone cancer, is a rare disease. Approximately 900 new cases of osteosarcoma are diagnosed each year in the United States (http://​www.​cancer.​org/​docroot/​home/​index.​asp) and 200 in France, including 150 in children (http://​www.​fnclcc.​fr/​sor/​SSP/​CancersEnfant/​PeauTissusSoutie​n/​Osteosarcome). Adjuvant and neoadjuvant chemotherapy have significantly improved the long-term survival rate for patients with osteosarcoma [1‐3]. Nevertheless, recurrent disease still occurs in about 30–40% of patients and more than 70% of them die of their tumor, despite second-line treatment. The standard therapy regimen of high-grade osteosarcoma includes induction by multiagent chemotherapy followed by surgical resection and postoperative chemotherapy [4]. The percentage of necrotic tissue following induction chemotherapy is classified with the Huvos grading system [5]. Patients with <90% tumor necrosis following induction therapy are classified as “poor responders” (PR) or Huvos grade I/II [6], while more than 90% necrosis corresponds to Huvos grade III and complete necrosis to Huvos grade IV. Good responders (GR) correspond to Huvos grade III/IV. The degree of necrosis remains the only reliable prognostic factor for the patients presenting with localized disease and is used to guide the choice of postoperative chemotherapy. Numerous clinical trials have attempted to increase the disease-free survival rate for poorly responding patients with intensified postoperative therapy. No survival benefit has been convincingly shown through the administration of more intensified therapy to poor responders [3, 7‐10]. This suggests that there may be an innate biological difference between good responsive and poor responsive tumors. Previous transcriptomic studies have shown that in chemoresistant tumors, the genes involved in osteoclastogenesis, extracellular matrix remodeling, bone development, tumor progression, drug resistance and angiogenesis are up-regulated [11‐17]. However, none of these molecular predictive factors can be used routinely. Therefore, there is a need to establish reliable predictive biomarkers for the response to chemotherapy at the time of diagnosis. The aim of this study was to identify molecular markers expressed differentially between “good” and “poor” responders to neoadjuvant chemotherapy in order to predict the response to chemotherapy in conventional osteosarcoma before beginning treatment, and to elucidate the mechanisms involved in this response. We identified several subsets of novel potential candidate genes. In particular, our data suggest that ERK1 and STAT3 expression are involved in the response to chemotherapy and that they could be therapeutic targets.

SSH is a molecular biology technique that enables the identification of differentially expressed genes between two groups with high sensitivity. By comparing PR to GR prior to chemotherapy among patients with an osteosarcoma, we found 126 clones. ERK1 and STAT3, the genes selected on the basis of their roles in tumorigenesis or chemoresistance, were further studied by QRT-PCR in an independent cohort. ERK1 and STAT3 expressions assessed by QRT-PCR and IHC were significantly linked to the response to chemotherapy. The protein encoded by ERK1 is a member of the MAP kinase family and acts in a signalling cascade that regulates various cellular processes such as proliferation, differentiation, and cell cycle progression in response to a variety of extracellular signals. We found ERK1/2 positivity score by IHC and ERK1/2 IHC high score (score 2 and 3) in 78% and 51% of our cohort, respectively. These results suggested that ERK1/2 pathway could be involved in osteosarcoma as it has supported by Pignochino et al. study that showed activated ERK 1/2 pathway in 66.6% of osteosarcoma samples. Moreover, the same team also showed that Sorafenib, a tyrosine kinase inhibitor, blocks tumor growth, angiogenesis and metastatic potential in preclinical models of osteosarcoma through a mechanism potentially involving the inhibition of ERK1/2[29]. No attempt to investigate the link between ERK1 expression and response to chemotherapy was made in vivo. However, our work suggested that ERK1 could be involved in drug resistance as reported recently by Si et al. with an approach by RNAi-mediated knockdown of ERK1/2 inhibiting cell proliferation and invasion and increasing chemosensitivity to cisplatin in human osteosarcoma U2-OS cells in vitro [30].

STAT3 is one of the transcription factors reported to play an important role in tumor survival, proliferation, angiogenesis and metastasis. In normal cells, STAT3 is activated transiently to maintain homeostasis. However, if STAT3 continues to be activated, the abnormal level of expression can trigger oncogenic pathways. Aberrant active STAT3 promotes uncontrolled growth and survival through dysregulation of expression of downstream targeted genes including survivin, Bcl-xL, Bcl-2, Mcl-1, c-Myc and cyclin D1. Constitutive activation of the STAT3 pathway has recently been shown in several malignancies, especially osteosarcoma [31]. It has recently been implicated in resistance to chemotherapy-induced apoptosis [32]. Furthermore, activation of STAT3 in several cancers has been found to be correlated with clinical outcome especially in osteosarcoma. A high level of expression of STAT3 by IHC in 76 biopsies of patients with an osteosarcoma was a poor prognostic factor for both overall survival and disease-free survival in univariate and multivariate analysis [33]. High staining with pSTAT3 was also of prognostic value in another series of 51 conventional osteosarcomas [34]. In addition, inhibition of STAT3 plays a role in proliferation, apoptosis and migration in osteosarcoma cells in vitro. The down-regulation of STAT3 by miR-125b suppresses in vitro proliferation and migration of osteosarcoma cells [35]. STAT3 inhibition by RNA interference induces inhibition of proliferation and apoptosis enhancement in osteosarcoma cells [33]. The novel curcumin analog FLLL32 decreases STAT3 DNA binding activity and expression, and induces apoptosis in osteosarcoma cell lines [36]. The small molecules, LLL12 and FLLL32, inhibit STAT3 phosphorylation and exhibit potent growth suppressive activity in osteosarcoma cells and tumor growth in mice [37]. In contrast, oncostatin M promotes STAT3 activation, VEGF production, and invasion in osteosarcoma cell lines [38]. Finally, STAT3 is involved in drug resistance in osteosarcoma cell lines. Ryu et al. recently showed that the STAT3 pathway was overexpressed in MDR osteosarcoma cells and that inhibitors of STAT3 such as CDDO-Me could reduce resistance to doxorubicin in these cell lines [31]. In our study, we have showed an expression of pSTAT3 in 58% of cases. This activated STAT3 pathway was correlated to poor response to chemotherapy. Thus, our results are consistent with the results of the literature in vitro through the analysis of patient samples.

The effects of EGFR are mediated by activation of downstream signal transduction cascades that include Janus tyrosine kinases (Jak), Signal Transducers and Activators of Transcription (STAT), Phophatidyl Inositol 3 Kinase (PI3K)/Akt and Ras/RAf/MAP kinase (ERK). The prognostic value of EGFR and its downstream signaling molecules such as STAT3 and ERK1 have been studied in many tumor types. Only one study [39] simultaneously examined the status of EGFR and four downstream molecules - pSTAT3, pERK1, pAkt, survivin - by IHC in 47 samples of conventional osteosarcomas. ERK1 and survivin expression were statistically correlated with survival. A high expression was negatively correlated with prognosis. Furthermore, EGFR expression was correlated with expression of ERK1 and it was observed a significant association of survivin expression with STAT3 and ERK activation. These results and ours support the idea that ERK is a downstream signaling molecule of EGFR and also suggest a link between the EGFR signaling pathway and drug resistance through ERK1 and STAT3 expression in conventional osteosarcoma.

We have shown that high pSTAT3 and pERK1 expression in the biopsies are suggestive of poor response to chemotherapy. The elevated positive predictive value of high score of both pSTAT3 and pERK1 in combination (91%) highly suggests that IHC test could be used at the time of diagnosis to stratifying patients enrolled in randomized trials. Our results also suggest that STAT3 and ERK1 inhibitors might be used in combination with common chemotherapeutic drugs in osteosarcoma in order to increase the response to chemotherapy and to improve the prognosis. Finally, other genes identified by SSH remain to be explored and a prospective validation phase on a larger cohort is still needed before these biomarkers could be used in clinical practice.