Abstract
Background/aimsA novel paradigm in tumor biology suggests that osteosarcoma (OS) chemo-resistance is driven by osteosarcoma stem cell-like cells (OSCs). As the sensitivity of only a few tumors to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) can be explained by the presence of EGFR tyrosine kinase (TK) domain mutations, there is a need to elucidate mechanisms of resistance to EGFR-targeted therapies in OS that do not harbor TK sensitizing mutations to develop new strategies to circumvent resistance to EGFR inhibitors.MethodsAs a measure of the characters of OSCs, serum-free cultivation, cell viability test with erlotinib, and serial transplantation in vivo was used. Western blot assays were used to detect the association between erlotinib resistance and transforming growth factor beta (TGFβ)-induced epithelial-to-mesenchymal transition (EMT) progression. By using TaqMan qPCR miRNA array, online prediction software, luciferase reporter assays and western blot analysis, we further elucidated the mechanisms.ResultsHere, CD166+ cells are found in 10 out of 10 tumor samples. We characterize that CD166+ cells from primary OS tissues bear hallmarks of OSCs and erlotinib-resistance. TGFβ-induced EMT-associated kinase switch is demonstrated to promote erlotinib-resistance of CD166+ OSCs. Further mechanisms study show that TGFβ-induced EMT decreases miR-499a expression through the direct binding of Snail1/Zeb1 to miR-499a promoter. Overexpression of miR-499a in CD166+ OSCs inhibits TGFβ-induced erlotinib-resistance in vitro and in vivo. SHKBP1, the direct target of miR-499a, regulates EGFR activity reduction occurring concomitantly with a TGFβ-induced EMT-associated kinase switch to an AKT-activated EGFR-independent state. TGFβ-induced activation of AKT co-opts an increased SHKBP1 expression, which further regulates EGFR activity. In clinic, the ratio of the expression levels of SHKBP1 and miR-499a is highly correlated with EMT and resistance to erlotinib.ConclusionTGFβ–miR-499a–SHKBP1 network orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors in CD166+ OSCs, implies that inhibition of TGFβ induced EMT-associated kinase switch may reverse the chemo-resistance of OSCs to EGFR inhibitors. We also suggest that an elevated SHKBP1/miR-499a ratio is a molecular signature that characterizes the erlotinib-resistant OS, which may have clinical value as a predictive biomarker.
Highlights
Osteosarcoma (OS) is the third most common cancer in adolescence and is the most frequent form of primary bone tumors [1]
We found that the levels of phosphorylated epidermal growth factor receptor (EGFR) and HER2 were markedly decreased in CD166+ osteosarcoma stem cell-like cells (OSCs), while the expression of p-AKT was significantly increased (Fig. 2). p-FGFR level was significantly increased (Fig. 2), which suggests that CD166+ OSCs exhibited a switch from EGFR to activation of an alternative tumor cell- specific RTKs (FGFR)
By further define the role of SHKBP1 in EGFR activity regulation, we demonstrated that SHKBP1 knockdown in CD166+ OSCs resulted in an increase of EGFR phosphorylation in response to treatment with EGF (Additional file 1: Figure S2B)
Summary
Osteosarcoma (OS) is the third most common cancer in adolescence and is the most frequent form of primary bone tumors [1]. The drug resistance induced recurrence and metastasis remarkably contribute to the failure in OS therapy [2, 3]. A rising number of observations indicates that cancer stem cells (CSCs), characterized by increased tumorigenicity, self-renewal ability and multipotency, have been described contributing to tumor progression and resistance to chemo-therapy [4, 5]. Crasto et al [7] used disulfiram as an ALDH inhibitor to target the CSC subpopulation in a murine osteosarcoma model. They found that a reduction in metastatic tumor burden after treatment, which was comparable to that achieved with doxorubicin chemotherapy. Therapeutics targeting OSCs may be an effective strategy to overcome chemo-resistance and preventing distant metastasis
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