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. 2019 May 28;38(1):226.
doi: 10.1186/s13046-019-1195-y.

The TGFβ-miR-499a-SHKBP1 pathway induces resistance to EGFR inhibitors in osteosarcoma cancer stem cell-like cells

Tian Wang  1 , Dexing Wang  1 , Lian Zhang  1 , Ping Yang  1 , Jing Wang  1 , Qi Liu  1 , Fei Yan  1 , Feng Lin  2
Affiliations

The TGFβ-miR-499a-SHKBP1 pathway induces resistance to EGFR inhibitors in osteosarcoma cancer stem cell-like cells

Tian Wang et al. J Exp Clin Cancer Res. .

Abstract

Background/aims: A 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.

Methods: As 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.

Results: Here, 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.

Conclusion: TGFβ-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.

Keywords: EGFR; Epithelial-to-mesenchymal transition; Osteosarcoma stem cell-like cells; SHKBP1; TGFβ; miR-499a.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
CD166+ cells from primary OS tissues display stem cell-like features and erlotinib resistant. a CD166+ cells were sorted from spheres by fluorescence-activated cell sorting. b Phase-contrast images of the ability of tumor spheres formation by seeding with CD166+ cells and CD166− cells in serum-free medium. Scale bar, 50 μm. c Immunofluorescent staining of CD166 (green) and CD133 (red) expression in CD166+ spheres (nuclei stained with DAPI). Scale bar, 10 μm. d With the presence of erlotinib, cell viability of CD166+ OSCs and CD166− cells were determined by MTT. Note: Columns, mean of three individual experiments; SD,** P < 0.01. e In vivo serial transplantation assay. A total of 10 4 CD166+ OSCs or CD166− cells from OS-1 or OS-5 in serum-free medium were injected s.c. into nude mice. Derived tumor xenografts were dissociated to single-cell suspension and then serially re-injected in mice (10 4 cells), generating secondary and then tertiary tumors. Tumor growth curves of primary and tertiary tumors are shown. Note: Columns, mean of three individual experiments; SD, *** P < 0.001; SD,** P < 0.01
Fig. 2
Fig. 2
CD166+ OSCs is associated with a kinase switch that enables EGFR-independent activation of AKT. Western-blotting was used with antibodies specific for phosphorylated and total EGFR, HER2, AKT, FGFR, and p-FAK, Vimentin, E-Cadherin. GAPDH was used as the control
Fig. 3
Fig. 3
TGFβ induces an EMT-associated kinase switch that promotes erlotinib resistance of CD166+ OSCs. a Tumor cell supernatants of CD166+ and CD166− cells from OS-1 or OS-5 were collected and differential levels of TGFβ production were analyzed by ELISA. Note: Columns, mean of three individual experiments; SD,** P < 0.01. b Immunoblot analysis was performed with antibodies against phosphorylated and total AKT, EGFR and Vimentin, E-Cadherin. GAPDH was used as the control. c With the presence of erlotinib, cell viability of CD166− cells with or without the treatment of TGFβ1 were determined by MTT. Note: Columns, mean of three individual experiments; SD,** P < 0.01. d Phase-contrast images of the ability of tumor spheres formation by seeding with CD166− cells with or without the treatment of TGFβ1 in serum-free medium. Scale bar, 50 μm. e Flow cytometry analysis of CD133 in CD166− cells (from OS-1) without the treatment of TGFβ1
Fig. 4
Fig. 4
miRNA profiling of CD166+ OSCs. a Venn analyses of the up- and downregulated miRNAs in CD166+ OSCs from primary OS tissues (left, yellow shaded; n = 3) and in CD166− cells with TGFβ1 (right, blue shaded), compared with CD166− cells, are shown. miRNAs that were significantly and commonly deregulated in both CD166+ OSCs and CD166− cells with TGFβ1 are shown in the overlapping area (green shaded). Only those miRNAs whose expression levels displayed greater than 2-fold decreases or increases were further studied. The table shows a summary of the significantly differentially expressed miRNAs in the overlapping area with fold change. b Relative expression of miR-499a in CD166+ OSCs, CD166− cells with or without TGFβ1 from OS-1 and OS-5 were examined by qPCR. Note: Columns, mean of three individual experiments; SD,**, P < 0.01
Fig. 5
Fig. 5
miR-499a directly targets SHKBP1 expression and SHKBP1 was negatively correlated with miR-499a level in OS tissues. a Illustration of SHKBP1 3’UTR as well as the seed sequence of miR-499a showed the predicted target region on the 3’UTR of SHKBP1 mRNA. b Dual-luciferase reporter assay with co-transfection SHKBP1 3’UTR plasmids and miR-499a mimics. The relative luciferase activity was obtained by firefly luciferase activity normalized against Renilla luciferase activity. Note: Columns, mean of three individual experiments; SD,**, P < 0.01. c Down-regulation of miR-499a was observed in OS tissues compared with that in adjacent ones by qPCR (left). Up-regulation of SHKBP1 was observed in OS tissues compared with that in adjacent ones by qPCR (right). Note: Columns, mean of three individual experiments; SD,**, P < 0.01
Fig. 6
Fig. 6
Inhibition of TGFβ signaling results in upregulation of miR-499a, decrease in SHKBP1 levels, and increased erlotinib sensitivity. a Relative expression of miR-499a and SHKBP1 in CD166− cells with or without TGFβ1 treatment were examined by qPCR. Note: Columns, mean of three individual experiments; SD,**, P < 0.01. b CD166+ OSCs treated with TGFβ1 or control vehicle for 21 day were exposed to LY294002 or erlotinib for 24 h. Immunoblot analysis was performed with antibodies against SHKBP1, AKT and GAPDH. c CD166+ OSCs treated with TGFβ1 or SB431542 were used for western-blot assays with antibodies against SHKBP1, AKT and GAPDH. d Relative expression of miR-499a in CD166+ OSCs with TGFβ1 or SB431542 were examined by qPCR. Note: Columns, mean of three individual experiments; SD,**, P < 0.01. e CD166+ OSCs were incubated with TGFβ (2 ng/mL) alone or in combination with either SB-431542 (10 m mol/L) or TGFβ -RII/Fc (20 ng/mL) for 7 days and then were treated with 1 m mol/L of erlotinib for an additional 72 h. Cell viability was assayed and values were set at 100% for untreated controls
Fig. 7
Fig. 7
Overexpression of miR-499a inhibits TGFβ-induced resistance in vitro and in vivo. a With increasing concentrations of erlotinib from 0.03 to 3 μmol/L, the percentage of viable cells of CD166+ OSCs infected with Lv-miR-499a, Lv-NC, si-SHKBP1 or si-NC were measured by MTT. Note: Columns, mean of three individual experiments; SD,**, P < 0.01. b Western-blotting was used with antibodies specific for Snail, Twist, and ZEB1 in CD166+ OSCs with or without infection of Lv-miR-499a. GAPDH was used as the control. c RNA was extracted from 5 OS directly xenografted tumors Levels of miR-499a were measured by qPCR. Note: Columns, mean of three individual experiments; SD,**, P < 0.01. d The potential of tumor initiation of CD166+ OSCs, Lv-NC-OSCs and si-SHKBP1- OSCs fractions by subcutaneous injection, and representative tumor volumes were measured following treatment with or without three cycles of erlotinib. Note: Columns, mean of three individual experiments; SD,**, P < 0.01
Fig. 8
Fig. 8
Evolution of erlotinib resistance in CD166+ OSCs
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