Selinexor (KPT-330) has antitumor activity against anaplastic thyroid carcinoma in vitro and in vivo and enhances sensitivity to doxorubicin

Abstract

Anaplastic thyroid carcinoma (ATC) is one of the most lethal malignancies having no effective treatment. Exportin-1 (XPO1) is the key mediator of nuclear export of many tumor suppressor proteins and is overexpressed in human cancers. In this study, we examined the therapeutic potential of selinexor (XPO1 inhibitor) against human ATC cells both in vitro and in vivo. Here, we showed that XPO1 is robustly expressed in primary ATC samples and human ATC cell lines. Silencing of XPO1 by either shRNA or selinexor significantly reduced cellular growth and induced cell cycle arrest, apoptosis of ATC cells by altering the protein expression of cancer-related genes. Moreover, selinexor significantly inhibited tumor growth of ATC xenografts. Microarray analysis showed enrichment of DNA replication, cell cycle, cell cycle checkpoint and TNF pathways in selinexor treated ATC cells. Importantly, selinexor decreased AXL and GAS6 levels in CAL62 and HTH83 cells and suppressed the phosphorylation of downstream targets of AXL signaling such as AKT and P70S6K. Finally, a combination of selinexor with doxorubicin demonstrated a synergistic decrease in the cellular proliferation of several ATC cells. These results provide a rationale for investigating the efficacy of combining selinexor and doxorubicin therapy to improve the outcome of ATC patients.

Figure 1

Endogenous expression of XPO1 in human ATC samples and cell lines, and effect of silencing of XPO1 in ATC cells. (A) Immunohistochemical analysis for detection of endogenous expression of XPO1 protein in thyroid tumors and benign thyroid tissues. Representative photographs showing nuclear staining (brown color) of XPO1 in different histological subtypes of thyroid carcinoma: papillary, follicular, ATC and benign thyroid (low or no reactivity) (original magnification, ×200; objective ×20). (B) Western blot analysis of ATC cell lines verified that the XPO1 antibody specifically recognizes a band of 123 kDa, corresponding to the size of XPO1 protein. (C) Indirect immunofluorescence assay showing nuclear localization of XPO1 protein in fixed/permeabilized ATC cell lines; DAPI stains nuclei. (D) ATC cells were transduced with either XPO1 shRNA or scrambled shRNA. XPO1 knockdown was confirmed in HTH83 and CAL62 cells by western blotting. GAPDH antibody was used to assure equal loading of lysates. Original and full-length blots are included in the Supplementary Information. XPO1 silencing resulted in decreased cell proliferation of HTH83 and CAL62 as measured using MTT assay. Results represent mean ± SD; n = 4. *P ≤ 0.01; **p ≤ 0.001; ***p ≤ 0.0001(Student’s t-test).

Figure 2

Selinexor significantly suppressed cellular growth and clonogenic ability of human ATC cells in vitro. (A) OGK-M, HTH83, CAL62 and T238 cells were cultured with increasing concentrations of selinexor (0–1,000 nM) for 24 h. Whole-cell lysates were prepared, and XPO1 protein expression was analyzed using western blot analysis. GAPDH was used as an internal control. Cell viability of eight human ATC cell lines. Original and full-length blots are included in the Supplementary Information. (B) Cells were treated with selinexor at indicated concentrations for 72 h, and growth inhibition was measured by MTT assay. Results are expressed as mean value ± SD; n = 4. (C and D) Representative photomicrograph displayed that selinexor treatment decreased clonogenic growth. ATC cells were exposed to indicated concentrations of selinexor for 48 h; cells were washed with drug-free medium and allowed to form colonies for 2 weeks followed by crystal violet staining and quantification. Data represent mean ± SD of four independent experiments done in triplicates. *p ≤ 0.01; **p ≤ 0.001; ***p ≤ 0.0001(Student’s t-test).

Figure 3

Inhibition of XPO1 induced cell cycle arrest and apoptosis of ATC cells in a dose-dependent manner. (A) Silencing of XPO1 in ATC cells resulted in an increased in G1 phase. (B) OGK-M, HTH83, CAL62, and T238 cells were treatment with either different concentrations of selinexor (0–1,000 nM) or diluent control (DMSO), stained with propidium iodide (PI) and analyzed by flow cytometric analysis. Histogram showed the proportion of cells in different phases of cell cycle. Data are presented as the mean of three independent experiments. (C) ATC cells were treated with selinexor at indicated concentrations for 24 h, stained with annexin V-FITC and PI, and subjected to flow cytometric analysis to evaluate the ability of selinexor to induce apoptosis. Histograms represent the percentage of apoptotic cells. Data are presented as mean ± SD of three independent experiments. *p ≤ 0.01; **p ≤ 0.001; ***p ≤ 0.0001for selinexor vs. controls. (D) OGK-M, HTH83, CAL62 and T238 cells were treated with either selinexor (1,000 nM) or DMSO for 24 h. Lysates were analyzed by western blot analysis for the indicated cell cycle and apoptosis proteins (GAPDH, internal loading control). Original and full-length blots are included in the Supplementary Information.

Figure 4

Selinexor inhibits ATC growth in vivo. (A) Immunodeficient mice received ATC cells (CAL62) subcutaneously. One week after implantation, mice began to receive either oral selinexor (10 mg/kg, 3 times per week for 4 weeks) or vehicle alone. Selinexor treatment significantly suppressed tumor volumes (mean ± SD of 6 tumors). *P ≤ 0.01; **P ≤ 0.001; ***p ≤ 0.0001(Student’s t-test). (B) Representative photograph showing decreased tumor growth in selinexor treated group compared to vehicle treated group (n = 6 for each group). Scale in cm. (Lower panel) Tumor weights from selinexor and vehicle treated groups (mean ± SD of 6 tumors, P = 0.000022). (C) At the end of the experiment, whole cell lysates (protein) were prepared from tumors resected from selinexor-treated and vehicle-treated mice and subjected to western blotting with indicated antibodies. (D and E) Tumor sections were subjected to immunohistochemical analysis. Tumors section from the selinexor-treated group showed weak staining for XPO1 (exportin 1), cell proliferation marker (Ki-67) and blood vessel marker (CD31) and strong staining for Tunel (a marker of apoptosis) as compared to vehicle-treated group original magnification, ×200; objective ×20). Columns (on the right) represent mean ± SD of three independent tumors. **P ≤ 0.001; ***p ≤ 0.0001 (Student’s t-test).

Figure 5

Effect of selinexor on gene expression profiling of ATC cells. (A) Volcano plot shows differentially expressed genes for OGK-M cells treated with either vehicle (DMSO) or 1,000 nM selinexor for 12 h. Red and green dots represent up-regulated and down-regulated genes, respectively. Grey dots represented no change. (B–E) Gene set enrichment analysis showed negative enrichment of DNA replication, cell cycle, cell cycle checkpoint and positive enrichment of TNF (tumor necrotic factor) target pathway. (F) qRT-PCR validation of 12 selected genes identified as differentially expressed by microarray. Expression of each gene was normalized to GAPDH as a reference (control value converted to 1). Figures are representative of 3 replicates. Data represent mean ± SD, n = 3. **p ≤ 0.001, ***p ≤ 0.0001. NES indicates normalized enrichment score; and q = false discovery rate.

Figure 6

Selinexor suppressed AXL signaling, and the drug effectively enhanced growth arrest of ATC cells in combination with doxorubicin. (A) HTH83 and CAL62 cells were cultured with either selinexor (1,000 nM) or diluent control (DMSO) for 24 h, and the whole-cell lysate was subjected to western blot analysis for AXL, p-AKT, total AKT, p-P70S6K and total P70S6K. (B and C) Proliferation study of the combination of selinexor with doxorubicin. Data represent means ± s.d.; n = 3. (D–F) Combinational growth inhibition of selinexor and doxorubicin on CAL62 and HTH83 cells displayed as CI. CI defines the interaction between selinexor and doxorubicin as plotted against a fraction of cell viability. CI < 1, CI = 1 and CI > 1 represent synergism, additive, and antagonism of the two compounds, respectively.