NKX6-1 mediates cancer stem-like properties and regulates sonic hedgehog signaling in leiomyosarcoma

Abstract

Background: Leiomyosarcoma (LMS), the most common soft tissue sarcoma, exhibits heterogeneous and complex genetic karyotypes with severe chromosomal instability and rearrangement and poor prognosis.

Methods: Clinical variables associated with NKX6-1 were obtained from The Cancer Genome Atlas (TCGA). NKX6-1 mRNA expression was examined in 49 human uterine tissues. The in vitro effects of NXK6-1 in LMS cells were determined by reverse transcriptase PCR, western blotting, colony formation, spheroid formation, and cell viability assays. In vivo tumor growth was evaluated in nude mice.

Results: Using The Cancer Genome Atlas (TCGA) and human uterine tissue datasets, we observed that NKX6-1 expression was associated with poor prognosis and malignant potential in LMS. NKX6-1 enhanced in vitro tumor cell aggressiveness via upregulation of cell proliferation and anchorage-independent growth and promoted in vivo tumor growth. Moreover, overexpression and knockdown of NKX6-1 were associated with upregulation and downregulation, respectively, of stem cell transcription factors, including KLF8, MYC, and CD49F, and affected sphere formation, chemoresistance, NOTCH signaling and Sonic hedgehog (SHH) pathways in human sarcoma cells. Importantly, treatment with an SHH inhibitor (RU-SKI 43) but not a NOTCH inhibitor (DAPT) reduced cell survival in NKX6-1-expressing cancer cells, indicating that an SHH inhibitor could be useful in treating LMS. Finally, using the TCGA dataset, we demonstrated that LMS patients with high expression of NKX6-1 and HHAT, an SHH pathway acyltransferase, had poorer survival outcomes compared to those without.

Conclusions: Our findings indicate that NKX6-1 and HHAT play critical roles in the pathogenesis of LMS and could be promising diagnostic and therapeutic targets for LMS patients.

Keywords: Chemoresistance; Leiomyosarcoma (LMS); NK6 homeobox 1 (NKX6-1); SHH inhibitor; Sonic hedgehog (SHH) signaling.

Fig. 1

RNA expression of NKX family members in LMS. The expression levels of 14 NKX family mRNAs in well-differentiated, conventional, and poorly differentiated LMS from TCGA. *p < 0.05

Fig. 2

NKX6-1 expression is upregulated in LMS tissues and correlates with malignancy. a Kaplan–Meier analysis of NKX3-2 and NKX6-1 in LMS patients from a dataset in The Cancer Genome Atlas (TCGA). b NKX6-1 mRNA levels in 49 human uterine tissues, including normal myometrium (n = 16), leiomyoma (n = 14), and LMS (n = 19). NKX6-1 mRNA c and protein d expression was determined by qRT-PCR and immunoblotting in MES-SA and MES-SA/DX5 cells. The NKX6-1/β-actin protein expression ratios were 0.31 and 1.19, respectively. In vitro growth curves e and analyses of resistance to cisplatin, lipodox, gemcitabine, and phyxol f in the MES-SA parental and MES-SA/DX5 daughter cell lines. *p < 0.05, **p < 0.01

Fig. 3

NKX6-1 promotes malignancy in LMS cells. Overexpression of NKX6-1 in MES-SA LMS cells increases proliferation and colony formation. Knockdown of NKX6-1 in MES-SA/DX5 cells, by contrast, decreases proliferation. a A cell proliferation assay was performed using the MTS assay. b Colony formation ability was assessed using the AIG assay. NKX6-1-overexpressing MES-SA cells c and NKX6-1 knockdown MES-SA/DX5 cells d were evaluated for resistance to cisplatin, gemcitabine, phyxol, and doxorubicin. The concentrations of cisplatin, gemcitabine, phyxol, and doxorubicin in c are 0.33, 0.01, 0.005 and 7.5 μM, respectively (the IC₅₀ in MES-SA-VC cells). The concentrations of cisplatin, gemcitabine, phyxol, and doxorubicin in d are 0.5, 0.05, 0.0125 and 30 μM, respectively (the IC₅₀ in MES-SA/DX5-Scramble cells). e In vivo tumor growth of NKX6-1-overexpressing MES-SA cells. *p < 0.05

Fig. 4

NKX6-1 promotes cancer stemness properties in LMS cells. Cells were seeded in ultralow attachment plates to assess sphere-forming ability. a Representative spheroid images from different NKX6-1 transfectants. NKX6-1-overexpressing MES-SA cells formed spheroids with round morphology (upper), and NKX6-1 knockdown MES-SA/DX5 cells formed clusters of loosely associated cells (lower). The scale bar represents 100 μm. b The number of spheres was quantified using ImageJ software. The total number of spheres increased upon NKX6-1 overexpression in MES-SA cells and decreased upon NKX6-1 knockdown in MES-SA/DX5 cells. The expression of cancer stemness-related genes correlated with sphere formation ability. c qRT-PCR was performed to evaluate stemness markers (including NESTIN, NANOG, KLF4, KLF8, MYC, OCT, SOX2, CD44, and CD49F) in MES-SA cells transfected with either NKX6-1 or vector control. d Expression of KLF8, MYC, and CD49F in MES-SA/DX5 cells transfected with either shNKX6-1 or scrambled control was determined by qRT-PCR. *p < 0.05

Fig. 5

NKX6-1 mediates the activation of stemness, SHH, and Notch signaling pathways. qRT-PCR analysis of Notch, WNT and SHH signaling. a qRT-PCR for NOTCH1, NOTCH2, WNT1, FZD1, SFRP5, SHH, IHH, and DHH in NKX6-1-overexpressing MES-SA cells. b qRT-PCR for NOTCH1, NOTCH2, and SHH in NKX6-1 knockdown MES-SA/DX5 cells. c Expression of SHH c and NOTCH d downstream genes was determined by qRT-PCR. *p < 0.05

Fig. 6

NKX6-1 activation enhances chemosensitivity to an SHH inhibitor. Dose–response curves (a and b, left panel) and growth curves (b, middle and right panel) of LMS cells and NKX6-1-overexpressing vs. -knockdown cells treated for 96 h with SHH (RU-SKI 43) and NOTCH (DAPT) inhibitors. c Immunohistochemical analysis of GLI1 protein expression in LMS tissue from four patients. The scale bar represents 200 μm. d Kaplan–Meier analysis of overall survival stratified by HHAT expression in LMS patients from the TCGA dataset. e Kaplan–Meier analysis of overall survival stratified by combined NKX6-1 and HHAT expression in LMS patients from the TCGA dataset. *p < 0.05

Fig. 7

Proposed model of NKX6-1-mediated stemness signaling and cancer stemness properties in LMS. NKX6-1 activates SHH and NOTCH but not WNT, resulting in enhanced malignant phenotypes and poor prognosis in LMS. Inhibition of SHH but not NOTCH inhibits cell growth, suggesting the potential of SHH inhibitors for the treatment of LMS