Hedgehog-Gli2 Signaling Promotes Chemoresistance in Ovarian Cancer Cells by Regulating MDR1

Abstract

Background: Cisplatin (DDP) resistance remains a key challenge in improving the clinical outcome of patients with ovarian cancer (OC). Gli2 overexpression can lead to DDP resistance in OC cells, but the specific underlying regulatory mechanism remains unclear. The membrane transporter encoding gene MDR1 positively regulates chemotherapy resistance in various cancer types. We evaluated MDR1 as a potential Gli2 downstream target and the contribution of the Gli2/MDR1 axis in promoting DDP resistance in OC cells.

Methods: To generate drug-resistant SKOV3/DDP cells, SKOV3 cells were grown for six months under continuous induction wherein the DDP concentration was steadily increased. Gli2 expression in OC cells with varying DDP sensitivities was detected using western blot. Cell counting kit-8 assays were used to assess the DDP sensitivity of SKOV3, SKOV3/DDP, A2780, and A2780/DDP cells and reversal of DDP resistance in SKOV3/DDP and A2780/DDP cells. Cell proliferation was analyzed using 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays. The transcriptional regulation of MDR1 by Gli2 was determined using luciferase reporter assays. Finally, xenograft OC tumors were generated in nude mice, which were then treated with intraperitoneal DDP or phosphate-buffered saline (PBS) injections to investigate if Gli2 affected DDP resistance in OC in vivo.

Results: DDP-resistant SKOV3/DDP and A2780/DDP cells showed higher expression of Gli2 and MDR1 as compared with that in DDP-sensitive OC cells. Gli2 knockdown in SKOV3/DDP cells significantly reduced MDR1 expression, whereas it increased DNA damage, thereby sensitizing OC cells to DDP. Similar results were obtained after targeting Gli2 expression with the Gli-antagonist 61 inhibitor (GANT61) in SKOV3/DDP and A2780/DDP cells. In cells stably overexpressing Gli2, treatment with gradient concentrations of verapamil, an MDR1 inhibitor, significantly inhibited MDR1 expression. Our findings indicate that downregulation of MDR1 expression may reverse OC cell resistance to DDP. Moreover, dual-luciferase reporter gene assays confirmed that MDR1 is a direct downstream target of Gli2, with Gli2 positively regulating MDR1 expression. Finally, subcutaneous xenotransplantation in nude mice demonstrated that Gli2 plays a key role in regulating OC drug resistance.

Conclusions: We identified a mechanism by which Hedgehog-Gli signaling regulates OC chemoresistance by modulating MDR1 expression. Hence, Gli2 and MDR1 are potential biomarkers and therapeutic targets in patients with chemoresistant OC.

Keywords: Gli2; MDR1; cisplatin resistance; hedgehog signaling; ovarian cancer; proliferation.

Figure 1

Gli2 is involved in ovarian cancer chemoresistance. (A–C) Western blot analysis of the expression of Gli2, MDR1 and γH2AX in SKOV3, SKOV3/DDP, A2780, and A2780/DDP cells. (D, E) Survival rates of SKOV3, SKOV3/DDP, A2780, and A2780/DDP cells after treatment with cisplatin (DDP) for 48 h detected using CCK-8 assays. (F, H) Proliferative capacity of SKOV3, SKOV3/DDP, A2780, and A2780/DDP cells after treatment with DDP for 48 h was identified using 5-ethynyl-2′-deoxyuridine (EdU) assays. (G, I) Bar chart of EdU-positive cells evaluated using the ImageJ software. ***P < 0.001.

Figure 2

Gli2 knockdown reversed cisplatin (DDP) resistance and inhibited proliferation of DDP-resistant ovarian cancer cells. (A, B) Western blot analysis of the protein expression levels of Gli2 in different groups after Gli2 knockdown in SKOV3/DDP cells. (C) Survival rate and IC₅₀ of SKOV3/DDP cells transfected with sh-control or sh-Gli2 #3 and #5 after 48 h DDP treatment determined using CCK-8 assays. (D–G) Western blot analysis of the expression levels of MDR1 and γH2AX in SKOV3/DDP cells transfected with empty plasmid or Gli2 specific short hairpin RNA following DDP treatment (0.5 µg/mL). n.s, not significant, * P < 0.05, **P < 0.01.

Figure 3

The Gli2 inhibitor promoted cell drug sensitivity and reduced the proliferative ability of cisplatin (DDP)-resistant ovarian cancer cells. (A–D) Western blot analysis of the protein expression levels of Gli2, MDR1, and γH2AX in SKOV3/DDP and A2780/DDP cells cultured with increasing concentrations of the Gli2 inhibitor Gli-antagonist 61 (GANT61) for 48 h. (E, F) Survival rate and IC₅₀ of SKOV3/DDP and A2780/DDP cells after 48 h of treatment with GANT61 and DDP determined using CCK-8 assays. (G–J) Proliferation capacity was detected by EdU assay after inhibition of SKOV3/DDP and A2780/DDP cells. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Reversal of cisplatin (DDP) resistance in ovarian cancer cells following treatment with MDR1 inhibitor verapamil. (A) Successful construction of stable cell line overexpressing Gli2 using lentiviral infection. (B) Survival rate and IC₅₀ of SKOV3/DDP cells after treatment with verapamil and DDP for 48 h determined using CCK-8 assays. (C–E) Western blot analysis of the protein expression levels of MDR1 and γH2AX in cells treated with gradient concentrations of verapamil for 48h. (F–G) Downregulation of MDR1 expression significantly inhibited cell proliferation, as detected using EdU assays. n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

MDR1 is the direct downstream target of Gli2. (A) Consensus sequence of Gli2-binding sites (GBS). The size of each base in the figure indicates its conservation. The larger the base, the stronger the conservation and more important its function. (B) Within the range of -2,002 to 0 in the promoter region of MDR1 and adjacent regions (the first base at the 5′ end of the MDR1 mRNA NM_000927.5 was recorded as +1), seven candidate GBS were predicted using software analysis. They are numbered separately, and the position and sequence are indicated. The consensus sequence with GBS is underlined in red with the corresponding sequence, and the base list differing from the GBS consensus sequence is in red font. (C) Schematic illustration of the distribution of candidate GBS within the MDR1 promoter and luciferase reporter constructs Frag-I, -II, –II-1, –II-2, –II-1-a, and –II-1-b containing the indicated GBS. (D–F) Gli2 activating effects on Frag-I, -II, –II-1, –II-2, –II-1-a, and –II-1-b reporter constructs detected using a dual-luciferase assay in Gli2-transfected and control HEK293 cells. (G) Wild-type and mutant sequences of the GBS-4 locus in Frag–II-1-a. (H) Compared with the wild-type, the activation effect of Gli2 on the mutant Frag–II-1-a was significantly reduced after GBS-4 point mutation. “Mut” indicates a mutation, and the letters after it denote different GBS sites. Data are represented as the mean ± SD of three independent experiments. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Gli2 knockdown trumps cisplatin (DDP) resistance in ovarian cancer in vivo. SKOV3 cells infected with sh-Control or sh-Gli2 were subcutaneously injected into nude mice. (A) Western blot analysis of the Gli2 protein expression after stable knockdown of Gli2 in SKOV3 cells. (B, C) SKOV3 cells infected with sh-Control or sh-Gli2 were subcutaneously injected into the skin of nude mice. The mice were injected intraperitoneally with 4 mg/kg DDP or phosphate-buffered saline (PBS) (control group) twice per week and photographed on day 32. Tumor volume (D) and tumor weight (E) data are shown as the mean ± SD (n = 5). (F) Following Gli2 knockdown, the expression levels of MDR1 and PCNA decreased, while those of γH2AX and Bcl2 increased in vivo. (G) Expression of Gli2, MDR1, and PCNA in SKOV3 sh-control and SKOV3 sh-Gli2 tumors determined using immunohistochemical analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

Schematic depiction of the role of Gli2 in cisplatin (DDP) resistance in ovarian cancer. Under normal circumstances, the Hh signaling pathway is inhibited and therefore, Gli2 cannot bind to the downstream target genes. This maintains DDP sensitivity in ovarian cancer cells and cell proliferation is inhibited. When the Hh signaling pathway is abnormally activated, Gli2 is overexpressed, enters the nucleus in the activated form of Gli2A and directly binds to the downstream target gene MDR1. This promotes DDP resistance in ovarian cancer cells, thereby significantly enhancing cell proliferation.