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. 2016 Oct 18;7(42):68229-68241.
doi: 10.18632/oncotarget.11920.

Galectin-3 Supports Stemness in Ovarian Cancer Stem Cells by Activation of the Notch1 Intracellular Domain

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Free PMC article

Galectin-3 Supports Stemness in Ovarian Cancer Stem Cells by Activation of the Notch1 Intracellular Domain

Hyeok Gu Kang et al. Oncotarget. .
Free PMC article

Abstract

Ovarian cancer is the most lethal gynecologic disease because usually, it is lately sensed, easily acquires chemoresistance, and has a high recurrence rate. Recent studies suggest that ovarian cancer stem cells (CSCs) are involved in these malignancies. Here, we demonstrated that galectin-3 maintains ovarian CSCs by activating the Notch1 intracellular domain (NICD1). The number and size of ovarian CSCs decreased in the absence of galectin-3, and overexpression of galectin-3 increased them. Overexpression of galectin-3 increased the resistance for cisplatin and paclitaxel-induced cell death. Silencing of galectin-3 decreased the migration and invasion of ovarian cancer cells, and overexpression of galectin-3 reversed these effects. The Notch signaling pathway was strongly activated by galectin-3 overexpression in A2780 cells. Silencing of galectin-3 reduced the levels of cleaved NICD1 and expression of the Notch target genes, Hes1 and Hey1. Overexpression of galectin-3 induced NICD1 cleavage and increased expression of Hes1 and Hey1. Moreover, overexpression of galectin-3 increased the nuclear translocation of NICD1. Interestingly, the carbohydrate recognition domain of galectin-3 interacted with NICD1. Overexpression of galectin-3 increased tumor burden in A2780 ovarian cancer xenografted mice. Increased expression of galectin-3 was detected in advanced stages, compared to stage 1 or 2 in ovarian cancer patients, suggesting that galectin-3 supports stemness of these cells. Based on these results, we suggest that targeting galectin-3 may be a potent approach for improving ovarian cancer therapy.

Keywords: Notch1; cancer stem cells; galectin-3; ovarian cancer.

Conflict of interest statement

CONFLICTS OF INTEREST

None.

Figures

Figure 1
Figure 1. Galectin-3 regulates cancersphere formation, which is a stem cell like phenotype, in ovarian cancer cells
(A and B) Detection of ovarian cancerspheres prepared by galectin-3-depleted SKOV3 and OVCRA429 cells or (C and D) galectin-3-overexpressed A2780 and OVCAR3 cells. (A and C) Galectin-3 shRNA containing lentiviruses (A) or galectin-3 overexpression vector containing lentiviruses (C) were infected in cells and stable galectin-3-depleted cells were selected by puromucin. These cells were cultured for 14 days and the morphology was taken by photographes. Scale bar represents 50 μm. (B and D) Galectin-3-depleted ovarian cancerspheres (B) or galectin-3-overexpressed (D) ovarian cancer spheres were collected and separated into single cells by trypsin treatment. The sphere forming cell numbers were calculated and prepared graphs by ratio of total seeded cell numbers. (E) Detection of CD133 expression level in galectin-3 overexpressed A2780 cells. The expression level was detected by real-time PCR analysis. (F) Detection of galectin-3 and CD133 expression level in OVCAR3 ovarian cancer spheres formed cells. The data are presented as the mean ± SD (n = 3). Significant differences are indicated by an asterisk (*p < 0.05), and the p values were calculated using the Student's t test.
Figure 2
Figure 2. Galectin-3 regulates cell proliferation and drug resistance in ovarian cancer cells
(A and B) (A) galectin-3 shRNA was transfected in SKOV3 cells and OVCAR429 cells, and (B) galectin-3 overexpression vector was transfected in A2780 cells and OVCAR3 cells. LacZ shRNA and PLL3.7 mock vector were used as the transfection control. Cell viability was analyzed by WST assays. (C and D) galectin-3 overexpression vector was transfected in A2780 cells and OVCAR3 cells. pLECE mock vector was used as a transfection control. After chemotherapeutic drugs, indicated paclitaxel, cisplatin, treatment for 48 hrs, cell viability was measured by WST assay. The data are presented as the mean ± SD (n = 3). Significant differences are indicated by an asterisk (*p < 0.05). The p values were calculated using the Student's t test.
Figure 3
Figure 3. Galectin-3 regulates invasion and migration of ovarian cancer cells
(AD) SKOV3 cells and OVCAR429 were transfected with galectin-3 siRNA. Scrambled RNA (scRNA) was used as a transfection control. (A) Detection of galectin-3 protein expression by western blot analysis, (B) Detection of the healing ability by wound healing assays, (C) Invasion activity and (D) migration assay by trans-filter well assays. (EH) A2780 and OVCAR3 cells was transfection with flag-tagged galectin-3 expression vector. pcDNA3.0 mock vector was used as a transfection control. (E) Detection of galectin-3 protein expression by western blot analysis, (F) Detection of the healing ability by wound healing assays, (G) Invasion activity and (H) migration assay by trans-filter well assays. Data are presented as mean ± SD (n = 3). The significant differences are indicated by asterisk (*p < 0.05), p values were calculated using the Student's t tests.
Figure 4
Figure 4. Galectin-3 regulates the Notch signaling and cancer stemness factors
(A and B) Stemness and dedifferentiation related proteins were detected by western blot analysis in (A) galectin-3 siRNA transfected SKOV3 cells and OVCAR429 cells and (B) galectin-3 overexpressing vector transfected A2780 cells and OVCAR3 cells. Scrambled RNA (scRNA) and pcDNA3.0 mock vector were used as the transfection control, respectively. GAPDH was used as a loading control for western blot analysis. (C and D) (C) RT and real-time PCR analysis of CD133, HEY1, HES1, CCND1, C-MYC, TCF, GLI1, GLI3, and LGALS3 abundance in scRNA or galectin-3 siRNA transfected SKOV3 cells. (D) Real-time PCR analysis of CD133, HEY1, HES1, CCND1, C-MYC, TCF, GLI1, GLI3, and LGALS3 abundance in galectin-3-overexpressing vector transfected A2780 cells. Scrambled RNA (scRNA) and pcDNA3.0 mock vector were used as the transfection control, respectively. The expression level of GAPDH was analyzed for normalization control.
Figure 5
Figure 5. Galectin-3 induces the cleavage and nuclear translocation of Notch1 intracellular domain (NICD1) and the expression of the Notch target genes HES1 and HEY1
(A) Detection of mRNA and protein expression of galectin-3, Notch1 and NICD1 in galectin-3 silenced SKOV3 cells by RT-PCR and immunoblotting, respectively. β-actin was used as a loading control. (B) Protein levels of NICD1 and galectin-3 were detected in the nuclear and cytosol fractions of SKOV3 cells that were transfected with scRNA or galectin-3 siRNA. GAPDH and Lamin A/C were used as loading controls. (C) Protein levels of galectin-3, NICD1, HES1, and HEY1 in SKOV3 cells transfected with scRNA or galectin-3 siRNA. GAPDH was used as a loading control. (D) Detection of mRNA and protein expression of galectin-3, Notch1 and NICD1 in A2780 cells transfected with control vector or with the galectin-3 expression vector by RT-PCR and immunoblotting, respectively. β-actin was used as a loading control. (E) Protein levels of NICD1 and galectin-3 were detected in the nuclear and cytosol fractions of A2780 cells that were transfected with control or galectin-3 expression vector. GAPDH and Lamin A/C were used as loading controls. (F) Protein levels of galectin-3, NICD1, HES1, and HEY1 in A2780 cells transfected with mock vector or galectin-3 overexpressing vector. GAPDH was used as a loading control. (G) Immunocytochemical analysis was performed for detection of co-localization NICD1 and galectin-3 after transfection with scRNA or galectin-3 siRNA in SKOV3 cells (upper) and transfection with mock vector or galectin-3 expression vector (lower) in A2780 cells. Scale bar represents 50 μm. DAPI staining was used for detection of nucleus and the procedure was described in “Materials and Methods”.
Figure 6
Figure 6. Galectin-3 interacts with NOTCH1 and NICD1 through its C-terminal domain
(A and B) Immunoprecipitation was performed using (A) antibodies against galectin-3 and Notch1 or (B) antibodies against galectin-3 and NICD1 to detect interactions between galectin-3 and NICD1 in the SKOV3 cells. (C) Schematic model of Flag-galectin-3 domain (full length: amino acids 1–250, and CRD truncations: amino acids 33–250, 63–250, and 111–250). (D) Immunoprecipitation of Flag-galectin-3 domains and NICD1 using transfection of the galectin-3 domains in A2780 cells. β-actin was used as the loading control.
Figure 7
Figure 7. Overexpression of galectin-3 increases the tumor growth in xenografted mouse tumors
A2780cells were transfected galectin-3 overexpression vectors and these cells were performed subcutaneous inoculation in nude mice. (A) Tumor formation was quantified by measuring the tumor volume 15 days after inoculation and represent as a graph (n = 3., left panel). The error bars indicate 95% confidence intervals; *p < 0.05 using two sided t-test. All statistical tests were two sided. Tumor formation was observed 41 days after inoculation and taken by photpgraphs (right panel) (B) Immunohistochemistry (IHC) analysis was performed to detect the expression level of galectin-3, NICD1, NANOG, OCT4, and SOX2 in tumors in in vivo mouse models. Method of IHC analysis was described in “Materials and Methods”. Scale bar presents 200 μm. (C) The expression level of galectin-3 was analyzed by stages in malignant tissues of ovarian cancer patients from TCGA database. The analysis method was described in “Materials and methods”.

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