Ganoderma lucidum extract (GLE) impairs breast cancer stem cells by targeting the STAT3 pathway

Abstract

The aggressive nature of triple negative breast cancer (TNBC) may be explained in part by the presence of breast cancer stem cells (BCSCs), a subpopulation of cells, which are involved in tumor initiation, progression, metastasis, recurrence, and therapy resistance. The signal transducer and activator of transcription 3 (STAT3) pathway participates in the development and progression of BCSCs, but its role in TNBC remains unclear. Here, we report that Ganoderma lucidum extract (GLE), a medicinal mushroom with anticancer activity, acts on BCSCs in vitro and in TNBC pre-clinical animal tumor models by downregulating the STAT3 pathway. We show that GLE significantly reduces TNBC cell viability, and down-regulates total and phosphorylated STAT3 expression. This is consistent with the reduction of OCT4, NANOG and SOX2 expression, reduction in the BCSC population by loss of the ALDH1 and CD44⁺/CD24^- population, the deformation of mammospheres, and the strong reduction in animal tumor volume and tumor weight. Analysis of the BCSC compartment in tumors revealed that GLE decreases the STAT3 pathway and the expression of OCT4, NANOG, and SOX2 in BCSCs. These findings demonstrate that the anti-cancer activity of GLE targets BCSCs of TNBC through the downregulation of the STAT3 pathway.

Keywords: Ganoderma lucidum extract; STAT3; aldehyde dehydrogenase; cancer stem cells; triple negative breast cancer.

Figure 1. GLE decreases cell viability in TNBC cell lines

(A) GLE significantly inhibited cell viability of MDA-MB-231 cells in a dose-dependent manner at 24 h, with a half inhibitory concentration (IC₅₀) of 0.96 mg/mL. (B) Live/Dead Staining kit used with the SUM-149 cells revealed that GLE significantly decreased the live cells and increased the dead cells at 24 h. (C) While in the MDA-MB-231 cells, GLE significantly decreased the live cell population. (D) Annexin V/PI staining showed that GLE significantly decreased live cells and increased late apoptotic cells at 24 h in the SUM-149 cell line. (E) While in the MDA-MB-231 cells, GLE significantly decreases the live cells and increases the dead cells. Columns represent mean ± SEM from 2 to 3 independent experiments. ^*P ≤ 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001, was considered statistically significant compared to vehicle.

Figure 2. GLE targets STAT3 and transcription factors involved in cancer cell stemness

mRNA expression was measured by qRT-PCR analysis and results were normalized to GAPDH. (A) GLE significantly decreases the gene expression of STAT3, OCT4, NANOG, and SOX2, when SUM-149 cells were treated with GLE at 24 h. (B) The same analyses were carried out for MDA-MB-231 cells at 24 h. Results show that GLE significantly decreases the gene expression of JAK2, STAT3, and SOX2, while increasing the expression of NANOG. (C) Western blot analyses of the selected proteins were analyzed, with results showing (D) GLE significantly decreasing the phosphorylation of STAT3 at Tyr705 and (E) the expression of NANOG in the SUM-149 cells at 24 h in comparison to vehicle. While in MDA-MB-231 cells, (F) GLE significantly decreases the phosphorylation of STAT3 at Tyr705, and (G) the expression of OCT4, NANOG, and SOX2 in MDA-MB-231 cells compared to vehicle. β-actin was used as a loading control. Columns represent mean ± SEM from 3 independent experiments. ^*P ≤ 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001, was considered statistically significant compared to vehicle.

Figure 3. GLE reduces ALDH1 activity, CD44⁺/CD24^– population, and deforms mammospheres in TNBC cells

(A) FACS images and quantified results of ALDH1 activity in MDA-MB-231 cells at 24 h reveal that GLE significantly decreases ALDH1 activity, compared to vehicle. (B) Representative flow cytometry dot plots of CD44 and CD24 expression in SUM-149 cells treated with GLE at 24 h show a significant increase in double-negative stained cells and a decrease in the CD44⁺/CD24^– (stem cell) population. (C) The statistical analyses performed in the MDA-MB-231 cells display that GLE significantly decreases the CD44⁺/CD24^– population at 24 h in comparison to vehicle. (D) TNBC cell lines were seeded to form mammospheres in non-serum non-adherent culture conditions in the absence (Vehicle) or presence of GLE, with results showing a decrease in size and deformation of the mammospheres at 24 h. (E) Quantification of the circularity for SUM-149 mammospheres shows that GLE significantly decreases the circularity at 24 h compared to vehicle, (F) while in the MDA-MB-231 mammospheres, GLE shows a tendency (P < 0.07) to decrease circularity compared to vehicle. (G) mRNA expression of STAT3 was measured by qRT-PCR analysis after mammospheres were treated with vehicle and GLE, with results showing that GLE significantly decreases the gene expression of STAT3 in both TNBC cell lines compared to vehicle. Results were normalized to β-actin. Columns represent mean ± SEM from 2 or 3 independent experiments. Data for the circularity analysis are from n = 20 replicates from 3 independent experiments. ^*P ≤ 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001, was considered statistically significant compared to vehicle.

Figure 4. GLE decreases CD44⁺/CD24^– tumor growth and inhibits the STAT3 signaling pathway in vivo

(A) Average mouse weights of each group. GLE treated group and vehicle treated group showed no significant differences in mouse weight. (B) The graph represents average tumor volumes of each group over time (10 weeks). The average tumor volume from GLE treated mice significantly decreased in comparison to the vehicle treated group. Bar represents tumor volume mean ± SEM (n = 7 mice, P < 0.05). (C) Average tumor weights of each group. The average tumor weight from GLE treated group was significant in comparison to the vehicle treated group (mean ± SEM, n = 7, p = 0.05). (D) Western blot assays of GLE effects in BCSCs tumors. (E) GLE decreases phospho-JAK2 (Tyr1007/1008), phospho-STAT3 (Tyr705), and total STAT3. (F) GLE does not change the expression of the three transcription factors (OCT4, NANOG, and SOX2) in BCSCs tumors. Total proteins were extracted from tumor tissues. Immunoblots were performed on three independent experiments with indicated antibodies. β-actin was used as a loading control. Columns represent mean ± SEM from 3 independent experiments. ^*P ≤ 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001, was considered statistically significant compared to vehicle.