Downregulation of cancer stem cell properties via mTOR signaling pathway inhibition by rapamycin in nasopharyngeal carcinoma

Abstract

Rapamycin, a mammalian target of rapamycin (mTOR) signaling inhibitor, inhibits cancer cell proliferation and tumor formation, including in nasopharyngeal carcinoma (NPC), which we proved in a previous study. However, whether rapamycin affects cancer stem cells (CSCs) is unclear. In examining samples of NPCs, we found regions of CD44-positive cancer cells co-expressing the stem cell biomarker OCT4, suggesting the presence of CSCs. Following this, we used double-label immunohistochemistry to identify whether the mTOR signaling pathway was activated in CD44-positive CSCs in NPCs. We used a CCK-8 assay and western blotting to explore whether the stem cell biomarkers CD44 and SOX2 and the invasion protein MMP-2 could be suppressed by treatment with rapamycin in cultured primary NPC cells and secondary tumors in BALB/c nude mice. Interestingly, we found that rapamycin inhibited mTOR signaling in addition to simultaneously downregulating the expression of CD44, SOX2 and MMP-2 and that it affected cell growth and tumor size and weight both in vitro and in vivo. Collectively, we confirmed for the first time that CSC properties are reduced and invasion potential is restrained in response to mTOR signaling inhibition in NPC. This evidence indicates that the targeted inhibition of CSC properties may provide a novel strategy to treat cancer.

Figure 1

CD44-positive cancer cells in NPC tissues and nasopharyngitis epithelial cells. (A) CD44 (red) was expressed in only a few nasopharyngitis epithelial cells, and OCT4 was rarely detected in these cells. (B) The merged image shows little co-expression of CD44 and OCT4 (green) in nasopharyngitis epithelial cells. (C) CD44 and OCT4 were expressed in select regions of NPC tissue sections, mainly in epithelial areas instead of lymph regions. Double-immunolabeling showed that some regions contained co-expression of CD44 and OCT4. (D) Cell nuclei were counterstained with DAPI (blue), and the right panel shows the merged color. The magnification is 50 μm.

Figure 2

mTOR signaling pathway activation in CD44-positive cancer cells and nasopharyngitis epithelial cells. (A) P-mTOR was expressed in a very small number of nasopharyngitis epithelial cells. (B) In these cells, there was sparse co-expression of OCT4 and P-mTOR, suggesting that the mTOR signaling pathway was almost inactive. (C and D) The orange color (in the right panel) represents the region containing co-expression of CD44 (red) and P-mTOR (green). It shows that the active mTOR protein, P-mTOR, was expressed in CSC-containing regions of NPC tissue sections, suggesting that the mTOR signaling pathway was activated mainly in NPC CSCs. Cell nuclei were counterstained with DAPI (blue).

Figure 3

CD44, a CSC biomarker, was expressed both in the NPC cell line CNE-2 and in 12th passage cultured primary NPC cells, and it was co-expressed with another CSC biomarker, OCT4, in cultured primary NPC cells. (A) Regions of CNE-2 cells and NPC cells expressing the CSC biomarker CD44. (B) Double-immunolabeling showed that CD44-positive cells (green) were CSCs, which co-expressed OCT4 (red) in 13th passage cultured primary NPC cells. Cell nuclei were counterstained with DAPI (blue).

Figure 4

Rapamycin inhibited cell growth by inhibiting mTOR signaling in cultured primary NPC cells as detected by CCK-8 assay and western blotting. CD44 and SOX2 were suppressed by rapamycin treatment, whereas OCT4 was less affected. (A) P-mTOR and a downstream effector, the phosphorylated 4E-BP1 protein (P-4E-BP1, active 4E-BP1), were gradually suppressed as the concentration of rapamycin increased (from 20 to 100 nM). In contrast, total mTOR and 4E-BP1 were not significantly affected in cultured primary NPC cells. (B) Various concentrations of rapamycin inhibited CD44 and SOX2, but OCT4 was rarely affected. (C) The number of NPC cells decreased gradually as the dose of rapamycin (from 10 to 100 nM) increased, demonstrating that NPC cell growth was inhibited by rapamycin. (D) The relative gray value ratios of P-mTOR to mTOR and P-4E-BP1 to 4E-BP1 at various concentrations of rapamycin, in which higher concentrations of rapamycin inhibited mTOR signaling more prominently compared to the normal control group (0 nM). (E) The relative expression levels of SOX2 to GAPDH and OCT4 to GAPDH, in which SOX2 was differentially inhibited by various concentrations of rapamycin, whereas OCT4 was not suppressed in cultured primary NPC cells. ^*P<0.05.

Figure 5

Regions of cultured primary NPC cells expressing the CSC biomarker CD44; the mTOR signaling pathway was also activated in these cells when they were injected subcutaneously into BALB/c nude mice. (A and B) CD44 and OCT4 co-expression in secondary tumor tissue sections (green, CD44; red, OCT4); (A) right panel shows co-expression in orange, while the merged color is shown in the right panel of (B-D). Double-immunostaining showed that the mTOR signaling pathway was also activated in NPC CSCs in vivo, in which CD44 (red) and P-mTOR (green) were co-expressed, suggesting that the mTOR signaling pathway was activated in CSCs of secondary NPCs.

Figure 6

The sizes and weights of secondary tumors in BALB/c nude mice were restrained following treatment with rapamycin compared to vehicle solution. (A and B) Various sizes of secondary tumors occurring in the two groups on the same day. (C) The growth curve of secondary tumors in mice from the 2nd to the 14th day after intraperitoneal injection with rapamycin or vehicle, which showed that the tumors were inhibited by treatment with rapamycin compared to control. (D) The weights of the tumors that were harvested from the two groups, which showed that the tumors were significantly inhibited in the rapamycin group compared with the vehicle group. ^*P<0.05. Veh, vehicle; Rap, rapamycin.

Figure 7

The CSC biomarkers CD44 and SOX2, as well as MMP-2, were suppressed in response to rapamycin-mediated inhibition of mTOR signaling in secondary NPCs in BALB/c nude mice, whereas OCT4 and MMP-9 were not inhibited by rapamycin. (A) CD44 and MMP-2 expression was inhibited in mice injected intraperitoneally with rapamycin compared to mice injected with control (vehicle), whereas OCT4 levels did not differ between the two groups. (B) The active form of mTOR, P-mTOR, was suppressed in NPC cells in the rapamycin group, and SOX2 was inhibited. However, MMP-9 and mTOR were not restrained by treatment with rapamycin compared to vehicle. (C and D) The relative expression levels of the above proteins in secondary tumor sections from both groups. ^*P<0.05.