mTOR plays critical roles in pancreatic cancer stem cells through specific and stemness-related functions

Abstract

Pancreatic cancer is characterized by near-universal mutations in KRAS. The mammalian target of rapamycin (mTOR), which functions downstream of RAS, has divergent effects on stem cells. In the present study, we investigated the significance of the mTOR pathway in maintaining the properties of pancreatic cancer stem cells. The mTOR inhibitor, rapamycin, reduced the viability of CD133(+) pancreatic cancer cells and sphere formation which is an index of self-renewal of stem-like cells, indicating that the mTOR pathway functions to maintain cancer stem-like cells. Further, rapamycin had different effects on CD133(+) cells compared to cyclopamine which is an inhibitor of the Hedgehog pathway. Thus, the mTOR pathway has a distinct role although both pathways maintain pancreatic cancer stem cells. Therefore, mTOR might be a promising target to eliminate pancreatic cancer stem cells.

Figure 1. The mTOR inhibitor rapamycin does not affect the frequency of CD133⁺ cells but significantly reduces the viability of pancreatic cancer cells.

(a) Capan-1M9 cells were treated with the inhibitors at the indicated concentrations for three days. The effects of the mTOR inhibitor rapamycin and the Hedgehog pathway inhibitor cyclopamine on CD133⁺ cell frequency and cell viability were determined by flow cytometry and the MTT assay, respectively. The results are presented as percentages of control values in untreated cells, showing the mean and s.d. of four replicates obtained in one representative experiment out of three. (b) Schematic representation of the effects of rapamycin and cyclopamine on cell viability of CD133⁺ and CD133⁻ cells. The vertical axis indicates the CD133⁺ cell frequency, and the horizontal axis indicates the total cell viability. The closed circles represent the CD133⁺ cells, and the open circles represent the CD133⁻ cells. (c) The mTOR inhibitor rapamycin suppresses the growth of Capan-1M9 pancreatic cancer cells. The cells were seeded at an initial density of 10⁵ cells per 35-mm dish. Rapamycin was administered two days after plating, and the cell numbers were then determined over a period of five days using trypsin/EDTA treatment. The vertical axis is shown on a logarithmic scale. All data are the mean ± s.d. *P < 0.01. (d) Morphology of Capan-1M9 cells untreated (left) and treated (right) with 10 nM rapamycin for three days. Scale bar, 200 μm.

Figure 2. Reduced CD133 expression does not affect cell viability after rapamycin treatment.

(a) Immunoblot detection of CD133 polypeptide in Capan-1M9 cells expressing the CD133 shRNA (shCD133M9). The full-length blots are shown in the supplemental Figure S4. (b) Capan-1M9 cells expressing (open bars) and not expressing (closed bars) the CD133 shRNA were treated with the indicated concentrations of rapamycin for three days. Cell viability was determined, as shown in Figure 1(a). All data are the mean ± s.d. *NS, not significant by a two-tailed Student's t-test. Rapamycin inhibits sphere formation in Capan-1M9 cells. (c) and (d) Representative photographs of spheres cultured in stem cell medium in the absence (c) or presence (d) of 10 nM rapamycin. Scale bar: 50 μm. (e) Single-cell suspensions (0.7 cells/100 μl) were plated in wells and cultured for eight days. The number of spheres formed per 96-well plate was determined in triplicate plates. All data are the mean ± s.d. *P < 0.01. (f) The cells were cultured as in (e), and the sizes of the spheres were determined. All data are the mean ± s.d. *P < 0.05.

Figure 3. mTOR inhibition reduces the self-renewal of cancer stem-like cells in PANC-1 cells.

(a) Rapamycin reduced the viability of PANC-1 cells in the two-dimensional culture. PANC-1 cells were treated with the indicated concentrations of rapamycin for three days. Cell viability was determined as indicated in Figure 1(a). Representative photographs of spheres (primary spheres) cultured in the stem cell medium in the absence (b) or presence of 10 nM (c) and 100 nM (d) rapamycin. Scale bar: 200 μm. (e) Rapamycin does not affect the number of primary spheres. The cells were cultured and assayed, as described in Figure 2(e). (f) Rapamycin reduces the size of primary spheres. The cells were cultured as in (e), and the results are indicated in Figure 3(f). (g) and (h) Representative photographs of secondary spheres. Single cells from primary spheres that were untreated (g) or treated with 100 nM rapamycin (h) were plated in 96 wells and then cultured without rapamycin for eight days. Scale bar: 200 μm. (i) Rapamycin reduces the self-renewal capacity of stem-like cells, as reflected by the formation of secondary spheres. Single cells from primary spheres were seeded in 96-well plates and cultured without rapamycin for eight days. The indicated results are the mean values and s.d. of the number of secondary spheres formed per 96-well plate. All data are the mean ± s.d. *P < 0.01.

Figure 4. mTOR inhibition reduces sphere growth in the recently established pancreatic cancer cell line PCK-2.

(a) Rapamycin reduced the viability of PCK-2 cells in the two-dimensional culture. Cell viability was determined, as indicated in Figure 1(a). Representative photographs of the spheres cultured in the stem cell medium in the absence (b) or presence (c) of 10 nM rapamycin. Scale bar: 50 μm. (d) Rapamycin does not affect the number of spheres. The cells were cultured, as shown in Figure 2(e). The indicated results are the mean values and s.d. of the number of spheres formed per 288-wells (three 96-well plates) in triplicate experiments. (e) The cells were cultured as in (d), and the sizes of the spheres were determined. All data are the mean ± s.d. *P < 0.05.

Figure 5. Rapamycin inhibits anchorage-independent colony formation in pancreatic cancer cells.

(a) Representative photographs of colonies in soft-agar in the absence (left) or presence (right) of 10 nM rapamycin. Scale bar: 400 μm. (b) Single cells in soft agar were seeded in 24-well plates and cultured for two weeks. The number of colonies formed per well is indicated as the mean ± s.d. from three wells in one representative experiment out of three. *P < 0.05.

Figure 6. Phosphorylation of mTOR effectors after rapamycin treatment.

(a) Phosphorylation and activation of mTOR downstream effectors in Capan-1M9 cells. (b) Phosphorylation and activation of Akt and ERK in Capan-1M9 cells. Capan-1M9 cells were cultured and treated with the indicated concentrations of rapamycin. The cell lysates were immunoblotted to detect the indicated proteins. The full-length blots are shown in the supplemental Figure S5.

Figure 7. mTOR inhibitor rapamycin impairs in vivo growth of xenografted pancreatic carcinoma in nude mice.

(a) Tumor growth of Capan-1M9 xenografts which were treated with vehicle (open circle), 1 mg/kg/day (closed circle), or 5 mg/kg/day (closed triangle) rapamycin. Tumor volume was shown as the mean and SD from 5 mice (10 tumors) from each group. Rapamycin treatment showed a significant effect. * P < 0.01. (b) The variance in the percentage of CD133⁺ cells after the flow cytometric analysis of xenografts.

Figure 8. Proposed model for mTOR function in pancreatic cancer stem cells.

The key genetic alteration driving the initiation of pancreatic ductal adenocarcinoma is activating KRAS mutations, which are found in > 90% of cases. mTOR functions downstream of KRAS to maintain the stemness of pancreatic CSCs. Therefore, the inhibition of mTOR by rapamycin reduced sphere formation and anchorage-independent cell growth and viability in CD133⁺ cells, which were likely CSCs. The Hedgehog pathway also functions to maintain pancreatic CSCs. However, the inhibition of these two pathways led to different cellular effects, suggesting that mTOR has specific function(s) in addition to the common function of maintaining the stemness of pancreatic CSCs. mTOR-mediated tumor suppressor responses have been reported in normal and cancer stem cells in another cell type, showing a candidate of mTOR specific function(s) in pancreatic cancer cells.