Knockdown of the sodium-dependent phosphate co-transporter 2b (NPT2b) suppresses lung tumorigenesis

Abstract

The sodium-dependent phosphate co-transporter 2b (NPT2b) plays an important role in maintaining phosphate homeostasis. In previous studies, we have shown that high dietary inorganic phosphate (Pi) consumption in mice stimulated lung tumorigenesis and increased NPT2b expression. NPT2b has also been found to be highly expressed in human lung cancer tissues. The association of high expression of NPT2b in the lung with poor prognosis in oncogenic lung diseases prompted us to test whether knockdown of NPT2b may regulate lung cancer growth. To address this issue, aerosols that contained small interfering RNA (siRNA) directed against NPT2b (siNPT2b) were delivered into the lungs of K-ras (LA1) mice, which constitute a murine model reflecting human lung cancer. Our results clearly showed that repeated aerosol delivery of siNPT2b successfully suppressed lung cancer growth and decreased cancer cell proliferation and angiogenesis, while facilitating apoptosis. These results strongly suggest that NPT2b plays a role lung tumorigenesis and represents a novel target for lung cancer therapy.

Figure 1. Expression levels of the NPT2 transporters in the lungs of WT and K-ras ^LA1 mice.

All wild-type mice (WT) and K-ras^LA1 mice were 10 week old female. (A) Western blot analysis of NPT2a, NPT2b, and NPT2c in the lungs of the WT and K-ras ^LA1 mice. (B) The bands-of-interest were further analyzed via densitometry. Each bar represents the mean±SEM (n = 3). **P<0.01 relative to corresponding control values. (C) Quantitative real-time PCR analysis of NPT2a, NPT2b, and NPT2c in the lungs of the WT and K-ras ^LA1 mice. Each bar represents the mean±SEM (n = 4). (WT = wild-type normal mice and K-ras = K-ras ^LA1 mice).

Figure 2. Tumor histopathology and expression level of NPT2b in the lungs of siNPT2b-delivered mice.

(A) Histological examination of the lungs of the 10-week-old K-ras ^LA1 mice (magnification×100 and ×200; the bars represent 100 µm and 50 µm each). (B) Tumor numbers in the lungs of the six K-ras ^LA1 mice. Each bar represents the mean±SEM (n = 6). (C) Western blot analysis of NPT2b in the lungs of the K-ras ^LA1 mice after the aerosol delivery. (D) Densitometric analysis was performed on the Western blot bands in five separate mice. Each bar represents the mean±SEM (n = 5). *P<0.05, **P<0.01 relative to Con or Scr. (E) Quantitative real-time PCR analysis of NPT2b in the lungs of the K-ras ^LA1 mice. Each bar represents the mean±SEM (n = 6). (Con = control; Scr = scrambled control; and siNPT2b = siNPT2b-delivered group).

Figure 3. Effects of the aerosol-delivered siNPT2b on the apoptotic proteins.

(A) Western blot analysis of the BAX and BAD proteins. (B) Densitometric analysis of the Western blots. (C) TUNEL assay on the tumor lesions of lungs of the K-ras ^LA1 mice. (D) Graph representing the number of TUNEL-positive cells. Each bar represents the mean±SEM (n = 4). *P<0.05, **P<0.01 relative to Con or Scr. (Con = control; Scr = scrambled control; and siNPT2b = siNPT2b-delivered group).

Figure 4. Effects of the aerosol-delivered siNPT2b on tumor cell proliferation and angiogenesis.

(A) Immunohistochemistry analysis of PCNA in the lungs (magnification×200; bar = 50 µm). (B) Graph representing the number of PCNA-positive cells. Each bar represents the mean±SEM (n = 4). (C) Immunohistochemistry analysis of VEGF (magnification×200; bar = 50 µm). (D) Graph representing the number of VEGF-positive cells. Each bar represents the mean±SEM (n = 4). (E) Immunohistochemistry analysis of NPT2b (magnification×400; bar = 50 µm). *P<0.05 was considered more significant than the corresponding control values. (F) Graph representing the number of NPT-2b-positive cells. Each bar represents the mean±SEM (n = 4). *P<0.05, **P<0.01 and ***P<0.001 relative to Con or Scr. (Con = control; Scr = scrambled control; and siNPT2b = siNPT2b-delivered group).