C9orf10/Ossa regulates the bone metastasis of established lung adenocarcinoma cell subline H322L-BO4 in a mouse model

Abstract

Lung cancer frequently metastasizes to the bones. An in vivo model is urgently required to identify potential therapeutic targets for the prevention and treatment of lung cancer with bone metastasis. We established a lung adenocarcinoma cell subline (H322L-BO4) that specifically showed metastasis to the leg bones and adrenal glands. This was achieved by repeated isolation of metastatic cells from the leg bones of mice. The cells were intracardially injected into nude mice. Survival was prolonged for mice that received H322L-BO4 cells versus original cells (H322L). H322L-BO4 cells did not exhibit obvious changes in general in vitro properties associated with the metastatic potential (e.g., cell growth, migration, and invasion) compared with H322L cells. However, the phosphorylation of chromosome 9 open reading frame 10/oxidative stress-associated Src activator (C9orf10/Ossa) was increased in H322L-BO4 cells. This result confirmed the increased anchorage independence through C9orf10/Ossa-mediated activation of Src family tyrosine kinase. Reduction of C9orf10/Ossa by shRNA reduced cells' metastasis to the leg bone and prolonged survival in mice. These findings indicate that H322L-BO4 cells can be used to evaluate the effect of candidate therapeutic targets against bone metastatic lung cancer cells. Moreover, C9orf10/Ossa may be a useful target for treatment of lung cancer with bone metastasis.

Keywords: C9orf10/Ossa/FAM120A; Src family kinase; anoikis resistance; bone metastasis; cancer metastasis; lung adenocarcinoma; mouse model; signal transduction.

FIGURE 1

Properties of H322L cells in preparation for establishing the H322L‐BO4 cell subline. (a) Morphology of H322 cells and H322 cells with luciferase (H322L). (b) Western blotting using an anti‐phosphotyrosine antibody in H322 and H322L cells. (c) Bioluminescent imaging of mice (abdomen side and back side) on 0, 7, and 14 days after intracardial injection of 1 × 10⁶ H322L cells. Back, backside. (d) Bioluminescent imaging detected metastatic H322L cells in the leg bones of mice. (e) Hematoxylin–eosin (H&E) staining of tissues obtained from the leg bones of mice (Magnification: 100× and 400×). Bo, bone tissue; T, tumor tissue.

FIGURE 2

Comparison of the properties of H322L and H322L‐BO4 cells. (a) Morphology of H322L cells and established H322L‐BO4 cells. BF, bright field; Phalloidin, phalloidin staining. (b) Cell proliferation assay using Tetra Color One for H322L and H322L‐BO4 cells. (c) Cell invasion and cell migration assay for H322L and H322L‐BO4 cells. (d) Left panel, bioluminescent imaging of mice at 28 days after intracardial injection of 1 × 10⁶ H322L and H322L‐BO4 cells in each mouse, Right panel, relative photon ratio at 7, 14, 21, and 28 days after intracardial injection of 1 × 10⁶ H322L and H322L‐BO4 cells in each mouse on the abdomen side. **p < .05. (e) Survival rate of each mouse (n = 8) after intracardial injection of 1 × 10⁶ H322L and H322L‐BO4 cells.

FIGURE 3

Characterization of established H322L‐BO4 cells. (a) Western blotting for protein and phosphorylation of SFK, p130cas, and C9orf10/Ossa in H322L and H322L‐BO4 cells. (b) Western blotting for activated SFK in H322L‐BO4 cells before and after treatment with H₂O₂. (c) Western blotting comparing SFK activation in H322L‐BO4 cells treated with shC9orf10/Ossa (Ossa#2, #4) and control shRNA (Ctr#1, #2). (d) Effects on anchorage independence using C9orf10/Ossa shRNA and soft agar assay in H322L‐BO4 cells. **p < .05. (e) Effects on cell proliferation using C9orf10/Ossa shRNA detected by Tetra Color One in H322L‐BO4 cells. C9orf10, chromosome 9 open reading frame 10; Ossa, oxidative stress‐associated Src activator; SFK, Src family tyrosine kinase.

FIGURE 4

Effect of shC9orf10/Ossa‐treated H322L‐BO4 cells on a metastatic mouse model. (a) Left panel, ratio of photon counts of the whole mouse body in bioluminescence imaging at 14, 21, and 28 days after intracardiac injection of 2 × 10⁶ H322L‐BO4 cells treated with control shRNA (Ctr #1) and C9orf10/Ossa shRNA (BO4 Ossa #2, #4). Right panel, bioluminescent imaging of the back side of mice at day 28 after intracardial injection of 1 × 10⁶ H322 H322L‐BO4 cells treated with control shRNA (Ctr #1) and C9orf10/Ossa shRNA (BO4 Ossa #2, #4). Back, backside. *p < .1; **p < .05. (b) Survival rate of each mouse (n = 10) after intracardial injection of 2 × 10⁶ H322L, control shRNA‐treated H322L‐BO4 cells (BO4 Ctr#1), and C9orf10/Ossa shRNA‐treated H322L‐BO4 cells (BO4 Ossa #2, #4). C9orf10, chromosome 9 open reading frame 10; Ossa, oxidative stress‐associated Src activator; SFK, Src family tyrosine kinase.