Periostin is a negative prognostic factor and promotes cancer cell proliferation in non-small cell lung cancer

Figure 1. Periostin expression in human lung cancer (A-D) Representative results of IHC for periostin in clinical specimens obtained from 189 lung cancer patients during surgical resection. Specimens were scored for the amount of anti-periostin staining, with scores of 0 (A) or 1 (B) considered low and scores of 2 (C) or 3 (D) considered high. Scale bar: 100 μm. (E-I) Survival curves for NSCLC according to IHC periostin-staining grades, for (E) total population and by population in (F) T1 stage (n=97), (G) T2, 3, or 4 (n=92), (H) N0 (n=152), and (I) N1 and N2 (n=37). The 5-year survival rate by low or high periostin level in each population was (E) 93.3% for low (n=106) and 67.5% for high (n=83); (F) 91.3% for low (n=58) and 81.3% for high (n=39); (G) 91.7% for low (n=48) and 55.3% for high (n=44); (H) 93.3% for low (n=92) and 81.3% for high (n=60); and (I) 92.8% for low (n=14) and 29.5% for high (n=23). N.S., not significant.

Figure 2. Periostin promotes lung-cancer proliferation in vivo Ex3LL cells were inoculated into the left thigh and observed for 4 weeks, followed by IHC for periostin of primary tumors and lung metastases. (A) Loupe images show a primary tumor generated in the left thigh and (D) metastasis in the lung in a periostin +/+ mouse. Magnified views are shown in (B), (C), (E), and (F). (G) Tumors resected 4 weeks after inoculation from a periostin+/+ mouse and (H) a periostin−/− mouse, and (I) average tumor volumes. (J and L) CT images of the lung from periostin+/+ and (L) periostin−/− mice 4 weeks after cell implantation. Arrows indicate metastatic tumor nodules. (K) Macroscopic images of lungs resected 4 weeks after inoculation from periostin+/+ and (M) periostin−/− mice; arrows indicate metastatic tumor nodules. (N) Microscopic images of the lung from periostin+/+ and (P) periostin−/− mice, showing metastatic lesions stained with H&E. (O) IHC for Ki67 in metastatic lesions in the lungs, showing the (R) average number of metastatic nodules and (S) area of metastasis, determined from CT images. (T) The Ki-67 index (counted in at least three fields) in metastatic lesions in the periostin+/+ and…

Figure 3. Ex3LL supernatant promotes periostin expression in NIH/3T3 cells (A and B) Periostin expression in NIH/3T3 cells, periostin+/+ fibroblasts, periostin−/− fibroblasts, and Ex3LL cells at steady state, determined by real-time PCR (A, n=3) and ELISA (B) N.D, not detected. (C) NIH/3T3 cells were cultured with the conditioned medium of NIH/3T3 (control) or Ex3LL (Ex3LL) cells, and the periostin expression was measured by real-time PCR (n=3).

Figure 4. Periostin promotes Ex3LL-cell proliferation and intracellular signaling in vitro (A) GFP fluorescence in Ex3LL-GFP cells co-cultured with periostin−/− or periostin+/+ fibroblasts (n=5). Values on the Y-axis are normalized to the value on day 0. (B) Effect of periostin stimulation on Ex3LL proliferation, assayed by MTT (n=5). Values on the Y-axis are normalized to the value on day 0. (C) Ex3LL cells were incubated for 24 h with medium containing 0.1% FBS and then treated with 1,000 ng/ml periostin for 5, 10, 30, or 60 min. The presence of ERK, AKT, FAK, and NF-κB and their phosphorylated forms [pERK (Thr202/Tyr204), pAKT (Thr308), pFAK (Tyr925), and p-NF-kB (Ser536)] in the Ex3LL cells was assessed by western blotting. Ratios of pERK to total ERK, based on band density analyzed with ImageJ software, are shown. (D) Ex3LL cell proliferation measured by MTT assay. Cells were incubated for 24 h with medium containing 0.1% FBS, and then treated with recombinant periostin with or without U0126. ****P < 0.001 (E) IHC for pERK and periostin in primary tumors in periostin+/+ and periostin−/− mice. Scale bar: 100 μm. (F) Ex3LL cells in 0.1% FBS with or without recombinant periostin were…