Effect of conditioned medium, IGF1, and IGF1R inhibitors on motility of PDAC cells.

<p>(A) Representative images of cell density in wound-healing assay. The number of PDAC cells migrating across the wound (broken line) was increased by conditioned medium (CM) from pancreas CAFs compared with that in control. IGF1 increased the number of migrating cancer cells. The IGF1R inhibitor picropodophyllin (PPP) and anti-IGF1R neutralizing antibody downregulated migration-stimulating activity by fibroblast CM. (B, C, D) CM from pancreas fibroblasts significantly stimulated the migratory activity of RWP-1 and MiaPaCa-2 cells, but not OCUP-AT, and Panc-1, under normoxia. Under hypoxia, the number of migrating cancer cells was significantly increased in all four cancer cell lines by CM from pancreas CAFs, and the migration-stimulating ability of CM in PDAC cells was inhibited by IGF1R inhibitors. Data are presented as mean ± SD.</p

IGF1R inhibitor inhibits invasion-stimulating activity of CM from fibroblasts.

<p>(A), Representative pictures of invading pancreas cancer cells, Panc-1. The number of invaded cells into pore membrane filter was increased in the presence of CM from pCaF-1 in compared to the control. IGF1R-neutralizing antibody and PPP inhibited the invasion induced by CM. (B), CM from pCaF-1 significantly stimulated the invasive behavior of pancreas cancer cells. IGF1R inhibitor, IGF1R-neutralizing antibody and PPP, significantly inhibited the invasion seen in these cells.</p

Effects of hypoxia on mRNA expression of <i>IGF1R</i> and <i>IGF1</i>, and production of IGF1 in cancer cells.

<p>(A) <i>IGF1R</i> mRNA expression was significantly higher in RWP-1, MiaPaCa-2, and OCUP-AT cells than in Panc-1 cells. The mRNA expression level of IGF1R was increased under hypoxia in all four cancer cell lines. (B) Cell surface expression of IGF1R by FACScan analysis. IGF1R expression level of Panc-1, RWP-1, OCUP-AT, and MiaPaCa-2 cells was higher in hypoxia than that in normoxia. (C) Western blot analysis. Three independent experiments were performed. IGF1R bands was calculated by β-actin as internal standard to normalize expression levels IGF1R level under hypoxia was high in 3 of four pancreas cancer cell lines, Panc-1, OCUP-AT, and RWP-1, in comparison with that under normoxia, while Panc-1 did not significantly increase under hypoxia. (D) IGF1 mRNA expression was significantly increased under hypoxia in both pCaF-1 and pCaF-2 cells. (E) The concentration of IGF1 in conditioned medium from pCaF-1 and pCaF-2 cells was 380 and 253 pg/ml under hypoxia. IGF1 production in PDAC cells was not detected.</p

Pancreatic Fibroblasts Stimulate the Motility of Pancreatic Cancer Cells through IGF1/IGF1R Signaling under Hypoxia

<div><p>Pancreatic ductal adenocarcinoma (PDAC) is characterized by its hypovascularity, with an extremely poor prognosis because of its highly invasive nature. PDAC proliferates with abundant stromal cells, suggesting that its invasive activity might be controlled by intercellular interactions between cancer cells and fibroblasts. Using four PDAC cell lines and two pancreas cancer-associated fibroblasts (CAFs), the expression of insulin-like growth factor-1 (IGF1) and IGF1 receptor (IGF1R) was evaluated by RT-PCR, FACScan, western blot, or ELISA. Correlation between IGF1R and the hypoxia marker carbonic anhydrase 9 (CA9) was examined by immunohistochemical staining of 120 pancreatic specimens. The effects of CAFs, IGF1, and IGF1R inhibitors on the motility of cancer cells were examined by wound-healing assay or invasion assay under normoxia (20% O₂) and hypoxia (1% O₂). IGF1R expression was significantly higher in RWP-1, MiaPaCa-2, and OCUP-AT cells than in Panc-1 cells. Hypoxia increased the expression level of IGF1R in RWP-1, MiaPaCa-2, and OCUP-AT cells. CA9 expression was correlated with IGF1R expression in pancreatic specimens. CAFs produced IGF1 under hypoxia, but PDAC cells did not. A conditioned medium from CAFs, which expressed αSMA, stimulated the migration and invasion ability of MiaPaCa-2, RWP-1, and OCUP-AT cells. The motility of all PDAC cells was greater under hypoxia than under normoxia. The motility-stimulating ability of CAFs was decreased by IGF1R inhibitors. These findings might suggest that pancreas CAFs stimulate the invasion activity of PDAC cells through paracrine IGF1/IGF1R signaling, especially under hypoxia. Therefore the targeting of IGF1R signaling might represent a promising therapeutic approach in IGF1R-dependent PDAC.</p></div

Hypoxia Stimulates the EMT of Gastric Cancer Cells through Autocrine TGFβ Signaling

<div><p>Epithelial mesenchymal transition (EMT) is considered to be correlated with malignancy of cancer cells and responsible for cancer invasion and metastasis. We previously reported that distant metastasis was associated with hypoxia in gastric cancer. We therefore investigated the effect of hypoxic condition on EMT of gastric cancer cells. Gastric cancer cells were cultured in normoxia (21% O₂) or hypoxia (1% O₂) for 24 h. EMT was evaluated as the percentage of spindle-shaped cells in total cells. Effect of transforming growth factor β1 (TGFβ1) or tyrosine kinase inhibitors on the EMT was evaluated. The expression level of <i>TGFβ1</i> and <i>TGFβR</i> was evaluated by real time RT-PCR. The TGFβ1 production from cancer cells was measured by ELISA. Hypoxia stimulated EMT of OCUM-2MD3 and OCUM-12 cells, but not that of OCUM-2M cells. The expression level of <i>TGFβ1</i> mRNA under hypoxia was significantly higher than that under normoxia in all of three cell lines. The expression level of <i>TGFβR</i> mRNA was significantly increased by hypoxia in OCUM-2MD3 cells, but not in OCUM-2M cells. TGFβR inhibitor, SB431542 or Ki26894, significantly suppressed EMT of OCUM-2MD3 and OCUM-12. TGFβ1 production from OCUM-2MD3 and OCUM-12 cells was significantly increased under hypoxia in comparison with that under normoxia. These findings might suggest that hypoxia stimulates the EMT of gastric cancer cells via autocrine TGFβ/TGFβR signaling.</p></div

Smad2 phosphorylation of gastric cancer cells under a hypoxic condition.

<p>Hypoxic condition for 24 h increased Smad2 phosphorylation of OCUM-2MD3 and OCUM-12 cells (arrowheads), but not that of OCUM-2M cells. Smad2/3 was used as an international control for p-Smad2.</p

Effects of neutralizing antibodies on cancer cell morphology under hypoxia.

<p>Effects of neutralizing antibodies were determined after 24 h of culture.</p><p>+; Partly effective, −; Not effective.</p><p>The culture medium was consisted of DMEM with 10% FBS.</p

Effects of hypoxic condition on the transcription factors associated with mesenchymal transition.

<p>(A) A hypoxia-target molecule, <i>VEGF-A</i> mRNA level in OCUM-2M, OCUM-2MD3, and OCUM-12 was increased under hypoxia. (B, C) Hypoxic condition significantly increased the expression level of <i>Twist</i> and <i>Zeb1</i> in OCUM-2MD3 and OCUM-12 cells, but not that in OCUM-2M cells. (D) The expression level of Snail1 was not affected by hypoxic condition.</p

Effects of inhibitors on EMT under hypoxic condition.

<p>(A, B) Hypoxia-induced EMT was significantly (p<0.01) inhibited by TGFβR inhibitors (Ki26894 and SB431542). Other inhibitors (Lapatinib, Sunitinib, and PHA665752) had no effect on EMT under hypoxia. (C) <i>Vimentin</i> mRNA expression was significantly increased under a hypoxic condition in OCUM-2M and OCUM-12 cells, and decreased with TGFβR inhibitors (Ki26894 and SB431542). (D)Vimentin protein was increased under hypoxia in OCUM-12 and OCUM-2MD3, and decreased with TGFβR inhibitors (Ki26894 and SB431542).</p

Effects of various growth factors on cancer cell morphology.

<p>+; Positive morphologic changes, −; No changes.</p><p>The culture medium was consisted of DMEM with 10% FBS.</p