Pancreatic RECK inactivation promotes cancer formation, epithelial-mesenchymal transition, and metastasis
Tomonori Masuda, … , Makoto Noda, Hiroshi Seno
Tomonori Masuda, … , Makoto Noda, Hiroshi Seno
Published September 15, 2023
Citation Information: J Clin Invest. 2023;133(18):e161847. https://doi.org/10.1172/JCI161847.
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Research Article Gastroenterology Oncology

Pancreatic RECK inactivation promotes cancer formation, epithelial-mesenchymal transition, and metastasis

Abstract

RECK is downregulated in various human cancers; however, how RECK inactivation affects carcinogenesis remains unclear. We addressed this issue in a pancreatic ductal adenocarcinoma (PDAC) mouse model and found that pancreatic Reck deletion dramatically augmented the spontaneous development of PDAC with a mesenchymal phenotype, which was accompanied by increased liver metastases and decreased survival. Lineage tracing revealed that pancreatic Reck deletion induced epithelial-mesenchymal transition (EMT) in PDAC cells, giving rise to inflammatory cancer-associated fibroblast–like cells in mice. Splenic transplantation of Reck-null PDAC cells resulted in numerous liver metastases with a mesenchymal phenotype, whereas reexpression of RECK markedly reduced metastases and changed the PDAC tumor phenotype into an epithelial one. Consistently, low RECK expression correlated with low E-cadherin expression, poor differentiation, metastasis, and poor prognosis in human PDAC. RECK reexpression in the PDAC cells was found to downregulate MMP2 and MMP3, with a concomitant increase in E-cadherin and decrease in EMT-promoting transcription factors. An MMP inhibitor recapitulated the effects of RECK on the expression of E-cadherin and EMT-promoting transcription factors and invasive activity. These results establish the authenticity of RECK as a pancreatic tumor suppressor, provide insights into its underlying mechanisms, and support the idea that RECK could be an important therapeutic effector against human PDAC.

Authors

Tomonori Masuda, Akihisa Fukuda, Go Yamakawa, Mayuki Omatsu, Mio Namikawa, Makoto Sono, Yuichi Fukunaga, Munemasa Nagao, Osamu Araki, Takaaki Yoshikawa, Satoshi Ogawa, Kenji Masuo, Norihiro Goto, Yukiko Hiramatsu, Yu Muta, Motoyuki Tsuda, Takahisa Maruno, Yuki Nakanishi, Toshihiko Masui, Etsuro Hatano, Tomoko Matsuzaki, Makoto Noda, Hiroshi Seno

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Figure 5

Effects of RECK reexpression on Reck-null PDAC cell lines.

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Effects of RECK reexpression on Reck-null PDAC cell lines. (A) RECK reex...
(A) RECK reexpression in Reck-null PDAC cell lines. Reck-null PDAC cells from KRC mice were transfected with control or RECK expression vector. Exogenous RECK expression was confirmed by RT-qPCR. Data are shown as the mean ± SEM. P = 0.0069, 2-tailed Student’s t test. (B) Effects of RECK on PDAC cell migration. Wound healing assay using Reck-null PDAC cell lines transfected with control or RECK expression vector. Left: Images of the scratched zone and recovered wound 24 hours after scratch (scale bar: 500 μm). Right: Data are shown as the mean ± SEM of the migrated area relative to control. P = 0.0079, 2-tailed Student’s t test. (C) Effects of RECK expression on PDAC cell invasion. Matrigel invasion assay using Reck-null PDAC cells (n = 3) transfected with control or RECK expression vector. Left: Images of migrated cells (scale bar: 50 μm). Right: Data are shown as the mean ± SEM of the invasion ratio. P = 0.0063, 2-tailed Student’s t test. (D–G) Effects of RECK on PDAC cell metastasis. (D) Morphology of resected liver (scale bar: 5 mm) and CK19 immunostaining (scale bar: 50 μm). (E) Quantitative assessments of liver metastasis. Ratio of liver weight to body weight and CK19-positive area. Data are shown as the mean ± SEM. P = 0.040 and P = 0.0016, respectively; 2-tailed Student’s t test. (F) Histology of liver lesions. H&E staining, or immunostaining for CK19 or RECK with nuclear counterstaining (blue) (scale bar: 50 μm). (G) Immunostaining for E-cadherin, N-cadherin, or Zeb1 (scale bar: 50 μm). (H) Effects of RECK on epithelial and mesenchymal marker gene expression in PDAC cells. RT-qPCR analysis of mRNA levels. Data are shown as the mean ± SEM. P = 0.022 (Zeb1), P = 0.008 (Snai2), P = 0.015 (Twist1); 2-tailed Student’s t test, *P < 0.05. (I) Effects of RECK on E-cadherin protein by immunoblot assay. Data are shown as the mean ± SEM of the densitometry measurements of the immunoblot band. P = 0.042, 2-tailed Student’s t test.