Autophagy Is Required for Activation of Pancreatic Stellate Cells, Associated With Pancreatic Cancer Progression and Promotes Growth of Pancreatic Tumors in Mice

Sho Endo, Kohei Nakata, Kenoki Ohuchida, Shin Takesue, Hiromichi Nakayama, Toshiya Abe, Kazuhiro Koikawa, Takashi Okumura, Masafumi Sada, Kohei Horioka, Biao Zheng, Yusuke Mizuuchi, Chika Iwamoto, Masaharu Murata, Taiki Moriyama, Yoshihiro Miyasaka, Takao Ohtsuka, Kazuhiro Mizumoto, Yoshinao Oda, Makoto HashizumeMasafumi Nakamura

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

Background & Aims Pancreatic stellate cells (PSCs) change from a quiescent to activated state in the tumor environment and secrete extracellular matrix (ECM) molecules and cytokines to increase the aggressiveness of tumors. However, it is not clear how PSCs are activated to produce these factors, or whether this process can be inhibited. PSCs have morphologic and functional similarities to hepatic stellate cells, which undergo autophagy to promote fibrosis and tumor growth. We investigated whether autophagy activates PSCs, which promotes development of the tumor stroma and growth of pancreatic tumors in mice. Methods We used immunofluorescence microscopy and immunohistochemistry to analyze pancreatic tumor specimens from 133 patients who underwent pancreatectomy in Japan from 2000 to 2009. PSCs were cultured from pancreatic tumor tissues or tissues of patients with chronic pancreatitis; these were analyzed by immunofluorescence microscopy, immunoblots, quantitative reverse transcription polymerase chain reaction, and in assays for invasiveness, proliferation, and lipid droplets. Autophagy was inhibited in PSCs by administration of chloroquine or transfection with small interfering RNAs. Proteins were knocked down in immortalized PSCs by expression of small hairpin RNAs. Cells were transplanted into pancreatic tails of nude mice, and tumor growth and metastasis were quantified. Results Based on immunohistochemical analyses, autophagy was significantly associated with tumor T category (P =.018), histologic grade (P =.001), lymph node metastases (P <.001), stage (P =.009), perilymphatic invasion (P =.001), and perivascular invasion (P =.003). Autophagy of PSCs was associated with shorter survival times of patients with pancreatic cancer. PSC expression of microtubule-associated protein 1 light chain 3, a marker of autophagosomes, was associated with poor outcomes (shorter survival time, disease recurrence) for patients with pancreatic cancer (relative risk of shorter survival time, 1.56). Immunoblots showed that PSCs from pancreatic tumor samples expressed higher levels of markers of autophagy than PSCs from chronic pancreatitis samples. Inhibitors of autophagy increased the number of lipid droplets of PSCs, indicating a quiescent state of PSCs, and reduced their production of ECM molecules and interleukin 6, as well as their proliferation and invasiveness in culture. PSCs exposed to autophagy inhibitors formed smaller tumors in nude mice (P =.001) and fewer liver metastases (P =.018) with less peritoneal dissemination (P =.018) compared to PSCs not exposed to autophagy inhibitors. Conclusions Autophagic PSCs produce ECM molecules and interleukin 6 and are associated with shorter survival times and disease recurrence in patients with pancreatic cancer. Inhibitors of PSC autophagy might reduce pancreatic tumor invasiveness by altering the tumor stroma.

Original languageEnglish
Pages (from-to)1492-1506.e24
JournalGastroenterology
Volume152
Issue number6
DOIs
Publication statusPublished - May 2017

Fingerprint

Pancreatic Stellate Cells
Autophagy
Pancreatic Neoplasms
Growth
Neoplasms
Extracellular Matrix
Survival
Chronic Pancreatitis
Neoplasm Metastasis
Fluorescence Microscopy
Nude Mice
Small Interfering RNA
Interleukin-6

All Science Journal Classification (ASJC) codes

  • Hepatology
  • Gastroenterology

Cite this

Autophagy Is Required for Activation of Pancreatic Stellate Cells, Associated With Pancreatic Cancer Progression and Promotes Growth of Pancreatic Tumors in Mice. / Endo, Sho; Nakata, Kohei; Ohuchida, Kenoki; Takesue, Shin; Nakayama, Hiromichi; Abe, Toshiya; Koikawa, Kazuhiro; Okumura, Takashi; Sada, Masafumi; Horioka, Kohei; Zheng, Biao; Mizuuchi, Yusuke; Iwamoto, Chika; Murata, Masaharu; Moriyama, Taiki; Miyasaka, Yoshihiro; Ohtsuka, Takao; Mizumoto, Kazuhiro; Oda, Yoshinao; Hashizume, Makoto; Nakamura, Masafumi.

In: Gastroenterology, Vol. 152, No. 6, 05.2017, p. 1492-1506.e24.

Research output: Contribution to journalArticle

Endo, Sho ; Nakata, Kohei ; Ohuchida, Kenoki ; Takesue, Shin ; Nakayama, Hiromichi ; Abe, Toshiya ; Koikawa, Kazuhiro ; Okumura, Takashi ; Sada, Masafumi ; Horioka, Kohei ; Zheng, Biao ; Mizuuchi, Yusuke ; Iwamoto, Chika ; Murata, Masaharu ; Moriyama, Taiki ; Miyasaka, Yoshihiro ; Ohtsuka, Takao ; Mizumoto, Kazuhiro ; Oda, Yoshinao ; Hashizume, Makoto ; Nakamura, Masafumi. / Autophagy Is Required for Activation of Pancreatic Stellate Cells, Associated With Pancreatic Cancer Progression and Promotes Growth of Pancreatic Tumors in Mice. In: Gastroenterology. 2017 ; Vol. 152, No. 6. pp. 1492-1506.e24.
@article{caf5612bba1a496e84471adad9cac3ae,
title = "Autophagy Is Required for Activation of Pancreatic Stellate Cells, Associated With Pancreatic Cancer Progression and Promotes Growth of Pancreatic Tumors in Mice",
abstract = "Background & Aims Pancreatic stellate cells (PSCs) change from a quiescent to activated state in the tumor environment and secrete extracellular matrix (ECM) molecules and cytokines to increase the aggressiveness of tumors. However, it is not clear how PSCs are activated to produce these factors, or whether this process can be inhibited. PSCs have morphologic and functional similarities to hepatic stellate cells, which undergo autophagy to promote fibrosis and tumor growth. We investigated whether autophagy activates PSCs, which promotes development of the tumor stroma and growth of pancreatic tumors in mice. Methods We used immunofluorescence microscopy and immunohistochemistry to analyze pancreatic tumor specimens from 133 patients who underwent pancreatectomy in Japan from 2000 to 2009. PSCs were cultured from pancreatic tumor tissues or tissues of patients with chronic pancreatitis; these were analyzed by immunofluorescence microscopy, immunoblots, quantitative reverse transcription polymerase chain reaction, and in assays for invasiveness, proliferation, and lipid droplets. Autophagy was inhibited in PSCs by administration of chloroquine or transfection with small interfering RNAs. Proteins were knocked down in immortalized PSCs by expression of small hairpin RNAs. Cells were transplanted into pancreatic tails of nude mice, and tumor growth and metastasis were quantified. Results Based on immunohistochemical analyses, autophagy was significantly associated with tumor T category (P =.018), histologic grade (P =.001), lymph node metastases (P <.001), stage (P =.009), perilymphatic invasion (P =.001), and perivascular invasion (P =.003). Autophagy of PSCs was associated with shorter survival times of patients with pancreatic cancer. PSC expression of microtubule-associated protein 1 light chain 3, a marker of autophagosomes, was associated with poor outcomes (shorter survival time, disease recurrence) for patients with pancreatic cancer (relative risk of shorter survival time, 1.56). Immunoblots showed that PSCs from pancreatic tumor samples expressed higher levels of markers of autophagy than PSCs from chronic pancreatitis samples. Inhibitors of autophagy increased the number of lipid droplets of PSCs, indicating a quiescent state of PSCs, and reduced their production of ECM molecules and interleukin 6, as well as their proliferation and invasiveness in culture. PSCs exposed to autophagy inhibitors formed smaller tumors in nude mice (P =.001) and fewer liver metastases (P =.018) with less peritoneal dissemination (P =.018) compared to PSCs not exposed to autophagy inhibitors. Conclusions Autophagic PSCs produce ECM molecules and interleukin 6 and are associated with shorter survival times and disease recurrence in patients with pancreatic cancer. Inhibitors of PSC autophagy might reduce pancreatic tumor invasiveness by altering the tumor stroma.",
author = "Sho Endo and Kohei Nakata and Kenoki Ohuchida and Shin Takesue and Hiromichi Nakayama and Toshiya Abe and Kazuhiro Koikawa and Takashi Okumura and Masafumi Sada and Kohei Horioka and Biao Zheng and Yusuke Mizuuchi and Chika Iwamoto and Masaharu Murata and Taiki Moriyama and Yoshihiro Miyasaka and Takao Ohtsuka and Kazuhiro Mizumoto and Yoshinao Oda and Makoto Hashizume and Masafumi Nakamura",
year = "2017",
month = "5",
doi = "10.1053/j.gastro.2017.01.010",
language = "English",
volume = "152",
pages = "1492--1506.e24",
journal = "Gastroenterology",
issn = "0016-5085",
publisher = "W.B. Saunders Ltd",
number = "6",

}

TY - JOUR

T1 - Autophagy Is Required for Activation of Pancreatic Stellate Cells, Associated With Pancreatic Cancer Progression and Promotes Growth of Pancreatic Tumors in Mice

AU - Endo, Sho

AU - Nakata, Kohei

AU - Ohuchida, Kenoki

AU - Takesue, Shin

AU - Nakayama, Hiromichi

AU - Abe, Toshiya

AU - Koikawa, Kazuhiro

AU - Okumura, Takashi

AU - Sada, Masafumi

AU - Horioka, Kohei

AU - Zheng, Biao

AU - Mizuuchi, Yusuke

AU - Iwamoto, Chika

AU - Murata, Masaharu

AU - Moriyama, Taiki

AU - Miyasaka, Yoshihiro

AU - Ohtsuka, Takao

AU - Mizumoto, Kazuhiro

AU - Oda, Yoshinao

AU - Hashizume, Makoto

AU - Nakamura, Masafumi

PY - 2017/5

Y1 - 2017/5

N2 - Background & Aims Pancreatic stellate cells (PSCs) change from a quiescent to activated state in the tumor environment and secrete extracellular matrix (ECM) molecules and cytokines to increase the aggressiveness of tumors. However, it is not clear how PSCs are activated to produce these factors, or whether this process can be inhibited. PSCs have morphologic and functional similarities to hepatic stellate cells, which undergo autophagy to promote fibrosis and tumor growth. We investigated whether autophagy activates PSCs, which promotes development of the tumor stroma and growth of pancreatic tumors in mice. Methods We used immunofluorescence microscopy and immunohistochemistry to analyze pancreatic tumor specimens from 133 patients who underwent pancreatectomy in Japan from 2000 to 2009. PSCs were cultured from pancreatic tumor tissues or tissues of patients with chronic pancreatitis; these were analyzed by immunofluorescence microscopy, immunoblots, quantitative reverse transcription polymerase chain reaction, and in assays for invasiveness, proliferation, and lipid droplets. Autophagy was inhibited in PSCs by administration of chloroquine or transfection with small interfering RNAs. Proteins were knocked down in immortalized PSCs by expression of small hairpin RNAs. Cells were transplanted into pancreatic tails of nude mice, and tumor growth and metastasis were quantified. Results Based on immunohistochemical analyses, autophagy was significantly associated with tumor T category (P =.018), histologic grade (P =.001), lymph node metastases (P <.001), stage (P =.009), perilymphatic invasion (P =.001), and perivascular invasion (P =.003). Autophagy of PSCs was associated with shorter survival times of patients with pancreatic cancer. PSC expression of microtubule-associated protein 1 light chain 3, a marker of autophagosomes, was associated with poor outcomes (shorter survival time, disease recurrence) for patients with pancreatic cancer (relative risk of shorter survival time, 1.56). Immunoblots showed that PSCs from pancreatic tumor samples expressed higher levels of markers of autophagy than PSCs from chronic pancreatitis samples. Inhibitors of autophagy increased the number of lipid droplets of PSCs, indicating a quiescent state of PSCs, and reduced their production of ECM molecules and interleukin 6, as well as their proliferation and invasiveness in culture. PSCs exposed to autophagy inhibitors formed smaller tumors in nude mice (P =.001) and fewer liver metastases (P =.018) with less peritoneal dissemination (P =.018) compared to PSCs not exposed to autophagy inhibitors. Conclusions Autophagic PSCs produce ECM molecules and interleukin 6 and are associated with shorter survival times and disease recurrence in patients with pancreatic cancer. Inhibitors of PSC autophagy might reduce pancreatic tumor invasiveness by altering the tumor stroma.

AB - Background & Aims Pancreatic stellate cells (PSCs) change from a quiescent to activated state in the tumor environment and secrete extracellular matrix (ECM) molecules and cytokines to increase the aggressiveness of tumors. However, it is not clear how PSCs are activated to produce these factors, or whether this process can be inhibited. PSCs have morphologic and functional similarities to hepatic stellate cells, which undergo autophagy to promote fibrosis and tumor growth. We investigated whether autophagy activates PSCs, which promotes development of the tumor stroma and growth of pancreatic tumors in mice. Methods We used immunofluorescence microscopy and immunohistochemistry to analyze pancreatic tumor specimens from 133 patients who underwent pancreatectomy in Japan from 2000 to 2009. PSCs were cultured from pancreatic tumor tissues or tissues of patients with chronic pancreatitis; these were analyzed by immunofluorescence microscopy, immunoblots, quantitative reverse transcription polymerase chain reaction, and in assays for invasiveness, proliferation, and lipid droplets. Autophagy was inhibited in PSCs by administration of chloroquine or transfection with small interfering RNAs. Proteins were knocked down in immortalized PSCs by expression of small hairpin RNAs. Cells were transplanted into pancreatic tails of nude mice, and tumor growth and metastasis were quantified. Results Based on immunohistochemical analyses, autophagy was significantly associated with tumor T category (P =.018), histologic grade (P =.001), lymph node metastases (P <.001), stage (P =.009), perilymphatic invasion (P =.001), and perivascular invasion (P =.003). Autophagy of PSCs was associated with shorter survival times of patients with pancreatic cancer. PSC expression of microtubule-associated protein 1 light chain 3, a marker of autophagosomes, was associated with poor outcomes (shorter survival time, disease recurrence) for patients with pancreatic cancer (relative risk of shorter survival time, 1.56). Immunoblots showed that PSCs from pancreatic tumor samples expressed higher levels of markers of autophagy than PSCs from chronic pancreatitis samples. Inhibitors of autophagy increased the number of lipid droplets of PSCs, indicating a quiescent state of PSCs, and reduced their production of ECM molecules and interleukin 6, as well as their proliferation and invasiveness in culture. PSCs exposed to autophagy inhibitors formed smaller tumors in nude mice (P =.001) and fewer liver metastases (P =.018) with less peritoneal dissemination (P =.018) compared to PSCs not exposed to autophagy inhibitors. Conclusions Autophagic PSCs produce ECM molecules and interleukin 6 and are associated with shorter survival times and disease recurrence in patients with pancreatic cancer. Inhibitors of PSC autophagy might reduce pancreatic tumor invasiveness by altering the tumor stroma.

UR - http://www.scopus.com/inward/record.url?scp=85018835771&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85018835771&partnerID=8YFLogxK

U2 - 10.1053/j.gastro.2017.01.010

DO - 10.1053/j.gastro.2017.01.010

M3 - Article

C2 - 28126348

AN - SCOPUS:85018835771

VL - 152

SP - 1492-1506.e24

JO - Gastroenterology

JF - Gastroenterology

SN - 0016-5085

IS - 6

ER -