title: Autophagy Is Required for Activation of Pancreatic Stellate Cells, Associated With Pancreatic Cancer Progression and Promotes Growth of Pancreatic Tumors in Mice.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Sho
authors.initials: S
authors.lastname: Endo
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. Electronic address: knakata@surg1.med.kyushu-u.ac.jp.
authors.forename: Kohei
authors.initials: K
authors.lastname: Nakata
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; Department of Advanced Medical Initiatives, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. Electronic address: kenoki@surg1.med.kyushu-u.ac.jp.
authors.forename: Kenoki
authors.initials: K
authors.lastname: Ohuchida
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Shin
authors.initials: S
authors.lastname: Takesue
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Nakayama
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Toshiya
authors.initials: T
authors.lastname: Abe
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Koikawa
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Okumura
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Sada
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Horioka
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Biao
authors.initials: B
authors.lastname: Zheng
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; Department of Anatomical Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Mizuuchi
authors.affiliation: Department of Advanced Medical Initiatives, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Chika
authors.initials: C
authors.lastname: Iwamoto
authors.affiliation: Department of Advanced Medical Initiatives, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Murata
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Taiki
authors.initials: T
authors.lastname: Moriyama
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Miyasaka
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Ohtsuka
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Mizumoto
authors.affiliation: Department of Anatomical Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Yoshinao
authors.initials: Y
authors.lastname: Oda
authors.affiliation: Department of Advanced Medical Initiatives, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Hashizume
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. Electronic address: mnaka@surg1.med.kyushu-u.ac.jp.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Nakamura
mesh_terms: D000818:Animals; D001343:Autophagy / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D002738:Chloroquine / Q000494:pharmacology; D018450:Disease Progression; D005109:Extracellular Matrix / Q000378:metabolism*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D015850:Interleukin-6 / Q000378:metabolism*; D066292:Lipid Droplets; D008207:Lymphatic Metastasis; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D060787:Neoplasm Grading; D009361:Neoplasm Invasiveness; D009367:Neoplasm Staging; D009368:Neoplasm Transplantation; D010190:Pancreatic Neoplasms / Q000737:chemistry / Q000000981:diagnostic imaging / Q000473:pathology* / Q000503:physiopathology*; D058954:Pancreatic Stellate Cells / Q000378:metabolism / Q000502:physiology*; D050500:Pancreatitis, Chronic / Q000000981:diagnostic imaging / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D015996:Survival Rate; D014162:Transfection
chemical_list: D015850:Interleukin-6; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; D002738:Chloroquine
keywords: Chloroquine; Cytokine; Tumor Progression; Tumor-Stromal Interaction