Cancer Res Treat.  2016 Jan;48(1):345-354. 10.4143/crt.2014.247.

Forkhead Transcription Factor FOXO1 Inhibits Angiogenesis in Gastric Cancer in Relation to SIRT1

Affiliations
  • 1Department of Anatomy, Seoul National University College of Medicine, Seoul, Korea. dslanat@snu.ac.kr
  • 2Department of Tumour Biology, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.
  • 3Department of Pharmacology, Seoul National University College of Medicine, Seoul, Korea.
  • 4Ischemic/Hypoxic Disease Institute Medical Research Center, Seoul National University College of Medicine, Seoul, Korea.
  • 5Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.
  • 6Department of Pathology, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea.
  • 7Department of Anatomy, Dankook University School of Medicine, Cheonan, Korea.
  • 8Department of Biomedical Sciences, Inha University College of Medicine, Incheon, Korea.

Abstract

PURPOSE
We previously reported that forkhead transcription factors of the O class 1 (FOXO1) expression in gastric cancer (GC) was associated with angiogenesis-related molecules. However, there is little experimental evidence for the direct role of FOXO1 in GC. In the present study, we investigated the effect of FOXO1 on the tumorigenesis and angiogenesis in GC and its relationship with SIRT1.
MATERIALS AND METHODS
Stable GC cell lines (SNU-638 and SNU-601) infected with a lentivirus containing FOXO1 shRNA were established for animal studies as well as cell culture experiments. We used xenograft tumors in nude mice to evaluate the effect of FOXO1 silencing on tumor growth and angiogenesis. In addition, we examined the association between FOXO1 and SIRT1 by immunohistochemical tissue array analysis of 471 human GC specimens and Western blot analysis of xenografted tumor tissues.
RESULTS
In cell culture, FOXO1 silencing enhanced hypoxia inducible factor-1alpha (HIF-1alpha) expression and GC cell growth under hypoxic conditions, but not under normoxic conditions. The xenograft study showed that FOXO1 downregulation enhanced tumor growth, microvessel areas, HIF-1alpha activation and vascular endothelial growth factor (VEGF) expression. In addition, inactivated FOXO1 expression was associated with SIRT1 expression in human GC tissues and xenograft tumor tissues.
CONCLUSION
Our results indicate that FOXO1 inhibits GC growth and angiogenesis under hypoxic conditions via inactivation of the HIF-1alpha-VEGF pathway, possibly in association with SIRT1. Thus, development of treatment modalities aiming at this pathway might be useful for treating GC.

Keyword

Stomach neoplasms; Angiogenesis modulating agents; Human FOXO1 protein; Human SIRT1 protein

MeSH Terms

Angiogenesis Modulating Agents
Animals
Anoxia
Blotting, Western
Carcinogenesis
Cell Culture Techniques
Cell Line
Down-Regulation
Forkhead Transcription Factors
Heterografts
Humans
Lentivirus
Mice
Mice, Nude
Microvessels
RNA, Small Interfering
Stomach Neoplasms*
Tissue Array Analysis
Transcription Factors*
Vascular Endothelial Growth Factor A
Angiogenesis Modulating Agents
Forkhead Transcription Factors
RNA, Small Interfering
Transcription Factors
Vascular Endothelial Growth Factor A

Figure

  • Fig. 1. Effect of forkhead transcription factors of the O class 1 (FOXO1) expression on hypoxia inducible factor-1α(HIF-1α) expression in cultured gastric cancer (GC) cells. (A) Western blot analysis shows that the protein contents of FOXO1 varied in GC cell lines. (B) FOXO1 expression in GC cells (SNU-638 and SNU-601) was silenced by infection with lentiviral particles containing non-targeting shRNA (shCtrl) or FOXO1 shRNA (shFOXO1). The protein expression of FOXO1 was determined by Western blot analysis. (C) FOXO1 transcriptional activity was determined by luciferase reporter assay. *p < 0.05, compared to shCtrl cells. (D) Protein expressions of FOXO1 and HIF-1α were measured by Western blot analysis after exposure to normoxia (N) or hypoxia (H) for 8 hours.

  • Fig. 2. Cell viability of gastric cancer (GC) cell lines (SNU-638 and SNU-601) was evaluated by crystal violet assay. (A) Two GC cell lines were cultured for 72 hours and cell growth rates were compared between the cell lines. *p < 0.05, compared to SNU-601 cells. (B, C) GC cells expressing forkhead transcription factors of the O class 1 (FOX1) shRNA (shFOXO1) show higher cell viability at 24 hours of hypoxia exposure than those expressing control shRNA (shCtrl). *p < 0.05, compared to shCtrl cells.

  • Fig. 3. Forkhead transcription factors of the O class 1 (FOXO1) shRNA (shFOXO1) promotes tumor growth and angiogenesis in subcutaneous gastric cancer (GC) xenografts. (A) SNU-638 cells expressing control shRNA (shCtrl) or shFOXO1 were subcutaneously injected into the left flanks of nude mice. Representative photos of mice were taken after killing at day 48 (left). Tumors were harvested, then weighed (right) (n=5 per group). *p < 0.05, compared to shCtrl tumors. The arrows indicate xenografted tumors in mouse flanks. (B) Tissue sections were obtained from the xenograft tumors and immunostained for FOXO1, CD31, hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) (left). Microvessel area were quantified by measuring areas of blood vessels immunostained for CD31 (right). *p < 0.05, compared to shCtrl cells. (C) Protein expressions of FOXO1, HIF-1α–VEGF, and SIRT1 in xenograft tumors were measured by Western blot analysis.

  • Fig. 4. Representative immunohistochemical findings in human gastric cancer (GC) tissue specimens. (A) GC cells showing cytoplasmic phospho-FOXO1Ser256 (pFOXO1) expression with or without nuclear staining. (B) GC cells showing nuclear SIRT1 expression. (C) GC cells showing nuclear hypoxia inducible factor-1α (HIF-1α) expression with or without cytoplasmic staining. (D) GC cells without cytoplasmic pFOXO1 expression. (E) GC cells without nuclear SIRT1 expression. (F) GC cells without nuclear HIF-1α expression (A-F, ×400).


Reference

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