Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Implication of High Mobility Group Box 1 (HMGB1) in Multicellular Tumor Spheroid (MTS) Culture-induced Epithelial-mesenchymal Transition

Multicellular tumor spheroid (MTS) 배양에 의한 EMT에서 HMGB1의 역할

  • Lee, Su Yeon (Department of Molecular Biology, College of Natural Sciences, Pusan National University) ;
  • Ju, Min Kyung (Department of Molecular Biology, College of Natural Sciences, Pusan National University) ;
  • Jeon, Hyun Min (Department of Molecular Biology, College of Natural Sciences, Pusan National University) ;
  • Kim, Cho Hee (Department of Molecular Biology, College of Natural Sciences, Pusan National University) ;
  • Park, Hye Gyeong (Nanobiotechnology Center, Pusan National University) ;
  • Kang, Ho Sung (Department of Molecular Biology, College of Natural Sciences, Pusan National University)
  • 이수연 (부산대학교 자연과학대학 분자생물학과) ;
  • 주민경 (부산대학교 자연과학대학 분자생물학과) ;
  • 전현민 (부산대학교 자연과학대학 분자생물학과) ;
  • 김초희 (부산대학교 자연과학대학 분자생물학과) ;
  • 박혜경 (한국나노바이오테크놀러지센터) ;
  • 강호성 (부산대학교 자연과학대학 분자생물학과)
  • Received : 2018.08.31
  • Accepted : 2018.11.16
  • Published : 2019.01.30
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Abstract

As tumors develop, they encounter microenvironmental stress, such as hypoxia and glucose depletion, due to poor vascular function, thereby leading to necrosis, which is observed in solid tumors. Necrotic cells are known to release cellular cytoplasmic contents, such as high mobility group box 1 (HMGB1), into the extracellular space. The release of HMGB1, a proinflammatory and tumor-promoting cytokine, plays an important role in promoting inflammation and metabolism during tumor development. Recently, HMGB1 was shown to induce the epithelial-mesenchymal transition (EMT) and metastasis. However, the underlying mechanism of the HMGB1-induced EMT, invasion, and metastasis is unclear. In this study, we showed that noninvasive breast cancer cells MCF-7 formed tightly packed, rounded spheroids and that the cells in the inner regions of a multicellular tumor spheroid (MTS), an in vitro model of a solid tumor, led to necrosis due to an insufficient supply of O2 and glucose. In addition, after 7 d of MTS culture, the EMT was induced via the transcription factor Snail. We also showed that HMGB1 receptors, including RAGE, TLR2, and TLR4, were induced by MTS culture. RAGE, TLR2, and TLR4 shRNA inhibited MTS growth, supporting the idea that RAGE/TLR2/TLR4 play critical roles in MTS growth. They also prevented MTS culture-induced Snail expression, pointing to RAGE/TLR2/TLR4-dependent Snail expression. RAGE, TLR2, and TLR4 shRNA suppressed the MTS-induced EMT. In human cancer tissues, high levels of RAGE, TLR2, and TLR4 were detected. These findings demonstrated that the HMGB-RAGE/TLR2/TLR4-Snail axis played a crucial role in the growth of the MTS and MTS culture-induced EMT.

암조직의 내부에서 hypoxia와 glucose depletion 등의 microenvironmental stress를 받게 되면 necrosis가 유도되고, 실제로 암 조직 내부에서 necrotic core 형성이 관찰된다. Necrotic cells은 high mobility group box 1(HMGB1)를 extracellular space로 방출하는 것으로 알려져 있다. 방출된 HMGB1은 tumor-promoting cytokine으로 작용함으로써 tumor development 시 inflammation, metabolism 및 metastasis에 기여한다. 본 연구에서 non-invasive breast cancer cells MCF-7이 solid tumor의 in vitro model인 multicellular tumor spheroid (MTS) 배양을 통해 완전한 구형의 MTS를 형성하며 MTS가 성장함에 따라 inner region에 necrosis가 유도됨을 밝혔다. 또한 MCF-7 세포의 MTS 배양은 Snail 의존적으로 epithelial-mesenchymal transition (EMT)를 유도함을 관찰하였다. HMGB1의 cell surface receptors인 RAGE, TLR2, TLR4 발현이 MTS 배양에 의해 증가됨을 발견하였다. RAGE, TLR2, TLR4 를 knockdown한 결과 MTS 성장을 억제할 뿐만 아니라 MTS에 의해 증가되는 Snail 발현을 억제함을 밝혔다. 이는 MTS-induced Snail 발현이 RAGE/TLR2/TLR4의존적으로 조절되며 RAGE/TLR2/TLR4-Snail이 MTS 성장에 관여하는 것으로 보인다. 또한 Snail, RAGE, TLR2, TLR4 shRNA는 MTS 배양에 의해 유도되는 EMT를 억제함을 밝혔다. 실제 인간 암조직에서 정상조직에 비해 RAGE, TLR2, TLR4 유전자의 발현이 높음을 관찰하였다. 따라서 HMGB1이 RAGE/TLR2/4-Snail axis를 통해 MTS 배양에 따른 성장 및 EMT에 중요하게 작용할 것으로 예상된다.

Keywords

SMGHBM_2019_v29n1_9_f0001.png 이미지

Fig. 1. MCF-7 cells acquire the potential to exhibit EMT during MTS culture.

SMGHBM_2019_v29n1_9_f0002.png 이미지

Fig. 2. RAGE/TLR2/4 signaling is involved in MTS culture-induced EMT via Snail activation.

SMGHBM_2019_v29n1_9_f0003.png 이미지

Fig. 3. The expression of RAGE, and TLR2/4 in human tumors.

Table 1. shRNA target sequences used in this paper

SMGHBM_2019_v29n1_9_t0001.png 이미지

Table 2. Primer sequences used in this study

SMGHBM_2019_v29n1_9_t0002.png 이미지

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