CHOSUN

Insight into the roles of interleukin-33 and LPIN1 in epithelial cell transformation and mammary tumorigenesis

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Author(s)
김진영
Issued Date
2016
Keyword
Interleukin-33, LPIN1
Abstract
The tumor microenvironment is now recognized as an important participant of tumor progression and response to treatment. There is increasing interest in developing novel therapies targeting the microenvironment, particularly as it relates to invasive and metastatic progression. The changes of the tumor microenvironment have been closely correlated to cancer-mediated inflammation. In addition, obesity can result in an inflammatory environment that can contribute to tumorigenesis. It is now clear that breast cancer cells in the tumor microenvironment play an important role in cancer development. However, the role of the inflammatory cytokine interleukin-33 and LPIN1 in tumor growth and development of breast cancer remains unclear. In this paper, we propose that interleukin-33 and LPIN1 has a crucial role to play in controlling tumorigenesis and epithelial cell proliferation in the breast. Cytokines of the interleukin-1 (IL-1) family, such as IL-1α/β and IL-18, have pleiotropic activities in innate and adaptive immune responses in host defense and diseases. Insight into their biological functions helped develop novel therapeutic approaches to treat human inflammatory diseases. IL-33 is an important member of the IL-1 family of cytokines and is a ligand of the ST2 receptor, a member of the IL-1 receptor family. However, the role of the IL-33/ST2 axis in tumor growth and metastasis of breast cancer remains unclear. Here, we demonstrate that is a critical tumor promoter during epithelial cell proliferation and tumorigenesis in the breast. IL-33 dose- and time-dependently increased Cancer Osaka Thyroid (COT) phosphorylation via ST2-COT interaction in normal epithelial and breast cancer cells. The IL-33/ST2/COT cascade induced the activation of the MEK-ERK, JNK-cJun, and STAT3 signaling pathways, followed by increased AP-1 and stat3 transcriptional activity. When small interfering RNAs of ST2 and COT were introduced into cells, IL-33-induced AP-1 and stat3 activity were significantly decreased, unlike that in the control cells. The inhibition of COT activity resulted in decreased IL-33-induced epithelial cell transformation, and knockdown of IL-33, ST2 and COT in breast cancer cells attenuated tumorigenicity of breast cancer cells. Consistent with these observations, ST2 levels were positively correlated with COT expression in human breast cancer. Here, we demonstrate that LPIN1 promotes epithelial cell transformation and tumorigenesis in breast and LPIN1 colocalizes and interacts with IRS1. In addition, LPIN1 overexpression in SK-BR3 and MDA-MB231 cells increased endogenous IRS1 levels by IGF-1. In contrast, silencing of LPIN1 attenuated the IRS1 levels increased by IGF-1. IRS1 mRNA level was not affected by knockdown of LPIN1. In contrast, overexpression of LPIN1 increases the half-life of IRS1 by cycloheximide. These results indicate that LPIN1 might be associated with IRS1 stability. Therefore, LPIN1 overexpression inhibited the ubiquitination of IRS1 that LPIN1 promotes IRS1 stability via inhibition of its ubiquitination. In addition, the lysosomal inhibition by chloroquine substantially enhanced the elevated IRS1 levels by LPIN1 overexpression. These results indicate that LPIN1 inhibits IRS1 degradation by the proteasomal and lysosomal pathways. Moreovere, LPIN1 contribute to the regulation of RAF1 and MAPK signaling cascade. To determine the regulatory role of LPIN1 on the phosphorylation of RAF1 and its downstream kinases, FLAG-LPIN1 overexpressing SK-BR3 and MDA-MB231 cells were exposed to IGF-1. The levels of RAF1, MEK1/2, ERK1/2, and p90RSK phosphorylation in LPIN1 overexpressing cells were enhanced compared with the control cells. In contrast, we observed the downregulated RAF1, MEK, ERK, p90RSK signaling cascade in human LPIN1-silenced cells. Propranolol and NT157 decreased the IGF-1-induced phosphorylation of RAF1 as well as its downstream kinases. In addition, propranolol and NT157 significantly inhibited the IGF-1-promoted c-fos and AP-1 promoter activity. Moreover, demonstrated that a profound reduction in the weight and volume of tumors was mediated by treatment of propranolol and NT157, compared with only IGF-1-promoted tumor. Thus, LPIN1 promotes breast tumorigenesis via upregulation of IRS1 stability. In conclusion this study, we demonstrate that role of the interleukin-33 and LPIN1 in supporting cancer-associated in the tumor microenvironment.|종양 미세환경은 종양의 발생과 성장, 침습과 전이에 미치는 영향에 주요한 인자로 인식되고 있다. 종양 미세환경의 변화는 암을 매개한 염증과 상관관계가 있으며, 비만도 마찬가지로 염증 환경에서 종양형성에 기여한다.이러한 염증과 비만은 여러가지 기전을 통하여 유방종양을 일으킨다. 그러나 유방암의 생성 및 발달과정에서 염증 사이토카인 Interleukin-33 (IL-33)과 지질 생합성 조절 효소인 LPIN1의 역할은 알려지지 않았다. 본 연구에서는 유방에서의 상피세포의 증식과 암 형성을 조절하는 데 있어 IL-33과 LPIN1이 중요한 역할을 수행하고 있다고 제안한다.
IL-1α/β, IL-18과 같은 interleukin-1 (IL-1) family의 cytokine들은 생체방어와 질환에 있어서 선천적 면역반응에 대한 다면활성을 가지고 있다. 그러므로 다양한 interleukin의 생물학적 기능에 대한 인식은 인간의 염증성 질환을 치료하는데 새로운 치료학적 방법을 제시해왔다. IL-33은 IL-1 interleukin family의 구성원으로 분류되었으며, ST2 수용체에 결합하는 특징을 가지고 있다. 그러나 유방암의 종양성장 및 전이에서 IL-33/ST2 axis의 역할은 알려지지 않았다. 본 연구에서는 유방에서의 암 형성과 내피세포의 증식을 조절하는 데 있어 IL-33이 중요한 역할을 수행하고 있다고 제안하였다. 정상 상피세포와 유방암 세포에서 ST2-COT 상호작용을 통해 IL-33의 농도 및 시간 의존적으로 COT의 인산화를 증가시켰다. IL-33/ST2/COT cascade는 MEK-ERK, JNK-cJun 과 STAT3의 활성화를 유도함으로써, AP-1 및 STAT3 전사활성을 증가시켰다. 그리고 ST2 및 COT의 siRNA가 세포내로 도입되었을 때, IL-33에 의해 유도되는 AP-1과 STAT3 활성은 대조군에 비하여 유의성 있게 감소하였다. COT의 활성을 억제한 결과 상피세포에서의 형질전환이 감소되었다. 유방암 세포에서 IL-33, ST2 및 COT의 knockdown은 유방암 세포에서의 종양형성을 억제시켰다. 이러한 관찰들과 일관성을 보이며, ST2와 COT의 발현 수준은 인간 유방암 조직에서 유의적으로 증가하였음을 규명하였다.
나아가 본 연구에서는 유방에서 암 형성과 내피세포의 증식을 조절하는 데 있어 LPIN1이 IRS1과 단백-단백 상호작용을 하는 분자 생물학적 기전을 규명하였다. 또한, 유방암 세포주인 SK-BR3와 MDA-MB231 세포에서 LPIN1을 과다발현 시켰을 때 IGF-1에 의해 내인성 IRS1의 발현을 대조군에 비하여 증가시켰다. 반면에 LPIN1의 kockdown은 IGF-1이 유도하는 IRS1의 발현을 억제시켰다. LPIN1의 kockdown에 의해 IRS1의 mRNA 발현에 영향을 미치지 않았으나, LPIN1의 과다발현은 cycloheximide 처리에 의해 감소되는 IRS1 발현을 억제하였다. 이러한 결과는 LPIN1이 IGF-1 의존적으로 IRS1 stability에 영향을 끼치는 것으로 사료된다. 그러므로 In vitro에서 LPIN1이 IRS1의 ubiquitination에 미치는 영향을 살펴본 결과 LPIN1의 과다발현은 ubiquitination의 억제를 통해 IRS1의 proteasome 의존적 단백질 분해를 억제시키는 것으로 보인다. 또한, chloroquine에 의해 lysosome의 활성 억제는 LPIN1의 과다발현에 의한 증가된 IRS1 발현을 대조군에 비하여 더욱 상승시켰다. 이러한 결과들은 LPIN1이 lysosome과 proteasome 의존적 경로에 의하여 IRS1 단백질의 분해를 억제하는 것으로 사료된다. 나아가, LPIN1은 IRS1에 의해 유도한 MAPK 경로에 영향을 미치는지 알아본 결과 LPIN1 과다발현 세포에서 IGF-1에 의한 RAF1, MEK1/2, ERK1/2, p90RSK의 인산화를 대조군에 비하여 더욱 증가시킨 반면에, LPIN1 kockdown은 IGF-1에 의해 증가된 RAF1, MEK1/2, ERK1/2, p90RSK의 인산화를 억제하였다. LIPIN1 억제제인 propranolol과 IRS1 억제제인 NT157은 농도 의존적으로 IGF-1에 의한 RAF1 뿐만아니라 downstream kinases의 인산화를 감소시켰다. 또한, SK-BR3와 MDA-MB231 세포에서 propranolol과 NT157는 IGF-1에 의해 촉진된 c-fos와 AP-1 promoter 활성을 억제시켰다. 나아가 xenograft model에서 IGF-1에 의하여 형성된 종양이 propranolol과 NT157를 처리하였을 때 억제되었다. 본 연구에서는 LPIN1이 IRS1 stability의 upregulation을 통하여 유방암 생성에 중요한 조절현상으로 규명하였다. 위의 결과를 종합적으로 볼 때, Interleukin-33과 LPIN1은 종양 미세환경에서 발암 및 암화 진행에 밀접한 관계가 있는 것으로 사료한다
Alternative Title
Interleukin-33과 LPIN1이 상피세포 형질전환과 유방암 발생에 미치는 영향
Alternative Author(s)
Kim, Jin Young
Affiliation
조선대학교 일반대학원
Department
일반대학원 약학과
Advisor
최홍석
Awarded Date
2016-02
Table Of Contents
I. Introduction ------------------------------------------------------------------------------ 1

1. Tumor microenvironment and breast cancer progression --------------------- 1
2. Cytokines involved in tumor microenvironment -------------------------------- 2
3. Significance of interleukin in breast cancer -------------------------------------- 3
4. Novel cytokine: IL-33------------------------------------------------------------------ 4
4.1 Biological function of IL-33 -------------------------------------------------------- 4
4.2 IL-33 receptor, ST2-----------------------------------------------------------------5
4.3 IL-33 involved in tumor microenvironment---------------------------------------6
4.4 IL-33/ST2 signaling pathway ------------------------------------------------------7
4.5 The role of IL-33 in tumourigenesis-----------------------------------------------8
5. LPIN1------------------------------------------------------------------------------------10
5.1 The LPIN1 proteins family and function ---------------------------------------10
5.2 LPIN1 proteins as PAP enzymes required for lipid synthesis--------------11
5.3 Regulation of LPIN1 proteins activity and expression------------------------14
5.4 The role of LPIN1 in insulin signaling and tumourigenesis -------------------15
6. The role of mitogen-activated protein (MAP) kinase in breast cancer tumourigenesis------------------------------------------------------------------------17
7. Role of STAT3 in tumorigenesis ----------------------------------------------------19
8. Intracellular protein degradation systems; Ubiquitin-proteasome system(UPS) and autophagy------------------------------------------------------- 21
8.1 The ubiquitin-proteasome system (UPS) -----------------------------------------21
8.2 Autophagy system------------------------------------------------------------------- 22
8.3 Diseases related with dysregulation of proteosomal and lysosomal proteolysis---------------------------------------------------------------------------25
9. Activator protein-1 (AP-1) in tumorigenesis ------------------------------------27
9.1 AP-1 family proteins, structures, and functions ---------------------------------27
9.2 Jun family proteins, structures, and functions ---------------------------------28
9.3 Fos family proteins, structures, and functions --------------------------------29

II. Materials & Methods ---------------------------------------------------------------- 32
1. Cell lines and establishment of stable cell lines ------------------------------------ 32
2. Reagents and antibodies ---------------------------------------------------------------- 33
3. Construction of mammalian expression and small interfering RNA (siRNAs)--33
4. RNA Isolation and Semiquantitative RT-PCR---------------------------------------34
5. Immunofluorescence staining-----------------------------------------------------------35
6. Cell proliferation assay (BrdU incorporation) ---------------------------------------35
7. Immunoblot analysis Measurement of cytokines------------------------------------35
8. Measurement of cytokines --------------------------------------------------------------36
9. Anchorage-independent cellular transformation assay------------------------------36
10. Reporter gene assays -------------------------------------------------------------------36
11. Tumor samples --------------------------------------------------------------------------37
12. Immunohistochemical staining analysis---------------------------------------------37
13. Tumorigenicity assay in nude mice---------------------------------------------------38
14. Statistical analysis ----------------------------------------------------------------------39

Ⅲ. Results ------------------------------------------------------------------------------------- 40

Part I: Interleukin-33/ST2 axis promotes epithelial cell transformation breast
tumorigenesis via upregulation of COT activity------------------------------------ 40
1. IL-33 promotes anchorage-independent transformation and tumorigenesis in breast ------------------------------------------------------------------------------------ 40
2. ST2 mediates IL-33-induced COT phosphorylation via the interaction of ST2
with COT ------------------------------------------------------------------------------- 49
3. COT mediates the MEK1/2-ERK1/2 and JNK1/2-cJun signaling pathways induced by IL-33 --------------------------------------------------------------------- 56
4. COT mediates STAT3 phosphorylation via its interaction with STAT3 -------- 60
5. COT is required for IL-33-induced AP-1 and stat3 activation ------------------ 65
6. An IL-33/ST2/COT axis promotes epithelial cell transformation and breastmammary tumorigenesis ------------------------------------------------------70
Part II: LPIN1 in regulating IRS1 protein stability and breast tumorigenesis---77
7. LPIN1 promotes epithelial cell transformation and tumorigenesis in breast----77
8. LPIN1 colocalizes and interacts with IRS1 in breast cancer cells----------------84
9. LPIN1 participates in the upregulation of IRS1 levels induced by IGF-1-------91
10. LPIN1 inhibits IRS1 degradation by the proteasomal and autophagic/lysosomal pathways------------------------------------------------------98
11. Activation of RAF1 is associated with increased IRS1 stability by LPIN1--105
12. Epithelial cell transformation and breast tumor growth is efficiently inhibited
by propranolol and NT157-----------------------------------------------------------114

IV. Discussion--------------------------------------------------------------------------------122
V. References---------------------------------------------------------------------------------135
Abstract-----------------------------------------------------------------------------------159
Degree
Doctor
Publisher
조선대학교 대학원
Citation
김진영. (2016). Insight into the roles of interleukin-33 and LPIN1 in epithelial cell transformation and mammary tumorigenesis.
Type
Dissertation
URI
https://oak.chosun.ac.kr/handle/2020.oak/12606
http://chosun.dcollection.net/common/orgView/200000265193
Appears in Collections:
General Graduate School > 4. Theses(Ph.D)
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