Induction of paraptosis by targeting proteostasis exploiting the dysregulated translation of cancer
- 주제(키워드) proteostasis , oncogene , translation , ISR , proteostasis , cancer
- 주제(DDC) 570
- 발행기관 아주대학교
- 지도교수 최경숙
- 발행년도 2022
- 학위수여년월 2022. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000031712
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Most cellular processes are modified during tumorigenesis, leading to unlimited proliferation potential and resistance to apoptotic cues in cancer cells. Among these processes, the dysregulation of the protein synthesis is commonly observed, and it is believed to support the transformed phenotype of cancers. At the same time, paradoxically, cancer cells are well known to suffer from numerous stress conditions limiting protein translation and proliferation, including hypoxia, nutrient limitation, proteotoxic stress, and oncogenic stress. Thus, these backgrounds push cancer cells to adapt to diverse stresses and to depend on the maintenance of protein homeostasis, to support their continuous and unregulated proliferation. Here, I show that these cancerous characters come at a significant cost for cancer cells under proteotoxic stress conditions induced by inhibition of the degradation pathway. Under stress conditions, following eIF2α phosphorylation, integrated stress response (ISR) is activated, reducing canonical cap-dependent translation and inducing alternative translation pathways. I found that the inhibition of VCP, an essential protein for cellular proteostasis via promoting several protein degradation pathways, induces excessive proteotoxic stress and death selectively in cancer cells via oncogenic Akt-eIF3d-mediated translation rebound. I further found that the co-treatment of ISRIB, an ISR inhibitor, with bortezomib (Bz), a proteasome inhibitor (PI), enhanced the proteotoxic stress and paraptotic cell death in turn cancer cells. Overall, this study offers novel insights into how to kill cancer cells harboring oncogenic activation via a non-oncogene targeting regimen and the possibility of paraptosis as a potent anti-cancer strategy. Additionally, this data provide an understanding of the relationship between cancerous features and eIF3d-mediated translation as well as between less sensitive properties of the solid tumor to Bz and the role of ISR.
more목차
I. INTRODUCTION 1
II. MATERIALS AND METHODS 15
A. Chemicals and antibodies 15
B. Cell culture 16
C. Cell viability assay 17
D. In vivo tumor growth inhibition assay 17
E. Immunoblot analyses 18
F. Immunofluorescence microscopy 19
G. Transmission electron microscopy 19
H. Small interfering RNA-mediated knockdown of the genes 20
I. Live-cell imaging 20
J. Transcription signature and dependency analysis 21
K. RNA-seq and Ingenuity Pathway Analysis (IPA) 21
L. shRNA generation 22
M. RNA isolation and quantitative real-time RT-PCR (qRT-PCR) 26
N. Morphological examination of the endoplasmic reticulum (ER) and mitochondria 28
O. Generation of MCF10A cell lines stably expressing HRasG12V and KRasG12V 28
P. Surface sensing of translation (SUnSET) assay 29
Q. Isobologram analysis 29
R. Statistical analysis 30
III. RESULTS 31
Part1. VCP inhibition preferentially kills cancer cells due to enhanced proteotoxic stress by Akt-mediated translational rebound 31
ABSTRACT 31
1. INTRODUCTION 32
2. RESULTS 41
2.1. VCP inhibition demonstrates an anti-cancer effect by inducing paraptosis. 41
2.2. VCP inhibition is preferentially cytotoxic to cancer cells, compared to non-transformed cells. 67
2.3. To live or to die in response to VCP inhibition may be determined by the cellular sustainability of translational suppression. 75
2.4. Oncogene-driven Akt activation is critical for the translational rebound and subsequent paraptosis in response to VCP inhibition. 85
2.5. eIF3d is critically involved in the translational rebound in VCP inhibition-mediated paraptosis. 98
2.6. ATF4-mediated DDIT4 upregulation critically contributes to the paraptosis induced by VCP inhibition. 105
3. DISCUSSION 120
Part2. The combination of bortezomib with ISRIB triggers paraptosis in breast cancer cells via enhanced translation and subsequent proteotoxic stress 128
ABSTRACT 128
1. INTRODUCTION 129
2. RESULTS 132
2.1. ISR is induced in both bortezomib-sensitive multiple myeloma cells and bortezomib-insensitive breast cancer cells. 132
2.2. ISRIB protects bortezomib-sensitive multiple myeloma cells from apoptosis, but enhances bortezomib-mediated cytotoxicity in breast cancer cells. 137
2.3. ISRIB plus bortezomib triggers paraptosis in breast cancer cells. 141
2.4. ISRIB plus bortezomib triggers paraptosis by inducing irresolvable proteotoxic stress due to enhanced translation. 144
3. DISCUSSION 149
VI. REFERENCES 153
국문 요약 171
