1. Grp78/PERK/Nrf2 Signaling Activation Mediated Metabolic Reprogramming of Cancer-initiating Cells
Speaker : Ching-Wen Chang (張瀞文)Ph.D. Adviser: Jeng-Fan Lo (羅正汎) Ph.D.
National Yang-Ming University (國立陽明大學)
2017/05/07

2. Cancer initiating cells, CICs
Tumorigenicity
Cancer progression and metastasis
Chemo-resistance and radio-resistance
Stem cell properties:
Rare cells
Self-Renewal ability
Differentiation
The Journal of Clinical Investigation, 2010

3. Distinct stemness and tumorigenic properties of ROSLow, ROSMedi, and ROSHigh cells.
Cancer Res; 74(21) November 1, 2014

5. Nrf2 acts as a central node to regulate the ROS scavenger enzymes within the CICs, and predicts the survival of HNSCC patients A B

6. Nrf2 acting as a central node in maintenance of low ROS levels within the CICs and predicts the survival of HNSCC patients A B

7. Nrf2 entry into the nucleus is important for maintenance of low ROS levels within the CICs.
The PE shows staining with Nrf2.
The DAPI shows nuclear staining by DAPI

8. Nrf2 knockdown abrogating cancer-initiating cell properties

9. Nrf2 overexpression enhancing cancer-initiating cell characteristics
Stable Nrf2-overexpressing cells
In selection medium
2weeks
Increased nuclear localization of Nrf2

10. Nrf2 activates genes involved in the intracellular redox state
Up-expression genes in ROSLow cell by RNA-Seq
Nrf2-target
Genes by
ChIP-Seq
Candidate Genes
HN-CICs
Nrf2-regulated genes involving in low level of ROS

11. Nrf2 activates genes involved in the metabolic pathway

12. Nrf2 activating glycolytic pathway related genes to mediate the intracellular redox state
Glycolytic phenotype maintains a high redox homeostasis state in cancer cells and may be responsible for their malignant behavior
El Sayed et al., 2013

13. HN-CICs may rely on glycolytic pathway through Nrf2-mediated transcriptional activation

14. Nrf2 controls metabolic activity in HN-CICs

15. A positively correlation between the nuclear Nrf2 and Grp78/p-PERK expression in ROSLow cells
Oncogene (2013)
ROSLow cells RNA-seq analysis

16. Grp78/p-PERK/Nrf2 signaling activating Nrf2 to enhance stemness properties in sphere cells

17. Grp78/p-PERK/Nrf2 signaling activating Nrf2 to enhance glycolytic gene expression in sphere cells

18. Kaplan–Meier analysis showing the overall survival of HNSCC patients according to the expression of p-Nrf2 protein and p-PERK protein

19. Pearson’s correlation analysis of Nrf2 and its target genes expression values derived from TCGA dataset

20. Conclusions

23. Reactive Oxygen Species and Stem cells
A low level of reactive oxygen species selects for primitive hematopoietic stem cells that may reside in the low-oxygenic niche.
Association of reactive oxygen species levels and radioresistance in cancer stem cells.
Drug-induced senescence generates chemoresistant stem-like cells with low reactive oxygen species.
Blood. 2007
Nature. 2009
所以接下來我們就想探討在CICs有沒有特殊的機制來maintain stemness
在之前的研究中已經知道ROS具有調控stem cell的能力
像是在hematopoietic stem cells 具有較低的ROS特性來維持stemness
之後再2009年的nature中發現cancer stem cells中也有一群較低ROS的細胞具有放射治療的抗性
而這群細胞也具有化療抗性
J Biol Chem. 2011

24. Summary of top ten canonical pathways toxicology pathway lists
ROSLow cells RNA-seq analysis
Toxicity Lists in sphere cells (Proteomics)

25. Summary of top ten canonical pathways toxicology pathway lists
Toxicity Lists in sphere cells (Microarray)

26. Nrf2 knockdown abrogating in vitro malignancy and in vivo tumorigenic properties

27. Nrf2 overexpression enhancing cancer-initiating cell characteristics

28. Immunoblots showing the expression of HK2, PFKFB3, Nanog and Oct4 proteins in sh-HK2 or sh-PFKFB3 sphere cells.

31. Identification of ROS scavenging expression of HN-CICs by microarray analysis
parental
SAS cells
(HNSCC)
SAS
sphere cells
(HN-CICs)
RNA isolation
Microarray analysis
SAS Parental
SAS –Sphere cells-2weeks
SAS –Sphere cells-3weeks
SAS –Sphere cells-5weeks
SAS –Sphere cells-9weeks
Information was
provided
by Dr. Yu-Syuan Chen

32. HN-CICs may rely on glycolytic pathway and impaired oxidative phosphorylation, consistent with a ‘‘Warburg effect’’ profile