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Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH
Published on: September 20, 2020
Estrogen response element-GFP (ERE-GFP) introduced MCF-7 cells demonstrated the coexistence of multiple
Natsu Fujiki1, Hiromi Konno, Yosuke Kaneko
1Department of Molecular and Functional Dynamics, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai 980-8575, Japan.
Abstract:
The acquisition of estrogen-deprivation resistance and estrogen receptor (ER) signal-independence in ER-positive breast cancer is one of the crucial steps in advancing the aggressiveness of breast cancer; however, this has not yet been elucidated in detail. To address this issue, we established several estrogen-deprivation-resistant (EDR) breast cancer cell lines from our unique MCF-7 cells, which had been stably transfected with an ERE-GFP reporter plasmid. Three cell lines with high ER activity and another 3 cell lines with no ER activity were established from cell cloning by monitoring GFP expression in living cells. The former three ERE-GFP-positive EDR cell lines showed the overexpression of ER and high expression of several ER-target genes. Further analysis of intracellular signaling factors revealed a marked change in the phosphorylation status of ERα on Ser167 and Akt on Thr308 by similar mechanisms reported previously; however, we could not find any changes in MAP-kinase factors. Comprehensive phospho-proteomic analysis also indicated the possible contribution of the Akt pathway to the phosphorylation of ERα. On the other hand, constitutive activation of c-Jun N-terminal kinase (JNK) was observed in ERE-GFP-negative EDR cells, and the growth of these cells was inhibited by a JNK inhibitor. An IGF1R-specific inhibitor diminished the phosphorylation of JNK, which suggested that a novel signaling pathway, IGF1R-JNK, may be important for the proliferation of ER-independent MCF-7 cells. These results indicate that ER-positive breast cancer cells can acquire resistance by more than two mechanisms at a time, which suggests that multiple mechanisms may occur simultaneously. This finding also implies that breast cancers with different resistance mechanisms can concomitantly occur and mingle in an individual patient, and may be a cause of the recurrence of cancer.
Insights
Estrogen-deprivation resistance in breast cancer can develop through multiple pathways, involving estrogen receptor (ER) signaling or ER-independent mechanisms like IGF1R-JNK. Understanding these diverse resistance mechanisms is crucial for treating recurrent ER-positive breast cancer.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Estrogen receptor (ER)-positive breast cancer resistance to endocrine therapy is a significant clinical challenge.
- Mechanisms underlying acquired estrogen-deprivation resistance (EDR) and ER signal-independence remain incompletely understood.
- Understanding these resistance pathways is critical for improving treatment strategies and patient outcomes.
Purpose of the Study:
- To elucidate the molecular mechanisms of estrogen-deprivation resistance (EDR) in ER-positive breast cancer.
- To establish and characterize novel EDR cell lines with varying ER activity.
- To identify key signaling pathways involved in ER-dependent and ER-independent resistance.
Main Methods:
- Development of unique MCF-7 breast cancer cell lines stably transfected with an ERE-GFP reporter plasmid.
- Cell cloning and monitoring of GFP expression to establish EDR cell lines with high ER activity (ERE-GFP-positive) and no ER activity (ERE-GFP-negative).
- Analysis of intracellular signaling factors, including ERα and Akt phosphorylation, MAP-kinase activity, comprehensive phospho-proteomics, and JNK pathway activation.
Main Results:
- ERE-GFP-positive EDR cells exhibited ER overexpression, increased ER-target gene expression, and altered phosphorylation of ERα (Ser167) and Akt (Thr308).
- ERE-GFP-negative EDR cells showed constitutive activation of c-Jun N-terminal kinase (JNK), with growth inhibition observed upon JNK inhibitor treatment.
- IGF1R-specific inhibitor reduced JNK phosphorylation, suggesting a novel IGF1R-JNK signaling pathway in ER-independent MCF-7 cells.
Conclusions:
- ER-positive breast cancer cells can acquire resistance through multiple, potentially simultaneous, mechanisms.
- Distinct signaling pathways, including ERα/Akt and IGF1R/JNK, contribute to different forms of estrogen-deprivation resistance.
- The co-occurrence of diverse resistance mechanisms in individual patients may drive cancer recurrence and necessitate combination therapies.
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