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Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis
Published on: November 18, 2019
Proteomic Profiling of Iron-Treated Ovarian Cells Identifies AKT Activation that Modulates the CLEAR Network
Stephanie Rockfield1, Jennifer Guergues1,2, Nabila Rehman1
1Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 East Fowler Avenue, ISA2015, Tampa, FL, 33620, USA.
Abstract:
Although iron is essential for cell survival, dysregulated levels can contribute to cancer development or even cell death. The underlying mechanisms mediating these events remain unclear. Herein, proteomic alterations are assessed in iron-treated ovarian cell lines using reverse phase protein array (RPPA) technology and potential functional responses via ingenuity pathway analysis (IPA). Using these approaches, upregulation of pathways modulating organismal death with alterations in mTOR, MAPK, and AKT signaling in HEY ovarian cancer cells in contrast to T80 non-malignant ovarian cells is noted. Since modulation of cell death is mediated in part via microphthalmia-associated transcription factor (MiTF) family, which regulates lysosomal biogenesis and autophagosome formation by upregulating expression of coordinated lysosomal expression and regulation (CLEAR) network, expression changes in these factors in response to iron are investigated. Increased transcription factor EB (TFEB) in T80 (relative to HEY), accompanied by its nuclear translocation and increased CLEAR network gene expression with iron, is identified. Inhibition of AKT alters these responses in contrast to mTOR inhibition, which has little effect. Collectively, these findings support use of RPPA/IPA technology to predict functional responses to iron and further implicate AKT pathway and MiTF members in iron-induced cellular responses in ovarian cells.
Insights
Iron dysregulation impacts ovarian cell survival and cancer. This study reveals AKT pathway and MiTF family involvement in iron-induced cellular responses, using proteomic and pathway analysis technologies.
Area of Science:
- Cellular biology
- Molecular oncology
- Biochemistry
Background:
- Iron is vital for cell survival, but abnormal iron levels are linked to cancer development and cell death.
- Mechanisms underlying iron's role in cell fate, particularly in ovarian cells, are not fully understood.
Purpose of the Study:
- To investigate proteomic and functional alterations in iron-treated ovarian cells.
- To elucidate the role of the microphthalmia-associated transcription factor (MiTF) family and related pathways in iron-mediated cellular responses.
Main Methods:
- Utilized reverse phase protein array (RPPA) technology to assess proteomic changes.
- Employed Ingenuity Pathway Analysis (IPA) to predict functional responses.
- Investigated expression and localization of transcription factor EB (TFEB) and CLEAR network genes.
Main Results:
- Identified differential pathway alterations, including mTOR, MAPK, and AKT signaling, between ovarian cancer (HEY) and non-malignant (T80) cells upon iron treatment.
- Observed increased TFEB expression, nuclear translocation, and CLEAR network gene upregulation in T80 cells compared to HEY cells in response to iron.
- Found that AKT inhibition modulated these responses, while mTOR inhibition had minimal effect.
Conclusions:
- RPPA and IPA are effective tools for predicting functional responses to iron in ovarian cells.
- The AKT pathway and MiTF family members, including TFEB, play significant roles in iron-induced cellular responses and iron metabolism in ovarian cells.
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