MEMO1 binds iron and modulates iron homeostasis in cancer cells
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eLife assessment
Dolgova et al present a well-written and important manuscript focused on the mechanism of MEMO1 function in tumor cells. The authors explore whether the mechanism of MEMO1 overexpression in breast cancer, especially TNBC, is related to regulating iron given evidence that MEMO1 binds multiple proteins in the iron regulation pathway. While the data is in part compelling, the claims are based on indirect evidence for a central role of MEMO1 in tumorogenesis and perhaps metastasis via its effect on iron homeostasis.
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Abstract
Mediator of ERBB2-driven cell motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 ( TFR 2), mitoferrin-2 ( SLC25A28 ), and the global iron response regulator IRP1 ( ACO1 ). These interactions indicate that cells with high-MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.
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eLife assessment
Dolgova et al present a well-written and important manuscript focused on the mechanism of MEMO1 function in tumor cells. The authors explore whether the mechanism of MEMO1 overexpression in breast cancer, especially TNBC, is related to regulating iron given evidence that MEMO1 binds multiple proteins in the iron regulation pathway. While the data is in part compelling, the claims are based on indirect evidence for a central role of MEMO1 in tumorogenesis and perhaps metastasis via its effect on iron homeostasis.
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Reviewer #1 (Public Review):
Dolgova et al present a well-written manuscript focused on the mechanism of MEMO1 function in tumor cells. They use genome-wide analyses to predict function based on MEMO1 structure in yeast, identify MEMO1 expression in a screen of cancer cell lines, and demonstrate a correlation between MEMO1 expression and severity of disease in primary breast cancer cells. The authors focus on a breast cancer model as it overexpresses MEMO1 and melanoma as a control and uses CRISPR-Cas9 knockdown of MEMO1 in breast and melanoma cell lines and concurrently knockdown selected genes in the iron homeostasis pathway. In this data, MEMO1 appears to interact with elements involved in iron trafficking and sensing and its overexpression leads to possible hypersensitivity while knockout/knockdown leads to resistance to lipid …
Reviewer #1 (Public Review):
Dolgova et al present a well-written manuscript focused on the mechanism of MEMO1 function in tumor cells. They use genome-wide analyses to predict function based on MEMO1 structure in yeast, identify MEMO1 expression in a screen of cancer cell lines, and demonstrate a correlation between MEMO1 expression and severity of disease in primary breast cancer cells. The authors focus on a breast cancer model as it overexpresses MEMO1 and melanoma as a control and uses CRISPR-Cas9 knockdown of MEMO1 in breast and melanoma cell lines and concurrently knockdown selected genes in the iron homeostasis pathway. In this data, MEMO1 appears to interact with elements involved in iron trafficking and sensing and its overexpression leads to possible hypersensitivity while knockout/knockdown leads to resistance to lipid oxidation. They also interrogate the effect of iron chelation on mitochondrial morphology and ferroptosis. In addition, they evaluate iron and copper binding loci and resolve MEMO1 structure. The work is of high quality. However, there are some inaccuracies regarding the known function of some iron-related elements. Furthermore, it is unresolved whether controlling iron per se (by modulating other importers and transporters or limiting iron availability in culture) recapitulates or ameliorates their findings, currently attributed specifically to the mechanism of action of MEMO1. In addition, the authors make claims that they have not substantiated about overexpression of MEMO1 by extrapolating from data about MEMO1 knockdown or knockout. Finally, the results show only indirect evidence for a central role for MEMO1 via regulation of iron trafficking and more targeted approaches are necessary to increase confidence in the claims.
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Reviewer #2 (Public Review):
While the hypothesis that MEMO1 plays a key role in cell iron homeostasis remains to be directly tested, the data presented herein clearly support further delineation of the underlying mechanisms. The key findings in this regard are the facts, as established herein, that: 1) MEMO1 binds ferrous iron (the appropriate valence state for cell iron) along with glutathione (Fig. 5A); 2) the structure of MEMO1 in complex with Fe(II)-GSH reveals the coordination site within the protein for this complex (Fig. 5B/c); 3) oxidative stress and sensitivity to ferroptosis correlate with MEMO1 protein abundance in a consistent fashion (Fig. 4); and 4) while the effect is limited, there are data that indicate a relation between cell iron content and MEMO1 abundance (Fig. 4A/B).
Experimentally, it is thorough and …
Reviewer #2 (Public Review):
While the hypothesis that MEMO1 plays a key role in cell iron homeostasis remains to be directly tested, the data presented herein clearly support further delineation of the underlying mechanisms. The key findings in this regard are the facts, as established herein, that: 1) MEMO1 binds ferrous iron (the appropriate valence state for cell iron) along with glutathione (Fig. 5A); 2) the structure of MEMO1 in complex with Fe(II)-GSH reveals the coordination site within the protein for this complex (Fig. 5B/c); 3) oxidative stress and sensitivity to ferroptosis correlate with MEMO1 protein abundance in a consistent fashion (Fig. 4); and 4) while the effect is limited, there are data that indicate a relation between cell iron content and MEMO1 abundance (Fig. 4A/B).
Experimentally, it is thorough and well-documented and offers a new look at a protein that has been at the edges of iron metabolism (and copper, but I agree with the authors that this is not likely to be the case). This work and its subject will stimulate much further research.
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Reviewer #3 (Public Review):
The goal of this manuscript is to determine the function of MEMO1 (mediator of ERBB2-driven cell motility 1), an evolutionarily conserved protein with many putative functions but none that have been firmly established. The authors take an unbiased, bioinformatics approach to identify genetic interactions between MEMO1 and other genes in cancer cell lines. Notably, they uncovered multiple links to genes with relevance to cellular iron homeostasis. They then explore these genetic links through a variety of experiments. First, they use shRNA-mediated gene knockdown to confirm the functional interaction between MEMO1 and interacting genes at the level of protein expression and cell proliferation. Second, they analyze the impact of altered MEMO1 levels on iron levels, mitochondrial morphology, and sensitivity to …
Reviewer #3 (Public Review):
The goal of this manuscript is to determine the function of MEMO1 (mediator of ERBB2-driven cell motility 1), an evolutionarily conserved protein with many putative functions but none that have been firmly established. The authors take an unbiased, bioinformatics approach to identify genetic interactions between MEMO1 and other genes in cancer cell lines. Notably, they uncovered multiple links to genes with relevance to cellular iron homeostasis. They then explore these genetic links through a variety of experiments. First, they use shRNA-mediated gene knockdown to confirm the functional interaction between MEMO1 and interacting genes at the level of protein expression and cell proliferation. Second, they analyze the impact of altered MEMO1 levels on iron levels, mitochondrial morphology, and sensitivity to ferroptosis. Third, they determine the crystal structure of MEMO1, both wild-type and mutant forms, and demonstrate that MEMO1 binds iron as well as copper.
There are notable strengths to this manuscript. I appreciated the unbiased, bioinformatics approach they took to identify genes that interact with MEMO1 and the ensuing approaches they took to explore the potential relevance of MEMO1 to cancer cell iron homeostasis. The methods employed are varied and state-of-the-art and address different aspects of MEMO1's potential role in cellular iron biology. There are some weaknesses. One is that direct protein-protein interactions are not assessed between MEMO1 and TFR2, one of the key genes shown to genetically interact with MEMO1 in cancer cell lines. This limits the authors' ability to more strongly assign a function for MEMO1 in cellular iron homeostasis. They do show that MEMO1 binds to iron, but how does this finding relate to the MEMO1-TFR2 interaction?
The authors conclude that MEMO1 is an iron-binding protein that regulates iron homeostasis in cancer cells. To this end, I agree that the authors have generated adequate evidence in support of this conclusion. The impact of this paper is that it will direct the field to focus on the relevance of MEMO1 to iron homeostasis. While this manuscript does not firmly establish the specific role of MEMO1 in iron homeostasis, future studies should be able to address that knowledge gap.
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