Widespread multi-targeted therapy resistance via drug-induced secretome fucosylation
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Evaluation Summary:
This manuscript provides a comprehensive unbiased analysis of the fucosylation secretome and correlates with drug response in cancer. It uses a combination of bioinformatic based analyses of multiple datasets and cell based data to identify changes in the secretome and correlates this to drug responses to several targeted therapies.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Cancer secretome is a reservoir for aberrant glycosylation. How therapies alter this post-translational cancer hallmark and the consequences thereof remain elusive. Here we show that an elevated secretome fucosylation is a pan-cancer signature of both response and resistance to multiple targeted therapies. Large-scale pharmacogenomics revealed that fucosylation genes display widespread association with resistance to these therapies. In both cancer cell cultures and patients, targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins less than 60 kDa. Label-free proteomics of N-glycoproteomes revealed that fucosylation of the antioxidant PON1 is a critical component of the therapy-induced secretome. Core fucosylation in the Golgi impacts PON1 stability and folding prior to secretion, promoting a more degradation-resistant PON1. Non-specific and PON1-specific secretome de-N-glycosylation both limited the expansion of resistant clones in a tumor regression model. Our findings demonstrate that core fucosylation is a common modification indirectly induced by targeted therapies that paradoxically promotes resistance.
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Evaluation Summary:
This manuscript provides a comprehensive unbiased analysis of the fucosylation secretome and correlates with drug response in cancer. It uses a combination of bioinformatic based analyses of multiple datasets and cell based data to identify changes in the secretome and correlates this to drug responses to several targeted therapies.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Aldonza MB, et. al. aim to understand how fucosylation of the cancer cell secretome affects the response to therapies. To begin, the authors analyze publicly available cell-based expression and drug sensitivity data, and conclude that high expression of FUT8, a fucosylation gene, results in resistance to specific therapies, including RTKs, EGFRi, and MAPK/ERK pathway inhibitors. The authors explore this further through the generation of 16 drug resistant cell lines, using inhibitors of EGFR, HER2, BRAF, MET, and ALK. The authors identify that fucosylated proteins, between 40-60kD are upregulated in drug resistant CM and patient serum, which the authors identify as PON1, a protein with paraoxonase enzymatic activity and component of HDL. The authors show that PON1 fucosylation, at N253, resulted in increased …
Reviewer #1 (Public Review):
Aldonza MB, et. al. aim to understand how fucosylation of the cancer cell secretome affects the response to therapies. To begin, the authors analyze publicly available cell-based expression and drug sensitivity data, and conclude that high expression of FUT8, a fucosylation gene, results in resistance to specific therapies, including RTKs, EGFRi, and MAPK/ERK pathway inhibitors. The authors explore this further through the generation of 16 drug resistant cell lines, using inhibitors of EGFR, HER2, BRAF, MET, and ALK. The authors identify that fucosylated proteins, between 40-60kD are upregulated in drug resistant CM and patient serum, which the authors identify as PON1, a protein with paraoxonase enzymatic activity and component of HDL. The authors show that PON1 fucosylation, at N253, resulted in increased stability and secretion of the protein. Blocking PON1 fucosylation, through knockdown of PON3 or expression of PON1-N253G, blocked the outgrowth of resistant cells. Exploration into the mechanism of how PON1 was contributing to therapy resistance, lead the authors to explore the UPR and ATF6 as potential downstream targets of PON1. Finally, the authors use transcriptomics to also look for potential downstream effectors of fucosylation. These data are interesting and show a potential mechanism of resistance to EGFR/MAPK pathway inhibitors via secreted factors.
While the conclusions of the paper are mostly well supported, the manuscript is dense and sometimes difficult to follow. Some additional clarification is needed.
1. The authors use analysis of many publicly available datasets to set up and frame their story of how fucosylation contributes to widespread therapy resistance. However, the data in these figures (1A, 1C, and 1D) in fact show that expression of FUT genes are correlated with a range of therapeutic response, not widespread resistance to all therapy which the authors claim. However, the depth in which the authors explore EGFR and MAPK pathway inhibition is a strength, and the authors show a potential mechanism to how fucosylation is contributing to resistance to these therapeutics.
2. The authors use extensive cell models to test their hypotheses, however, they do not perform any in vivo experiments. It would be important for the authors to test their mechanism (modulation of FUT8 or PON1 through shRNA or overexpression) in vivo to determine if modulation of these factors changed tumor growth inhibition or the time to development of resistance.
3. As stated above, FUT8 expression was correlated with resistance to specific therapies, including RTKs, EGFRi, and MAPK/ERK pathway inhibitors. How does treatment with these inhibitors increase PON1 fucosylation? This is an important question that remains unanswered. Discussion of why fucosylation of PON1 is contributing specifically to the resistance of these inhibitors would be of interest to readers and should at least be commented on in the discussion.
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Reviewer #2 (Public Review):
In this study, the authors study therapy-induced fucosylated secretome signatures at a pan-cancer level. The authors initially profiled therapy-associated changes in fucosylation-related genes and their methylation statuses by querying a number of online datasets including CCLE and GDSC. They found that fucosylation-related gene expression is correlated with various therapies. Further, they identified fucosyltransferase 8 (FUT8) as a key therapy-associated fucosylation gene, the expression of which correlated with reduced responsiveness. In vitro, the authors demonstrated that targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins <60 kDa. They further established that the fucosylation of these secreted proteins is regulated by the fucose salvage SLC35C1-FUT8 pathway and is …
Reviewer #2 (Public Review):
In this study, the authors study therapy-induced fucosylated secretome signatures at a pan-cancer level. The authors initially profiled therapy-associated changes in fucosylation-related genes and their methylation statuses by querying a number of online datasets including CCLE and GDSC. They found that fucosylation-related gene expression is correlated with various therapies. Further, they identified fucosyltransferase 8 (FUT8) as a key therapy-associated fucosylation gene, the expression of which correlated with reduced responsiveness. In vitro, the authors demonstrated that targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins <60 kDa. They further established that the fucosylation of these secreted proteins is regulated by the fucose salvage SLC35C1-FUT8 pathway and is enriched in the Golgi prior to secretion. The authors further demonstrated that the upregulation of core fucosylation of the secreted proteins correlated with drug resistance in a patient cohort and drug resistance clones. By the label-free proteomics of N65 glycoproteomes the authors discovered antioxidant PON1 as a critical component of the therapy-induced secretome. The authors showed that the core fucosylation at N253 is required for PON1 stability and secretion, and further that PON1 fucosylation is mediated by PON3 in the Golgi, which establishes the Golgi redox homeostasis. The transcriptomic analysis demonstrated that genes negatively regulating the response to stimulus and cell communication act as modulators upon inhibition of secretome PON1 fucosylation.
General Comments:
This is a novel study where the authors study the fucosylated secretome that is induced during therapy, which promotes resistance. The authors conducted a detailed in-vitro study by generating the resistance clones and comprehensively analyzing PON1 fucosylation. Although the authors have delineated the effects of fucosylation on the stability and secretion of PON1, how fucosylated PON1 promotes therapeutic resistance, remains unclear. Moreover, the study would have benefitted from in vivo validation of the author's findings to delineate the contribution of fucosylated PON1 to the development of therapeutic resistance (and perhaps other tumorigenic capacities not observed in the in vitro assays). Nonetheless, this study highlights novel contributions of fucosylation specifically to therapeutic-stress induced adaptations that promote resistance. Further, the findings reported herein lay the foundation for subsequent assessment of fucosylation/fucosylated secretome-related novel therapeutic targets and biomarkers that are predictive of therapeutic responsiveness.
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Reviewer #3 (Public Review):
This is a data rich manuscript that provides a novel basis for drug resistance. The authors show an increased fucosylation of secreted proteins is a hallmark of drug resistance to multiple targeted therapies. Through unbiased approaches the authors demonstrate that fucosylated PON1 is secreted and is degradation resistant. Furthermore, PON1 is essential in driving drug resistance through decreasing tumor inflammation and oxidative stress. This is a robust and rigorous study that provides a novel and druggable mechanism for drug resistance in cancer.
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