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Cancer homeostasis depends on a balance between activated oncogenic pathways driving tumorigenesis and engagement of stress response programs that counteract the inherent toxicity of such aberrant signaling. Although inhibition of oncogenic signaling pathways has been explored extensively, there is increasing evidence that overactivation of the same pathways can also disrupt cancer homeostasis and cause lethality. We show here that inhibition of protein phosphatase 2A (PP2A) hyperactivates multiple oncogenic pathways and engages stress responses in colon cancer cells. Genetic and compound screens identify combined inhibition of PP2A and WEE1 as synergistic in multiple cancer models by collapsing DNA replication and triggering premature mitosis followed by cell death. This combination also suppressed the growth of patient-derived tumors in vivo. Remarkably, acquired resistance to this drug combination suppressed the ability of colon cancer cells to form tumors in vivo. Our data suggest that paradoxical activation of oncogenic signaling can result in tumor-suppressive resistance.

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The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has antiproliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin-, nucleolus- and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription, rRNA expression and chromatin-remodeling proteins. The global transcriptional rate and nucleolus area increased along with nucleolar disorganization upon 24 h of FGF2 stimulation. FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing. RNA Pol I inhibition partially reversed the growth arrest induced by FGF2, indicating that changes in rRNA expression might be crucial for triggering the antiproliferative effect. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes and modulates the main cell transcription site, the nucleolus.

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The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has antiproliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin-, nucleolus- and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription, rRNA expression and chromatin-remodeling proteins. The global transcriptional rate and nucleolus area increased along with nucleolar disorganization upon 24 h of FGF2 stimulation. FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing. RNA Pol I inhibition partially reversed the growth arrest induced by FGF2, indicating that changes in rRNA expression might be crucial for triggering the antiproliferative effect. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes and modulates the main cell transcription site, the nucleolus.

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The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has antiproliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin-, nucleolus-, and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription, rRNA expression, and chromatin remodeling proteins. The global transcriptional rate and nucleolus area increased along with nucleolar disorganization upon 24 h of FGF2 stimulation. FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing. RNA Pol I inhibition partially reversed the growth arrest induced by FGF2, indicating that changes in rRNA expression may be crucial for triggering the antiproliferative effect. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes and modulates the main cell transcription site, the nucleolus.

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Malignant transformation involves an orchestrated rearrangement of cell cycle regulation mechanisms that must balance autonomic mitogenic impulses and deleterious oncogenic stress. Human papillomavirus (HPV) infection is highly prevalent in populations around the globe, whereas the incidence of cervical cancer is 0.15%. Since HPV infection primes cervical keratinocytes to undergo malignant transformation, we can assume that the balance between transforming mitogenic signals and oncogenic stress is rarely attained. We showed that highly transforming mitogenic signals triggered by HRasG12V activity in E6E7–HPV–keratinocytes generate strong replication and oxidative stresses. These stresses are counteracted by autophagy induction that buffers the rapid increase of ROS that is the main cause of genotoxic stress promoted by the oncoprotein. As a result, autophagy creates a narrow window of opportunity for malignant keratinocytes to emerge. This work shows that autophagy is crucial to allow the transition of E6E7 keratinocytes from an immortalized to a malignant state caused by HRasG12V.

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In malignant transformation, cellular stress-response pathways are dynami-cally mobilized to counterbalance oncogenic activity, keeping cancer cellsviable. Therapeutic disruption of this vulnerable homeostasis might changethe outcome of many human cancers, particularly those for which no effec-tive therapy is available. Here, we report the use of fibroblast growth factor2 (FGF2) to demonstrate that further mitogenic activation disrupts cellularhomeostasis and strongly sensitizes cancer cells to stress-targeted therapeu-tic inhibitors. We show that FGF2 enhanced replication and proteotoxicstresses in a K-Ras-driven murine cancer cell model, and combinations ofFGF2 and proteasome or DNA damage response-checkpoint inhibitorstriggered cell death. CRISPR/Cas9-mediated K-Ras depletion suppressedthe malignant phenotype and prevented these synergic toxicities in thesemurine cells. Moreover, in a panel of human Ewing’s sarcoma family tumorcells, sublethal concentrations of bortezomib (proteasome inhibitor) or VE-821 (ATR inhibitor) induced cell death when combined with FGF2. Sus-tained MAPK-ERK1/2 overactivation induced by FGF2 appears to under-lie these synthetic lethalities, as late pharmacological inhibition of thispathway restored cell homeostasis and prevented these described synergies.Our results highlight how mitotic signaling pathways which are frequentlyoverridden in malignant transformation might be exploited to disrupt therobustness of cancer cells, ultimately sensitizing them to stress-targeted ther-apies. This approach provides a new therapeutic rationale for human can-cers, with important implications for tumors still lacking effectivetreatment, and for those that frequently relapse after treatment with avail-able therapies.

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In malignant transformation, cellular stress-response pathways are dynami-cally mobilized to counterbalance oncogenic activity, keeping cancer cellsviable. Therapeutic disruption of this vulnerable homeostasis might changethe outcome of many human cancers, particularly those for which no effec-tive therapy is available. Here, we report the use of fibroblast growth factor2 (FGF2) to demonstrate that further mitogenic activation disrupts cellularhomeostasis and strongly sensitizes cancer cells to stress-targeted therapeu-tic inhibitors. We show that FGF2 enhanced replication and proteotoxicstresses in a K-Ras-driven murine cancer cell model, and combinations ofFGF2 and proteasome or DNA damage response-checkpoint inhibitorstriggered cell death. CRISPR/Cas9-mediated K-Ras depletion suppressedthe malignant phenotype and prevented these synergic toxicities in thesemurine cells. Moreover, in a panel of human Ewing’s sarcoma family tumorcells, sublethal concentrations of bortezomib (proteasome inhibitor) or VE-821 (ATR inhibitor) induced cell death when combined with FGF2. Sus-tained MAPK-ERK1/2 overactivation induced by FGF2 appears to under-lie these synthetic lethalities, as late pharmacological inhibition of thispathway restored cell homeostasis and prevented these described synergies.Our results highlight how mitotic signaling pathways which are frequentlyoverridden in malignant transformation might be exploited to disrupt therobustness of cancer cells, ultimately sensitizing them to stress-targeted ther-apies. This approach provides a new therapeutic rationale for human can-cers, with important implications for tumors still lacking effectivetreatment, and for those that frequently relapse after treatment with avail-able therapies.

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Fibroblast growth factor 2 (FGF2) is a well-known cell proliferation promoter; however, it can also induce cell cycle arrest. To gain insight into the molecular mechanisms of this antiproliferative effect, for the first time, the early systemic proteomic differences induced by this growth factor in a K-Ras-driven mouse tumor cell line using a quantitative proteomics approach are investigated. More than 2900 proteins are quantified, indicating that terms associated with metabolism, RNA processing, replication, and transcription are enriched among proteins differentially expressed upon FGF2 stimulation. Proteomic trend dynamics indicate that, for proteins mainly associated with DNA replication and carbohydrate metabolism, an FGF2 stimulus delays their abundance changes, whereas FGF2 stimulation accelerates other metabolic programs. Transcription regulatory network analysis indicates master regulators of FGF2 stimulation, including two critical transcription factors, FOSB and JUNB. Their expression dynamics, both in the Y1 cell line (a murine model of adenocarcinoma cells) and in two other human cell lines (SK-N-MC and UM-UC-3) also susceptible to FGF2 antiproliferative effects, are investigated. Both protein expression levels depend on fibroblast growth factor receptor (FGFR) and src signaling. JUNB and FOSB knockdown do not rescue cells from the growth arrest induced by FGF2; however, FOSB knockdown rescue cells from DNA replication delay, indicating that FOSB expression underlies one of the FGF2 antiproliferative effects, namely, S-phase progression delay.

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Fibroblast growth factor 2 (FGF2) is a well-known cell proliferation promoter; however, it can also induce cell cycle arrest. To gain insight into the molecular mechanisms of this antiproliferative effect, for the first time, the early systemic proteomic differences induced by this growth factor in a K-Ras-driven mouse tumor cell line using a quantitative proteomics approach are investigated. More than 2900 proteins are quantified, indicating that terms associated with metabolism, RNA processing, replication, and transcription are enriched among proteins differentially expressed upon FGF2 stimulation. Proteomic trend dynamics indicate that, for proteins mainly associated with DNA replication and carbohydrate metabolism, an FGF2 stimulus delays their abundance changes, whereas FGF2 stimulation accelerates other metabolic programs. Transcription regulatory network analysis indicates master regulators of FGF2 stimulation, including two critical transcription factors, FOSB and JUNB. Their expression dynamics, both in the Y1 cell line (a murine model of adenocarcinoma cells) and in two other human cell lines (SK-N-MC and UM-UC-3) also susceptible to FGF2 antiproliferative effects, are investigated. Both protein expression levels depend on fibroblast growth factor receptor (FGFR) and src signaling. JUNB and FOSB knockdown do not rescue cells from the growth arrest induced by FGF2; however, FOSB knockdown rescue cells from DNA replication delay, indicating that FOSB expression underlies one of the FGF2 antiproliferative effects, namely, S-phase progression delay.

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Fibroblast growth factor 2 (FGF2) is a well-known cell proliferation promoter; however, it can also induce cell cycle arrest. To gain insight into the molecular mechanisms of this antiproliferative effect, for the first time, the early systemic proteomic differences induced by this growth factor in a K-Ras-driven mouse tumor cell line using a quantitative proteomics approach are investigated. More than 2900 proteins are quantified, indicating that terms associated with metabolism, RNA processing, replication, and transcription are enriched among proteins differentially expressed upon FGF2 stimulation. Proteomic trend dynamics indicate that, for proteins mainly associated with DNA replication and carbohydrate metabolism, an FGF2 stimulus delays their abundance changes, whereas FGF2 stimulation accelerates other metabolic programs. Transcription regulatory network analysis indicates master regulators of FGF2 stimulation, including two critical transcription factors, FOSB and JUNB. Their expression dynamics, both in the Y1 cell line (a murine model of adenocarcinoma cells) and in two other human cell lines (SK-N-MC and UM-UC-3) also susceptible to FGF2 antiproliferative effects, are investigated. Both protein expression levels depend on fibroblast growth factor receptor (FGFR) and src signaling. JUNB and FOSB knockdown do not rescue cells from the growth arrest induced by FGF2; however, FOSB knockdown rescue cells from DNA replication delay, indicating that FOSB expression underlies one of the FGF2 antiproliferative effects, namely, S-phase progression delay.

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Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU- and EdU-incorporated parasites with the respective standard detection approaches: indirect immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and "click" chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re-estimated the phases G1, S, G2 + M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring.

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Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis , Trypanosoma brucei , and Trypanosoma cruzi to monitor their DNA replication. We used BrdU- and EdU-incorporated parasites with the respective standard detection approaches: indirect immunofluorescence to detect BrdU after standard denaturation (2 M HC l) and "click" chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HC l. Using a new value for HC l concentration, we re-estimated the phases G1, S, G2 + M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring.

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Extracellular protein disulfide isomerase (PDIA1) pool mediates thrombosis and vascular remodeling, however its externalization mechanisms remain unclear. We performed systematic pharmacological screening of secretory pathways affecting extracellular PDIA1 in endothelial cells (EC). We identified cell-surface (csPDIA1) and secreted non-particulated PDIA1 pools in EC. Such Golgi bypass also occurred for secreted PDIA1 in EC at baseline or after PMA, thrombin or ATP stimulation. Inhibitors of Type I, II and III unconventional routes, secretory lysosomes and recycling endosomes, including syntaxin-12 deletion, did not impair EC PDIA1 externalization This suggests predominantly Golgi-independent unconventional secretory route(s), which were GRASP55-independent. Also, these data reinforce a vesicular-type traffic for PDIA1. We further showed that PDIA1 traffic is ATP-independent, while actin or tubulin cytoskeletal disruption markedly increased EC PDIA1 secretion. Clathrin inhibition enhanced extracellular soluble PDIA1, suggesting dynamic cycling. Externalized PDIA1 represents <2% of intracellular PDIA1. PDIA1 was robustly secreted by physiological levels of arterial laminar shear in EC and supported alpha 5 integrin thiol oxidation. Such results help clarify signaling and homeostatic mechanisms involved in multiple (patho)physiological extracellular PDIA1 functions.

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Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH) binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH). We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA.

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The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of ß-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47 (phox) phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47 (phox) mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts.

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