Due to its fundamental role in all eukaryotic cells, a deeper understanding of the molecular mech... more Due to its fundamental role in all eukaryotic cells, a deeper understanding of the molecular mechanisms underlying ubiquitination is of central importance. Being responsible for chain specificity and substrate recognition, E3 ligases are the selective elements of the ubiquitination process. In this review, we discuss different cellular pathways regulated by one of the first identified E3 ligase, NEDD4, focusing on its pathophysiological role, its known targets and modulators. In addition, we highlight small molecule inhibitors that act on NEDD4 and discuss new strategies to effectively target this E3 enzyme.
HIGHLIGHTS ERGIC-53 binds ERp44 and accelerates its exit from the ER Genetic or pharmacological i... more HIGHLIGHTS ERGIC-53 binds ERp44 and accelerates its exit from the ER Genetic or pharmacological inhibition of ERGIC-53 impairs ERp44 retrieval function ERp44 and ERGIC-53 form a functional couple needed for postER QC
In addition, by stimulating secretion of lactate, mutp53 induces extracellular acidification regu... more In addition, by stimulating secretion of lactate, mutp53 induces extracellular acidification regulating the fine crosstalk between cancer and stromal cells (Cordani et al., 2016). Mutant p53 can also affect steroid biosynthesis, in particular by acting on the mevalonate pathway-this has a profound oncogenic impact, also mediated by activation of YAP/TAZ (Di Agostino et al., 2015) and ID4-regulated angiogenesis (Fontemaggi et al., 2009). More recently, it was discovered that mutant p53 promotes transcription of the ER localized enzyme ectonucleside triphosphate diphosphohydrolase (ENTPD5). This leads to stimulation of the calnexin/calreticulin-dependent quality control that could help cells to survive in proteotoxic conditions. Importantly, by upregulating ENTPD5 mutant p53 promotes N-glycosylation, folding, and maturation of secreted growth factors and membrane receptors, in turn enhancing cancer cell proliferation and/or survival (Vogiatzi et al., 2016). 2.3 Targeting mutant p53 Given its central role in cancer progression, invasion, and metastasis, targeting mutant p53 function may reveal potential strategies to develop novel anti-cancer therapies (Figure 3). Considering that mutp53 stability is key to its GOF, one approach is to stimulate its degradation. As for wt p53, MDM2 seems to be a major determinant of mutant p53 levels. Therefore, specifically and selectively activating MDM2-induced degradation may be a strategy to eliminate mutant p53 (Terzian et al., 2008). Mutant p53 is stabilized by interaction with the HSP90 machinery, which includes HSP90, HSP70 and other cochaperones (Peng et al., 2001). This complex binds MDM2 and CHiP, blocking their capacity to induce mutant p53 degradation. Disruption of this complex or inhibition of chaperones activity in cultured cells, induces the release of MDM2 and CHiP from inhibition and triggered mutant
In the past decades, many studies reported the presence of endoplasmic reticulum (ER)-resident pr... more In the past decades, many studies reported the presence of endoplasmic reticulum (ER)-resident proteins in the cytosol. However, the mechanisms by which these proteins relocate and whether they exert cytosolic functions remain unknown. We find that a subset of ER luminal proteins accumulates in the cytosol of glioblastoma cells isolated from mouse and human tumors. In cultured cells, ER protein reflux to the cytosol occurs upon ER proteostasis perturbation. Using the ER luminal protein anterior gradient 2 (AGR2) as a proof of concept, we tested whether the refluxed proteins gain new functions in the cytosol. We find that refluxed, cytosolic AGR2 binds and inhibits the tumor suppressor p53. These data suggest that ER reflux constitutes an ER surveillance mechanism to relieve the ER from its contents upon stress, providing a selective advantage to tumor cells through gainof-cytosolic functions-a phenomenon we name ER to Cytosol Signaling (ERCYS).
ABSTRACTIn the past decades many studies reported Endoplasmic Reticulum (ER) resident proteins to... more ABSTRACTIn the past decades many studies reported Endoplasmic Reticulum (ER) resident proteins to localize to the cytosol but the mechanisms by which this occurs and whether these proteins exert cytosolic functions remain unknown. We found that select ER luminal proteins accumulate in the cytosol of glioblastoma cells isolated from mouse and human tumors. In cultured cells ER protein reflux to the cytosol occurs upon proteostasis perturbation. As such we investigated whether refluxed proteins gain new functions in the cytosol thus providing advantage to tumor cells. Using the ER luminal protein AGR2 as a model, we showed that it is refluxed to the cytosol where it binds and inhibits the tumor suppressor p53. We named this phenomenon ER to Cytosol Signaling (ERCYS) as an ER surveillance mechanism conserved in Eukaryotes to relieve the ER from its contents upon stress and to provide selective advantage to tumor cells through gain-of-cytosolic functions.
Glucocorticoids (GCs) are commonly used as therapeutic agents for immune-mediated diseases and le... more Glucocorticoids (GCs) are commonly used as therapeutic agents for immune-mediated diseases and leukemia. However, considerable inter-individual differences in efficacy have been reported. Several reports indicate that the inhibitor of mTOR rapamycin can reverse GC resistance, but the molecular mechanism involved in this synergistic effect has not been fully defined. In this context, we explored the differential miRNA expression in a GC-resistant CCRF-CEM cell line after treatment with rapamycin alone or in co-treatment with methylprednisolone (MP). The expression analysis identified 70, 99 and 96 miRNAs that were differentially expressed after treatment with MP, rapamycin and their combination compared to non-treated controls, respectively. Two pathways were exclusively altered as a result of the co-treatment: the MAPK and ErbB pathways. We validated the only miRNA upregulated specifically by the co-treatment associated with the MAPK signaling, miR-331-3p. Looking for miR-331-3p tar...
Similar to other RNA viruses, SARS-CoV-2 must (1) enter a target/host cell, (2) reprogram it to e... more Similar to other RNA viruses, SARS-CoV-2 must (1) enter a target/host cell, (2) reprogram it to ensure its replication, (3) exit the host cell, and (4) repeat this cycle for exponential growth. During the exit step, the virus hijacks the sophisticated machineries that host cells employ to correctly fold, assemble, and transport proteins along the exocytic pathway. Therefore, secretory pathway–mediated assemblage and excretion of infective particles represent appealing targets to reduce the efficacy of virus biogenesis, if not to block it completely. Here, we analyze and discuss the contribution of the molecular machines operating in the early secretory pathway in the biogenesis of SARS-CoV-2 and their relevance for potential antiviral targeting. The fact that these molecular machines are conserved throughout evolution, together with the redundancy and tissue specificity of their components, provides opportunities in the search for unique proteins essential for SARS-CoV-2 biology tha...
Discrimination between properly folded proteins and those that do not reach this state is necessa... more Discrimination between properly folded proteins and those that do not reach this state is necessary for cells to achieve functionality. Eukaryotic cells have evolved several mechanisms to ensure secretory protein quality control, which allows efficiency and fidelity in protein production. Among the actors involved in such process, both endoplasmic reticulum (ER) and the Golgi complex play prominent roles in protein synthesis, biogenesis and secretion. ER and Golgi functions ensure that only properly folded proteins are allowed to flow through the secretory pathway while improperly folded proteins have to be eliminated to not impinge on cellular functions. Thus, complex quality control and degradation machineries are crucial to prevent the toxic accumulation of improperly folded proteins. However, in some instances, improperly folded proteins can escape the quality control systems thereby contributing to several human diseases. Herein, we summarize how the early secretory pathways co...
The endoplasmic reticulum (ER) is a multifunctional organelle that constitutes the entry into the... more The endoplasmic reticulum (ER) is a multifunctional organelle that constitutes the entry into the secretory pathway. The ER contributes to the maintenance of cellular calcium homeostasis, lipid synthesis and productive secretory, and transmembrane protein folding. Physiological, chemical, and pathological factors that compromise ER homeostasis lead to endoplasmic reticulum stress (ER stress). To cope with this situation, cells activate an adaptive signaling pathway termed the unfolded protein response (UPR) that aims at restoring ER homeostasis. The UPR is transduced through post-translational, translational, post-transcriptional, and transcriptional mechanisms initiated by three ER-resident sensors, inositol-requiring protein 1a, activating transcription factor 6a, and PRKR-like endoplasmic reticulum kinase. Determining the in and out of ER homeostasis control and UPR activation still represents a challenge for the community. Hence, standardized criteria and methodologies need to be proposed for monitoring ER homeostasis and ER stress in different model systems. Here, we summarize the pathways that are activated during ER stress and provide approaches aimed at assess ER homeostasis and stress in vitro and in vivo mammalian systems that can be used by researchers to plan and interpret experiments. We recommend the use of multiple assays to verify ER stress because no individual assay is guaranteed to be the most appropriate one.
Human anterior gradient proteins AGR2 and AGR3 are overexpressed in a variety of adenocarcinomas ... more Human anterior gradient proteins AGR2 and AGR3 are overexpressed in a variety of adenocarcinomas and are often secreted in cancer patients' specimens, which suggests a role for AGR proteins in intra and extracellular compartments. Although these proteins exhibit high sequence homology, AGR2 is predominantly described as a pro-oncogene and a potential prognostic biomarker. However, little is known about the function of AGR3. Therefore, the aim of the present study was to investigate the role of AGR3 in breast cancer. The results demonstrated that breast cancer cells secrete AGR3. Furthermore, it was revealed that extracellular AGR3 (eAGR3) regulates tumor cell adhesion and migration. The current study indicated that the pharmacological and genetic perturbation of Src kinase signaling, through treatment with Dasatinib (protein kinase inhibitor) or investigating cells that express a dominant-negative form of Src, significantly abrogated eAGR3-mediated breast cancer cell migration. Therefore, the results indicated that eAGR3 may control tumor cell migration via activation of Src kinases. The results of the present study indicated that eAGR3 may serve as a microenvironmental signaling molecule in tumor-associated processes.
Missense mutations in the TP53 gene are frequent in human cancers, giving rise to mutant p53 prot... more Missense mutations in the TP53 gene are frequent in human cancers, giving rise to mutant p53 proteins that can acquire oncogenic properties. Gain of function mutant p53 proteins can enhance tumour aggressiveness by promoting cell invasion, metastasis and chemoresistance. Accumulating evidences indicate that mutant p53 proteins can also modulate cell homeostatic processes, suggesting that missense p53 mutation may increase resistance of tumour cells to intrinsic and extrinsic cancer-related stress conditions, thus offering a selective advantage. Here we provide evidence that mutant p53 proteins can modulate the Unfolded Protein Response (UPR) to increase cell survival upon Endoplasmic Reticulum (ER) stress, a condition to which cancer cells are exposed during tumour formation and progression, as well as during therapy. Mechanistically, this action of mutant p53 is due to enhanced activation of the pro-survival UPR effector ATF6, coordinated with inhibition of the pro-apoptotic UPR effectors JNK and CHOP. In a triple-negative breast cancer cell model with missense TP53 mutation, we found that ATF6 activity is necessary for viability and invasion phenotypes. Together, these findings suggest that ATF6 inhibitors might be combined with mutant p53-targeting drugs to specifically sensitise cancer cells to endogenous or chemotherapy-induced ER stress.
Tumor cells are exposed to intrinsic and environmental challenges that trigger endoplasmic reticu... more Tumor cells are exposed to intrinsic and environmental challenges that trigger endoplasmic reticulum (ER) homeostasis alteration, in turn leading to ER stress. To cope with this, tumor cells engage an adaptive signaling pathway, the unfolded protein response (UPR) thus promoting the acquisition of malignant features. As such, glioblastoma multiforme (GBM), the most aggressive primary brain tumors, exhibit constitutive UPR signals to sustain growth. Herein, we showed that signaling elicited by one of the UPR sensors, IRE1, promotes GBM tumor invasion, angiogenesis and infiltration by macrophages. Hence, high IRE1 activity in tumors predicts worse outcome. We further dissect IRE1-dependent mechanisms that shape the brain tumor immune microenvironment towards myeloid cells. We identify an IRE1-dependent signaling pathway that directly controls the expression/release of proinflammatory chemokines (CXCL2, IL6, IL8) leading to tumor cell-mediated chemoattraction of neutrophils and macroph...
The still largely obscure molecular events in the glioblastoma oncogenesis, a primary brain tumor... more The still largely obscure molecular events in the glioblastoma oncogenesis, a primary brain tumor characterized by an inevitably dismal prognosis, impel for investigation. The importance of Long noncoding RNAs as regulators of gene expression has recently become evident. Among them, H19 has a recognized oncogenic role in several types of human tumors and was shown to correlate to some oncogenic aspects of glioblastoma cells. Here we, hypothesyze that in glioblastoma H19 exerts its function through the interaction with the catalytic subunit of the PRC2 complex, EZH2. By employing a factor analysis on a SAGE dataset of 12 glioblastoma samples, we show that H19 expression in glioblastoma tissues correlates with that of several genes involved in glioblastoma growth and progression. H19 knock-down reduces viability, migration and invasiveness of two distinct human glioblastoma cell lines. Most importantly, we provide a mechanistic perspective about the role of H19 in glioblastoma cells, by showing that its expression is inversely linked to that of NKD1, a negative regulator of Wnt pathway, suggesting that H19 might regulate NKD1 transcription via EZH2-induced H3K27 trimethylation of its promoter. Indeed, we showed that H19 binds EZH2 in glioblastoma cells, and that EZH2 binding to NKD1 and other promoters is impaired by H19 silencing. In this work we describe H19 as part of an epigenetic modulation program executed by EZH2, that results in the repression of Nkd1. We believe that our results can provide a new piece to the complex puzzle of H19 function in glioblastoma.
The tumor suppressor DAB2IP contributes to modulate the network of information established betwee... more The tumor suppressor DAB2IP contributes to modulate the network of information established between cancer cells and tumor microenvironment. Epigenetic and post-transcriptional inactivation of this protein is commonly observed in multiple human malignancies, and can potentially favor progression of tumors driven by a variety of genetic mutations. Performing a high-throughput screening of a large collection of human microRNA mimics, we identified miR-149-3p as a negative post-transcriptional modulator of DAB2IP. By efficiently downregulating DAB2IP, this miRNA enhances cancer cell motility and invasiveness, facilitating activation of NF-kB signaling and promoting expression of pro-inflammatory and pro-angiogenic factors. In addition, we found that miR-149-3p secreted by prostate cancer cells induces DAB2IP downregulation in recipient vascular endothelial cells, stimulating their proliferation and motility, thus potentially remodeling the tumor microenvironment. Finally, we found that ...
Proceedings of the National Academy of Sciences, 2017
Significance Understanding the molecular factors that shape the impact of hyperinsulinemia on can... more Significance Understanding the molecular factors that shape the impact of hyperinsulinemia on cancer progression would improve treatment of obese and diabetic patients. This work contributes to such understanding by showing that gain-of-function p53 mutations can modify the response of cancer cells to insulin, increasing proliferation and motility through enhanced activation of AKT.
Glioblastoma multiforme (GBM) is the most common and deadliest primary brain tumor, driving patie... more Glioblastoma multiforme (GBM) is the most common and deadliest primary brain tumor, driving patients to death within 15 months after diagnosis (short term survivors, ST), with the exception of a small fraction of patients (long term survivors, LT) surviving longer than 36 months. Here we present deep sequencing data showing that peritumoral (P) areas differ from healthy white matter, but share with their respective frankly tumoral (C) samples, a number of mRNAs and microRNAs representative of extracellular matrix remodeling, TGFβ and signaling, of the involvement of cell types different from tumor cells but contributing to tumor growth, such as microglia or reactive astrocytes. Moreover, we provide evidence about RNAs differentially expressed in ST vs LT samples, suggesting the contribution of TGF-β signaling in this distinction too. We also show that the edited form of miR-376c-3p is reduced in C vs P samples and in ST tumors compared to LT ones. As a whole, our study provides new ...
Glioblastoma multiforme (GBM) is the most common and most malignant primary brain tumour. Despite... more Glioblastoma multiforme (GBM) is the most common and most malignant primary brain tumour. Despite multimodal aggressive treatment, the prognosis of GBM patients is poor and the median survival time after diagnosis is still in the range of just 12 months, with the interesting exception of a small fraction (less than 10%) of patients, referred to as long-term survivors (LTS), who survive longer than 36 months. In general, GBM recurrence occurs in peritumoral tissue in about 90% of patients, making this area a crucial one for studies about the molecular pathways perturbed early in GBM malignant evolution. By SAGE analysis, we have studied the transcriptome and miRNome of GBM tissues from 13 patients classified as either STS or LTS. For each patient, we have analysed one sample representing the center of the tumour (C) and another excised from the tumour-free peri-tumoral area (P). We describe a group of RNAs (coding and noncoding) found to be commonly modulated in P as well as in C samples compared to healthy white matter, that we think may represent early markers of GBM pathogenesis, and we also present RNAs that differentiate ST from LT tumors. Among upregulated RNAs in both comparisons, we have found a remarkable enrichment of mesenchymal-stem-like markers. For these comparisons, we will also show the relevant pathways affected by the modulated RNAs. Our data show that some genes and molecular pathways known to be perturbed in glioblastoma are already disrupted in the apparently tumor-free peri-tumor area, where it is conceivable that early tumor development occurs, predisposing cells to recurrence. Moreover, our results differentiating STS from LTS may help unravel the basis of this still elusive distinction.
Breast cancer is the most frequently diagnosed malignancy in women, and mutations in the tumor su... more Breast cancer is the most frequently diagnosed malignancy in women, and mutations in the tumor suppressor p53 are commonly detected in the most aggressive subtypes. The majority of gene alterations are missense substitutions, leading to expression of mutant forms of the p53 protein that are frequently detected at high levels in cancer cells. P53 mutants not only lose the physiological tumor-suppressive activity of the wild-type p53 protein but also acquire novel powerful oncogenic functions, referred to as gain of function, that may actively confer a selective advantage during tumor progression. Some of the best-characterized oncogenic activities of mutant p53 are mediated by its ability to form aberrant protein complexes with other transcription factors or proteins not directly related to gene transcription. The set of cellular proteins available to interact with mutant p53 is dependent on cell type and extensively affected by environmental signals, so the prognostic impact of p53 ...
Due to its fundamental role in all eukaryotic cells, a deeper understanding of the molecular mech... more Due to its fundamental role in all eukaryotic cells, a deeper understanding of the molecular mechanisms underlying ubiquitination is of central importance. Being responsible for chain specificity and substrate recognition, E3 ligases are the selective elements of the ubiquitination process. In this review, we discuss different cellular pathways regulated by one of the first identified E3 ligase, NEDD4, focusing on its pathophysiological role, its known targets and modulators. In addition, we highlight small molecule inhibitors that act on NEDD4 and discuss new strategies to effectively target this E3 enzyme.
HIGHLIGHTS ERGIC-53 binds ERp44 and accelerates its exit from the ER Genetic or pharmacological i... more HIGHLIGHTS ERGIC-53 binds ERp44 and accelerates its exit from the ER Genetic or pharmacological inhibition of ERGIC-53 impairs ERp44 retrieval function ERp44 and ERGIC-53 form a functional couple needed for postER QC
In addition, by stimulating secretion of lactate, mutp53 induces extracellular acidification regu... more In addition, by stimulating secretion of lactate, mutp53 induces extracellular acidification regulating the fine crosstalk between cancer and stromal cells (Cordani et al., 2016). Mutant p53 can also affect steroid biosynthesis, in particular by acting on the mevalonate pathway-this has a profound oncogenic impact, also mediated by activation of YAP/TAZ (Di Agostino et al., 2015) and ID4-regulated angiogenesis (Fontemaggi et al., 2009). More recently, it was discovered that mutant p53 promotes transcription of the ER localized enzyme ectonucleside triphosphate diphosphohydrolase (ENTPD5). This leads to stimulation of the calnexin/calreticulin-dependent quality control that could help cells to survive in proteotoxic conditions. Importantly, by upregulating ENTPD5 mutant p53 promotes N-glycosylation, folding, and maturation of secreted growth factors and membrane receptors, in turn enhancing cancer cell proliferation and/or survival (Vogiatzi et al., 2016). 2.3 Targeting mutant p53 Given its central role in cancer progression, invasion, and metastasis, targeting mutant p53 function may reveal potential strategies to develop novel anti-cancer therapies (Figure 3). Considering that mutp53 stability is key to its GOF, one approach is to stimulate its degradation. As for wt p53, MDM2 seems to be a major determinant of mutant p53 levels. Therefore, specifically and selectively activating MDM2-induced degradation may be a strategy to eliminate mutant p53 (Terzian et al., 2008). Mutant p53 is stabilized by interaction with the HSP90 machinery, which includes HSP90, HSP70 and other cochaperones (Peng et al., 2001). This complex binds MDM2 and CHiP, blocking their capacity to induce mutant p53 degradation. Disruption of this complex or inhibition of chaperones activity in cultured cells, induces the release of MDM2 and CHiP from inhibition and triggered mutant
In the past decades, many studies reported the presence of endoplasmic reticulum (ER)-resident pr... more In the past decades, many studies reported the presence of endoplasmic reticulum (ER)-resident proteins in the cytosol. However, the mechanisms by which these proteins relocate and whether they exert cytosolic functions remain unknown. We find that a subset of ER luminal proteins accumulates in the cytosol of glioblastoma cells isolated from mouse and human tumors. In cultured cells, ER protein reflux to the cytosol occurs upon ER proteostasis perturbation. Using the ER luminal protein anterior gradient 2 (AGR2) as a proof of concept, we tested whether the refluxed proteins gain new functions in the cytosol. We find that refluxed, cytosolic AGR2 binds and inhibits the tumor suppressor p53. These data suggest that ER reflux constitutes an ER surveillance mechanism to relieve the ER from its contents upon stress, providing a selective advantage to tumor cells through gainof-cytosolic functions-a phenomenon we name ER to Cytosol Signaling (ERCYS).
ABSTRACTIn the past decades many studies reported Endoplasmic Reticulum (ER) resident proteins to... more ABSTRACTIn the past decades many studies reported Endoplasmic Reticulum (ER) resident proteins to localize to the cytosol but the mechanisms by which this occurs and whether these proteins exert cytosolic functions remain unknown. We found that select ER luminal proteins accumulate in the cytosol of glioblastoma cells isolated from mouse and human tumors. In cultured cells ER protein reflux to the cytosol occurs upon proteostasis perturbation. As such we investigated whether refluxed proteins gain new functions in the cytosol thus providing advantage to tumor cells. Using the ER luminal protein AGR2 as a model, we showed that it is refluxed to the cytosol where it binds and inhibits the tumor suppressor p53. We named this phenomenon ER to Cytosol Signaling (ERCYS) as an ER surveillance mechanism conserved in Eukaryotes to relieve the ER from its contents upon stress and to provide selective advantage to tumor cells through gain-of-cytosolic functions.
Glucocorticoids (GCs) are commonly used as therapeutic agents for immune-mediated diseases and le... more Glucocorticoids (GCs) are commonly used as therapeutic agents for immune-mediated diseases and leukemia. However, considerable inter-individual differences in efficacy have been reported. Several reports indicate that the inhibitor of mTOR rapamycin can reverse GC resistance, but the molecular mechanism involved in this synergistic effect has not been fully defined. In this context, we explored the differential miRNA expression in a GC-resistant CCRF-CEM cell line after treatment with rapamycin alone or in co-treatment with methylprednisolone (MP). The expression analysis identified 70, 99 and 96 miRNAs that were differentially expressed after treatment with MP, rapamycin and their combination compared to non-treated controls, respectively. Two pathways were exclusively altered as a result of the co-treatment: the MAPK and ErbB pathways. We validated the only miRNA upregulated specifically by the co-treatment associated with the MAPK signaling, miR-331-3p. Looking for miR-331-3p tar...
Similar to other RNA viruses, SARS-CoV-2 must (1) enter a target/host cell, (2) reprogram it to e... more Similar to other RNA viruses, SARS-CoV-2 must (1) enter a target/host cell, (2) reprogram it to ensure its replication, (3) exit the host cell, and (4) repeat this cycle for exponential growth. During the exit step, the virus hijacks the sophisticated machineries that host cells employ to correctly fold, assemble, and transport proteins along the exocytic pathway. Therefore, secretory pathway–mediated assemblage and excretion of infective particles represent appealing targets to reduce the efficacy of virus biogenesis, if not to block it completely. Here, we analyze and discuss the contribution of the molecular machines operating in the early secretory pathway in the biogenesis of SARS-CoV-2 and their relevance for potential antiviral targeting. The fact that these molecular machines are conserved throughout evolution, together with the redundancy and tissue specificity of their components, provides opportunities in the search for unique proteins essential for SARS-CoV-2 biology tha...
Discrimination between properly folded proteins and those that do not reach this state is necessa... more Discrimination between properly folded proteins and those that do not reach this state is necessary for cells to achieve functionality. Eukaryotic cells have evolved several mechanisms to ensure secretory protein quality control, which allows efficiency and fidelity in protein production. Among the actors involved in such process, both endoplasmic reticulum (ER) and the Golgi complex play prominent roles in protein synthesis, biogenesis and secretion. ER and Golgi functions ensure that only properly folded proteins are allowed to flow through the secretory pathway while improperly folded proteins have to be eliminated to not impinge on cellular functions. Thus, complex quality control and degradation machineries are crucial to prevent the toxic accumulation of improperly folded proteins. However, in some instances, improperly folded proteins can escape the quality control systems thereby contributing to several human diseases. Herein, we summarize how the early secretory pathways co...
The endoplasmic reticulum (ER) is a multifunctional organelle that constitutes the entry into the... more The endoplasmic reticulum (ER) is a multifunctional organelle that constitutes the entry into the secretory pathway. The ER contributes to the maintenance of cellular calcium homeostasis, lipid synthesis and productive secretory, and transmembrane protein folding. Physiological, chemical, and pathological factors that compromise ER homeostasis lead to endoplasmic reticulum stress (ER stress). To cope with this situation, cells activate an adaptive signaling pathway termed the unfolded protein response (UPR) that aims at restoring ER homeostasis. The UPR is transduced through post-translational, translational, post-transcriptional, and transcriptional mechanisms initiated by three ER-resident sensors, inositol-requiring protein 1a, activating transcription factor 6a, and PRKR-like endoplasmic reticulum kinase. Determining the in and out of ER homeostasis control and UPR activation still represents a challenge for the community. Hence, standardized criteria and methodologies need to be proposed for monitoring ER homeostasis and ER stress in different model systems. Here, we summarize the pathways that are activated during ER stress and provide approaches aimed at assess ER homeostasis and stress in vitro and in vivo mammalian systems that can be used by researchers to plan and interpret experiments. We recommend the use of multiple assays to verify ER stress because no individual assay is guaranteed to be the most appropriate one.
Human anterior gradient proteins AGR2 and AGR3 are overexpressed in a variety of adenocarcinomas ... more Human anterior gradient proteins AGR2 and AGR3 are overexpressed in a variety of adenocarcinomas and are often secreted in cancer patients' specimens, which suggests a role for AGR proteins in intra and extracellular compartments. Although these proteins exhibit high sequence homology, AGR2 is predominantly described as a pro-oncogene and a potential prognostic biomarker. However, little is known about the function of AGR3. Therefore, the aim of the present study was to investigate the role of AGR3 in breast cancer. The results demonstrated that breast cancer cells secrete AGR3. Furthermore, it was revealed that extracellular AGR3 (eAGR3) regulates tumor cell adhesion and migration. The current study indicated that the pharmacological and genetic perturbation of Src kinase signaling, through treatment with Dasatinib (protein kinase inhibitor) or investigating cells that express a dominant-negative form of Src, significantly abrogated eAGR3-mediated breast cancer cell migration. Therefore, the results indicated that eAGR3 may control tumor cell migration via activation of Src kinases. The results of the present study indicated that eAGR3 may serve as a microenvironmental signaling molecule in tumor-associated processes.
Missense mutations in the TP53 gene are frequent in human cancers, giving rise to mutant p53 prot... more Missense mutations in the TP53 gene are frequent in human cancers, giving rise to mutant p53 proteins that can acquire oncogenic properties. Gain of function mutant p53 proteins can enhance tumour aggressiveness by promoting cell invasion, metastasis and chemoresistance. Accumulating evidences indicate that mutant p53 proteins can also modulate cell homeostatic processes, suggesting that missense p53 mutation may increase resistance of tumour cells to intrinsic and extrinsic cancer-related stress conditions, thus offering a selective advantage. Here we provide evidence that mutant p53 proteins can modulate the Unfolded Protein Response (UPR) to increase cell survival upon Endoplasmic Reticulum (ER) stress, a condition to which cancer cells are exposed during tumour formation and progression, as well as during therapy. Mechanistically, this action of mutant p53 is due to enhanced activation of the pro-survival UPR effector ATF6, coordinated with inhibition of the pro-apoptotic UPR effectors JNK and CHOP. In a triple-negative breast cancer cell model with missense TP53 mutation, we found that ATF6 activity is necessary for viability and invasion phenotypes. Together, these findings suggest that ATF6 inhibitors might be combined with mutant p53-targeting drugs to specifically sensitise cancer cells to endogenous or chemotherapy-induced ER stress.
Tumor cells are exposed to intrinsic and environmental challenges that trigger endoplasmic reticu... more Tumor cells are exposed to intrinsic and environmental challenges that trigger endoplasmic reticulum (ER) homeostasis alteration, in turn leading to ER stress. To cope with this, tumor cells engage an adaptive signaling pathway, the unfolded protein response (UPR) thus promoting the acquisition of malignant features. As such, glioblastoma multiforme (GBM), the most aggressive primary brain tumors, exhibit constitutive UPR signals to sustain growth. Herein, we showed that signaling elicited by one of the UPR sensors, IRE1, promotes GBM tumor invasion, angiogenesis and infiltration by macrophages. Hence, high IRE1 activity in tumors predicts worse outcome. We further dissect IRE1-dependent mechanisms that shape the brain tumor immune microenvironment towards myeloid cells. We identify an IRE1-dependent signaling pathway that directly controls the expression/release of proinflammatory chemokines (CXCL2, IL6, IL8) leading to tumor cell-mediated chemoattraction of neutrophils and macroph...
The still largely obscure molecular events in the glioblastoma oncogenesis, a primary brain tumor... more The still largely obscure molecular events in the glioblastoma oncogenesis, a primary brain tumor characterized by an inevitably dismal prognosis, impel for investigation. The importance of Long noncoding RNAs as regulators of gene expression has recently become evident. Among them, H19 has a recognized oncogenic role in several types of human tumors and was shown to correlate to some oncogenic aspects of glioblastoma cells. Here we, hypothesyze that in glioblastoma H19 exerts its function through the interaction with the catalytic subunit of the PRC2 complex, EZH2. By employing a factor analysis on a SAGE dataset of 12 glioblastoma samples, we show that H19 expression in glioblastoma tissues correlates with that of several genes involved in glioblastoma growth and progression. H19 knock-down reduces viability, migration and invasiveness of two distinct human glioblastoma cell lines. Most importantly, we provide a mechanistic perspective about the role of H19 in glioblastoma cells, by showing that its expression is inversely linked to that of NKD1, a negative regulator of Wnt pathway, suggesting that H19 might regulate NKD1 transcription via EZH2-induced H3K27 trimethylation of its promoter. Indeed, we showed that H19 binds EZH2 in glioblastoma cells, and that EZH2 binding to NKD1 and other promoters is impaired by H19 silencing. In this work we describe H19 as part of an epigenetic modulation program executed by EZH2, that results in the repression of Nkd1. We believe that our results can provide a new piece to the complex puzzle of H19 function in glioblastoma.
The tumor suppressor DAB2IP contributes to modulate the network of information established betwee... more The tumor suppressor DAB2IP contributes to modulate the network of information established between cancer cells and tumor microenvironment. Epigenetic and post-transcriptional inactivation of this protein is commonly observed in multiple human malignancies, and can potentially favor progression of tumors driven by a variety of genetic mutations. Performing a high-throughput screening of a large collection of human microRNA mimics, we identified miR-149-3p as a negative post-transcriptional modulator of DAB2IP. By efficiently downregulating DAB2IP, this miRNA enhances cancer cell motility and invasiveness, facilitating activation of NF-kB signaling and promoting expression of pro-inflammatory and pro-angiogenic factors. In addition, we found that miR-149-3p secreted by prostate cancer cells induces DAB2IP downregulation in recipient vascular endothelial cells, stimulating their proliferation and motility, thus potentially remodeling the tumor microenvironment. Finally, we found that ...
Proceedings of the National Academy of Sciences, 2017
Significance Understanding the molecular factors that shape the impact of hyperinsulinemia on can... more Significance Understanding the molecular factors that shape the impact of hyperinsulinemia on cancer progression would improve treatment of obese and diabetic patients. This work contributes to such understanding by showing that gain-of-function p53 mutations can modify the response of cancer cells to insulin, increasing proliferation and motility through enhanced activation of AKT.
Glioblastoma multiforme (GBM) is the most common and deadliest primary brain tumor, driving patie... more Glioblastoma multiforme (GBM) is the most common and deadliest primary brain tumor, driving patients to death within 15 months after diagnosis (short term survivors, ST), with the exception of a small fraction of patients (long term survivors, LT) surviving longer than 36 months. Here we present deep sequencing data showing that peritumoral (P) areas differ from healthy white matter, but share with their respective frankly tumoral (C) samples, a number of mRNAs and microRNAs representative of extracellular matrix remodeling, TGFβ and signaling, of the involvement of cell types different from tumor cells but contributing to tumor growth, such as microglia or reactive astrocytes. Moreover, we provide evidence about RNAs differentially expressed in ST vs LT samples, suggesting the contribution of TGF-β signaling in this distinction too. We also show that the edited form of miR-376c-3p is reduced in C vs P samples and in ST tumors compared to LT ones. As a whole, our study provides new ...
Glioblastoma multiforme (GBM) is the most common and most malignant primary brain tumour. Despite... more Glioblastoma multiforme (GBM) is the most common and most malignant primary brain tumour. Despite multimodal aggressive treatment, the prognosis of GBM patients is poor and the median survival time after diagnosis is still in the range of just 12 months, with the interesting exception of a small fraction (less than 10%) of patients, referred to as long-term survivors (LTS), who survive longer than 36 months. In general, GBM recurrence occurs in peritumoral tissue in about 90% of patients, making this area a crucial one for studies about the molecular pathways perturbed early in GBM malignant evolution. By SAGE analysis, we have studied the transcriptome and miRNome of GBM tissues from 13 patients classified as either STS or LTS. For each patient, we have analysed one sample representing the center of the tumour (C) and another excised from the tumour-free peri-tumoral area (P). We describe a group of RNAs (coding and noncoding) found to be commonly modulated in P as well as in C samples compared to healthy white matter, that we think may represent early markers of GBM pathogenesis, and we also present RNAs that differentiate ST from LT tumors. Among upregulated RNAs in both comparisons, we have found a remarkable enrichment of mesenchymal-stem-like markers. For these comparisons, we will also show the relevant pathways affected by the modulated RNAs. Our data show that some genes and molecular pathways known to be perturbed in glioblastoma are already disrupted in the apparently tumor-free peri-tumor area, where it is conceivable that early tumor development occurs, predisposing cells to recurrence. Moreover, our results differentiating STS from LTS may help unravel the basis of this still elusive distinction.
Breast cancer is the most frequently diagnosed malignancy in women, and mutations in the tumor su... more Breast cancer is the most frequently diagnosed malignancy in women, and mutations in the tumor suppressor p53 are commonly detected in the most aggressive subtypes. The majority of gene alterations are missense substitutions, leading to expression of mutant forms of the p53 protein that are frequently detected at high levels in cancer cells. P53 mutants not only lose the physiological tumor-suppressive activity of the wild-type p53 protein but also acquire novel powerful oncogenic functions, referred to as gain of function, that may actively confer a selective advantage during tumor progression. Some of the best-characterized oncogenic activities of mutant p53 are mediated by its ability to form aberrant protein complexes with other transcription factors or proteins not directly related to gene transcription. The set of cellular proteins available to interact with mutant p53 is dependent on cell type and extensively affected by environmental signals, so the prognostic impact of p53 ...
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