1,720,997 research outputs found
Strategies to Overcome Resistance to Osimertinib in EGFR-Mutated Lung Cancer
Full text linkNon-small-cell lung cancer (NSCLC) represents the most common type of lung cancer. The majority of patients with lung cancer characterized by activating mutations in the epidermal growth factor receptor (EGFR), benefit from therapies entailing tyrosine kinase inhibitors (TKIs). In this regard, osimertinib, a third-generation EGFR TKI, has greatly improved the outcome for patients with EGFR-mutated lung cancer. The AURA and FLAURA trials displayed the superiority of the third-generation TKI in both first- and second-line settings, making it the drug of choice for treating patients with EGFR-mutated lung cancer. Unfortunately, the onset of resistance is almost inevitable. On-target mechanisms of resistance include new mutations (e.g., C797S) in the kinase domain of EGFR, while among the off-target mechanisms, amplification of MET or HER2, mutations in downstream signaling molecules, oncogenic fusions, and phenotypic changes (e.g., EMT) have been described. This review focuses on the strategies that are currently being investigated, in preclinical and clinical settings, to overcome resistance to osimertinib, including the use of fourth-generation TKIs, PROTACs, bispecific antibodies, and ADCs, as monotherapy and as part of combination therapies
PDIA3: a versatile and pleiotropic protein disulfide isomerase
Full text linkIntroduction
PDIA3, also known as ERp57 or GRP58, is a member of the protein disulfide isomerase family; its structure is characterized by four thioredoxin-like domains: a, b, b’ and a’; a and a’ domains contain the redox active site while b and b’ domains are redox inactive. PDIA3 is localized predominantly in the ER (Endoplasmic Reticulum), where it is involved in the correct folding of newly synthesized glycoproteins and in the assembly of the MHC class I complex, but it is also present in the cytosol, in the nucleus and on the cell surface. PDIA3 is a multifunctional protein disulfide isomerase with a wide range of functions. It has been shown that this protein is involved in the cellular response to stress as well as in several diseases such as cancer, prion disorders, Alzheimer’s and Parkinson’s diseases.
Considering this and given that PDIA3 is able to interact with a number of macromolecules and small ligands, such as green tea catechins, in the first part of my research I focused my attention on finding molecules that can interact with and modulate PDIA3 activity. This study, previously started in our lab analyzing the major catechins present in the extracts of green tea, has been expanded to various classes of flavonoids to verify if their activity was in some way connected to the modulation of PDIA3 functions. Flavonoids are a large class of plant secondary metabolites of low molecular weight present in fruits, vegetables and in products such as tea and red wine. Their basic structure shows two benzene rings (A and B) linked by the heterocyclic pyran ring, and, according to type and position of substituents on the central structure, they can be divided into different classes. These molecules show antioxidant, anti-inflammatory, antithrombotic, antiviral and antitumor activity. However, for many of them the molecular and cellular basis of their activities are not well known. They can act on different targets affecting regulation of cell signalling and cell cycle, free radical scavenging, inhibition of angiogenesis, initiation of DNA repair mechanisms, apoptotic induction and inhibition of metastasis. For this reason, we undertook a screening study for assessing the interaction and impact on PDIA3 protein activity of several types of flavonoids.
Alzheimer’s disease (AD) is a common neurodegenerative disorder in humans, characterized by deposition in the brain of β amyloid (Aβ) plaques and neurofibrillary tangles (NFTs). Aβ plaques are constituted by Aβ1-40 and Aβ1-42 peptides, which are the results of the APP (amyloid precursor protein) proteolytic cleavage and are thought to be the main cause of the Alzheimer development. Aβ is present in the plaques of Alzheimer’s patients only as a naked peptide, while it is complexed to PDIA3 and calreticulin in the cerebrospinal fluid (CSF) of healthy individuals. It has also been reported the beneficial effect of diosgenin on the memory deficit in an AD mice model and on retrieval of axonal and presynaptic degeneration in the cerebral cortex and hippocampus, detecting PDIA3 as a target of diosgenin. Interestingly, the diosgenin-induced axonal growth was significantly inhibited in primary cortical neurons after PDIA3 knockdown. Given the evidences of PDIA3 involvement in AD, in the second part of my PHD I decided to better investigate PDIA3 role in amyloid beta deposits and in Alzheimer’s disease.
The last part of my research, started in the Children’s Hospital of Philadelphia in Yair Argon’s laboratory, has been focused on ER stress and UPR (Unfolded Protein Response). In particular I studied one of the sensors of UPR, IRE1. IRE1 is a transmembrane receptor kinase located on the surface of the ER. This protein consists of different domains: the luminal domain, the transmembrane domain (TM), the linker domain, the kinase domain and the endoribonuclease domain (KEN). When ER stress occurs, BIP dissociates from the luminal domain of IRE1; interactions of the stress-sensing luminal domains of two IRE1 monomers promotes trans-autophosphorylation of the kinase and RNase domains on the cytoplasmic side of the ER membrane Phosphorylation triggers conformational changes in IRE1, which stabilize the dimer. This rearrangement in the RNase domain places the residues necessary for the catalysis in the correct orientation. Moreover IRE1 can also arrange in higher-order oligomers. The result is the cleavage of an intron from the XBP1 mRNA, leading to the translation of a potent transcription factor, which regulates the expression of several genes encoding for ER chaperones. Activated IRE1 can also cleave a subset of mRNAs encoding proteins targeted to the ER, leading to mRNA degradation; this process has been called regulated IRE1-dependent decay (RIDD). RIDD regulates many physiological processes, including degradation of mRNAs encoding a subset of ER or secretory proteins prone to misfolding, and regulation of lipid metabolism genes. RIDD is also responsible for the activation of apoptosis through degradation of several miRNAs’ precursors, such as miR-17. Moreover IRE1, through its kinase domain, activates the c-Jun N-terminal kinases (JNKs) via the formation of a complex with the E3 ubiquitin ligase TRAF2 (TNF receptor-associated factor 2) and the apoptosis signal regulating kinase 1 (ASK1), leading to apoptosis. It has been demonstrated in several studies that alteration in IRE1 function occurs in different diseases such as cancer, diabetes, inflammatory and neurodegenerative diseases.
It still not entirely known how IRE1 regulates cell fate; the common thinking is that XBP1 splicing has mostly a prosurvival effect, whereas RIDD shows a proapoptotic output. Nevertheless, the precise mechanisms of these actions need to be further investigated. For this reason the main goal of this research was to understand how the different IRE1 activities are related to each other. Moreover we wanted to investigate if they are related to protein dimerization and clustering, and how phosphorylation affects those activities.
METHODOLOGIES
PDIA3-flavonoids interaction was investigated by quenching analysis of protein intrinsic fluorescence. This analysis was extended to the PDIA3 a’ domain. Disulfide reductase activity of PDIA3 was monitored by sensitive fluorescent assay using dieosin glutathione disulfide (DiE-GSSG) as fluorogenic probe. Flavonoid’s effect on the DNA binding properties of PDIA3 was also evaluated by EMSA analysis.
To investigate PDIA3 involvement in AD, the neuroblastoma cell line SH-SY5Y, a model of neuronal cell, was treated with the 25-35 fragment of the amyloid β peptide. PDIA3 protein levels were analyzed through western blot analysis; mRNA levels through real time-PCR. Immunofluorescence studies were conducted to follow PDIA3 localization under Aβ treatment. To assess PDIA3 levels in the culture media proteins were precipitated with trichloroacetic acid (TCA) and analyzed through western blot.
To study IRE1 activities we used an IRE1GFP construct in a TetOn inducible expression system. We established a stable IRE1-/- HAP1 cell line, in which the expression of WT-IRE1GFP and its mutants was regulated by adjusting the doxycycline concentration. IRE1-complemented IRE1 -/-HAP1s were generated by two consecutive transductions with lentiviral particles carrying pLVX-Tet-OnTM or pLVX-Tight-PuroTM plasmids (Clontech). XBP1 splicing assay and cell living image were performed under ER stress.
RESULTS AND CONCLUSIONS
In the first part of this study, the interaction of different flavonoids with PDIA3 and their effect on protein reductase activity were evaluated. Two molecules, eupatorin and eupatorin-5-methyl ether, showed the highest affinity for PDIA3 with a Kd near to 1.0x10-5 M. They also showed a noticeable inhibitory effect on disulphide reductase activity of PDIA3, but they did not significantly affect its DNA binding activity. The backbone structure of these two flavones is characterized by a more stable conformation where B and A rings are almost parallel. This structure, associated with a definite degree of polarity, due to the presence of several methoxyl- groups, seems to be an important feature to determine a good affinity toward PDIA3. We can hypothesize that flavones interact with a region of the protein involving the tryptophan residues close to the redox site and given that PDIA3 does not contain any evident deep cavity or slot where this kind of ligands can bind, the binding of flavonoids may occur mainly via a flat interaction with the protein surface. Therefore, the planarity of the molecule as well as the number and specific position of its functional groups (hydroxyl-, methoxyl- and carbohydrates) will definitively play a major role to determine the affinity for the protein. In conclusion, eupatorin and eupatorin-5-methyl ether represent leading compounds for the binding to PDIA3 and for the inhibition of its redox activity. Further experiments are required to better characterize the effect of flavonoids on PDIA3 and to understand if some of the biological activities of these compounds are depending on the interaction with PDIA3. Since these flavones and PDIA3 are both involved in proliferative and carcinogenic processes, our in vitro findings on their interaction suggest that some of the biological effects of flavones could be mediated by modulation of PDIA3 activity. Additionally, this study will help to define and identify compounds to be used as selective inhibitors/modulators of PDIA3 biological activities.
Regarding the implications of PDIA3 in β-amyloid deposits and Alzheimer’s disease, we observed that β-amyloid peptide fragment 25-35 induced a decrease in PDIA3 protein but not in its mRNA levels. We demonstrated that this decrease was not a consequence of ER stress, since we proved that this specific fragment of the amyloid β peptide did not cause activation of the unfolded protein response. We also proved that this decrease was not due to protein degradation through proteasome. Moreover we observed a delocalization of PDIA3 toward the plasma membrane following Aβ treatment. Considering our data and since in literature evidences about PDIA3 extracellular presence can be found, we hypothesized that PDIA3 can be secreted under Aβ treatment. In this study we indeed showed that PDIA3 is secreted by SH-SY5Y, with a significant increase after 1 hour of Aβ25-35 treatment. We also observed that PDIA3 secretion seemed not to be dependent on the classical secretion pathway Golgi-mediated. An explanation for this observation could be that PDIA3 is released from the cell within exosomes. This is not totally unlikely since PDIA3 is present in the ExoCarta Database as an exosome-associated protein. From data presented in this work, our hypothesis is that β-amyloid peptide induces a PDIA3 delocalization and secretion in the extracellular fluid as a defence mechanism carried out by the cell to counteract the toxic action of Aβ. Considering the work of Erickson et al., it could be that PDIA3 pursues this aim through direct binding to amyloid β peptide in order to prevent its aggregation and keep it in solution.
In the study of IRE1 activities we observed that, in order to have activation of the RNase domain of IRE1 and clustering, a functional luminal domain is required. Interestingly we found out that if the RNase domain of IRE1 is somehow inhibited, with a mutation or using a chemical inhibitor, IRE1 persists in clusters in the ER membrane. This means that IRE1 clustering does not require the endoribonuclease activity of IRE1. Moreover this led us to hypothesize that IRE1 clustering can be related to other activities, such as RIDD or activation of the JNK pathway. Another interesting finding in this study was that a stimulus coming from the cytosol induces XBP1 splicing but not IRE1 clustering. Indeed, the flavonoid luteolin, through direct binding to the interphase between the kinase and endoribonuclease domains, triggers the splicing of XBP1 mRNA but it does not cause IRE1 redistribution in clusters in the ER membrane. This could be explained by the finding that luteolin induces a different degree in IRE1 phosphorylation if compared with the one induced by a common ER stress activator, such as thapsigargin. Our hypothesis is that luteolin induces phosphorylation only in the activation loop, which is sufficient to have XBP1 splicing, but in order to have IRE1 clustering, phosphorylation in additional residues is required. More studies are needed to confirm our thinking and, more important, the next step will be to investigate the other IRE1 activities, RIDD and JNK-pathway activation, in order to relate every single IRE1 activity to its dimeric or oligomeric state. Moreover we want to better understand how the phosphorylation state of IRE1 affects its activities. Last, during the course of this study on IRE1, we discovered by chance a new IRE1 mutant, L827P. This mutation, not present in any of the catalytic domains of IRE1, has proven to led to the complete suppression of its endoribonuclease activity. We are now interested in better characterizing the mechanism of action of this mutant with the intent to develop, in the future, small peptides that can inhibit IRE1. Since it has been proved that IRE1 is involved in pathologies, such as multiple myeloma, it can be a valid and promising pharmacological target
Strategies to Overcome Resistance to Osimertinib in EGFR-Mutated Lung Cancer
No full textNon-small-cell lung cancer (NSCLC) represents the most common type of lung
cancer. The majority of patients with lung cancer characterized by activating mutations
in the epidermal growth factor receptor (EGFR), benefit from therapies entailing tyrosine
kinase inhibitors (TKIs). In this regard, osimertinib, a third-generation EGFR TKI, has
greatly improved the outcome for patients with EGFR-mutated lung cancer. The AURA
and FLAURA trials displayed the superiority of the third-generation TKI in both first- and
second-line settings, making it the drug of choice for treating patients with EGFR-mutated
lung cancer. Unfortunately, the onset of resistance is almost inevitable. On-target mechanisms of resistance include new mutations (e.g., C797S) in the kinase domain of EGFR,
while among the off-target mechanisms, amplification of MET or HER2, mutations in downstream signaling molecules, oncogenic fusions, and phenotypic changes (e.g., EMT) have
been described. This review focuses on the strategies that are currently being investigated,
in preclinical and clinical settings, to overcome resistance to osimertinib, including the use
of fourth-generation TKIs, PROTACs, bispecific antibodies, and ADCs, as monotherapy
and as part of combination therapies
L858R emerges as a potential biomarker predicting response of lung cancer models to anti-EGFR antibodies: Comparison of osimertinib vs. cetuximab
Full text linkEGFR-specific tyrosine kinase inhibitors (TKIs), especially osimertinib, have changed lung cancer therapy, but secondary mutations confer drug resistance. Because other EGFR mutations promote dimerization-independent active conformations but L858R strictly depends on receptor dimerization, we herein evaluate the therapeutic potential of dimerization-inhibitory monoclonal antibodies (mAbs), including cetuximab. This mAb reduces viability of cells expressing L858R-EGFR and blocks the FOXM1-aurora survival pathway, but other mutants show no responses. Unlike TKI-treated patient-derived xenografts, which relapse post osimertinib treatment, cetuximab completely prevents relapses of L858R+ tumors. We report that osimertinib's inferiority associates with induction of mutagenic reactive oxygen species, whereas cetuximab's superiority is due to downregulation of adaptive survival pathways (e.g., HER2) and avoidance of mutation-prone mechanisms that engage AXL, RAD18, and the proliferating cell nuclear antigen. These results identify L858R as a predictive biomarker, which may pave the way for relapse-free mAb monotherapy relevant to a large fraction of patients with lung cancer
Measurement and clinical significance of biomarkers of oxidative stress in humans
Full text linkOxidative stress is the result of the imbalance between reactive oxygen species (ROS) formation and enzymatic and nonenzymatic antioxidants. Biomarkers of oxidative stress are relevant in the evaluation of the disease status and of the health-enhancing effects of antioxidants. We aim to discuss the major methodological bias of methods used for the evaluation of oxidative stress in humans. There is a lack of consensus concerning the validation, standardization, and reproducibility of methods for the measurement of the following: (1) ROS in leukocytes and platelets by flow cytometry, (2) markers based on ROS-induced modifications of lipids, DNA, and proteins, (3) enzymatic players of redox status, and (4) total antioxidant capacity of human body fluids. It has been suggested that the bias of each method could be overcome by using indexes of oxidative stress that include more than one marker. However, the choice of the markers considered in the global index should be dictated by the aim of the study and its design, as well as by the clinical relevance in the selected subjects. In conclusion, the clinical significance of biomarkers of oxidative stress in humans must come from a critical analysis of the markers that should give an overall index of redox status in particular conditions
STAT3/ERP57/TPX2 axis and process of “androgen escape” in prostate cancer
No full textThe mechanisms of Prostate Cancer (PCa) progression through hormone-dependent to hormone refractory form is still unclear. Many data indicate that JAK/STAT signaling contributes to tumor resistance and STAT3 hyperactivation is observed in a variety of human cancers. Moreover, several authors suggested that ERp57 (GRP58/PDIA3), a disulfide isomerases protein, is associated with modulation of STAT3 activity. We investigate the role of STAT3-ERp57-TPX2 axis in the ormone-responsive and androgen-refractory tumor using human PCa cell lines, LNCaP (androgen-sensitive) and PC3 (androgen-refractory), untreated and stimulated with IL-6 and EGF. Immunoblotting and CoIP analysis were performed to confirm STAT3 activation and ERp57-STAT3 interaction. To investigate the physiological relevance of STAT3-ERp57-TPX2 axis, we inhibited STAT3 or ERp57 activity and the expression levels of TPX2 was monitored by qRT-PCR. The results showed that increased STAT3-ERp57 complex association determines an TPX2 overexpression. In conclusion, this study showed that STAT3-ERp57-TPX2 axis is correlated with tumor progression and it suggests a functional role of STAT3/ERp57 complex in the Androgen Escape
Comparative analysis of the interaction between different flavonoids and pdia3
Full text linkFlavonoids, plant secondary metabolites present in fruits, vegetables, and products such as tea and red wine, show antioxidant, anti-inflammatory, antithrombotic, antiviral, and antitumor activity. PDIA3 is a member of the protein disulfide isomerase family mainly involved in the correct folding of newly synthetized glycoproteins. PDIA3 is associated with different human pathologies such as cancer, prion disorders, Alzheimer's disease, and Parkinson's diseases and it has the potential to be a pharmacological target. The interaction of different flavonoids with PDIA3 was investigated by quenching fluorescence analysis and the effects on protein activity were evaluated. A higher affinity was observed for eupatorin-5-methyl ether and eupatorin which also inhibit reductase activity of PDIA3 but do not significantly affect its DNA binding activity. The use of several flavonoids differing in chemical structure and functional groups allows us to make some consideration about the relationship between ligand structure and the affinity for PDIA3. The specific flavone backbone conformation and the degree of polarity seem to play an important role for the interaction with PDIA3. The binding site is probably similar but not equivalent to that of green tea catechins, which, as previously demonstrated, can bind to PDIA3 and prevent its interaction with DNA
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