RESUMO
Cancer cells are commonly subjected to endoplasmic reticulum (ER) stress. To gain survival advantage, cancer cells exploit the adaptive aspects of the unfolded protein response such as upregulation of the ER luminal chaperone GRP78. The finding that when overexpressed, GRP78 can escape to other cellular compartments to gain new functions regulating homeostasis and tumorigenesis represents a paradigm shift. Here, toward deciphering the mechanisms whereby GRP78 knockdown suppresses EGFR transcription, we find that nuclear GRP78 is prominent in cancer and stressed cells and uncover a nuclear localization signal critical for its translocation and nuclear activity. Furthermore, nuclear GRP78 can regulate expression of genes and pathways, notably those important for cell migration and invasion, by interacting with and inhibiting the activity of the transcriptional repressor ID2. Our study reveals a mechanism for cancer cells to respond to ER stress via transcriptional regulation mediated by nuclear GRP78 to adopt an invasive phenotype.
Assuntos
Núcleo Celular , Chaperona BiP do Retículo Endoplasmático , Humanos , Carcinogênese , Movimento Celular , Transformação Celular NeoplásicaRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 global pandemic, utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) for viral entry. However, other host factors might also play important roles in SARS-CoV-2 infection, providing new directions for antiviral treatments. GRP78 is a stress-inducible chaperone important for entry and infectivity for many viruses. Recent molecular docking analyses revealed putative interaction between GRP78 and the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (SARS-2-S). Here we report that GRP78 can form a complex with SARS-2-S and ACE2 on the surface and at the perinuclear region typical of the endoplasmic reticulum in VeroE6-ACE2 cells and that the substrate-binding domain of GRP78 is critical for this interaction. In vitro binding studies further confirmed that GRP78 can directly bind to the RBD of SARS-2-S and ACE2. To investigate the role of GRP78 in this complex, we knocked down GRP78 in VeroE6-ACE2 cells. Loss of GRP78 markedly reduced cell surface ACE2 expression and led to activation of markers of the unfolded protein response. Treatment of lung epithelial cells with a humanized monoclonal antibody (hMAb159) selected for its safe clinical profile in preclinical models depleted cell surface GRP78 and reduced cell surface ACE2 expression, as well as SARS-2-S-driven viral entry and SARS-CoV-2 infection in vitro. Our data suggest that GRP78 is an important host auxiliary factor for SARS-CoV-2 entry and infection and a potential target to combat this novel pathogen and other viruses that utilize GRP78 in combination therapy.
Assuntos
Enzima de Conversão de Angiotensina 2/genética , Proteínas de Choque Térmico/genética , Interações Hospedeiro-Patógeno/genética , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Sítios de Ligação , Chlorocebus aethiops , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Chaperona BiP do Retículo Endoplasmático , Regulação da Expressão Gênica , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/metabolismo , Humanos , Mutação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/metabolismo , Resposta a Proteínas não Dobradas , Células VeroRESUMO
Despite new advances on the functions of ER chaperones at the cell surface, the translocation mechanisms whereby these chaperones can escape from the ER to the cell surface are just emerging. Previously we reported that in many cancer types, upon ER stress, IRE1α binds to and triggers SRC activation resulting in KDEL receptor dispersion from the Golgi and suppression of retrograde transport. In this study, using a combination of molecular, biochemical, and imaging approaches, we discovered that in colon and lung cancer, upon ER stress, ER chaperones, such as GRP78 bypass the Golgi and unconventionally traffic to the cell surface via endosomal transport mediated by Rab GTPases (Rab4, 11 and 15). Such unconventional transport is driven by membrane fusion between ER-derived vesicles and endosomes requiring the v-SNARE BET1 and t-SNARE Syntaxin 13. Furthermore, GRP78 loading into ER-derived vesicles requires the co-chaperone DNAJC3 that is regulated by ER-stress induced PERK-AKT-mTOR signaling.
Assuntos
Membrana Celular/metabolismo , Neoplasias do Colo/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Choque Térmico/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutagênese Sítio-Dirigida , Mutação , Transporte Proteico , Transdução de Sinais , Células Tumorais CultivadasRESUMO
The discovery that endoplasmic reticulum (ER) luminal chaperones such as GRP78/BiP can escape to the cell surface upon ER stress where they regulate cell signaling, proliferation, apoptosis, and immunity represents a paradigm shift. Toward deciphering the mechanisms, we report here that, upon ER stress, IRE1α binds to and triggers tyrosine kinase SRC activation, leading to ASAP1 phosphorylation and Golgi accumulation of ASAP1 and Arf1-GTP, resulting in KDEL receptor dispersion from the Golgi and suppression of retrograde transport. At the cell surface, GRP78 binds to and acts in concert with a glycosylphosphatidylinositol-anchored protein, CD109, in blocking TGF-ß signaling by promoting the routing of the TGF-ß receptor to the caveolae, thereby disrupting its binding to and activation of Smad2. Collectively, we uncover a SRC-mediated signaling cascade that leads to the relocalization of ER chaperones to the cell surface and a mechanism whereby GRP78 counteracts the tumor-suppressor effect of TGF-ß.
Assuntos
Antígenos CD/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Proteínas de Choque Térmico/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Quinases da Família src/metabolismo , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos CD/genética , Chaperona BiP do Retículo Endoplasmático , Ativação Enzimática/fisiologia , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/genética , Humanos , Células MCF-7 , Proteínas de Neoplasias/genética , Transporte Proteico/fisiologia , Proteína Smad2/genética , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal disease in critical need of new therapeutic strategies. Here, we report that the stress-inducible 78-kDa glucose-regulated protein (GRP78/HSPA5), a key regulator of endoplasmic reticulum homeostasis and PI3K/AKT signaling, is overexpressed in the acini and PDAC of Pdx1-Cre;KrasG12D/+;p53f/+ (PKC) mice as early as 2 mo, suggesting that GRP78 could exert a protective effect on acinar cells under stress, as during PDAC development. The PKC pancreata bearing wild-type Grp78 showed detectable PDAC by 3 mo and rapid subsequent tumor growth. In contrast, the PKC pancreata bearing a Grp78f/+ allele (PKC78f/+ mice) expressing about 50% of GRP78 maintained normal sizes during the early months, with reduced proliferation and suppression of AKT, S6, ERK, and STAT3 activation. Acinar-to-ductal metaplasia (ADM) has been identified as a key tumor initiation mechanism of PDAC. Compared with PKC, the PKC78f/+ pancreata showed substantial reduction of ADM as well as pancreatic intraepithelial neoplasia-1 (PanIN-1), PanIN-2, and PanIN-3 and delayed onset of PDAC. ADM in response to transforming growth factor α was also suppressed in ex vivo cultures of acinar cell clusters isolated from mouse pancreas bearing targeted heterozygous knockout of Grp78 (c78f/+ ) and subjected to 3D culture in collagen. We further discovered that GRP78 haploinsufficiency in both the PKC78f/+ and c78f/+ pancreata leads to reduction of epidermal growth factor receptor, which is critical for ADM initiation. Collectively, our studies establish a role for GRP78 in ADM and PDAC development.
Assuntos
Carcinoma Ductal Pancreático/genética , Transdiferenciação Celular , Proteínas de Choque Térmico/genética , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Células Acinares/metabolismo , Animais , Carcinogênese , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Chaperona BiP do Retículo Endoplasmático , Feminino , Haploinsuficiência , Proteínas de Choque Térmico/metabolismo , Masculino , Metaplasia , Camundongos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fator de Crescimento Transformador alfa/metabolismoRESUMO
The 78-kDa glucose regulated protein (GRP78) commonly upregulated in a wide variety of tumors is an important prognostic marker and a promising target for suppressing tumorigenesis and treatment resistance. While GRP78 is well established as a major endoplasmic reticulum (ER) chaperone with anti-apoptotic properties and a master regulator of the unfolded protein response, its new role as a regulator of oncoprotein expression is just emerging. MYC is dysregulated in about 70 % of human cancers and is the most commonly activated oncoprotein. However, despite recent advances, therapeutic targeting of MYC remains challenging. Here we identify GRP78 as a new target for suppression of MYC expression. Using multiple MYC-dependent cancer models including head and neck squamous cell carcinoma and their cisplatin-resistant clones, breast and pancreatic adenocarcinoma, our studies revealed that GRP78 knockdown by siRNA or inhibition of its activity by small molecule inhibitors (YUM70 or HA15) reduced c-MYC expression, leading to onset of apoptosis and loss of cell viability. This was observed in 2D cell culture, 3D spheroid and in xenograft models. Mechanistically, we determined that the suppression of c-MYC is at the post-transcriptional level and that YUM70 and HA15 treatment potently upregulated the eukaryotic translation inhibitor 4E-BP1, which targets eIF4E critical for c-MYC translation initiation. Furthermore, knock-down of 4E-BP1 via siRNA rescued YUM70-mediated c-MYC suppression. As YUM70 is also capable of suppressing N-MYC expression, this study offers a new approach to suppress MYC protein expression through knockdown or inhibition of GRP78.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Chaperona BiP do Retículo Endoplasmático , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico , Proteínas Proto-Oncogênicas c-myc , Humanos , Chaperona BiP do Retículo Endoplasmático/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/antagonistas & inibidores , Camundongos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias/genética , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Regulação para Cima/efeitos dos fármacosRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has given rise to many new variants with increased transmissibility and the ability to evade vaccine protection. The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum (ER) chaperone that has been recently implicated as an essential host factor for SARS-CoV-2 entry and infection. In this study, we investigated the efficacy of YUM70, a small molecule inhibitor of GRP78, to block SARS-CoV-2 viral entry and infection in vitro and in vivo. Using human lung epithelial cells and pseudoviral particles carrying spike proteins from different SARS-CoV-2 variants, we found that YUM70 was equally effective at blocking viral entry mediated by original and variant spike proteins. Furthermore, YUM70 reduced SARS-CoV-2 infection without impacting cell viability in vitro and suppressed viral protein production following SARS-CoV-2 infection. Additionally, YUM70 rescued the cell viability of multi-cellular human lung and liver 3D organoids transfected with a SARS-CoV-2 replicon. Importantly, YUM70 treatment ameliorated lung damage in transgenic mice infected with SARS-CoV-2, which correlated with reduced weight loss and longer survival. Thus, GRP78 inhibition may be a promising approach to augment existing therapies to block SARS-CoV-2, its variants, and other viruses that utilize GRP78 for entry and infection.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Camundongos , Humanos , SARS-CoV-2/fisiologia , Chaperona BiP do Retículo Endoplasmático , Internalização do Vírus , Glicoproteína da Espícula de Coronavírus , Pandemias , PulmãoRESUMO
KRAS is the most commonly mutated oncogene in human cancers with limited therapeutic options, thus there is a critical need to identify novel targets and inhibiting agents. The 78-kDa glucose-regulated protein GRP78, which is upregulated in KRAS cancers, is an essential chaperone and the master regulator of the unfolded protein response (UPR). Following up on our recent discoveries that GRP78 haploinsufficiency suppresses both KRASG12D-driven pancreatic and lung tumorigenesis, we seek to determine the underlying mechanisms. Here, we report that knockdown of GRP78 via siRNA reduced oncogenic KRAS protein level in human lung, colon, and pancreatic cancer cells bearing various KRAS mutations. This effect was at the post-transcriptional level and is independent of proteasomal degradation or autophagy. Moreover, targeting GRP78 via small molecule inhibitors such as HA15 and YUM70 with anti-cancer activities while sparing normal cells significantly suppressed oncogenic KRAS expression in vitro and in vivo, associating with onset of apoptosis and loss of viability in cancer cells bearing various KRAS mutations. Collectively, our studies reveal that GRP78 is a previously unidentified regulator of oncogenic KRAS expression, and, as such, augments the other anti-cancer activities of GRP78 small molecule inhibitors to potentially achieve general, long-term suppression of mutant KRAS-driven tumorigenesis.
Assuntos
Chaperona BiP do Retículo Endoplasmático , Proteínas Proto-Oncogênicas p21(ras) , Carcinogênese , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Glucose , Humanos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Interferente PequenoRESUMO
The 78 kilodalton glucose-regulated protein (GRP78) is a major endoplasmic reticulum (ER) molecular chaperone with antiapoptotic properties and a key regulator of the unfolded protein response (UPR). ER-stress induction of GRP78 in cancer cells represents a major pro-survival branch of the UPR. Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal disease and high level of GRP78 is associated with aggressive disease and poor survival. Recently, we reported that PDAC exhibited high level of ER stress and that GRP78 haploinsufficiency is sufficient to suppress pancreatic tumorigenesis in mice, suggesting the utility of inhibitors of GRP78 expression in combating pancreatic cancer. Screening of clinically relevant compound libraries revealed that cardiac glycosides (CGs) can inhibit ER-stress induction of GRP78 in pancreatic and other types of human cancers. Using the FDA-approved CG compound Lanatoside C (LanC) and human pancreatic cancer cell lines as model systems, we discovered that LanC preferably suppressed ER stress induction of GRP78 and to a lesser extent GRP94. The suppression is at the post-transcriptional level and dependent on the Na+/K+-ATPase ion pump. Overexpression of GRP78 mitigates apoptotic activities of LanC in ER stressed cells. Our study revealed a new function of CGs as inhibitor of stress induction of GRP78, and that this suppression at least in part contributes to the apoptotic activities of CGs in human pancreatic cancer cells in vitro. These findings support further investigation into CGs as potential antineoplastic agents for pancreatic and other cancers which depend on GRP78 for growth and survival.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Chaperona BiP do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Lanatosídeos/farmacologia , Neoplasias Pancreáticas/metabolismo , Glicosídeos Cardíacos/farmacologia , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático/metabolismo , Humanos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Neoplasias PancreáticasRESUMO
Lung cancer is the leading cause of cancer mortality worldwide and KRAS is the most commonly mutated gene in lung adenocarcinoma (LUAD). The 78-kDa glucose-regulated protein GRP78/BiP is a key endoplasmic reticulum chaperone protein and a major pro-survival effector of the unfolded protein response (UPR). Analysis of the Cancer Genome Atlas database and immunostain of patient tissues revealed that compared to normal lung, GRP78 expression is generally elevated in human lung cancers, including tumors bearing the KRASG12D mutation. To test the requirement of GRP78 in human lung oncogenesis, we generated mouse models containing floxed Grp78 and Kras Lox-Stop-Lox G12D (KrasLSL-G12D) alleles. Simultaneous activation of the KrasG12D allele and knockout of the Grp78 alleles were achieved in the whole lung or selectively in lung alveolar epithelial type 2 cells known to be precursors for adenomas that progress to LUAD. Here we report that GRP78 haploinsufficiency is sufficient to suppress KrasG12D-mediated lung tumor progression and prolong survival. Furthermore, GRP78 knockdown in human lung cancer cell line A427 (KrasG12D/+) leads to activation of UPR and apoptotic markers and loss of cell viability. Our studies provide evidence that targeting GRP78 represents a novel therapeutic approach to suppress mutant KRAS-mediated lung tumorigenesis.
Assuntos
Chaperona BiP do Retículo Endoplasmático/metabolismo , Neoplasias Pulmonares/patologia , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Resposta a Proteínas não Dobradas , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Progressão da Doença , Chaperona BiP do Retículo Endoplasmático/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Transdução de SinaisRESUMO
GRP78, a major molecular chaperone, is critical for the folding and maturation of membrane and secretory proteins and serves as the master regulator of the unfolded protein response. Thus, GRP78 is frequently upregulated in highly proliferative cells to cope with elevated protein synthesis and metabolic stress. IGF-1 is a potent regulator of cell growth, metabolism and survival. Previously we discovered that GRP78 is a novel downstream target of IGF-1 signaling by utilizing mouse embryonic fibroblast model systems where the IGF-1 receptor (IGF-1R) was either overexpressed (R+) or knockout (R-). Here we investigated the mechanisms whereby GRP78 is upregulated in the R+ cells. Our studies revealed that suppression of PI3K/AKT/mTOR downstream of IGF-1R signaling resulted in concurrent decrease in GRP78 and the transcription factor ATF4. Through knock-down and overexpression studies, we established ATF4 as the essential downstream nodal of the PI3K/AKT/mTOR signaling pathway critical for GRP78 transcriptional upregulation mediated by IGF-1R.
Assuntos
Fibroblastos/metabolismo , Proteínas de Choque Térmico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptor IGF Tipo 1/metabolismo , Animais , Linhagem Celular , Chaperona BiP do Retículo Endoplasmático , Fibroblastos/citologia , Camundongos , Serina-Treonina Quinases TOR/metabolismoRESUMO
In many cancers, combination therapy regimens are successfully improving response and survival rates, but the challenges of toxicity remain. GRP78, the master regulator of the unfolded protein response, is emerging as a target that is upregulated in tumors, specifically following treatment, and one that impacts tumor cell survival and disease recurrence. Here, we show IT-139, an antitumor small molecule inhibitor, suppresses induction of GRP78 from different types of endoplasmic reticulum (ER) stress in a variety of cancer cell lines, including those that have acquired therapeutic resistance, but not in the non-cancer cells being tested. We further determined that IT-139 treatment exacerbates ER stress while at the same time suppresses GRP78 induction at the transcriptional level. Our studies revealed a differential effect of IT-139 on chaperone protein family expression at multiple levels in different cancer cell lines. In xenograft studies, IT-139 decreased BRAF inhibitor upregulation of GRP78 expression in the tumor, while having minimal effect on GRP78 expression in the adjacent normal cells. The preferential decrease in GRP78 levels in tumor cells over normal cells, supported by the manageable safety profile seen in the Phase 1 clinical trial, reinforce the value IT-139 brings to combination therapies as it continues its clinical development.