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The mTORC2 pathway plays a critical role in promoting tumor progression in human colorectal cancer (CRC). The regulatory mechanisms for this signaling pathway are only partially understood. We previously identified UBXN2A as a novel tumor suppressor protein in CRCs and hypothesized that UBXN2A suppresses the mTORC2 pathway, thereby inhibiting CRC growth and metastasis. We first used murine models to show that haploinsufficiency of UBXN2A significantly increases colon tumorigenesis. Induction of UBXN2A reduces AKT phosphorylation downstream of the mTORC2 pathway, which is essential for a plethora of cellular processes, including cell migration. Meanwhile, mTORC1 activities remain unchanged in the presence of UBXN2A. Mechanistic studies revealed that UBXN2A targets Rictor protein, a key component of the mTORC2 complex, for 26S proteasomal degradation. A set of genetic, pharmacological, and rescue experiments showed that UBXN2A regulates cell proliferation, apoptosis, migration, and colon cancer stem cells (CSCs) in CRC. CRC patients with a high level of UBXN2A have significantly better survival, and high-grade CRC tissues exhibit decreased UBXN2A protein expression. A high level of UBXN2A in patient-derived xenografts and tumor organoids decreases Rictor protein and suppresses the mTORC2 pathway. These findings provide new insights into the functions of an ubiquitin-like protein by inhibiting a dominant oncogenic pathway in CRC.
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Neoplasias do Colo , Humanos , Camundongos , Animais , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Neoplasias do Colo/patologia , Linhagem Celular Tumoral , Células-Tronco Neoplásicas/patologia , Transdução de Sinais , Fatores de Transcrição/genética , Carcinogênese/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ubiquitinas/metabolismoRESUMO
BACKGROUND: Neointimal hyperplasia (NH) is a common pathological response to vascular injury and mediated primarily by vascular smooth muscle cell (VSMC) migration and proliferation. The COP9 signalosome (CSN) is formed by 8 canonical subunits (CSN1 through CSN8) with its deneddylation activity residing in CSN5. Each or some of CSN subunits may have deneddylation-independent function. Despite strong evidence linking the CSN to cell cycle regulation in cancer cells, the role of the CSN in vascular biology remains obscure. METHODS: Neointimal CSN5 expression in the lung tissue of pulmonary hypertension (PAH) patients was assessed with immunohistochemistry. Adult mice with smooth muscle cell-restricted CSN5 knockout (CSN5-SMKO) or CSN8 hypomorphism (CSN8-hypo) and cultured mouse VSMCs were studied to determine the role and governing mechanisms of the CSN in NH. NH was induced by ligation of the left common carotid artery (LCCA) and PDGF-BB stimulation was used to mimic the vascular injury in cell cultures. RESULTS: Remarkably higher CSN5 levels were detected in the neointimal VSMCs of the pulmonary arteries of human PAH. LCCA ligation induced NH and significantly increased the mRNA and protein levels of CSN subunits in the LCCA wall of adult wild type mice. CSN5-SMKO impaired Cullin deneddylation and the nuclear export of p27 in vessel walls and markedly inhibited VSMC proliferation in mice. On the contrary, CSN8-hypo significantly exacerbated NH and VSMC proliferation in vivo and in cellulo . Cytoplasmic CSN5 mini-complexes and the nuclear export of p27 were significantly increased in CSN8-hypo mouse vessels and cultured CSN8-hypo VSMCs. Nuclear export inhibition with leptomycin attenuated the PDGF-BB-induced increases in VSMC proliferation in both CSN8-hypo and control VSMCs. Further, genetically disabling CSN5 nuclear export but not disabling CSN5 deneddylase activity suppressed the hyperproliferation and restored p27 nuclear localization in CSN8 hypomorphic VSMCs. Interestingly, CSN deneddylase inhibition by CSN5i-3 did not alter the hyperproliferation of cultured CSN8-hypo VSMCs but suppressed wild type VSMC proliferation in cellulo and in vivo and blocked neointimal formation in wild type mice. CONCLUSION: The CSN promotes VSMC proliferation and NH in injured vessels through deneddylation activity and CSN5-mediated nuclear export.
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This study is focused on the selective delivery and release of the plant-based anticancer compound eugenol (EUG) in colorectal cancer cells (CRC). EUG is an apoptotic and anti-growth compound in diverse malignant tumors, including CRC. However, EUG's rapid metabolization, excretion, and side effects on normal cells at higher dosages are major limitations of its therapeutic potential. To address this problem, we developed a "smart" enzyme-responsive nanoparticle (eNP) loaded with EUG that exposes tumors to a high level of the drug while keeping its concentration low among healthy cells. We demonstrated that EUG induces apoptosis in CRC cells irrespective of their grades in a dose- and time-dependent manner. EUG significantly decreases cancer cell migration, invasion, and the population of colon cancer stem cells, which are key players in tumor metastasis and drug resistance. The "smart" eNPs-EUG show a high affinity to cancer cells with rapid internalization with no affinity toward normal colon epithelial cells. NPs-EUG enhanced the therapeutic efficacy of EUG measured by a cell viability assay and showed no toxicity effect on normal cells. The development of eNPs-EUG is a promising strategy for innovative anti-metastatic therapeutics.
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Mortalin (GRP75, HSPA9A), a heat shock protein (HSP), regulates a wide range of cellular processes, including cell survival, growth, and metabolism. The regulatory functions of mortalin are mediated through a diverse set of protein partners associated with different cellular compartments, which allows mortalin to perform critical functions under physiological conditions, including mitochondrial protein quality control. However, alteration of mortalin's activities, its abnormal subcellular compartmentalization, and its protein partners turn mortalin into a disease-driving protein in different pathological conditions, including cancers. Here, mortalin's contributions to tumorigenic pathways are explained. Pathology information based on mortalin's RNA expression extracted from The Cancer Genome Atlas (TCGA) transcriptomic database indicates that mortalin has an independent prognostic value in common tumors, including lung, breast, and colorectal cancer (CRC). Subsequently, the binding partners of mortalin reported in different cellular models, from yeast to mammalian cells, and its regulation by post-translational modifications are discussed. Finally, we focus on colorectal cancer and discuss how mortalin and its tumorigenic downstream protein targets are regulated by a ubiquitin-like protein through the 26S proteasomal degradation machinery. A broader understanding of the function of mortalin and its positive and negative regulation in the formation and progression of human diseases, particularly cancer, is essential for developing new strategies to treat a diverse set of human diseases critically associated with dysregulated mortalin.
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A high-throughput drug screen revealed that veratridine (VTD), a natural plant alkaloid, induces expression of the anti-cancer protein UBXN2A in colon cancer cells. UBXN2A suppresses mortalin, a heat shock protein, with dominant roles in cancer development including epithelial-mesenchymal transition (EMT), cancer cell stemness, drug resistance, and apoptosis. VTD-dependent expression of UBXN2A leads to the deactivation of mortalin in colon cancer cells, making VTD a potential targeted therapy in malignant tumors with high levels of mortalin. VTD was used clinically for the treatment of hypertension in decades past. However, the discovery of newer antihypertensive drugs and concerns over potential neuro- and cardiotoxicity ended the use of VTD for this purpose. The current study aims to determine the safety and efficacy of VTD at doses sufficient to induce UBXN2A expression in a mouse model. A set of flow-cytometry experiments confirmed that VTD induces both early and late apoptosis in a dose-dependent manner. In vivo intraperitoneal (IP) administration of VTD at 0.1 mg/kg every other day (QOD) for 4 weeks effectively induced expression of UBXN2A in the small and large intestines of mice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays on tissues collected from VTD-treated animals demonstrated VTD concentrations in the low pg/mg range. To address concerns regarding neuro- and cardiotoxicity, a comprehensive set of behavioral and cardiovascular assessments performed on C57BL/6NHsd mice revealed that VTD generates no detectable neurotoxicity or cardiotoxicity in animals receiving 0.1 mg/kg VTD QOD for 30 days. Finally, mouse xenograft experiments in athymic nude mice showed that VTD can suppress tumor growth. The main causes for the failure of experimental oncologic drug candidates are lack of sufficient safety and efficacy. The results achieved in this study support the potential utility of VTD as a safe and efficacious anti-cancer molecule.
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Colorectal cancer (CRC) is one of the most widely diagnosed cancers worldwide. Despite notable improvements in therapeutic strategies available to CRC patients, late stages of CRC have a higher incidence rate of drug resistance, which is associated with a higher mortality rate. The development of therapeutic strategies that use nanoparticles as a drug delivery system has become one of the most promising potential approaches for cancer therapy. Previous studies have shown that a natural plant alkaloid, veratridine (VTD), suppresses colon cancer cell migration and invasion, two essential factors in tumor metastasis, through activation of the gene that encodes the tumor-suppressor protein UBXN2A. The goal of this study is to develop a nanoassembly to selectively deliver VTD to cancer cells and release it on demand while leaving normal cells intact. We packaged the targeted therapy anticancer molecule VTD inside mesoporous silica nanoparticles (MSNs) impermeable to the blood-brain barrier (BBB) and with selective affinity to CRC cells and sealed the VTD-loaded nanoparticles with an enzymatically cleavable protein. The particles will deliver and release VTD only at the targeted colorectal tumor sites. Since the enzyme MMP-7 protease is dominantly secreted by CRC cells, the release triggered by the enzymes will increase VTD concentration at tumor cells, enhancing the efficiency of the new therapy. We have proven the selective affinity of two types of VTD-carrying particles to CRC cells and enzyme- or acid-triggered VTD release. Negatively surface-charged MSNs showed significant affinity toward positively charged cancer cells but not negatively charged normal fibroblast colon cells, making VTD-MSNs a promising anticancer drug with minimal side effects.
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Neoplasias do ColoRESUMO
Targeting the ubiquitin-proteasome system (UPS) - in particular, the proteasome complex - has emerged as an attractive novel cancer therapy. While several proteasome inhibitors have been successfully approved by the Food and Drug Administration for the treatment of hematological malignancies, the clinical efficacy of these inhibitors is unexpectedly lower in the treatment of solid tumors due to the functional and structural heterogeneity of proteasomes in solid tumors. There are ongoing trials to examine the effectiveness of compound and novel proteasome inhibitors that can target solid tumors either alone or in combination with conventional chemotherapeutic agents. The modest therapeutic efficacy of proteasome inhibitors such as bortezomib in solid malignancies demands further research to clarify the exact effects of these proteasome inhibitors on different proteasomes present in cancer cells. The structural, cellular localization and functional analysis of the proteasome complexes in solid tumors originated from different tissues provides new insights into the diversity of proteasomes' responses to inhibitors. In this study, we used an optimized iodixanol gradient ultracentrifugation to purify a native form of proteasome complexes with their intact associated protein partners enriched within distinct cellular compartments. It is therefore possible to isolate proteasome subcomplexes with far greater resolution than sucrose or glycerol fractionations. We have identified differences in the catalytic activities, subcellular distribution, and inhibitor sensitivity of cytoplasmic proteasomes isolated from human colon, breast, and pancreatic cancer cell lines. Our developed techniques and generated results will serve as a valuable guideline for investigators developing a new generation of proteasome inhibitors as an effective targeted therapy for solid tumors.
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Neurite outgrowth involves reciprocal signaling interactions between tumor cells and nerves where invading tumor cells have acquired the ability to respond to pro-invasive signals within the nerve environment. Neurite outgrowth could serve as a mechanism leading to invasion of cancer cells into the nerve sheath and subsequent metastasis. Snail transcription factor can promote migration and invasion of prostate cancer cells. We hypothesized that prostate cancer cell interaction with nerve cells will be mediated by Snail expression within prostate cancer cells. For this study we utilized various prostate cancer cell lines: C4-2 non-silencing (NS, control); C4-2 Snail shRNA, (stable Snail knockdown); LNCaP Neo (empty vector control) and LNCaP Snail (stably over-expressing Snail). Cancer cell adhesion and migration towards nerve cells (snF96.2 or NS20Y) was examined by co-culture assays. Conditioned media (CM) collected from C4-2 cells was cultured with nerve cells (PC-12 or NS20Y) for 48 h followed by qualitative or quantitative neurite outgrowth assay. Our results showed that cancer cells expressing high levels of Snail (LNCaP Snail/C4-2 NS) displayed significantly higher migration adherence to nerve cells, compared to cells with lower levels of Snail (LNCaP Neo/C4-2 Snail shRNA). Additionally, LNCaP Snail or C4-2 NS (Snail-high) CM led to a higher neurite outgrowth compared to the LNCaP Neo or C4-2 Snail shRNA (Snail-low). In conclusion, Snail promotes migration and adhesion to nerve cells, as well as neurite outgrowth via secretion of soluble factors. Therefore, targeting cancer cell interaction with nerves may contribute to halting prostate cancer progression/metastasis.
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Crescimento Neuronal/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Animais , Adesão Celular/genética , Comunicação Celular/genética , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/genética , Inativação Gênica , Humanos , Masculino , Camundongos , Neurônios/metabolismo , Neoplasias da Próstata/patologia , RatosRESUMO
Overexpression of oncoproteins is a major cause of treatment failure using current chemotherapeutic drugs. Drug-induced degradation of oncoproteins is feasible and can improve clinical outcomes in diverse types of cancers. Mortalin-2 (mot-2) is a dominant oncoprotein in several tumors, including colorectal cancer (CRC). In addition to inactivating the p53 tumor suppressor protein, mot-2 enhances tumor cell invasion and migration. Thus, mot-2 is considered a potential therapeutic target in several cancer types. The current study investigated the biological role of a ubiquitin-like protein called UBXN2A in the regulation of mot-2 turnover. An orthogonal ubiquitin transfer technology followed by immunoprecipitation, in vitro ubiquitination, and Magnetic Beads TUBE2 pull-down experiments revealed that UBXN2A promotes carboxyl terminus of the HSP70-interacting protein (CHIP)-dependent ubiquitination of mot-2. We subsequently showed that UBXN2A increases proteasomal degradation of mot-2. A subcellular compartmentalization experiment revealed that induced UBXN2A decreases the level of mot-2 and its chaperone partner, HSP60. Pharmacological upregulation of UBXN2A using a small molecule, veratridine (VTD), decreases the level of mot-2 in cancer cells. Consistent with the in vitro results, UBXN2A+/- mice exhibited selective elevation of mot-2 in colon tissues. An in vitro Anti-K48 TUBE isolation approach showed that recombinant UBXN2A enhances proteasomal degradation of mot-2 in mouse colon tissues. Finally, we observed enhanced association of CHIP with the UBXN2A-mot-2 complex in tumors in an azoxymethane/dextran sulfate sodium-induced mouse CRC model. The existence of a multiprotein complex containing UBXN2A, CHIP, and mot-2 suggests a synergistic tumor suppressor activity of UBXN2A and CHIP in mot-2-enriched tumors. This finding validates the UBXN2A-CHIP axis as a novel and potential therapeutic target in CRC.
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Proteínas de Choque Térmico HSP70/metabolismo , Proteínas Mitocondriais/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Chaperonina 60/metabolismo , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Feminino , Haploinsuficiência/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Complexos Multiproteicos/metabolismo , Fenótipo , Estabilidade Proteica , Especificidade por Substrato , UbiquitinaçãoRESUMO
The ubiquitination pathway and proteasomal degradation machinery dominantly regulate p53 tumor suppressor protein stability, localization, and functions in both normal and cancerous cells. Selective E3 ubiquitin ligases dominantly regulate protein levels and activities of p53 in a large range of physiological conditions and in response to cellular changes induced by exogenous and endogenous stresses. The regulation of p53's functions by E3 ubiquitin ligases is a complex process that can lead to positive or negative regulation of p53 protein in a context- and cell type-dependent manner. Accessory proteins bind and modulate E3 ubiquitin ligases, adding yet another layer of regulatory control for p53 and its downstream functions. This review provides a comprehensive understanding of p53 regulation by selective E3 ubiquitin ligases and their potential to be considered as a new class of biomarkers and therapeutic targets in diverse types of cancers.
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Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Humanos , Modelos Biológicos , Ligação ProteicaRESUMO
The UBXD family is a diverse group of UBX (ubiquitin-regulatory X) domain-containing proteins in mammalian cells. Members of this family contain a UBX domain typically located at the carboxyl-terminal of the protein. In contrast to the UBX domain shared by all members of UBXD family, the amino-terminal domains are diverse and appear to carry out different roles in a subcellular localization-dependent manner. UBXD proteins are principally associated with the endoplasmic reticulum (ER), where they positively or negatively regulate the ER-associated degradation machinery (ERAD). The distinct protein interaction networks of UBXD proteins allow them to have specific functions independent of the ERAD pathway in a cell type- and tissue context-dependent manner. Recent reports have illustrated that a number of mammalian members of the UBXD family play critical roles in several proliferation and apoptosis pathways dysregulated in selected types of cancer. This review covers recent advances that elucidate the therapeutic potential of selected members of the UBXD family that can contribute to tumor growth.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Degradação Associada com o Retículo Endoplasmático , Neoplasias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/análise , Animais , Apoptose , Proliferação de Células , Humanos , NF-kappa B/metabolismo , Neoplasias/patologia , Transdução de SinaisRESUMO
BACKGROUND: Targeted drugs modulate selective pathways activated or repressed only in cancer cells, resulting in a higher response to chemotherapy with less severe side effects. The use of a selected member of the heat shock protein 70 family (HSP70) as an effective therapeutic target in the treatment of colorectal cancer (CRC) will be the focus of this review. METHODS: We generated two main questions for this study: 1) What are the current and potential future molecular therapies in CRC? 2) Can selective members of the HSP70 family advance drug design and drug discovery for treatment of CRC patients? We discuss related articles based on their significance and translational contributions to the existing literature. RESULTS: The first part of this review discusses molecularly targeted agents that are currently used successfully in the clinic for the treatment of patients with CRC and highlights several novel targeted agents that are being investigated in ongoing trials. The second part of this review focuses on the unique tumorigenic functions of heat shock proteins, particularly mortalin-2, an essential heat shock protein for mitochondrial biogenesis in normal cells and a dominant oncoprotein in colon cancer cells. Basic and clinical studies have justified mortalin-2 as a potential molecular target, and its inhibition could dramatically improve patients' responses to standard chemotherapies. CONCLUSION: Further understanding of the contributions of HSP70 family members to CRC at the molecular level, combined with translation of new concepts into effective targeted therapies, are anticipated to improve clinical outcomes and increase the therapeutic synergy with combination treatment with cytotoxic agents.
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Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Proteínas de Choque Térmico HSP70/metabolismo , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/química , Antineoplásicos/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Humanos , Proteínas Oncogênicas/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Ubiquitinas/química , Ubiquitinas/metabolismoRESUMO
The relatively low success rates of current colorectal cancer (CRC) therapies have led investigators to search for more specific treatments. Vertebrate models of colorectal cancer are essential tools for the verification of new therapeutic avenues such as gene therapy. The evaluation of colorectal cancer in mouse models has been limited due to the lack of an accurate quantitative and longitudinal noninvasive method. This work introduces a method of three-dimensional micro-ultrasound reconstruction and microbubble administration for the comprehensive and longitudinal evaluation of CRC progression. This approach enabled quantification of both tumor volume and relative vascularity using a well-established inducible murine model of colon carcinogenesis. This inducible model recapitulated the adenocarcinoma sequence that occurs in human CRC allowing systematic in situ evaluation of the ultrasound technique. The administration of intravenous microbubbles facilitated enhancement of colon vascular contrast and quantification of relative vascularity of the mid and distal colon of the mouse in three dimensions. In addition, two-dimensional imaging in the sagittal orientation of the colon using Non-Linear Contrast Mode enabled calculation of relative blood volume and perfusion as the microbubbles entered the colon microvasculature. Quantitative results provided by the outlined protocol represent a noninvasive tool that can more accurately define CRC development and progression. This ultrasound technique will allow the practical and economical longitudinal study of murine CRC in both basic and preclinical studies.
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Overexpression of the oncoprotein mortalin in cancer cells and its protein partners enables mortalin to promote multiple oncogenic signaling pathways and effectively antagonize chemotherapy-induced cell death. A UBX-domain-containing protein, UBXN2A, acts as a potential mortalin inhibitor. This current study determines whether UBXN2A effectively binds to and occupies mortalin's binding pocket, resulting in a direct improvement in the tumor's sensitivity to chemotherapy. Molecular modeling of human mortalin's binding pocket and its binding to the SEP domain of UBXN2A followed by yeast two-hybrid and His-tag pull-down assays revealed that three amino acids (PRO442, ILE558, and LYS555) within the substrate-binding domain of mortalin are crucial for UBXN2A binding to mortalin. As revealed by chase experiments in the presence of cycloheximide, overexpression of UBXN2A seems to interfere with the mortalin-CHIP E3 ubiquitin ligase and consequently suppresses the C-terminus of the HSC70-interacting protein (CHIP)-mediated destabilization of p53, resulting in its stabilization in the cytoplasm and upregulation in the nucleus. Overexpression of UBXN2A causes a significant inhibition of cell proliferation and the migration of colon cancer cells. We silenced UBXN2A in the human osteosarcoma U2OS cell line, an enriched mortalin cancer cell, followed by a clinical dosage of the chemotherapeutic agent 5-fluorouracil (5-FU). The UBXN2A knockout U2OS cells revealed that UBXNA is essential for the cytotoxic effect achieved by 5-FU. UBXN2A overexpression markedly increased the apoptotic response of U2OS cells to the 5-FU. In addition, silencing of UBXN2A protein suppresses apoptosis enhanced by UBXN2A overexpression in U2OS. The knowledge gained from this study provides insights into the mechanistic role of UBXN2A as a potent mortalin inhibitor and as a potential chemotherapy sensitizer for clinical application.
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Antimetabólitos Antineoplásicos/farmacologia , Fluoruracila/farmacologia , Proteínas de Choque Térmico HSP70/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Ubiquitinas/metabolismo , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Proteínas de Choque Térmico HSP70/química , Humanos , Simulação de Acoplamento Molecular , Neoplasias/genética , Neoplasias/patologia , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Ligação Proteica , Domínios Proteicos , Interferência de RNA , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinas/química , Ubiquitinas/genéticaRESUMO
The subcellular localization, expression level, and activity of anti-cancer proteins alter in response to intrinsic and extrinsic cellular stresses to reverse tumor progression. The purpose of this study is to determine whether UBXN2A, an activator of the p53 tumor suppressor protein, has different subcellular compartmentalization in response to the stress of DNA damage. We measured trafficking of the UBXN2A protein in response to two different DNA damage stresses, UVB irradiation and the genotoxic agent Etoposide, in colon cancer cell lines. Using a cytosol-nuclear fractionation technique followed by western blot and immunofluorescence staining, we monitored and quantitated UBXN2A and p53 proteins as well as p53's downstream apoptotic pathway. We showed that the anti-cancer protein UBXN2A acts in the early phase of cell response to two different DNA damage stresses, being induced to translocate into the cytoplasm in a dose- and time-dependent manner. UVB-induced cytoplasmic UBXN2A binds to mortalin-2 (mot-2), a known oncoprotein in colon tumors. UVB-dependent upregulation of UBXN2A in the cytoplasm decreases p53 binding to mot-2 and activates apoptotic events in colon cancer cells. In contrast, the shRNA-mediated depletion of UBXN2A leads to significant reduction in apoptosis in colon cancer cells exposed to UVB and Etoposide. Leptomycin B (LMB), which was able to block UBXN2A nuclear export following Etoposide treatment, sustained p53-mot-2 interaction and had partially antagonistic effects with Etoposide on cell apoptosis. The present study shows that nucleocytoplasmic translocation of UBXN2A in response to stresses is necessary for its anti-cancer function in the cytoplasm. In addition, LMB-dependent suppression of UBXN2A's translocation to the cytoplasm upon stress allows the presence of an active mot-2 oncoprotein in the cytoplasm, resulting in p53 sequestration as well as activation of other mot-2-dependent growth promoting pathways.
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Neuronal nicotinic acetylcholine receptors (nAChRs) containing the α3 subunit are known for their prominent role in normal ganglionic transmission while their involvement in the mechanisms underlying nicotine addiction and smoking-related disease has been emerging only in recent years. The amount of information available on the maturation and trafficking of α3-containing nAChRs is limited. We previously showed that UBXN2A is a p97 adaptor protein that facilitates the maturation and trafficking of α3-containing nAChRs. Further investigation of the mechanisms of UBXN2A actions revealed that the protein interacts with CHIP (carboxyl terminus of Hsc70 interacting protein), whose ubiquitin E3 ligase activity regulates the degradation of several disease-related proteins. We show that CHIP displays E3 ligase activity toward the α3 nAChR subunit and contributes to its ubiquitination and subsequent degradation. UBXN2A interferes with CHIP-mediated ubiquitination of α3 and protects the nicotinic receptor subunit from endoplasmic reticulum associated degradation (ERAD). UBXN2A also cross-talks with VCP/p97 and HSC70/HSP70 proteins in a complex where α3 is likely to be targeted by CHIP. Overall,we identify CHIP as an E3 ligase for α3 and UBXN2A as a protein that may efficiently regulate the stability of CHIP's client substrates.
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Regulação Enzimológica da Expressão Gênica/fisiologia , Receptores Nicotínicos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Retículo Endoplasmático/fisiologia , Degradação Associada com o Retículo Endoplasmático , Complexo de Golgi/fisiologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células PC12 , Complexo de Endopeptidases do Proteassoma , Ratos , Receptores Nicotínicos/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitinação/fisiologiaRESUMO
Veratridine (VTD), an alkaloid derived from the Liliaceae plant shows anti-tumor effects; however, its molecular targets have not been thoroughly studied. Using a high-throughput drug screen, we found that VTD enhances transactivation of UBXN2A, resulting in upregulation of UBXN2A in the cytoplasm, where UBXN2A binds and inhibits the oncoprotein mortalin-2 (mot-2). VTD-treated cancer cells undergo cell death in UBXN2A- and mot-2-dependent manners. The cytotoxic function of VTD is grade-dependent, and the combined treatment with a sub-optimal dose of the standard chemotherapy, 5-Fluorouracil (5-FU) and etoposide, demonstrated a synergistic effect, resulting in higher therapeutic efficacy. VTD influences the CD44+ stem cells, possibly through UBXN2A-dependent inhibition of mot-2. The VTD-dependent expression of UBXN2A is a potential candidate for designing novel strategies for colon cancer treatment because: 1) In 50% of colon cancer patients, UBXN2A protein levels in tumor tissues are significantly lower than those in the adjacent normal tissues. 2) Cytoplasmic expression of the mot-2 protein is very low in non-cancerous cells; thus, VTD can produce tumor-specific toxicity while normal cells remain intact. 3) Finally, VTD or its modified analogs offer a valuable adjuvant chemotherapy strategy to improve the efficacy of 5-FU-based chemotherapy for colon cancer patients harboring WT-p53.
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Neoplasias da Mama/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas Mitocondriais/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Veratridina/química , Animais , Antineoplásicos/química , Apoptose , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Citoplasma/metabolismo , Progressão da Doença , Elementos Facilitadores Genéticos , Etoposídeo/química , Feminino , Fluoruracila/química , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Células HCT116 , Humanos , Receptores de Hialuronatos/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Extratos Vegetais/química , Análise Serial de Proteínas , Proteína Supressora de Tumor p53/metabolismoRESUMO
Recent advances in trans-differentiation of one type cell to another have made it possible to directly convert Huntington's disease (HD) patient fibroblasts into neurons by modulation of cell-lineage-specific transcription factors or RNA processing. However, this possibility has not been examined. Here, we demonstrate that HD patient-derived fibroblasts can be directly trans-differentiated into neuron-like cells by knockdown of the expression of a single gene encoding the polypyrimidine-tract-binding protein. The directly converted HD neuron-like cells were positive in expression of Tuj1, NeuN, DARPP-32, and γ-aminobutyric acid and exhibited neuritic breakdown, abnormal neuritic branching, increased cell death, and aggregation of mutant huntingtin. These observations indicate that the neuron-like cells directly converted from HD patient fibroblasts recapitulate the major aspects of neuropathological characteristics of HD and thus provide an additional model for understanding the disorder and validation of therapeutic reagents.
Assuntos
Reprogramação Celular , Fibroblastos/patologia , Doença de Huntington/patologia , Neuritos/patologia , Neurônios/patologia , Morte Celular , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Mutação , Proteínas do Tecido Nervoso/genética , Neuritos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologiaRESUMO
In response to DNA damage or replication stress, proliferating cells are arrested at different cell cycle stages for DNA repair by downregulating the activity of both the cyclin-dependent kinases (CDKs) and other important cell cycle kinases, including Polo-like kinase 1 (PLK1) . The signaling pathway to inhibit CDKs is relatively well understood, and breast cancer gene 1 (BRCA1) and other DNA damage response (DDR) factors play a key role in this process. However, the DNA damage-induced inhibition of PLK1 is still largely a mystery. Here we show that DNA damage and replication stress stimulate the association between BRCA1 and PLK1. Most importantly, we demonstrate that BRCA1 downregulates the kinase activity of PLK1 by modulating the dynamic interactions of Aurora A, hBora, and PLK1. Together with previous findings, we propose that in response to replication stress and DNA damage, BRCA1 plays a critical role in downregulating the kinase activity of both CDKs and PLK1.
Assuntos
Proteína BRCA1/genética , Proteínas de Ciclo Celular/genética , Quinases Ciclina-Dependentes/genética , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Estresse Fisiológico/genética , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Proteína BRCA1/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/metabolismo , Dano ao DNA , Humanos , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Quinase 1 Polo-LikeRESUMO
Breast cancer gene 1 (BRCA1) deficient cells not only are hypersensitive to double-strand breaks but also are hypersensitive to UV irradiation and other agents that cause replication blockade; however, the molecular mechanisms behind these latter sensitivities are largely unknown. Here, we report that BRCA1 promotes cell survival by directly regulating the DNA damage tolerance pathway in response to agents that create cross-links in DNA. We show that BRCA1 not only promotes efficient mono- and polyubiquitination of proliferating cell nuclear antigen (PCNA) by regulating the recruitment of replication protein A, Rad18, and helicase-like transcription factor to chromatin but also directly recruits translesion polymerases, such as Polymerase eta and Rev1, to the lesions through protein-protein interactions. Our data suggest that BRCA1 plays a critical role in promoting translesion DNA synthesis as well as DNA template switching.