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The heterogeneity of protein-rich inclusions and its significance in neurodegeneration is poorly understood. Standard patient-derived iPSC models develop inclusions neither reproducibly nor in a reasonable time frame. Here, we developed screenable iPSC "inclusionopathy" models utilizing piggyBac or targeted transgenes to rapidly induce CNS cells that express aggregation-prone proteins at brain-like levels. Inclusions and their effects on cell survival were trackable at single-inclusion resolution. Exemplar cortical neuron α-synuclein inclusionopathy models were engineered through transgenic expression of α-synuclein mutant forms or exogenous seeding with fibrils. We identified multiple inclusion classes, including neuroprotective p62-positive inclusions versus dynamic and neurotoxic lipid-rich inclusions, both identified in patient brains. Fusion events between these inclusion subtypes altered neuronal survival. Proteome-scale α-synuclein genetic- and physical-interaction screens pinpointed candidate RNA-processing and actin-cytoskeleton-modulator proteins like RhoA whose sequestration into inclusions could enhance toxicity. These tractable CNS models should prove useful in functional genomic analysis and drug development for proteinopathies.
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Triple-negative breast cancer (TNBC) is responsible for a disproportionate number of breast cancer patient deaths due to extensive molecular heterogeneity, high recurrence rates and lack of targeted therapies. Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT pathway occurs in approximately 50% of TNBC patients. Here, we performed a genome-wide CRISPR/Cas9 screen with PI3Kα and AKT inhibitors to find targetable synthetic lethalities in TNBC. Cholesterol homeostasis was identified as a collateral vulnerability with AKT inhibition. Disruption of cholesterol homeostasis with pitavastatin synergized with AKT inhibition to induce TNBC cytotoxicity in vitro, in mouse TNBC xenografts and in patient-derived, estrogen receptor (ER)-negative breast cancer organoids. Neither ER-positive breast cancer cell lines nor ER-positive organoids were sensitive to combined AKT inhibitor and pitavastatin. Mechanistically, TNBC cells showed impaired sterol regulatory element-binding protein 2 (SREBP-2) activation in response to single agent or combination treatment with AKT inhibitor and pitavastatin, which was rescued by inhibition of the cholesterol trafficking protein Niemann-Pick C1 (NPC1). NPC1 loss caused lysosomal cholesterol accumulation, decreased endoplasmic reticulum cholesterol levels, and promoted SREBP-2 activation. Taken together, these data identify a TNBC-specific vulnerability to the combination of AKT inhibitors and pitavastatin mediated by dysregulated cholesterol trafficking. These findings support combining AKT inhibitors with pitavastatin as a therapeutic modality in TNBC. .
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Small molecule-mediated proteasomal degradation of proteins is a powerful tool for synthetic regulation of biological activity. To control Cas9 activity in cells, we engineered an anti-CRISPR protein, AcrIIA4, fused to a degradation (dTAG) or small molecule assisted shutoff (SMASh) tag. Co-expression of the tagged AcrIIA4 along with Cas9 and riboswitch-regulated sgRNAs enables precise tunable control of CRISPR activity by small molecule addition.
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Immune evasion is a significant contributor to tumor evolution, and the immunoinhibitory axis PD-1/PD-L1 is a frequent mechanism employed to escape tumor immune surveillance. To identify cancer drivers involved in immune evasion, we performed a CRISPR-Cas9 screen of tumor suppressor genes regulating the basal and interferon (IFN)-inducible cell surface levels of PD-L1. Multiple regulators of PD-L1 were identified, including IRF2, ARID2, KMT2D, and AAMP. We also identified CTCF and the cohesin complex proteins, known regulators of chromatin architecture and transcription, among the most potent negative regulators of PD-L1 cell surface expression. Additionally, loss of the cohesin subunit RAD21 was shown to up-regulate PD-L2 and MHC-I surface expression. PD-L1 and MHC-I suppression by cohesin were shown to be conserved in mammary epithelial and myeloid cells. Comprehensive examination of the transcriptional effect of STAG2 deficiency in epithelial and myeloid cells revealed an activation of strong IFN and NF-κB expression signatures. Inhibition of JAK-STAT or NF-κB pathways did not result in rescue of PD-L1 up-regulation in RAD21-deficient cells, suggesting more complex or combinatorial mechanisms at play. Discovery of the PD-L1 and IFN up-regulation in cohesin-mutant cells expands our understanding of the biology of cohesin-deficient cells as well as molecular regulation of the PD-L1 molecule.
Assuntos
Antígeno B7-H1/metabolismo , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias/metabolismo , Antígeno B7-H1/genética , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Humanos , Janus Quinases/genética , Janus Quinases/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Regulação para Cima , CoesinasRESUMO
ECT2 is an activator of RHO GTPases that is essential for cytokinesis. In addition, ECT2 was identified as an oncoprotein when expressed ectopically in NIH/3T3 fibroblasts. However, oncogenic activation of ECT2 resulted from N-terminal truncation, and such truncated ECT2 proteins have not been found in patients with cancer. In this study, we observed elevated expression of full-length ECT2 protein in preneoplastic colon adenomas, driven by increased ECT2 mRNA abundance and associated with APC tumor-suppressor loss. Elevated ECT2 levels were detected in the cytoplasm and nucleus of colorectal cancer tissue, suggesting cytoplasmic mislocalization as one mechanism of early oncogenic ECT2 activation. Importantly, elevated nuclear ECT2 correlated with poorly differentiated tumors, and a low cytoplasmic:nuclear ratio of ECT2 protein correlated with poor patient survival, suggesting that nuclear and cytoplasmic ECT2 play distinct roles in colorectal cancer. Depletion of ECT2 reduced anchorage-independent cancer cell growth and invasion independent of its function in cytokinesis, and loss of Ect2 extended survival in a Kras G12D Apc-null colon cancer mouse model. Expression of ECT2 variants with impaired nuclear localization or guanine nucleotide exchange catalytic activity failed to restore cancer cell growth or invasion, indicating that active, nuclear ECT2 is required to support tumor progression. Nuclear ECT2 promoted ribosomal DNA transcription and ribosome biogenesis in colorectal cancer. These results support a driver role for both cytoplasmic and nuclear ECT2 overexpression in colorectal cancer and emphasize the critical role of precise subcellular localization in dictating ECT2 function in neoplastic cells. SIGNIFICANCE: ECT2 overexpression and mislocalization support its role as a driver in colon cancer that is independent from its function in normal cell cytokinesis.
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Neoplasias Colorretais/genética , Genômica/métodos , Proteínas Proto-Oncogênicas/metabolismo , Idoso , Animais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Masculino , CamundongosRESUMO
Understanding the genetic control of human embryonic stem cell function is foundational for developmental biology and regenerative medicine. Here we describe an integrated genome-scale loss- and gain-of-function screening approach to identify genetic networks governing embryonic stem cell proliferation and differentiation into the three germ layers. We identified a deep link between pluripotency maintenance and survival by showing that genetic alterations that cause pluripotency dissolution simultaneously increase apoptosis resistance. We discovered that the chromatin-modifying complex SAGA and in particular its subunit TADA2B are central regulators of pluripotency, survival, growth, and lineage specification. Joint analysis of all screens revealed that genetic alterations that broadly inhibit differentiation across multiple germ layers drive proliferation and survival under pluripotency-maintaining conditions and coincide with known cancer drivers. Our results show the power of integrated multilayer genetic screening for the robust mapping of complex genetic networks.
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Células-Tronco Embrionárias Humanas , Diferenciação Celular/genética , Células-Tronco Embrionárias , Mutação com Ganho de Função , Camadas Germinativas , HumanosRESUMO
During tumorigenesis, tumors must evolve to evade the immune system and do so by disrupting the genes involved in antigen processing and presentation or up-regulating inhibitory immune checkpoint genes. We performed in vivo CRISPR screens in syngeneic mouse tumor models to examine requirements for tumorigenesis both with and without adaptive immune selective pressure. In each tumor type tested, we found a marked enrichment for the loss of tumor suppressor genes (TSGs) in the presence of an adaptive immune system relative to immunocompromised mice. Nearly one-third of TSGs showed preferential enrichment, often in a cancer- and tissue-specific manner. These results suggest that clonal selection of recurrent mutations found in cancer is driven largely by the tumor's requirement to avoid the adaptive immune system.
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Carcinogênese , Inativação Gênica , Genes Supressores de Tumor , Evasão da Resposta Imune , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Quimiocina CCL2/metabolismo , Feminino , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Evasão da Resposta Imune/genética , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Transplante de Neoplasias , Neoplasias Experimentais/patologia , Seleção Genética , Microambiente TumoralRESUMO
Genomics has provided a detailed structural description of the cancer genome. Identifying oncogenic drivers that work primarily through dosage changes is a current challenge. Unrestrained proliferation is a critical hallmark of cancer. We constructed modular, barcoded libraries of human open reading frames (ORFs) and performed screens for proliferation regulators in multiple cell types. Approximately 10% of genes regulate proliferation, with most performing in an unexpectedly highly tissue-specific manner. Proliferation drivers in a given cell type showed specific enrichment in somatic copy number changes (SCNAs) from cognate tumors and helped predict aneuploidy patterns in those tumors, implying that tissue-type-specific genetic network architectures underlie SCNA and driver selection in different cancers. In vivo screening confirmed these results. We report a substantial contribution to the catalog of SCNA-associated cancer drivers, identifying 147 amplified and 107 deleted genes as potential drivers, and derive insights about the genetic network architecture of aneuploidy in tumors.
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Aneuploidia , Neoplasias/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Mapeamento Cromossômico , Cromossomos/genética , Fator de Transcrição E2F1/antagonistas & inibidores , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Feminino , Biblioteca Gênica , Genômica , Humanos , Queratinas/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Oncogenes , Fases de Leitura Aberta/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Activating mutations in the KRAS oncogene are highly prevalent in tumors, especially those of the colon, lung, and pancreas. To better understand the genetic dependencies that K-Ras mutant cells rely upon for their growth, we employed whole-genome CRISPR loss-of-function screens in two isogenic pairs of cell lines. Since loss of essential genes is uniformly toxic in CRISPR-based screens, we also developed a small hairpin RNA (shRNA) library targeting essential genes. These approaches uncovered a large set of proteins whose loss results in the selective reduction of K-Ras mutant cell growth. Pathway analysis revealed that many of these genes function in the mitochondria. For validation, we generated isogenic pairs of cell lines using CRISPR-based genome engineering, which confirmed the dependency of K-Ras mutant cells on these mitochondrial pathways. Finally, we found that mitochondrial inhibitors reduce the growth of K-Ras mutant tumors in vivo, aiding in the advancement of strategies to target K-Ras-driven malignancy.
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Proliferação de Células/fisiologia , Genes ras/genética , Proteínas Proto-Oncogênicas/metabolismo , Animais , Linhagem Celular , Proliferação de Células/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Feminino , Células HCT116 , Humanos , Hidrazonas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Minociclina/análogos & derivados , Minociclina/farmacologia , Mutação/genética , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Proteínas Proto-Oncogênicas/genética , Tigeciclina , Triazóis/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cellular senescence is a terminal stress-activated program controlled by the p53 and p16(INK4a) tumor suppressor proteins. A striking feature of senescence is the senescence-associated secretory phenotype (SASP), a pro-inflammatory response linked to tumor promotion and aging. We have identified the transcription factor GATA4 as a senescence and SASP regulator. GATA4 is stabilized in cells undergoing senescence and is required for the SASP. Normally, GATA4 is degraded by p62-mediated selective autophagy, but this regulation is suppressed during senescence, thereby stabilizing GATA4. GATA4 in turn activates the transcription factor NF-κB to initiate the SASP and facilitate senescence. GATA4 activation depends on the DNA damage response regulators ATM and ATR, but not on p53 or p16(INK4a). GATA4 accumulates in multiple tissues, including the aging brain, and could contribute to aging and its associated inflammation.
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Envelhecimento/genética , Autofagia/genética , Senescência Celular/genética , Dano ao DNA , Fator de Transcrição GATA4/metabolismo , Inflamação/genética , Proteínas Adaptadoras de Transdução de Sinal , Envelhecimento/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Encéfalo/metabolismo , Ciclo Celular/genética , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina , Fibroblastos , Fator de Transcrição GATA4/genética , Perfilação da Expressão Gênica , Humanos , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The Ras-like small GTPases RalA and RalB are well validated effectors of RAS oncogene-driven human cancer growth, and pharmacologic inhibitors of Ral function may provide an effective anti-Ras therapeutic strategy. Intriguingly, although RalA and RalB share strong overall amino acid sequence identity, exhibit essentially identical structural and biochemical properties, and can utilize the same downstream effectors, they also exhibit divergent and sometimes opposing roles in the tumorigenic and metastatic growth of different cancer types. These distinct biological functions have been attributed largely to sequence divergence in their carboxyl-terminal hypervariable regions. However, the role of posttranslational modifications signaled by the hypervariable region carboxyl-terminal tetrapeptide CAAX motif (C = cysteine, A = aliphatic amino acid, X = terminal residue) in Ral isoform-selective functions has not been addressed. We determined that these modifications have distinct roles and consequences. Both RalA and RalB require Ras converting CAAX endopeptidase 1 (RCE1) for association with the plasma membrane, albeit not with endomembranes, and loss of RCE1 caused mislocalization as well as sustained activation of both RalA and RalB. In contrast, isoprenylcysteine carboxylmethyltransferase (ICMT) deficiency disrupted plasma membrane localization only of RalB, whereas RalA depended on ICMT for efficient endosomal localization. Furthermore, the absence of ICMT increased stability of RalB but not RalA protein. Finally, palmitoylation was critical for subcellular localization of RalB but not RalA. In summary, we have identified striking isoform-specific consequences of distinct CAAX-signaled posttranslational modifications that contribute to the divergent subcellular localization and activity of RalA and RalB.
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Processamento de Proteína Pós-Traducional/fisiologia , Proteínas ral de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Endossomos/genética , Endossomos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Transporte Proteico/fisiologia , Proteínas ral de Ligação ao GTP/genéticaRESUMO
Since their discovery in 1986, Ral (Ras-like) GTPases have emerged as critical regulators of diverse cellular functions. Ral-selective guanine nucleotide exchange factors (RalGEFs) function as downstream effectors of the Ras oncoprotein, and the RalGEF-Ral signaling network comprises the third best characterized effector of Ras-dependent human oncogenesis. Because of this, Ral GTPases as well as their effectors are being explored as possible therapeutic targets in the treatment of RAS mutant cancer. The two Ral isoforms, RalA and RalB, interact with a variety of downstream effectors and have been found to play key and distinct roles in both normal and neoplastic cell physiology including regulation of vesicular trafficking, migration and invasion, tumor formation, metastasis, and gene expression. In this review we provide an overview of Ral biochemistry and biology, and we highlight recent discoveries.
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Diverse environmental cues converge on and are integrated by the mTOR signaling network to control cellular growth and homeostasis. The mammalian Tsc1-Tsc2 GTPase activating protein (GAP) heterodimer is a critical negative regulator of Rheb and mTOR activation. The RalGAPα-RalGAPß heterodimer shares sequence and structural similarity with Tsc1-Tsc2. Unexpectedly, we observed that C. elegans expresses orthologs for the Rheb and RalA/B GTPases and for RalGAPα/ß, but not Tsc1/2. This prompted our investigation to determine whether RalGAPs additionally modulate mTOR signaling. We determined that C. elegans RalGAP loss decreased lifespan, consistent with a Tsc-like function. Additionally, RalGAP suppression in mammalian cells caused RalB-selective activation and Sec5- and exocyst-dependent engagement of mTORC1 and suppression of autophagy. Unexpectedly, we also found that Tsc1-Tsc2 loss activated RalA/B independently of Rheb-mTOR signaling. Finally, RalGAP suppression caused mTORC1-dependent pancreatic tumor cell invasion. Our findings identify an unexpected crosstalk and integration of the Ral and mTOR signaling networks.
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Autofagia/genética , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/citologia , Senescência Celular/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Invasividade Neoplásica/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas ral de Ligação ao GTP/fisiologia , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Transdução de Sinais , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/metabolismoRESUMO
The high prevalence of KRAS mutations and importance of the RalGEF-Ral pathway downstream of activated K-ras in pancreatic ductal adenocarcinoma (PDAC) emphasize the importance of identifying novel methods by which to therapeutically target these pathways. It was recently demonstrated that phosphorylation of RalA S194 by Aurora A kinase (AAK) is critical for PDAC tumorigenesis. We sought to evaluate the AAK-selective inhibitor MLN8237 as a potential indirect anti-RalA-targeted therapy for PDAC. We used a site-specific phospho-S194 RalA antibody and determined that RalA S194 phosphorylation levels were elevated in a subset of PDAC cell lines and human tumors relative to unmatched normal controls. Effects of MLN8237 on anchorage-independent growth in PDAC cell lines and growth of patient-derived xenografts (PDX) were variable, with a subset of cell lines and PDX showing sensitivity. Surprisingly, RalA S194 phosphorylation levels in PDAC cell lines or PDX tumors did not correlate with MLN8237 responsiveness. However, we identified Ki67 as a possible early predictive biomarker for response to MLN8237 in PDAC. These results indicate that MLN8237 treatment may be effective for a subset of patients with PDAC independent of RalA S194 phosphorylation. Ki67 may be an effective pharmacodynamic biomarker to identify response early in the course of treatment.
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Azepinas/administração & dosagem , Carcinogênese/efeitos dos fármacos , Carcinoma Ductal Pancreático/tratamento farmacológico , Antígeno Ki-67/genética , Pirimidinas/administração & dosagem , Proteínas ral de Ligação ao GTP/genética , Aurora Quinase A/metabolismo , Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Humanos , Mutação , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Proteínas ral de Ligação ao GTP/metabolismo , Proteínas ras/genéticaRESUMO
The effectiveness of cancer therapeutics targeting signal transduction pathways is comprised of a diversity of mechanisms that drive de novo or acquired resistance. Two recent studies identify mTOR activation as a point of convergence of mechanisms that cause resistance to inhibitors of the Raf-MEK-ERK and PI3K signaling.
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Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Melanoma/enzimologia , Melanoma/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Complexos Multiproteicos/antagonistas & inibidores , Mutação/genética , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Feminino , HumanosRESUMO
Mutationally activated K-Ras can utilize a multitude of downstream effector proteins to promote oncogenesis. While the Raf and phosphoinositol 3-kinase effector pathways are the best-studied and validated, recent studies have established the critical importance of Ral guanine nucleotide exchange factor (RalGEF) activation of the RalA and RalB small GTPases in cancer biology. Due to recent evidence that the RalGEF-Ral pathway is necessary for the tumorigenic and metastatic potential of KRAS mutant pancreatic ductal adenocarcinoma (PDAC) tumor cells, we investigated whether or not Ral signaling was necessary for KRAS mutant colorectal cancer (CRC) tumor cell growth. As in PDAC, we found upregulated RalA and RalB activation in CRC tumor cell lines and tumors. Surprisingly we found antagonistic roles for RalA and RalB in the regulation of CRC tumor cell anchorage-independent growth. This observation contrasts with PDAC, where RalA but not RalB is necessary for PDAC tumor cell anchorage-independent growth. Our results emphasize cancer cell type differences in Ral function and hence the need for distinct Ral targeted therapeutic approaches in the treatment of CRC vs. PDAC.
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Neoplasias Colorretais/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Proliferação de Células , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Dados de Sequência Molecular , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Reto/metabolismo , Reto/patologia , Transdução de Sinais , Proteínas ral de Ligação ao GTP/química , Proteínas ras/genéticaRESUMO
Ras-like (Ral) small GTPases are regulated downstream of Ras and the noncanonical Ral guanine nucleotide exchange factor (RalGEF) effector pathway. Despite RalA and RalB sharing 82% sequence identity and utilization of shared effector proteins, their roles in normal and neoplastic cell growth have been shown to be highly distinct. Here, we determined that RalB function is regulated by protein kinase Cα (PKCα) phosphorylation. We found that RalB phosphorylation on Ser-198 in the C-terminal membrane targeting sequence resulted in enhanced RalB endomembrane accumulation and decreased RalB association with its effector, the exocyst component Sec5. Additionally, RalB phosphorylation regulated vesicular trafficking and membrane fusion by regulating v- and t-SNARE interactions. RalB phosphorylation regulated vesicular traffic of α5-integrin to the cell surface and cell attachment to fibronectin. In summary, our data suggest that phosphorylation by PKCα is critical for RalB-mediated vesicle trafficking and exocytosis.
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Exocitose/fisiologia , Proteína Quinase C-alfa/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Ativação Enzimática/fisiologia , Humanos , Fosforilação/fisiologia , Proteína Quinase C-alfa/genética , Transporte Proteico/fisiologia , Ratos , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas ral de Ligação ao GTP/genéticaRESUMO
Our recent studies implicated key and distinct roles for the highly related RalA and RalB small GTPases (82% sequence identity) in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis and invasive and metastatic growth, respectively. How RalB may promote PDAC invasion and metastasis has not been determined. In light of known Ral effector functions in regulation of actin organization and secretion, we addressed a possible role for RalB in formation of invadopodia, actin-rich membrane protrusions that contribute to tissue invasion and matrix remodeling. We determined that a majority of KRAS mutant PDAC cell lines exhibited invadopodia and that expression of activated K-Ras is both necessary and sufficient for invadopodium formation. Invadopodium formation was not dependent on the canonical Raf-MEK-ERK effector pathway and was instead dependent on the Ral effector pathway. However, this process was more dependent on RalB than on RalA. Surprisingly, RalB-mediated invadopodium formation was dependent on RalBP1/RLIP76 but not Sec5 and Exo84 exocyst effector function. Unexpectedly, the requirement for RalBP1 was independent of its best known function as a GTPase-activating protein for Rho small GTPases. Instead, disruption of the ATPase function of RalBP1 impaired invadopodium formation. Our results identify a novel RalB-mediated biochemical and signaling mechanism for invadopodium formation.
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Transportadores de Cassetes de Ligação de ATP/metabolismo , Extensões da Superfície Celular/enzimologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Extensões da Superfície Celular/ultraestrutura , Ativação Enzimática , Humanos , Invasividade Neoplásica/ultraestrutura , Neoplasias Pancreáticas/patologia , Transdução de SinaisRESUMO
The high frequency of RAS mutations in human cancers (33%) has stimulated intense interest in the development of anti-Ras inhibitors for cancer therapy. Currently, the major focus of these efforts is centered on inhibitors of components involved in Ras downstream effector signaling. In particular, more than 40 inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase cascade and phosphoinositide 3-kinase-AKT-mTOR effector signaling networks are currently under clinical evaluation. However, these efforts are complicated by the fact that Ras can utilize at least 9 additional functionally distinct effectors, with at least 3 additional effectors with validated roles in Ras-mediated oncogenesis. Of these, the guanine nucleotide exchange factors of the Ras-like (Ral) small GTPases (RalGEFs) have emerged as important effectors of mutant Ras in pancreatic, colon, and other cancers. In this review, we summarize the evidence for the importance of this effector pathway in cancer and discuss possible directions for therapeutic inhibition of aberrant Ral activation and signaling.
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Current approaches to block KRAS oncogene function focus on inhibition of K-Ras downstream effector signaling. We evaluated the antitumor activity of selumetinib (AZD6244, ARRY-142886), a potent and selective MEK1/2 inhibitor, on a panel of colorectal carcinoma (CRC) cells and found no inhibition of KRAS mutant CRC cell anchorage-independent growth. Although AKT activity was elevated in KRAS mutant cells, and PI3K inhibition did impair the growth of MEK inhibitor-insensitive CRC cell lines, concurrent treatment with selumetinib did not provide additional antitumor activity. Therefore, we speculated that inhibition of the Ral guanine exchange factor (RalGEF) effector pathway may be a more effective approach for blocking CRC growth. RalGEFs are activators of the related RalA and RalB small GTPases and we found activation of both in CRC cell lines and patient tumors. Interfering RNA stable suppression of RalA expression reduced CRC tumor cell anchorage-independent growth, but surprisingly, stable suppression of RalB greatly enhanced soft agar colony size and formation frequency. Despite their opposing activities, both RalA and RalB regulation of anchorage-independent growth required interaction with RalBP1/RLIP76 and components of the exocyst complex. Interestingly, RalA interaction with the Exo84 but not Sec5 exocyst component was necessary for supporting anchorage-independent growth, whereas RalB interaction with Sec5 but not Exo84 was necessary for inhibition of anchorage-independent growth. We suggest that anti-RalA-selective therapies may provide an effective approach for KRAS mutant CRC.