ABSTRACT: Mortalin [also known as heat shock protein family A (HSP70) member 9 (HSPA9) or glucose-regulated protein 75 (GRP75)] is a mitochondrial molecular chaperone that is often up-regulated and mislocalized in tumors with abnormal activation of the kinases MEK and ERK. Here, we found that mortalin depletion was selectively lethal to tumor and immortalized normal cells expressing the mutant kinase B-RafV600E or the chimeric protein ?Raf-1:ER and that MEK-ERK-sensitive regulation of the peptide-binding domain in mortalin was critical to cell survival or death. Proteomics screening identified adenine nucleotide translocase 3 (ANT3) as a previously unknown mortalin substrate and cell survival/death effector. Mechanistically, increased MEK-ERK signaling activity and mortalin function converged opposingly on the regulation of mitochondrial permeability. Specifically, whereas MEK-ERK activity increased mitochondrial permeability by promoting the interaction between ANT3 and the peptidyl-prolyl isomerase cyclophilin D (CypD), mortalin decreased mitochondrial permeability by inhibiting this interaction. Hence, mortalin depletion increased mitochondrial permeability in MEK-ERK-deregulated cells to an extent that triggered cell death. HSP70 inhibitor derivatives that effectively inhibited mortalin suppressed the proliferation of B-RafV600E tumor cells in culture and in vivo, including their B-Raf inhibitor-resistant progenies. These findings suggest that targeting mortalin has potential as a selective therapeutic strategy in B-Raf-mutant or MEK-ERK-driven tumors.
Project description:Dysregulated Raf/MEK/extracellular signal-regulated kinase (ERK) signaling, a common hallmark of tumorigenesis, can trigger innate tumor-suppressive mechanisms, which must be inactivated for carcinogenesis to occur. This innate tumor-suppressive signaling may provide a potential therapeutic target. Here we report that mortalin (HSPA9/GRP75/PBP74) is a novel negative regulator of Raf/MEK/ERK and may provide a target for the reactivation of tumor-suppressive signaling of the pathway in cancer. We found that mortalin is present in the MEK1/MEK2 proteome and is upregulated in human melanoma biopsy specimens. In different MEK/ERK-activated cancer cell lines, mortalin depletion induced cell death and growth arrest, which was accompanied by increased p21(CIP1) transcription and MEK/ERK activity. Remarkably, MEK/ERK activity was necessary for mortalin depletion to induce p21(CIP1) expression in B-Raf(V600E)-transformed cancer cells regardless of their p53 status. In contrast, in cell types exhibiting normal MEK/ERK status, mortalin overexpression suppressed B-Raf(V600E)- or ?Raf-1:ER-induced MEK/ERK activation, p21(CIP1) expression, and cell cycle arrest. Other HSP70 family chaperones could not effectively replace mortalin for p21(CIP1) regulation, suggesting a unique role for mortalin. These findings reveal a novel mechanism underlying p21(CIP1) regulation in MEK/ERK-activated cancer and identify mortalin as a molecular switch that mediates the tumor-suppressive versus oncogenic result of dysregulated Raf/MEK/ERK signaling. Our study also demonstrates that p21(CIP1) has dual effects under mortalin-depleted conditions, i.e., mediating cell cycle arrest while limiting cell death.
Project description:Although deregulation of MEK/extracellular signal-regulated kinase (ERK) activity is a key feature in cancer, high-magnitude MEK/ERK activity can paradoxically induce growth inhibition. Therefore, additional mechanisms may exist to modulate MEK/ERK activity in favor of tumor cell proliferation. We previously reported that mortalin/HSPA9 can facilitate proliferation of certain KRAS and BRAF tumor cells by modulating MEK/ERK activity. In this study, we demonstrated that mortalin can regulate MEK/ERK activity via protein phosphatase 1? (PP1?). We found that PP1? inhibition increases steady-state levels of phosphorylated MEK1/2 in various tumor cells expressing B-RafV600E or K-RasG12C/D Intriguingly, coimmunoprecipitation and in vitro binding assays revealed that mortalin facilitates PP1?-mediated MEK1/2 dephosphorylation by promoting PP1?-MEK1/2 interaction in an ATP-sensitive manner. The region spanning Val482 to Glu491 in the substrate-binding cavity and the substrate lid of mortalin were necessary for these physical interactions, which is consistent with conventional heat shock protein 70 (HSP70)-client interaction mechanisms. Nevertheless, mortalin depletion did not affect cellular PP1? levels or its regulatory phosphorylation, suggesting a nonconventional role for mortalin in promoting PP1?-MEK1/2 interaction. Of note, PP1? was upregulated in human melanoma and pancreatic cancer biopsy specimens in correlation with mortalin upregulation. PP1? may therefore have a role in tumorigenesis in concert with mortalin, which affects MEK/ERK activity in tumor cells.
Project description:The mitochondrial HSP70 chaperone mortalin (HSPA9/GRP75) is often upregulated and mislocalized in MEK/ERK-deregulated tumors. Here, we show that mortalin depletion can selectively induce death of immortalized normal fibroblasts IMR90E1A when combined with K-RasG12V expression, but not with wild-type K-Ras expression, and that K-RasG12V-driven MEK/ERK activity is necessary for this lethality. This cell death was attenuated by knockdown or inhibition of adenine nucleotide translocase (ANT), cyclophilin D (CypD), or mitochondrial Ca2+ uniporter (MCU), which implicates a mitochondria-originated death mechanism. Indeed, mortalin depletion increased mitochondrial membrane permeability and induced cell death in KRAS-mutated human pancreatic ductal adenocarcinoma (PDAC) and colon cancer lines, which were attenuated by knockdown or inhibition of ANT, CypD, or MCU, and occurred independently of TP53 and p21CIP1. Intriguingly, JG-98, an advanced MKT-077 derivative, phenocopied the lethal effects of mortalin depletion in K-RasG12V-expressing IMR90E1A and KRAS-mutated tumor cell lines in vitro. Moreover, JG-231, a JG-98 analog with improved microsomal stability effectively suppressed the xenograft of MIA PaCa-2, a K-RasG12C-expressing human PDAC line, in athymic nude mice. These data demonstrate that oncogenic KRAS activity sensitizes cells to the effects of mortalin depletion, suggesting that mortalin has potential as a selective therapeutic target for KRAS-mutated tumors.
Project description:Recent high-throughput-sequencing of cancer genomes has identified oncogenic mutations in the B-Raf genetic locus as one of the critical events in melanomagenesis. B-Raf encodes a serine/threonine kinase that regulates the MAPK/ERK kinase (MEK) and extracellular signal-regulated kinase (ERK) protein kinase cascade. In normal cells, the activity of B-Raf is tightly regulated and is required for cell growth and survival. B-Raf gain-of-function mutations in melanoma frequently lead to unrestrained growth, enhanced cell invasion and increased viability of cancer cells. Although it is clear that the invasive phenotypes of B-Raf mutated melanoma cells are stringently dependent on B-Raf-MEK-ERK activation, the downstream effector targets that are required for oncogenic B-Raf-mediated melanomagenesis are not well defined. miRNAs have regulatory functions towards the expression of genes that are important in carcinogenesis. We observed that miR-10b expression correlates with the presence of the oncogenic B-Raf (B-RafV600E) mutation in melanoma cells. While expression of miR-10b enhances anchorage-independent growth of B-Raf wild-type melanoma cells, miR-10b silencing decreases B-RafV600E cancer cell invasion in vitro. Importantly, the expression of miR-10b is required for B-RafV600E-mediated anchorage independent growth and invasion of melanoma cells in vitro. Taken together our results suggest that miR-10b is an important mediator of oncogenic B-RafV600E activity in melanoma.
Project description:Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the rearranged during transfection (RET) proto-oncogene. For therapy of advanced MTC, the Food and Drug Administration recently approved vandetanib and cabozantinib, the tyrosine kinase inhibitors targeting RET, vascular endothelial growth factor receptor, epidermal growth factor receptor and/or c-MET. Nevertheless, not all patients respond to these drugs, demanding additional therapeutic strategies. We found that mortalin (HSPA9/GRP75), a member of HSP70 family, is upregulated in human MTC tissues and that its depletion robustly induces cell death and growth arrest in MTC cell lines in culture and in mouse xenografts. These effects were accompanied by substantial downregulation of RET, induction of the tumor-suppressor TP53 and altered expression of cell cycle regulatory machinery and apoptosis markers, including E2F-1, p21(CIP1), p27(KIP1) and Bcl-2 family proteins. Our investigation of the molecular mechanisms underlying these effects revealed that mortalin depletion induces transient MEK/ERK (extracellular signal-regulated kinase) activation and altered mitochondrial bioenergetics in MTC cells, as indicated by depolarized mitochondrial membrane, decreased oxygen consumption and extracellular acidification and increased oxidative stress. Intriguingly, mortalin depletion induced growth arrest partly via the MEK/ERK pathway, whereas it induced cell death by causing mitochondrial dysfunction in a Bcl-2-dependent manner. However, TP53 was not necessary for these effects except for p21(CIP1) induction. Moreover, mortalin depletion downregulated RET expression independently of MEK/ERK and TP53. These data demonstrate that mortalin is a key regulator of multiple signaling and metabolic pathways pivotal to MTC cell survival and proliferation, proposing mortalin as a novel therapeutic target for MTC.
Project description:BACKGROUND: Colorectal cancers carrying the B-Raf V600E-mutation are associated with a poor prognosis. The purpose of this study was to identify B-RafV600E-mediated traits of cancer cells in a genetic in vitro model and to assess the selective sensitization of B-RafV600E-mutant cancer cells towards therapeutic agents. METHODS: Somatic cell gene targeting was used to generate subclones of the colorectal cancer cell line RKO containing either wild-type or V600E-mutant B-Raf kinase. Cell-biologic analyses were performed in order to link cancer cell traits to the BRAF-mutant genotype. Subsequently, the corresponding tumor cell clones were characterized pharmacogenetically to identify therapeutic agents exhibiting selective sensitivity in B-RafV600E-mutant cells. RESULTS: Genetic targeting of mutant BRAF resulted in restoration of sensitivity to serum starvation-induced apoptosis and efficiently inhibited cell proliferation in the absence of growth factors. Among tested agents, the B-Raf inhibitor dabrafenib was found to induce a strong V600E-dependent shift in cell viability. In contrast, no differential sensitizing effect was observed for conventional chemotherapeutic agents (mitomycin C, oxaliplatin, paclitaxel, etoposide, 5-fluorouracil), nor for the targeted agents cetuximab, sorafenib, vemurafenib, RAF265, or for inhibition of PI3 kinase. Treatment with dabrafenib efficiently inhibited phosphorylation of the B-Raf downstream targets Mek 1/2 and Erk 1/2. CONCLUSION: Mutant BRAF alleles mediate self-sufficiency of growth signals and serum starvation-induced resistance to apoptosis. Targeting of the BRAF mutation leads to a loss of these hallmarks of cancer. Dabrafenib selectively inhibits cell viability in B-RafV600E mutant cancer cells.
Project description:The lysyl oxidase gene (LOX) inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162-amino-acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibits Erk signaling, motility, and tumor formation in a breast cancer xenograft model; however, its mechanism of action is largely unknown. Here, a copurification-mass spectrometry approach was taken using ectopically expressed LOX-PP in HEK293T cells and the heat shock/chaperone protein Hsp70 identified. Hsp70 interaction with LOX-PP was confirmed using coimmunoprecipitation of intracellularly and bacterially expressed and endogenous proteins. The interaction was mapped to the Hsp70 peptide-binding domain and to LOX-PP amino acids 26 to 100. LOX-PP association reduced Hsp70 chaperone activities of protein refolding and survival after heat shock. LOX-PP interacted with the Hsp70 chaperoned protein c-Raf. With the use of ectopic expression of LOX-PP wild-type and deletion proteins, small interfering RNA (siRNA) knockdown, and Lox(-/-) mouse embryo fibroblasts, LOX-PP interaction with c-Raf was shown to decrease downstream activation of MEK and NF-?B, migration, and anchorage-independent growth and reduce its mitochondrial localization. Thus, the interaction of LOX-PP with Hsp70 and c-Raf inhibits a critical intermediate in Ras-induced MEK signaling and plays an important role in the function of this tumor suppressor.
Project description:Influenza A viruses (IAV) can cause severe global pandemic outbreaks. The currently licensed antiviral drugs are not very effective and prone to viral resistance. Thus, novel effective and broadly active drugs are urgently needed. We have identified the cellular Raf/MEK/ERK signaling cascade as crucial for IAV replication and suitable target for an antiviral intervention. Since this signaling cascade is aberrantly activated in many human cancers, several clinically approved inhibitors of Raf and MEK are now available. Here we explored the anti-IAV action of the licensed B-RafV600E inhibitor Vemurafenib. Treatment of B-RafWT cells with Vemurafenib induced a hyperactivation of the Raf/MEK/ERK cascade rather than inhibiting its activation upon IAV infection. Despite this hyperactivation, which has also been confirmed by others, Vemurafenib still strongly limited IAV-induced activation of other signaling cascades especially of p38 and JNK mitogen-activated protein kinase (MAPK) pathways. Most interestingly, Vemurafenib inhibited virus-induced apoptosis via impaired expression of apoptosis-inducing cytokines and led to hampered viral protein expression most likely due to the decreased activation of p38 and JNK MAPK. These multiple actions resulted in a profound and broadly active inhibition of viral replication, up to a titer reduction of three orders of a magnitude. Thus, while Vemurafenib did not act similar to MEK inhibitors, it displays strong antiviral properties via a distinct and multi-target mode of action.
Project description:Mutations in the BRAF proto-oncogene, which encodes the B-Raf kinase, are associated with more aggressive, less-differentiated and therapy-resistant colorectal cancers (CRC). However, the molecular mechanisms responsible for these correlations remain unknown. Here, we report the characterization of human isogenic CRC cell line models (Caco-2, HT29, Colo-205) in which we modulate the expression of the B-RafV600E oncoprotein either by conditional cDNA or shRNA expression. Using these models in conventional and three dimensional tissue culture systems, we demonstrate that genetic depletion of endogenous B-RafV600E decreases cellular motility and invasion, while it induces hallmarks of differentiated epithelia such as the formation of functional adherens and tight junctions. Importantly, these effects are recapitulated by exposing these lines to B-Raf (PLX4720, vemurafenib, dabrafenib) or MEK inhibitors (trametinib). Furthermore, loss of endogenous B-RafV600E in HT29 xenografts does not only stall tumor growth, but also induces epithelial structures with marked expression of Cdx-2, a prognostic marker and master regulator of intestinal morphogenesis. By performing the first transcriptome profiles of B-Raf inhibitor treated 3D cultures of a primary adenocarcinoma and a metastasis derived CRC cell line, we establish functional links between B-RafV600E and proteins of known and potentially new prognostic relevance. We propose that B-Raf/MEK/ERK pathway inhibitors could be used to induce CRC differentiation and thereby to limit metastatic disease. Overall design: To measure the time resolved gene responses, RNA was isolated from PLX4720 or DMSO treated Colo-205 and HT29 3D culture cell lysates at days 1,3 and 10 and days 1,3, and 8, respectively. The time points for HT29 cells were taken in biological duplicates.
Project description:Mutations in the BRAF proto-oncogene, which encodes the B-Raf kinase, are associated with more aggressive, less-differentiated and therapy-resistant colorectal cancers (CRC). However, the molecular mechanisms responsible for these correlations remain unknown. Here, we report the characterization of human isogenic CRC cell line models (Caco-2, HT29, Colo-205) in which we modulate the expression of the B-RafV600E oncoprotein either by conditional cDNA or shRNA expression. Using these models in conventional and three dimensional tissue culture systems, we demonstrate that genetic depletion of endogenous B-RafV600E decreases cellular motility and invasion, while it induces hallmarks of differentiated epithelia such as the formation of functional adherens and tight junctions. Importantly, these effects are recapitulated by exposing these lines to B-Raf (PLX4720, vemurafenib, dabrafenib) or MEK inhibitors (trametinib). Furthermore, loss of endogenous B-RafV600E in HT29 xenografts does not only stall tumor growth, but also induces epithelial structures with marked expression of Cdx-2, a prognostic marker and master regulator of intestinal morphogenesis. By performing the first transcriptome profiles of B-Raf inhibitor treated 3D cultures of a primary adenocarcinoma and a metastasis derived CRC cell line, we establish functional links between B-RafV600E and proteins of known and potentially new prognostic relevance. We propose that B-Raf/MEK/ERK pathway inhibitors could be used to induce CRC differentiation and thereby to limit metastatic disease. To measure the time resolved gene responses, RNA was isolated from PLX4720 or DMSO treated Colo-205 and HT29 3D culture cell lysates at days 1,3 and 10 and days 1,3, and 8, respectively. The time points for HT29 cells were taken in biological duplicates.