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MUC1-C overexpression has been associated with the progression of pancreatic tumors by promoting the aggressive and metastatic phenotypes. As MUC1 is a STAT3 target gene, STAT3 plays a major role in regulating MUC1-C expression. In this study, we report an alternative mechanism by which integrin-linked kinase (ILK) post-transcriptionally modulates the expression of MUC1-C by maintaining its protein stability in pancreatic cancer cells. We found that ILK acts in concert with STAT3 to facilitate IL-6-mediated upregulation of MUC1-C; ILK depletion was equally effective as STAT3 depletion in abolishing IL-6-induced MUC1-C overexpression without disturbing the phosphorylation or cellular distribution of STAT3. Conversely, ectopic expression of constitutively active ILK increased MUC1-C expression, though this increase was not noted with kinase-dead ILK. This finding suggests the requirement of the kinase activity of ILK in regulating MUC1-C stability, which was confirmed by using the ILK kinase inhibitor T315. Furthermore, our data suggest the involvement of protein kinase C (PKC)δ in mediating the suppressive effect of ILK inhibition on MUC1-C repression. For example, co-immunoprecipitation analysis indicated that ILK depletion-mediated MUC1-C phosphorylation was accompanied by increased phosphorylation of PKCδ at the activation loop Thr-507 and increased binding of PKCδ to MUC1-C. Conversely, ILK overexpression resulted in decreased PKCδ phosphorylation. From a mechanistic perspective, the present finding, together with our recent report that ILK controls the expression of oncogenic KRAS through a regulatory loop, underscores the pivotal role of ILK in promoting pancreatic cancer progression.
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The highly homeostasis-resistant nature of cancer cells leads to their escape from treatment and to liver metastasis, which in turn makes pancreatic ductal adenocarcinoma (PDAC) difficult to treat, especially the squamous/epithelial-to-mesenchymal transition (EMT)-like subtype. As the molecular mechanisms underlying tumour heterogeneity remain elusive, we investigated whether epigenetic regulation might explain inter-individual differences in the progression of specific subtypes. DNA methylation profiling performed on cancer tissues prior to chemo/radiotherapy identified one hypermethylated CpG site (CpG6882469) in the VAV1 gene body that was correlated with demethylation of two promoter CpGs (CpG6772370/CpG6772811) in both PDAC and peripheral blood. Transforming growth factor ß treatment induced gene-body hypermethylation, dissociation of DNMT1 from the promoter, and VAV1 expression via SMAD4 and mutant KrasG12D. Pharmacological inhibition of TGFß-VAV1 signalling decreased the squamous/EMT-like cancer cells, promoted nuclear VAV1 localization, and enhanced the efficacy of gemcitabine in prolonging the survival of KPfl/flC mice. Together, the three VAV1 CpGs serve as biomarkers for prognosis and early detection, and the TGFß-VAV1 axis represents a therapeutic target.
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Adenocarcinoma/metabolismo , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas c-vav/genética , Fator de Crescimento Transformador beta/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Animais , Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Progressão da Doença , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Regiões Promotoras Genéticas , Pirazóis/uso terapêutico , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
Integrin-linked kinase (ILK) is a mediator of aggressive phenotype in pancreatic cancer. On the basis of our finding that knockdown of either KRAS or ILK has a reciprocal effect on the other's expression, we hypothesized the presence of an ILK-KRAS regulatory loop that enables pancreatic cancer cells to regulate KRAS expression. This study aimed to elucidate the mechanism by which this regulatory circuitry is regulated and to investigate the translational potential of targeting ILK to suppress oncogenic KRAS signaling in pancreatic cancer. Interplay between KRAS and ILK and the roles of E2F1, c-Myc and heterogeneous nuclear ribonucleoprotein as intermediary effectors in this feedback loop was interrogated by genetic manipulations through small interfering RNA/short hairpin RNA knockdown and ectopic expression, western blotting, PCR, promoter-luciferase reporter assays, chromatin immunoprecipitation and pull-down analyses. In vivo efficacy of ILK inhibition was evaluated in two murine xenograft models. Our data show that KRAS regulated the expression of ILK through E2F1-mediated transcriptional activation, which, in turn, controlled KRAS gene expression via hnRNPA1-mediated destabilization of the G-quadruplex on the KRAS promoter. Moreover, ILK inhibition blocked KRAS-driven epithelial-mesenchymal transition and growth factor-stimulated KRAS expression. The knockdown or pharmacological inhibition of ILK suppressed pancreatic tumor growth, in part, by suppressing KRAS signaling. These studies suggest that this KRAS-E2F1-ILK-hnRNPA1 regulatory loop enables pancreatic cancer cells to promote oncogenic KRAS signaling and to interact with the tumor microenvironment to promote aggressive phenotypes. This regulatory loop provides a mechanistic rationale for targeting ILK to suppress oncogenic KRAS signaling, which might foster new therapeutic strategies for pancreatic cancer.
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Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/fisiologia , Neoplasias Pancreáticas/patologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Transdução de Sinais/fisiologia , Animais , Linhagem Celular Tumoral , Fator de Transcrição E2F1/fisiologia , Transição Epitelial-Mesenquimal , Ribonucleoproteína Nuclear Heterogênea A1 , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Ativação TranscricionalRESUMO
Methylated histone readers are critical for chromatin dynamics, transcription, and DNA repair. Human PHRF1 contains a plant homeodomain (PHD) that recognizes methylated histones and a RING domain, which ubiquitinates substrates. A recent study reveals that PHRF1 is a tumor suppressor that promotes TGF-ß cytostatic signaling through TGIF ubiquitination. Also, PHRF1 is a putative phosphorylation substrate of ataxia telangiectasia-mutated/ataxia telangiectasia and Rad3-related kinases; however, the role of PHRF1 in DNA damage response is unclear. Here we report a novel function of PHRF1 in modulating non-homologous end-joining (NHEJ). PHRF1 quickly localizes to DNA damage lesions upon genotoxic insults. Ablation of PHRF1 decreases the efficiency of plasmid-based end-joining, whereas PHRF1 overexpression leads to an elevated NHEJ in H1299 reporter cells. Immunoprecipitation and peptide pull-down assays verify that PHRF1 constitutively binds to di- and trimethylated histone H3 lysine 36 (H3K36) (H3K36me2 and H3K36me3) via its PHD domain. Substitution of S915DT917E to ADAE in PHRF1 decreases its affinity for NBS1. Both PHD domain and SDTE motif are required for its NHEJ-promoting activity. Furthermore, PHRF1 mediates PARP1 polyubiquitination for proteasomal degradation. These results suggest that PHRF1 may combine with H3K36 methylation and NBS1 to promote NHEJ and stabilize genomic integrity upon DNA damage insults.
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Dano ao DNA , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/genética , Proteínas do Grupo Polycomb/genética , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Genoma Humano , Células HEK293 , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Metilação , Dados de Sequência Molecular , Proteínas do Grupo Polycomb/metabolismoRESUMO
mu-Opioid receptors are the pharmacological targets of endogenous opioid peptides and morphine-like alkaloid drugs. Previous studies of transfected cells and peripheral neurons indicate that opioid receptors are rapidly internalized after activation by the alkaloid agonist etorphine but not after activation by morphine. To determine whether opioid receptors in the central nervous system are regulated by a similar process of agonist-selective internalization, mu-opioid receptors were examined in rat brain neurons after treatment of animals with opioid drugs. Internalized mu receptors were observed within 30 min after intraperitoneal injection of the alkaloid agonist etorphine, and this process was blocked by the antagonist naloxone. Colocalization of internalized opioid receptors with transferrin receptors in confocal optical sections indicated that receptor internalization observed in vivo is mediated by a membrane trafficking pathway similar to that observed previously in vitro using transfected human embryonic kidney 293 cells. Morphine failed to induce detectable rapid internalization of receptors, even when administered to animals at doses far in excess of those required to induce analgesia. To quantify these agonist-selective differences and to analyze an array of opioid ligands for their ability to trigger internalization, we used flow cytometry on stably transfected 293 cells. These studies indicated that the different effects of individual agonists are not correlated with their potencies for receptor activation and that a variety of clinically important agonists differ significantly in their relative abilities to stimulate the rapid internalization of opioid receptors.
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Encéfalo/metabolismo , Endocitose/efeitos dos fármacos , Entorpecentes/farmacologia , Receptores Opioides mu/metabolismo , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Humanos , Camundongos , Ratos , Receptores Opioides mu/efeitos dos fármacosRESUMO
The effects of administration of thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), luteinizing hormone (LH) or luteinizing hormone-releasing hormone (LHRH) into the lateral cerebral ventricle on thermoregulation and food and water intake were assessed in rats. Intracerebroventricular, but not intraperitoneal, injection of TSH, LH or LHRH produced hypothermia in rats at ambient temperatures of both 8 and 22 degrees C. The hypothermia in response to TSH injection was due to both decreased metabolic heat production and increased heat loss (cutaneous vasodilatation). The hypothermia in response to either LH or LHRH was due solely to decreased metabolic heat production. There was no change in respiratory evaporative heat loss in response to TSH, LH or LHRH injection. Furthermore, food but not water intake was greatly reduced following an intracerebroventricular injection of TSH or TRH in rats. On the other hand, intracerebroventricular administration of LH, but not LHRH, caused an increase in relative water intake (or water/food) in rats. However, intracerebroventricular administration of LH or LHRH had an insignificant effect on food intake. The data indicate that, in addition to their hormone actions, TSH, LH and their releasing hormones act through a central mechanism to influence some of the physiological or behavioral functions.