Marina Lasa

RhoA and p38 MAPK mediate apoptosis induced by cellular cholesterol depletion

Cholesterol is essential for cell viability, and homeostasis of cellular cholesterol is crucial to various cell functions. Here we examined the effect of cholesterol depletion on apoptosis and the mechanisms underlying this effect in NIH3T3 cells. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment resulted in a significant increase in cellular apoptosis and caspase-3 activation. This effect is not due to a deficiency of nonsterol isoprenoids, intermediate metabolites of the cholesterol biosynthetic pathway, but rather to low cholesterol levels, since addition of cholesterol together with LPDS and 25-HC nearly abolished apoptosis, whereas addition of farnesyl pyrophosphate or geranylgeranyl-pyrophosphate did not reverse the cell viability loss induced by LPDS plus 25-HC treatment. These effects were accompanied by an increase in ERK, JNK and p38 MAPK activity. However, only the inhibition of p38 MAPK with the specific inhibitor SB203580 or the overexpression of a kinase defective MKK6 resulted in a significant decrease in apoptosis and caspase-3 cleavage induced by cholesterol depletion. Furthermore, LPDS plus 25-HC increased RhoA activity, and this effect was reversed by addition of exogenous cholesterol. Finally, overexpression of the dominant negative N19RhoA inhibited p38 MAPK phosphorylation and apoptosis induced by low cholesterol levels. Together, our results demonstrate that cholesterol depletion induces apoptosis through a RhoA- and p38 MAPK-dependent mechanism.

Dual specificity phosphatase 1 expression inversely correlates with NF-κB activity and expression in prostate cancer and promotes apoptosis through a p38 MAPK dependent mechanism

Dual specificity phosphatase 1 (DUSP1) and the transcription factor NF‐κB are implicated in prostate cancer since their expression levels are altered along this disease, although there are no evidences up to date demonstrating a crosstalk between them. In this report, we show for the first time that DUSP1 over‐expression in DU145 cells promotes apoptosis and decreases NF‐κB activity by blocking p65/NF‐κB nuclear translocation. Moreover, although DUSP1 impairs TNF‐α‐induced p38 MAPK and JNK activation, only the specific inhibition of p38 MAPK exerts the same effects than DUSP1 over‐expression on both apoptosis and NF‐κB activity. Consistently, DUSP1 promotes apoptosis and decreases NF‐κB activity in cells in which p38 MAPK is induced by TNF‐α treatment. These results demonstrate that p38 MAPK is specifically involved in DUSP1‐mediated effects on both apoptosis and NF‐κB activity. Interestingly, we show an inverse correlation between DUSP1 expression and activation of both p65/NF‐κB and p38 MAPK in human prostate tissue specimens. Thus, most of apparently normal glands, benign prostatic hyperplasia and low‐grade prostatic intraepithelial neoplasia samples show high DUSP1 expression and low levels of both nuclear p65/NF‐κB and activated p38 MAPK. By contrast, DUSP1 expression levels are low or even absent in high‐grade prostatic intraepithelial neoplasia and prostatic adenocarcinoma samples, whereas nuclear p65/NF‐κB and activated p38 MAPK are highly expressed in the same samples. Overall, our results provide evidence for a role of DUSP1 in the apoptosis of prostate cancer cells, through a mechanism involving the inhibition of p38 MAPK and NF‐κB. Furthermore, our findings suggest that the ratio between DUSP1 and p65/NF‐κB expression levels, rather than the individual expression of both molecules, is a better marker for diagnostic purposes in prostate cancer.

Thyroid hormone-mediated activation of the ERK/dual specificity phosphatase 1 pathway augments the apoptosis of GH4C1 cells by down-regulating nuclear factor-κB activity

Thyroid hormone (T3) plays a crucial role in processes such as cell proliferation and differentiation, whereas its implication on cellular apoptosis has not been well documented. Here we examined the effect of T3 on the apoptosis of GH4C1 pituitary cells and the mechanisms underlying this effect. We show that T3 produced a significant increase in apoptosis in serum-depleted conditions. This effect was accompanied by a decrease in nuclear factor-kappaB (NF-kappaB)-dependent transcription, IkappaBalpha phosphorylation, translocation of p65/NF-kappaB to the nucleus, phosphorylation, and transactivation. Moreover, these effects were correlated with a T3-induced decrease in the expression of antiapoptotic gene products, such as members of the inhibitor of apoptosis protein and Bcl-2 families. On the other hand, ERK but not c-Jun N-terminal kinase or MAPK p38, was activated upon exposure to T3, and inhibition of ERK alone abrogated T3-mediated apoptosis. In addition, T3 increased the expression of the MAPK phosphatase, dual specificity phosphatase 1 (DUSP1), in an ERK-dependent manner. Interestingly, the suppression of DUSP1 expression abrogated T3-induced inhibition of NF-kappaB-dependent transcription and p65/NF-kappaB translocation to the nucleus, as well as T3-mediated apoptosis. Overall, our results indicate that T3 induces apoptosis in rat pituitary tumor cells by down-regulating NF-kappaB activity through a mechanism dependent on the ERK/DUSP1 pathway.

Thyroid Hormone Antagonizes Tumor Necrosis Factor-α Signaling in Pituitary Cells through the Induction of Dual Specificity Phosphatase 1

Pituitary function has been shown to be regulated by an increasing number of factors, including cytokines and hormones, such as TNFalpha and T(3). Both the proinflammatory cytokine TNFalpha and T(3) have been suggested to be involved in the maintenance of tissue homeostasis in the anterior pituitary gland. In this report we show that T(3) negatively interferes with MAPK p38 and nuclear factor-kappaB (NF-kappaB) activation by TNFalpha in GH4C1 cells. Our data demonstrate that MAPK p38 is specifically activated upon exposure to TNFalpha and that T(3) abolishes this activation in a time-dependent manner by a mechanism that involves the induction of the MAPK phosphatase, DUSP1. Our data show that the pool of up-regulated DUSP1 by T(3) is mainly localized to the cytosol, and that TNFalpha does not affect this localization. On the other hand, we show that T(3) impairs the activation of the NF-kappaB pathway induced by TNFalpha, producing a significant decrease in NF-kappaB-dependent transcription, phosphorylation of IkappaBalpha, translocation of p65/NF-kappaB to the nucleus, and p65/NF-kappaB transactivation potential. Interestingly, the overexpression of DUSP1 inhibits the NF-kappaB activation achieved by either TNFalpha or ectopic expression of the upstream inducer of MAPK p38. Conversely, DUSP1 depletion abrogates the inhibitory effect of T(3) on the induction of NF-kappaB-dependent transcription by TNFalpha. Overall, our results indicate that T(3) antagonizes TNFalpha signaling in rat pituitary tumor cells through the induction of DUSP1.

TGFβ induces epithelial‐mesenchymal transition of thyroid cancer cells by both the BRAF/MEK/ERK and Src/FAK pathways

The epithelial‐mesenchymal transition (EMT) is a crucial process in tumour progression, by which epithelial cells acquire a mesenchymal phenotype, increasing its motility and the ability to invade distant sites. Here, we describe the molecular mechanisms by which V600EBRAF, TGFβ and the Src/FAK complex cooperatively regulate EMT induction and cell motility of anaplastic thyroid cancer cells. Analysis of EMT marker levels reveals a positive correlation between TGFβ and Snail expression, with a concomitant downregulation of E‐cadherin, accompanied by an increase of cell migration and invasion. Furthermore, we show that V600EBRAF depletion by siRNA or inhibition of its activity by treatment with its inhibitor PLX4720 reverses the TGFβ‐mediated effects on Snail, E‐cadherin, migration and invasion. Moreover, V600EBRAF induces TGFβ secretion through a MEK/ERK‐dependent mechanism. In addition, TGFβ activates the Src/FAK complex, which in turn regulates the expression of Snail and E‐cadherin as well as cell migration. The inhibition of Src with the inhibitor SU6656 or abrogation of FAK expression with a specific siRNA reverses the TGFβ‐induced effects. Interestingly, we demonstrate that activation of the Src/FAK complex by TGFβ is independent of V600EBRAF signalling, since inhibition of this oncogene does not affect its phosphorylation. Our data strongly suggest that TGFβ induces EMT and aggressiveness of thyroid cancer cells by parallel mechanisms involving both the V600EBRAF/MEK/ERK and Src/FAK pathways independently. Thus, we describe novel functions for Src/FAK in mediating the EMT program and aggressiveness regulated by TGFβ, establishing the inhibition of these proteins as a possible effective approach in preventing tumour progression of V600EBRAF‐expressing thyroid tumours.

VHL promotes immune response against renal cell carcinoma via NF-κB–dependent regulation of VCAM-1

Vascular cell adhesion molecule 1 (VCAM-1) is an adhesion molecule assigned to the activated endothelium mediating immune cells adhesion and extravasation. However, its expression in renal carcinomas inversely correlates with tumor malignancy. Our experiments in clear cell renal cell carcinoma (ccRCC) cell lines demonstrated that von Hippel Lindau (VHL) loss, hypoxia, or PHD (for prolyl hydroxylase domain–containing proteins) inactivation decreased VCAM-1 levels through a transcriptional mechanism that was independent of the hypoxia-inducible factor and dependent on the nuclear factor &kgr;B signaling pathway. Conversely, VHL expression leads to high VCAM-1 levels in ccRCC, which in turn leads to better outcomes, possibly by favoring antitumor immunity through VCAM-1 interaction with the &agr;4&bgr;1 integrin expressed in immune cells. Remarkably, in ccRCC human samples with VHL nonmissense mutations, we observed a negative correlation between VCAM-1 levels and ccRCC stage, microvascular invasion, and symptom presentation, pointing out the clinical value of VCAM-1 levels as a marker of ccRCC progression.

Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1

Excitotoxicity, a critical process in neurodegeneration, induces oxidative stress and neuronal death through mechanisms largely unknown. Since oxidative stress activates protein kinase D1 (PKD1) in tumor cells, we investigated the effect of excitotoxicity on neuronal PKD1 activity. Unexpectedly, we find that excitotoxicity provokes an early inactivation of PKD1 through a dephosphorylation-dependent mechanism mediated by protein phosphatase-1 (PP1) and dual specificity phosphatase-1 (DUSP1). This step turns off the IKK/NF-κB/SOD2 antioxidant pathway. Neuronal PKD1 inactivation by pharmacological inhibition or lentiviral silencing in vitro, or by genetic inactivation in neurons in vivo, strongly enhances excitotoxic neuronal death. In contrast, expression of an active dephosphorylation-resistant PKD1 mutant potentiates the IKK/NF-κB/SOD2 oxidative stress detoxification pathway and confers neuroprotection from in vitro and in vivo excitotoxicity. Our results indicate that PKD1 inactivation underlies excitotoxicity-induced neuronal death and suggest that PKD1 inactivation may be critical for the accumulation of oxidation-induced neuronal damage during aging and in neurodegenerative disorders.Excitotoxicity due to excessive glutamate release causes oxidative stress and neuronal death, and is a feature of many brain diseases. Here the authors show that protein kinase D1 is inactivated by excitotoxicity in a model of stroke and that its activation can be neuroprotective.

Author Correction: Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1

The original version of this Article contained an error in the spelling of the author Álvaro Sebastián-Serrano, which was incorrectly given as Álvaro Sebastián Serrano. This has now been corrected in both the PDF and HTML versions of the Article.

PO-067 Autophagy modulates response to cisplatin in thyroid cancer cells

Introduction Thyroid cancer is the most common endocrine malignancy and, although the death rate of thyroid cancer is relatively low, the recurrence rate of this disease is high. One of the most prevalent mutation in thyroid cancer is V600EBRAF. This mutation causes hiperactivation of the ERK-MAPKs pathway, which leads to a greater cell proliferation, survival and invasion. Nevertheless, the treatment with BRAF inhibitors lacks of efficacy in thyroid cancer, due to development of resistance and important adverse effects in other tissues. Thus, new therapies are required to improve treatment efficiency and specificity in this type of tumours. On the other hand, autophagy is a process whereby the cells recycle their own cellular components and obtain energy, but its role in thyroid cancer is not clarified. In some cases, autophagy is related to a chemotherapy resistance response in cancer cells. We sought to characterise the role of autophagy on survival of anaplastic thyroid cancer cells with V600EBRAF, and to determine the effects of its inhibition on chemosensitivity to cisplatin. Material and methods Human anaplastic thyroid carcinoma cell line BHT-101 was treated with cisplatin in the presence of the autophagy-specific inhibitors bafilomycin A1 (BafA1) and 3-methyladenine (3MA). Cell viability was measured by MTT assay. Apoptosis and cell cycle were detected by flow cytometry. Western blotting assay was used to investigate autophagy markers levels before cisplatin treatment. Results and discussions Our results show that autophagy plays a protective role in these cells, since its inhibition with BafA1 and 3MA increases cell death. Cisplatin also inhibits cell proliferation and induces cell death, but at lower rate. In addition, cisplatin modulates the expression of autophagy-related proteins LC3-I/II and p62 in BHT101 cells, indicating an increase of autophagic flux, compatible with a self-protection mechanism. Finally, the combined treatment with cisplatin and autophagy inhibitors shows higher effects on cell viability than the individual treatments. Conclusion All this data, suggest that autophagy is a pro-survival mechanism in anaplastic thyroid cancer cells and that its inhibition enhances cisplatin treatment response. Thus, we establish the inhibition of this process as an effective approach in treating of V600EBRAF-expressing thyroid tumours.

Hepatitis C virus-mediated Aurora B kinase inhibition modulates inflammatory pathway and viral infectivity

BACKGROUND & AIMS Chronic hepatitis C is a leading cause of chronic liver disease, cirrhosis and hepatocellular carcinoma. DNA methylation and histone covalent modifications constitute crucial mechanisms of genomic instability in human disease, including liver fibrosis and hepatocellular carcinoma. The present work studies the consequences of HCV-induced histone modifications in early stages of infection. METHODS Human primary hepatocytes and HuH7.5 cells were transiently transfected with the core protein of hepatitis C virus (HCV) genotypes 1a, 1b, and 2a. Infectious genotype 2a HCV in culture was also used. RESULTS We show that HCV and core protein inhibit the phosphorylation of Serine 10 in histone 3. The inhibition is due to the direct interaction between HCV core and Aurora B kinase (AURKB) that results in a decrease of AURKB activity. HCV and core significantly downregulate NF-κB and COX-2 transcription, two proteins with anti-apoptotic and proliferative effects implicated in the control of the inflammatory response. AURKB depletion reduced HCV and core repression of NF-κB and COX-2 gene transcription and AURKB overexpression reversed the viral effect. AURKB abrogation increased HCV specific infectivity which was decreased when AURKB was overexpressed. CONCLUSIONS The core-mediated decrease of AURKB activity may play a role in the inflammatory pathway during the initial steps of viral infection, while ensuring HCV infectivity.