The impact of inflammation and acute phase activation in cancer cachexia

The development of cachexia in the setting of cancer or other chronic diseases is a significant detriment for patients. Cachexia is associated with a decreased ability to tolerate therapies, reduction in ambulation, reduced quality of life, and increased mortality. Cachexia appears intricately linked to the activation of the acute phase response and is a drain on metabolic resources. Work has begun to focus on the important inflammatory factors associated with the acute phase response and their role in the immune activation of cachexia. Furthermore, data supporting the liver, lung, skeletal muscle, and tumor as all playing a role in activation of the acute phase are emerging. Although the acute phase is increasingly being recognized as being involved in cachexia, work in understanding underlying mechanisms of cachexia associated with the acute phase response remains an active area of investigation and still lack a holistic understanding and a clear causal link. Studies to date are largely correlative in nature, nonetheless suggesting the possibility for a role for various acute phase reactants. Herein, we examine the current literature regarding the acute phase response proteins, the evidence these proteins play in the promotion and exacerbation of cachexia, and current evidence of a therapeutic potential for patients

Deficient LRRC8A-dependent volume-regulated anion channel activity is associated with male infertility in mice

Ion channel-controlled cell volume regulation is of fundamental significance to the physiological function of sperm. In addition to volume regulation, LRRC8A-dependent volume-regulated anion channel (VRAC) activity is involved in cell cycle progression, insulin signaling, and cisplatin resistance. Nevertheless, the contribution of LRRC8A and its dependent VRAC activity in the germ cell lineage remain unknown. By utilizing a spontaneous Lrrc8a mouse mutation (c.1325delTG, p.F443*) and genetically engineered mouse models, we demonstrate that LRRC8A-dependent VRAC activity is essential for male germ cell development and fertility. Lrrc8a-null male germ cells undergo progressive degeneration independent of the apoptotic pathway during postnatal testicular development. Lrrc8a-deficient mouse sperm exhibit multiple morphological abnormalities of the flagella (MMAF), a feature commonly observed in the sperm of infertile human patients. Importantly, we identified a human patient with a rare LRRC8A hypomorphic mutation (c.1634G>A, p.Arg545His) possibly linked to Sertoli cell-only syndrome (SCOS), a male sterility disorder characterized by the loss of germ cells. Thus, LRRC8A is a critical factor required for germ cell development and volume regulation in the mouse, and it might serve as a novel diagnostic and therapeutic target for SCOS patients

The stress protein p8 favors pancreatic cancer cell adaptation to hostile micro-environment : study of its mechanism of action

La protéine de stress p8 fut découverte et caractérisée dans notre laboratoire. Cette protéine est surexprimée dans l'adénocarcinome pancréatique et possède un rôle dans la progression tumorale. Pendant ce travail de thèse, nous nous sommes focalisé sur le rôle de p8 dans les phases initiales de la tumorigénèse pancréatique et sur ses mécanismes d'action, impliqués dans la progression tumorale. Nous avons montré que p8 joue un rôle essentiel dans l'apparition des lésions PanINs suite à l'activation de l'oncogène Kras. Au niveau cellulaire, nous avons montré que la protéine p8 protège les cellules tumorales pancréatiques contre le stress de la privation en nutriments via la régulation de l'expression de RelB et IER3. La voie p8, RelB et IER3 est impliquée dans l'inhibition de l'apoptose suite au stress. De plus, nous avons observé que ces trois protéines sont co-exprimées dans les adénocarcinomes humains et leur expression corrèle avec l'évolution de la maladie. Dans un second lieu, nous avons démontré le rôle essentiel de la protéine p8 dans la résistance des cellules tumorales pancréatiques à l'hypoxie et à la privation en glucose. Nous avons identifié la voie p8/Aurora KinaseA, qui en réponse au stress métabolique, réduit l'apparition des dommages à l'ADN en contrôlant l'expression des gènes liés au cycle cellulaire et à la réparation de l'ADN. De plus, nos recherches ont montré que p8 protège les cellules tumorales du stress métabolique en inhibant la mort cellulaire dépendante de l'autophagie. Nous espérons que nos résultats aideront à mieux cibler les cellules tumorales pancréatiques et leur caractère résistant au stress micro-environnemental extrême.The stress protein p8 was discovered and characterized in our laboratory. Over expressed in pancreatic adenocarcinoma, p8 is involved in tumor progression. During my PhD studies, we focused on the role of p8 in pancreatic cancer development and on its mechanisms of action. First, we demonstrated that p8 is essential for PanIN development following Kras oncogene activation. At the cellular level, we found that p8 protects pancreatic cancer cells upon nutrient starvation stress through the regulation of RelB and IER3 expression. p8, RelB and IER3-dependent cascade inhibits apoptosis after the starvation stress. Furthermore, we showed that these tree proteins are co expressed in human pancreatic adenocarcinoma. One the other hand, our study showed that p8 is involved in pancreatic cancer cells resistance to hypoxia and glucose starvation. We identified a p8/Aurora KinaseA pathway which, in response to such metabolic stress, reduces DNA damage by regulating cell cycle and DNA repair genes expression. Moreover, our studies demonstrated that p8/AURKA path protects cancer cells against metabolic stress by inhibiting autophagy-associated cell death. We expect that our data will help to get new therapeutics against pancreatic cancer

IER3 supports KRASG12D-dependent pancreatic cancer development by sustaining ERK1/2 phosphorylation

Activating mutations in the KRAS oncogene are prevalent in pancreatic ductal adenocarcinoma (PDAC). We previously demonstrated that pancreatic intraepithelial neoplasia (PanIN) formation, which precedes malignant transformation, associates with the expression of immediate early response 3 (Ier3) as part of a prooncogenic transcriptional pathway. Here, we evaluated the role of IER3 in PanIN formation and PDAC development. In human pancreatic cancer cells, IER3 expression efficiently sustained ERK1/2 phosphorylation by inhibiting phosphatase PP2A activity. Moreover, IER3 enhanced KrasG12D-dependent oncogenesis in the pancreas, as both PanIN and PDAC development were delayed in IER3-deficient KrasG12D mice. IER3 expression was discrete in healthy acinar cells, becoming highly prominent in peritumoral acini, and particularly high in acinar ductal metaplasia (ADM) and PanIN lesions, where IER3 colocalized with phosphorylated ERK1/2. However, IER3 was absent in undifferentiated PDAC, which suggests that the IER3-dependent pathway is an early event in pancreatic tumorigenesis. IER3 expression was induced by both mild and severe pancreatitis, which promoted PanIN formation and progression to PDAC in KrasG12D mice. In IER3-deficient mice, pancreatitis abolished KrasG12D-induced proliferation, which suggests that pancreatitis enhances the oncogenic effect of KRAS through induction of IER3 expression. Together, our data indicate that IER3 supports KRASG12D-associated oncogenesis in the pancreas by sustaining ERK1/2 phosphorylation via phosphatase PP2A inhibition.Fil: Garcia, Maria Noe. Inserm; Francia. Centre National de la Recherche Scientifique; Francia. Aix-Marseille Université; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Grasso, Daniel Hector. Aix-Marseille Université; Francia. Inserm; Francia. Centre National de la Recherche Scientifique; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Lopez Millian, Maria Belen. Aix-Marseille Université; Francia. Centre National de la Recherche Scientifique; Francia. Inserm; FranciaFil: Hamidi, Tewfik. Aix-Marseille Université; Francia. Centre National de la Recherche Scientifique; Francia. Inserm; FranciaFil: Loncle, Celine. Inserm; Francia. Centre National de la Recherche Scientifique; Francia. Aix-Marseille Université; FranciaFil: Tomasini, Richard. Aix-Marseille Université; Francia. Centre National de la Recherche Scientifique; Francia. Inserm; FranciaFil: Lomberk, Gwen. Mayo Clinic. Departments of Biochemistry and Molecular Biology, Biophysics, and Medicine; Estados UnidosFil: Porteu, Françoise. Inserm; Francia. Université de Paris XI; FranciaFil: Urrutia, Raul. Mayo Clinic. Departments of Biochemistry and Molecular Biology, Biophysics, and Medicine; Estados UnidosFil: Iovanna, Juan L.. Aix-Marseille Université; Francia. Centre National de la Recherche Scientifique; Francia. Inserm; Franci

Deciphering the Binding between Nupr1 and MSL1 and Their DNA-Repairing Activity

<div><p>The stress protein Nupr1 is a highly basic, multifunctional, intrinsically disordered protein (IDP). MSL1 is a histone acetyl transferase-associated protein, known to intervene in the dosage compensation complex (DCC). In this work, we show that both Nupr1 and MSL1 proteins were recruited and formed a complex into the nucleus in response to DNA-damage, which was essential for cell survival in reply to cisplatin damage. We studied the interaction of Nupr1 and MSL1, and their binding affinities to DNA by spectroscopic and biophysical methods. The MSL1 bound to Nupr1, with a moderate affinity (2.8 µM) in an entropically-driven process. MSL1 did not bind to non-damaged DNA, but it bound to chemically-damaged-DNA with a moderate affinity (1.2 µM) also in an entropically-driven process. The Nupr1 protein bound to chemically-damaged-DNA with a slightly larger affinity (0.4 µM), but in an enthalpically-driven process. Nupr1 showed different interacting regions in the formed complexes with Nupr1 or DNA; however, they were always disordered (“fuzzy”), as shown by NMR. These results underline a stochastic description of the functionality of the Nupr1 and its other interacting partners.</p></div

Interaction parameters of MSL1, Nupr1 and complexes at 25°C, in buffer acetate (10 mM, pH 4.5).

<p>Interaction parameters of MSL1, Nupr1 and complexes at 25°C, in buffer acetate (10 mM, pH 4.5).</p

Calorimetric titrations for Nupr1, MSL1 and chemically-damaged-DNA interactions.

<p>Binding isotherms (normalized injection heat <i>versus</i> molar ratio of the reactants in the cell) are shown: (A) Nupr1 interacting with etoposide-damaged DNA. (B) MSL1 interacting with etoposide-damaged-DNA. (C) MSL1 interacting with Nupr1. (D) MSL1 interacting with Nupr1 + etoposide-damaged-DNA complex. All the titrations were carried out at 25°C in 10 mM acetate buffer (pH 4.5). The binding experiments involving damaged DNA were analyzed considering an estimated molecular weight of 10000 kDa.</p

Cell survival and caspase 3/7 activity in response to cisplatin treatment and Nupr1 and MSL1 interaction.

<p>(A) MiaPaCa-2 cells were transfected with siNupr1 or siMSL1 and cultured in conventional media for an additional 24-h period; Nupr1 and MSL1 mRNA expression was measured by qRT-PCR, and proteins levels by western blot analysis. β-tubulin was used as a control of loading. (B) MiaPaCa-2 cells were plated on coverslips and transfected with siNupr1 or siMSL1, alone or together, and 24 h later treated with cisplatin (10 µM) for a 24 h-period. γ-H2AX staining was performed by immunofluorescence. The 40× magnification was used to count the number of γ-H2AX dots. Data are the means of 10 field counting with not less than 100 nucleus counted (* p≤0.05). (C) Proximity Ligation Assay (PLA) of Nupr1 and MSL1. Cells were plated on coverslips and transfected with pcDNA3-Nupr1-Flag and pcDNA4-MSL1-V5 constructs. The day after the experiment, cells were treated with cisplatin (5 µM or 10 µM) to induce DNA damage and 24 h later the PLA was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078101#s2" target="_blank">Material and methods</a> section. Red dots represent Nupr1/MSL1 interaction. DNA transfection with only pcDNA3-Nupr1-Flag construct was used as a negative control. (D) Nupr1 and MSL1 do not interact into the DNA damage sites. PLA was reproduced as in (B) and followed by γ-H2AX staining. A 20 fields of 40× magnification were used to count the number of γ-H2AX green dots, the number of PLA red dots and the number of co-localizing green and red dots. Data are the means of 20 field counting with not less than 100 nucleus counted (* p≤0.05). (E) MiaPaCa-2 cells were plated in six-well plates and treated with cisplatin (30 µM) for 24 h. Cell survival rate was estimated using a cell counter as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078101#s2" target="_blank">Material and Methods</a> section. Data are expressed as percentage of the control. Cells were plated in ninety-six-well plates treated with cisplatin as described above and caspase 3/7; we assayed the caspase acativity by using the Apo-ONE® homogenous caspase 3/7 assay. The normalization value was performed by using CellTiter-Blue® viability assay according to manufacturer’s instructions (* p≤0.05).</p

Fluorescence and CD spectra of MSL1.

<p>Fluorescence (A) and far-UV CD (B) spectra for MSL1 in 50 mM acetic/acetate buffer (pH 4.5). Conditions for both experiments were 20 µM of protein and 25°C. Thermal denaturation of MSL1 followed by fluorescence (C) and far-UV CD (D). Fluorescence lines represent the thermal denaturations of MSL1 at an emission wavelength of 315 nm, either at an excitation wavelength of 278 nm (blue line) or 295 nm (red line). Similar results were obtained when emission wavelength was monitored at 330 nm or 350 nm. Spectra were acquired in either 1 cm- (fluorescence) or 0.1 cm- (far-UV CD) path-length cells.</p