Molecular physiology of plasma cell differentiation

Plasma cells are central to an effective immune response, being the sole producers of the antibodies, yet they can cause severe disease in autoimmunity and multiple myeloma. Therefore their differentiation and survival must be tightly regulated. In this work I investigated some of the mechanisms regulating plasma cell differentiation and life span. The differentiation of a long lived B cell to a short lived plasma cell entails a profound structural and functional metamorphosis finalized to the massive production of immunoglobulins (Ig). Exuberant Ig synthesis causes several types of stress in differentiating plasma cells. My work deals with the characterization of the C/EBP transcription factor CHOP in plasma cell differentiation. Comparing differentiation of B cells harvested from chop-l- mice to wt cells I found a mild phenotype, consisting in an increased accumulation of intracellular IgM aggregates and a decreased secretion of this antibody class, in vitro and in vivo. These findings reveal a novel role for CHOP in ensuring optimal functionality of the secretory pathway in the course of plasma cell differentiation. CHOP is involved in the differentiation of various cell types, where it interacts with other members of the C/EBP family favoring or impeding differentiation and it is an important factor in the ER stress response named unfolded protein response (UPR), in which it plays a pro-apoptotic role in most of the systems tested. I extended my investigation on the functions of CHOP in B cells by examining the resistance to ER stress-induced apoptosis in wt and in chop-l- cells. Surprisingly, I observed that in B cells CHOP expression in the UPR plays an anti-apoptotic function. Altogether my data suggest a cell-type specific role for CHOP in B cells and add information on the multi-faceted role of this transcription factor. Most plasma cells exhibit a short life span. The mechanisms at the basis of plasma cell apoptosis are still obscure. I propose that multiple forms of stress, linked to the massive antibody production, contribute to plasma cell death

The miR-223 host non-coding transcript linc-223 induces IRF4 expression in acute myeloid leukemia by acting as a competing endogenous RNA

Alterations in genetic programs required for terminal myeloid differentiation and aberrant proliferation characterize acute myeloid leukemia (AML) cells. Here, we identify the host transcript of miR-223, linc-223, as a novel functional long non-coding RNA (lncRNA) in AML. We show that from the primary nuclear transcript, the alternative production of miR-223 and linc-223 is finely regulated during monocytic differentiation. Moreover, linc-223 expression inhibits cell cycle progression and promotes monocytic differentiation of AML cells. We also demonstrate that endogenous linc-223 localizes in the cytoplasm and acts as a competing endogenous RNA for miR-125-5p, an oncogenic microRNA in leukemia. In particular, we show that linc-223 directly binds to miR-125-5p and that its knockdown increases the repressing activity of miR-125-5p resulting in the downregulation of its target interferon regulatory factor 4 (IRF4), which it was previously shown to inhibit the oncogenic activity of miR-125-5p in vivo. Furthermore, data from primary AML samples show significant downregulation of linc-223 in different AML subtypes. Therein, these findings indicate that the newly identified lncRNA linc-223 may have an important role in myeloid differentiation and leukemogenesis, at least in part, by cross-talking with IRF4 mRNA

Antisense to Epstein Barr virus-encoded LMP1 does not affect the transcription of viral and cellular proliferation-related genes, but induces phenotypic effects on EBV-transformed B lymphocytes

It is generally accepted that Epstein-Barr virus (EBV) latent genes EBNA-2, EBNA-3A, -3C, EBNA-LP and LMP1 are essential for growth transformation and immortalization of B lymphocytes. Among these genes, LMP1 plays a key role in the survival and dissemination of the infected B cells by inducing anti-apoptotic genes and surface expression of several activation antigens and adhesion molecules. We have previously shown that antisense oligodeoxynucleotides directed to LMP1 mRNA, effectively suppress LMP1 gene expression and substantially reduce B95.8 cell proliferation. In this study, we have used antisense LMP1 oligomers to investigate whether LMP1 suppression might influence the expression of latent EBV genes with oncogenic potential, anti-apoptotic genes, or affect the phenotype of EBV-infected B95.8 cells. Our data show that LMP1 suppression does not affect the transcription of EBNA-2, EBNA-3A, -3B and -3C genes, or that of bcl-2 and mcl-1 anti-apoptotic genes. In contrast, consistent modifications in the expression of CD39, CD54, CD23, CD11 and CD10 molecules were observed in B95.8 cells after treatment with antisense LMP1. Our findings support the possibility for using LMP1 antisense oligomers as therapeutics in EBV-associated tumors

C/EBPα-p30 protein induces expression of the oncogenic long non-coding RNA UCA1 in acute myeloid leukemia

Accumulating evidences indicate that different long non-coding RNAs (lncRNAs) might play a relevant role in tumorigenesis, with their expression and function already associated to cancer development and progression. CCAAT/enhancer-binding protein-α (CEBPA) is a critical regulator of myeloid differentiation whose inactivation contributes to the development of acute myeloid leukemia (AML). Mutations in C/EBPα occur in around 10% of AML cases, leading to the expression of a 30-kDa dominant negative isoform (C/EBPα-p30). In this study, we identified the oncogenic urothelial carcinoma associated 1 (UCA1) lncRNA as a novel target of the C/EBPα-p30. We show that wild-type C/EBPα and C/EBPα-p30 isoform can bind the UCA1 promoter but have opposite effects on UCA1 expression. While wild-type C/EBPα represses, C/EBPα-p30 can induce UCA1 transcription. Notably, we also show that UCA1 expression increases in cytogenetically normal AML cases carrying biallelic CEBPA mutations. Furthermore, we demonstrate that UCA1 sustains proliferation of AML cells by repressing the expression of the cell cycle regulator p27kip1. Thus, we identified, for the first time, an oncogenic lncRNA functioning in concert with the dominant negative isoform of C/EBPα in AML

Retinoic acid-induced differentiation sensitizes myeloid progenitors cells to ER stress

The clonal expansion of hematopoietic myeloid precursors blocked at different stages of differentiation characterizes the acute myeloid leukemia (AML) phenotype. A subtype of AML, acute promyelocytic leukemia (APL), characterized by the chimeric protein PML-RARα is considered a paradigm of differentiation therapy. In this leukemia subtype the all-trans-retinoic acid (RA)-based treatments are able to induce PML-RARα degradation and leukemic blast terminal differentiation [1-2]. Granulocytic differentiation of APL cells driven by RA triggers a physiological Unfolded Protein Response (UPR), a series of pathways emanating from the ER in case of ER stress, which ensues when higher protein folding activity is required as during differentiation. We show here that, although mild, the ER stress induced by RA is sufficient to render human APL cell lines and primary blasts very sensitive to low doses of Tunicamycin (Tm), an ER stress inducing drug, at doses that are not toxic in the absence of RA. Importantly only human progenitors cells derived from APL patients resulted sensitive to the combined treatment with RA and Tm whereas those obtained from healthy donors were not affected. We also show that the UPR pathway downstream of PERK plays a major protective role against ER stress in differentiating cells and, by using a specific PERK inhibitor, we potentiated the toxic effect of the combination of RA and Tm. In conclusion, our findings identify the ER stress-related pathways as potential targets in the search for novel therapeutic strategies in AML

A novel role of c-FLIP protein in regulation of ER stress response

Cellular-Flice-like Inhibitory Protein (c-FLIP) is an apoptosis modulator known to inhibit the extrinsic apoptotic pathway thus blocking Caspase-8 processing in the Death Inducing Signalling Complex (DISC). We previously demonstrated that c-FLIP localizes at the Endoplasmic Reticulum (ER) and that c-FLIP-deficient Mouse Embryonic Fibroblasts (MEFs) display an enlarged ER morphology. In the present study, we have addressed the consequences of c-FLIP ablation in the ER stress response by investigating the effects of pharmacologically-induced ER stress in Wild Type (WT) and c-FLIP-/- MEFs. Surprisingly, c-FLIP-/- MEFs were found to be strikingly more resistant than WT MEFs to ER stress-mediated apoptosis. Analysis of Unfolded Protein Response (UPR) pathways revealed that Pancreatic ER Kinase (PERK) and Inositol-Requiring Enzyme 1 (IRE1) branch signalling is compromised in c-FLIP-/- cells when compared with WT cells. We found that c-FLIP modulates the PERK pathway by interfering with the activity of the serine threonine kinase AKT. Indeed, c-FLIP-/- MEFs display higher levels of active AKT than WT MEFs upon ER stress, while treatment with a specific AKT inhibitor of c-FLIP-/- MEFs subjected to ER stress restores the PERK but not the IRE1 pathway. Importantly, the AKT inhibitor or dominant negative AKT transfection sensitizes c-FLIP-/- cells to ER stress-induced cell death while the expression of a constitutively active AKT reduces WT cells sensitivity to ER stress-induced death. Thus, our results demonstrate that c-FLIP modulation of AKT activity is crucial in controlling PERK signalling and sensitivity to ER stress, and highlight c-FLIP as a novel molecular player in PERK and IRE1-mediated ER stress response

Evaluación de las propiedades funcionales y la capacidad antioxidante de harina de remolacha (Beta vulgaris)

The influence of drying temperatures (40, 70 and 100 ° C) and of different ethanol / water extraction mixtures on the beet quality parameters (Beta vulgaris) was studied. The ratio of rehydration (RR), water retention capacity (CRA) and non-enzymatic browning index (IPNE) was evaluated. The antioxidant capacity of the extracts was determined from the content of total flavonoids (CFT), the percentage of the free radical DPPH residual (% DPPH * residual) and the presence of the betaxanthine and betacyanine pigments. The highest values ​​of RR and IPNE were obtained for 100 ° C and that of CRA for 40 ° C. The lowest residual% DPPH *, corresponded to the 50:50 hydroalcoholic mixture, in samples dried at 40 ° C. For this mixture, the highest CFT was obtained, for all drying temperatures. In the 50:50 mixture, the betaxanthine and betacyanine pigments were detected, for temperatures of 40 and 70 ° C. It is concluded that the greatest extraction of flavonoids was produced for the 50:50 ethanol / water mixture, for the three drying temperatures, which coincides with the higher absorbance values ​​obtained for betaxanthine and betacyanin.Se estudió la influencia de las temperaturas de secado (40, 70 y 100 ºC)  y de diferentes mezclas extractoras etanol/agua, en los parámetros de calidad de la remolacha (Beta vulgaris). Se evaluó la relación de rehidratación (RR), de la capacidad de retención de agua (CRA) y del índice de pardeamiento no enzimático (IPNE). La capacidad antioxidante de los extractos, se determinó a partir del contenido de flavonoides totales (CFT), del porcentaje del radical libre DPPH residual (%DPPH* residual) y de la presencia de los pigmentos betaxantina y la betacianina. Los mayores valores de RR e IPNE se obtuvieron para 100°C y el de CRA para 40°C. Los menores %DPPH* residual, correspondieron a la mezcla hidroalcóholica 50:50, en las muestras secadas a 40ºC. Para dicha mezcla se obtuvo el mayor CFT, para todas las temperaturas de secado. En la mezcla 50:50, se detectaron los pigmentos betaxantina y betacianina, para las temperaturas de 40 y 70°C. Se concluye que la mayor extracción de flavonoides se produjo para la mezcla etanol/agua 50:50, para las tres temperaturas de secado, lo que coincide con los mayores valores de absorbancia obtenidos para la betaxantina y betacianina

Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

Background: As crucial regulators of the immune response against pathogens, macrophages have been extensively shown also to be important players in several diseases, including cancer. Specifically, breast cancer macrophages tightly control the angiogenic switch and progression to malignancy. ID4, a member of the ID (inhibitors of differentiation) family of proteins, is associated with a stem-like phenotype and poor prognosis in basal-like breast cancer. Moreover, ID4 favours angiogenesis by enhancing the expression of pro-angiogenic cytokines interleukin-8, CXCL1 and vascular endothelial growth factor. In the present study, we investigated whether ID4 protein exerts its pro-angiogenic function while also modulating the activity of tumour-associated macrophages in breast cancer. Methods: We performed IHC analysis of ID4 protein and macrophage marker CD68 in a triple-negative breast cancer series. Next, we used cell migration assays to evaluate the effect of ID4 expression modulation in breast cancer cells on the motility of co-cultured macrophages. The analysis of breast cancer gene expression data repositories allowed us to evaluate the ability of ID4 to predict survival in subsets of tumours showing high or low macrophage infiltration. By culturing macrophages in conditioned media obtained from breast cancer cells in which ID4 expression was modulated by overexpression or depletion, we identified changes in the expression of ID4-dependent angiogenesis-related transcripts and microRNAs (miRNAs, miRs) in macrophages by RT-qPCR. Results: We determined that ID4 and macrophage marker CD68 protein expression were significantly associated in a series of triple-negative breast tumours. Interestingly, ID4 messenger RNA (mRNA) levels robustly predicted survival, specifically in the subset of tumours showing high macrophage infiltration. In vitro and in vivo migration assays demonstrated that expression of ID4 in breast cancer cells stimulates macrophage motility. At the molecular level, ID4 protein expression in breast cancer cells controls, through paracrine signalling, the activation of an angiogenic programme in macrophages. This programme includes both the increase of angiogenesis-related mRNAs and the decrease of members of the anti-angiogenic miR-15b/107 group. Intriguingly, these miRNAs control the expression of the cytokine granulin, whose enhanced expression in macrophages confers increased angiogenic potential. Conclusions: These results uncover a key role for ID4 in dictating the behaviour of tumour-associated macrophages in breast cancer

Argonaute 2 drives miR-145-5p-dependent gene expression program in breast cancer cells

To perform their regulatory functions, microRNAs (miRNAs) must assemble with any of the four mammalian Argonaute (Ago) family of proteins, Ago1–4, into an effector complex known as the RNA-induced silencing complex (RISC). While the mature miRNA guides the RISC complex to its target mRNA, the Ago protein represses mRNA translation. The specific roles of the various Ago members in mediating miRNAs activity, however, haven’t been clearly established. In this study, we investigated the contribution of Ago2, the only human Ago protein endowed with nuclease activity, to the function of tumor-suppressor miR-145-5p in breast cancer (BC). We show that miR-145-5p and Ago2 protein are concomitantly downregulated in BC tissues and that restoration of miR-145-5p expression in BC cells leads to Ago2 protein induction through the loosening of Ago2 mRNA translational repression. Functionally, miR-145-5p exerts its inhibitory activity on cell migration only in presence of Ago2, while, upon Ago2 depletion, we observed increased miR-145/Ago1 complex and enhanced cell motility. Profiling by microarray of miR-145-5p target mRNAs, in BC cells depleted or not of Ago2, revealed that miR-145-5p drives Ago2-dependent and -independent activities. Our results highlight that the Ago2 protein in cancer cells strictly dictates miR-145-5p tumor suppressor activity