RUOLO DI VAV1 NELLA MATURAZIONE DI PROMIELOCITI TUMORALI APL-DERIVATI

Vav1, one of the genetic markers that correlates with the differentiation of hematopoietic cells, belongs to a family of multidomain signal transduction proteins involved in the regulation of diverse cellular responses such as proliferation, differentiation, survival and migration. Normal and tumoral myeloid precursors are characterized by a low expression of this protein, that increases in amount and in tyrosine phosphorylation level during agonist-induced granulocytic differentiation. In blasts derived from acute promyelocytic leukemia (APL), the M3 subtype of acute myeloid leukemias, Vav1 plays a crucial role in the overcoming of the differentiation blockade. In particular, this protein promotes differentiation, potentiates ATRA-induced maturation and is involved in regulating expression of genes up-regulated by ATRA. It has also been demonstrated that in differentiating APL-derived cells Vav1 is tyrosine phosphorylated by Syk and this event is crucial for the maturation-related changes of cell morphology. Starting from these notions, the first aim of this study was to evaluate a possible role for Vav1 in modulating protein expression during ATRA-treatment of APL-derived cells. With this purpose, high-resolution 2-DE coupled with mass spectra analysis was performed on HL-60 and NB4 promyelocytes induced to differentiate with ATRA under conditions in which either the amount or the tyrosine phosphorylation of Vav1 were reduced. In both cell lines, the down-regulation of Vav1 affected the expression level of various proteins, including cell cycle/apoptosis- and cytoskeletonrelated proteins, that may constitute a common part of the ATRA-induced maturation pathway. These data indicate for Vav1 an unprecedented role in the differentiation of myeloid cells as a regulator of protein expression. A second aim of this work was to identify tyrosine residue(s) of Vav1 phosphorylated during ATRA treatment and involved in the overcoming of the differentiation blockade of APL-derived cells. Vav1 was then immunoprecipitated from differentiating NB4 cells and subjected to mass spectra analysis, allowing to identify the Y745 residue phosphorylated after ATRA treatment. The mutation of the residue Y745 of Vav1 impaired the completion of maturation program of NB4 cells, in terms of CD11b expression and migration capability. Since a functional role for Y745 has not been described so far, the tyrosine phosphorylation of this residue could be at the basis of a role of Vav1 alternative to the best known activity as guanosine exchange factor. As previously reported, the Syk-dependent tyrosine phosphorylation of Vav1 plays a role in the regulation of cell morphology of differentiating HL-60 cells. Since Vav1 interacts with Syk by means of its SH2 domain, where 4 tyrosines are present, the last part of this study was aimed to establish if these residues are possible substrates of Syk and to determine their functional role during ATRA-induced granulocytic differentiation. With this aim, HL-60 cells were transfected with plasmids containing mutated forms of Vav1 and induced to differentiate with ATRA. The role of each tyrosine residue on the differentiation process was studied by evaluating both CD11b expression and migration capability. The results indicate that both Y672 and Y711 are possible Syk substrates since their mutation, as well as Syk inhibition, have no effect on CD11b expression, but affect migration capability of ATRA-treated cells. The data reported in this work have considerably contributed to define the involvement of Vav1 in the maturation process of APL-derived cells, even though further analysis will be necessary to clearly establish its functional role

Vav1 sustains the in vitro differentiation of normal and tumor precursors to insulin producing cells induced by all-trans retinoic acid (ATRA)

All-trans retinoic acid (ATRA) promotes the development and the function of insulin producing cells and induces partial differentiation of pancreatic tumor cells. A number of evidences clearly indicate that the ATRA mediated signaling may have a substantial role in therapeutic approaches based on restoration of functional beta-cells. Among the proteins up-regulated by ATRA, Vav1 is involved in maturation and function of haematopoietic cells and is essential for retinoids induced differentiation of tumor promyelocytes. The presence of Vav1 in solid tissues, including pancreas, is considered ectopic and no role in the differentiation of human epithelial cells has so far been described. We demonstrated here that Vav1 sustains the maturation to beta-cells of the normal precursors human Biliary Tree Stem/progenitor Cells (hBTSCs) induced by a differentiation medium containing ATRA and that, in the mature normal pancreas, insulin-producing cells express variable levels of Vav1. Using pancreatic ductal adenocarcinoma (PDAC)-derived cells, we also revealed that the ATRA induced up-modulation of Vav1 is essential for the retinoid-induced trans-differentiation of neoplastic cells into insulin producing cells. The results of this study identify Vav1 as crucial molecule in ATRA induced maturation of insulin producing cells and suggest this protein as a marker for new strategies ended to restore functional beta-cells

PLC-βeta2 plays a phenotype dependent role in the malignant potential induced by hypoxia in breast cancer cells

Hypoxia plays a crucial role in malignant progression of solid tumors, including breast cancer, since neoplastic cells adapt to low oxygen availability by modulating the expression of genes involved in survival, proliferation, metabolic reprogramming, stem cell maintenance, EMT, angiogenesis, invasion and metastasis (1). Among the signaling molecules deregulated in breast tumors, the beta2 isoform of the phosphoi- nositide-dependent phospholipase C (PLC-β2) is expressed in the large majority of primary invasive tumors from all histological subtypes in which it strongly correlates with malignancy and with a poor prognosis (2). PLC-β2 is also expressed in breast tumor- derived cells, in which it improves proliferation and motility and sustains invasion capability (3). A decreased PLC-β2 expression is induced by hypoxia in the BT-474 and MCF7 cell lines, that is correlated with the hypoxia-induced modulation of the EMT markers E-cadherin and Vimentin, as well as of the stem cell marker CD133. In contrast, hypoxia induced the increase of PLC-β2 levels in MDA-MB-231 cells, in which it supports the hypoxia-related reorganization of actin cytoskeleton. In all examined cell lines, the prevention of the effects of hypoxia on PLC-β2 also reduced the recovery of HIF-1α, in turn able to exert a phenotype-related role in modulating EMT and expression of CD133 during the cell response to low oxygen. Our data highlighted a peculiar effect of low oxygen availability on PLC-β2 expression in breast tumor cells with different phenotypes and allocate PLC-β2 in the complex and interconnected transcriptional activity induced by hypoxia. Our results also suggest that the forced modulation of PLC-β2 programmed on the basis of the tumor phenotype may prevent malignant progression of breast neoplasia as a consequence of intra-tumoral hypoxia

In triple negative breast tumor cells, PLC-β2 promotes the conversion of CD133high to CD133low phenotype and reduces the CD133-related invasiveness

Beyond its possible relationship with stemness of tumor cells, CD133/prominin is a highly glycosylated trans-membrane protein that correlates with tumor size, metastasis and clinical stage of triple negative breast cancers (TNBC), that represent 20% of all breast tumors and have a particularly worse clinical outcome than other tumor subtypes [1]. The correlation between the levels of CD133 expression and the biology of breast tumor cells was studied in CD133low and CD133high cell subpopulations isolated from MDA-MB-231 cells (ER-, PR-, HER2-). High expression of CD133 characterizes a small percentage of cells with larger adhesion area, lower proliferation rate, higher invasion capability and increased expression of proteins involved in metastasis and drug resistance of breast cancers. PLC-b2 expression, that plays a crucial role in malignancy of breast tumor cells [2, 3], inversely correlates with the levels of CD133 and has a role in inducing the CD133high cells to CD133low cells conversion. The forced up-regulation of PLC-b2 counteracts the invasiveness of CD133high MDAMB- 231, suggesting that, in TNBC, the de-regulation of this PLC isozyme is responsible of the switch from an early to a mature tumoral phenotype also by reducing the expression of CD133. These data might contribute to identify unexplored key steps in TNBC malignancy, to be considered for potential therapeutic strategies

Correlation between the surface expression of CD133 and the phenotype of breast tumor cells

Originally considered a marker of hematopoietic stem cells, CD133/prominin is a highly glycosylated trans-membrane protein expressed in various tissues, such as breast, in which it seems to regulate ductal branching but not regenerative capacity [1]. CD133 is also expressed in various solid tumors, including breast cancer, in which CD133-positivity seems to identify a restricted subgroup of tumor stem cells [2]. CD133 expression was heterogeneous in different breast carcinomas but, in triple- negative (ER-, PR-, HER2-) invasive ductal breast carcinoma, CD133 correlates with tumor size, metastasis and clinical stage [3]. In order to establish a correlation between the surface recognition of CD133 and the phenotype of tumor cells, the highly invasive breast-derived MDA-MB-231 cells (ER-, PR-, HER2-) were subjected to immunomagnetic separation of CD133+ and CD133- subpopulations, which were analyzed for malignant properties. In comparison to CD133- cells, the expression of CD133 characterizes cells with a larger adhesion area, lower proliferation rate and reduced migration speed. This phenotype correlates with altered expression of malignancy-associated proteins and with a peculiar pattern of PLC, in turn involved in proliferation and motility of breast tumor cells (4-6). This suggests that, in triple negative ductal breast tumor-derived cells, the expression of CD133 characterizes a small subset of cells with a less undifferentiated phenotype. The reduced expression of CD133 at membrane level may constitute a marker of the switch of tumor cells from a less malignant to a mature phenotype since it correlates with the de-regulation of proteins involved in cell proliferation, motility and invasion

Low oxygen availability and malignant evolution of non-invasive breast tumors: potential protective role of all-trans retinoic acid (ATRA)

Reduced oxygen availability plays a crucial role in malignancy of solid tumors, including breast cancer. Even if the presence of hypoxic areas is common in all breast lesions, no data clearly correlate low oxygenation with the acquisition of malignant features by non-invasive cells, particularly by cells from ductal carcinoma in situ (DCIS), the most frequently diagnosed tumor in women in industrialized countries [1]. We demonstrated that 96 hours of culture under moderate hypoxia is sufficient to induce in DCIS-derived cells motility and epithelial-to-mesenchymal transition (EMT) and to enlarge the number of cells expressing the stem cell marker CD133, indicative of their increased malignant potential. Administration of all-trans retinoic acid (ATRA), a well-known anti-leukemic drug with an anti-tumor role in invasive breast tumor cells [2], supports the epithelial phenotype of DCIS-derived cells cultured under hypoxia and reduces the number of CD133 positive cells, abrogating almost completely the effects of poor oxygenation. In DCIS-derived cells, as in leukemic cells, ATRA up-regulates the β2 isoform of PI-PLC (PLC-β2), ectopically expressed in invasive breast tumors in which counteracts the effects of hypoxia on both EMT and CD133 levels [3]. This suggests that the mechanisms triggered by ATRA in non-invasive breast tumor cells cultured under hypoxia may, at least in part, depend on PLC-β2 activity. Overall, we assigned to hypoxia a role in increasing the malignant potential of DCIS-derived cells and identified in ATRA, currently used in treating acute promyelocytic leukemia (APL), an agonist potentially useful in preventing malignant progression of non-invasive breast lesions with hypoxic areas

Experimental Observation of Surface Charge Inversion in a Biological Nanopore in Presence of Monovalent and Multivalent Cations

Surface CD133 and EpCAM, proliferation and invasiveness in breast derived cell lines. In A, representative cytofluorimetrical evaluation of CD133 and EpCAM surface levels in MCF7 and MDA-MB-468 cells after labelling with a PE-conjugated anti-CD133 antibody or with a FITC-conjugated anti-EpCAM antibody. The staining with isotype matched antibodies (IgG) is used as a control. The expression of each antigen is shown on a biparametric dot plot and the percentage and MFI of positive cells are indicated at the upper right of each panel. In B, MDA-MB-231, MCF7 and MDA-MB-468 cells were subjected to dynamic monitoring of proliferation and invasion through Matrigel using the xCELLigence RTCA system. Cell Index (CI) is reported and error bars indicate ÂąSD. The correspondent Slope analysis, that describes the steepness, incline, gradient, and changing rate of the CI curves over time, is shown on the right. The data were collected from three separate experiments (PDF 364 kb

Vav1 is ectopically expressed in breast tumors in which reduces the efficiency of the metastatic process

Vav1, normally restricted to hematopoietic cells, results ectopically expressed in solid tumors, including breast cancer (Sebban et al., 2013) in which, contrarily to other neoplasias, seems to be higher in tumors from patients who remained diseasefree than in patients who developed recurrence (Lane et al., 2008). The significance of Vav1 expression in breast tumors was evaluated by immunohistochemical analysis on TMAs containing invasive breast tumors from patients without lymph node involvement. Our findings indicate that Vav1 is expressed in almost all investigated cancers and shows a peculiar localization inside the nucleus of tumor cells. High amounts of nuclear Vav1 positively correlates with low incidence of relapse, regardless phenotype and molecular subtype of the neoplasia. Experiments performed with breast tumor-derived cells showing different morphology, immunoprofile and invasive properties indicated that Vav1 negatively modulates their invasiveness in vitro and their metastatic efficiency in vivo, possibly by affecting the expression of genes involved in invasion and/or metastasis of breast cancer. Since the high heterogeneity of breast tumors makes difficult to predict the evolution of early neoplasias, the evaluation of nuclear Vav1 levels may help in profiling and management of early breast cancer patients. In addition, Vav1 may serve as a target for new therapies designed to prevent breast cancer progression