Ervin Nika

Vav1 in differentiation of tumoral promyelocytes

The multidomain protein Vav1, in addition to promote the acquisition of maturation related properties by normal hematopoietic cells, is a key player in the ATRA- and PMA-induced completion of the differentiation program of tumoral myeloid precursors derived from APL. This review is focussed on the role of Vav1 in differentiating promyelocytes, as part of interconnected networks of functionally related proteins ended to regulate different aspects of myeloid maturation. The role of Vav1 in determining actin cytoskeleton reorganization alternative to the best known function as a GEF for small G proteins is discussed, as well as the binding of Vav1 with cytoplasmic and nuclear signaling molecules which provides a new perspective in the modulation of nuclear architecture and activity. In particular, new hints are provided on the ability of Vav1 to determine the nuclear amount of proteins implicated in modulating mRNA production and stability and in regulating the ATRA-dependent protein expression also by direct interaction with transcription factors known to drive the ATRA-induced maturation of myeloid cells. The reviewed findings summarize the major advances in the understanding of additional, non conventional functions connected with the vast interactive potential of Vav1.

Vav1 is a crucial molecule in monocytic/macrophagic differentiation of myeloid leukemia-derived cells

Vav1 is a critical signal transducer for both the development and function of normal hematopoietic cells, in which it regulates the acquisition of maturation-related properties, including adhesion, motility, and phagocytosis. Vav1 is also important for the agonist-induced maturation of acute promyelocytic leukemia (APL)-derived promyelocytes, in which it promotes the acquisition of a mature phenotype by playing multiple functions at both cytoplasmic and nuclear levels. We investigated the possible role of Vav1 in the differentiation of leukemic precursors to monocytes/macrophages. Tumoral promyelocytes in which Vav1 was negatively modulated were induced to differentiate into monocytes/macrophages with phorbol-12-myristate-13-acetate (PMA) and monitored for their maturation-related properties. We found that Vav1 was crucial for the phenotypical differentiation of tumoral myeloid precursors to monocytes/macrophages, in terms of CD11b expression, adhesion capability and cell morphology. Confocal analysis revealed that Vav1 may synergize with actin in modulating nuclear morphology of PMA-treated adherent cells. Our data indicate that, in tumoral promyelocytes, Vav1 is a component of lineage-specific transduction machineries that can be recruited by various differentiating agents. Since Vav1 plays a central role in the completion of the differentiation program of leukemic promyelocytes along diverse hematopoietic lineages, it can be considered a common target for developing new therapeutic strategies for the various subtypes of myeloid leukemias.

Nuclear proteome analysis reveals a role of Vav1 in modulating RNA processing during maturation of tumoral promyelocytes

Vav1 is a key molecule in the ATRA-induced acquisition of a mature phenotype by tumoral myeloid precursors. Since ATRA acts throughout events that require extensive changes of nuclear architecture and activity and considering that Vav1 accumulates inside the nuclear compartment of differentiating APL-derived cells, the possible role of this protein in modulating the nuclear proteome was investigated. Membrane-depleted nuclei purified from NB4 cells induced to differentiate with ATRA in the presence of forcedly down-modulated Vav1 were subjected to 2D-DIGE followed by mass spectra analysis. The obtained data demonstrated that, in NB4 cells treated with ATRA, Vav1 is involved in determining the nuclear amount of proteins involved in molecular complexes with DNA and may participate to RNA processing by carrying in the nucleus molecules involved in modulating mRNA production and stability, like hnRNPs and SR proteins. Our results provide the first evidence that, at least in maturation of tumoral myeloid precursors, Vav1 is part of interconnected networks of functionally related proteins ended to regulate different aspects of gene expression. Since defects in mRNA processing are common in tumor development, our data suggest that Vav1 is a potential target molecule for developing new anti-cancer strategies.

A network including PU.1, Vav1 and miR-142-3p sustains ATRA-induced differentiation of acute promyelocytic leukemia cells - a short report.

PurposeReduced expression of miR-142-3p has been found to be associated with the development of various subtypes of myeloid leukemia, including acute promyelocytic leukemia (APL). In APL-derived cells, miR-142-3p expression can be restored by all-trans retinoic acid (ATRA), which induces the completion of their maturation program. Here, we aimed to assess whether PU.1, essential for ATRA-induced gene transcription, regulates the expression of miR-142-3p in APL-derived cells and, based on the established cooperation between PU.1 and Vav1 in modulating gene expression, to evaluate the role of Vav1 in restoring the expression of miR-142-3p.MethodsATRA-induced increases in PU.1 and Vav1 expression in APL-derived NB4 cells were counteracted with specific siRNAs, and the expression of miR-142-3p was measured by quantitative real-time PCR (qRT-PCR). The recruitment of PU.1 and/or Vav1 to the regulatory region of miR-142 was assessed by quantitative chromatin immunoprecipitation (Q-ChIP). Synthetic inhibitors or mimics for miR-142-3p were used to assess whether this miRNA plays a role in regulating the expression of PU.1 and/or Vav1.ResultsWe found that the expression of miR-142-3p in differentiating APL-derived NB4 cells is dependent on PU.1, and that Vav1 is essential for the recruitment of this transcription factor to its cis-binding element on the miR-142 promoter. In addition, we found that in ATRA-treated NB4 cells miR-142-3p sustains agonist-induced increases in both PU.1 and Vav1.ConclusionsOur results suggest the existence of a Vav1/PU.1/miR-142-3p network that supports ATRA-induced differentiation in APL-derived cells. Since selective regulation of miRNAs may play a role in the future treatment of hematopoietic malignancies, our results may provide a basis for the development of new therapeutic strategies to restore the expression of miR-142-3p.

hnRNP K in PU.1-containing complexes recruited at the CD11b promoter: a distinct role in modulating granulocytic and monocytic differentiation of AML-derived cells.

PU.1 is essential for the differentiation of haemopoietic precursors and is strongly implicated in leukaemogenesis, yet the protein interactions that regulate its activity in different myeloid lineages are still largely unknown. In the present study, by combining fluorescent EMSA (electrophoretic mobility-shift assay) with MS, we reveal the presence of hnRNP K (heterogeneous nuclear ribonucleoprotein K) in molecular complexes that PU.1 forms on the CD11b promoter during the agonist-induced maturation of AML (acute myeloid leukaemia)-derived cells along both the granulocytic and the monocytic lineages. Although hnRNP K and PU.1 act synergistically during granulocytic differentiation, hnRNP K seems to have a negative effect on PU.1 activity during monocytic maturation. Since hnRNP K acts as a docking platform, integrating signal transduction pathways to nucleic acid-directed processes, it may assist PU.1 in activating or repressing transcription by recruiting lineage-specific components of the transcription machinery. It is therefore possible that hnRNP K plays a key role in the mechanisms underlying the specific targeting of protein-protein interactions identified as mediators of transcriptional activation or repression and may be responsible for the block of haemopoietic differentiation.

Vav1 is necessary for PU.1 mediated upmodulation of miR‐29b in acute myeloid leukaemia‐derived cells

It has been recently demonstrated that high pre‐treatment levels of miR‐29b positively correlated with the response of patients with acute myeloid leukaemia (AML) to hypomethylating agents. Upmodulation of miR‐29b by restoring its transcriptional machinery appears indeed a tool to improve therapeutic response in AML. In cells from acute promyelocytic leukaemia (APL), miR‐29b is regulated by PU.1, in turn upmodulated by agonists currently used to treat APL. We explored here the ability of PU.1 to also regulate miR‐29b in non‐APL cells, in order to identify agonists that, upmodulating PU.1 may be beneficial in hypomethylating agents‐based therapies. We found that PU.1 may regulate miR‐29b in the non‐APL Kasumi‐1 cells, showing the t(8;21) chromosomal rearrangement, which is prevalent in AML and correlated with a relatively low survival. We demonstrated that the PU.1‐mediated contribution of the 2 miR‐29b precursors is cell‐related and almost completely dependent on adequate levels of Vav1. Nuclear PU.1/Vav1 association accompanies the transcription of miR‐29b but, at variance with the APL‐derived NB4 cells, in which the protein is required for the association of PU.1 with both miRNA promoters, Vav1 is part of molecular complexes to the PU.1 consensus site in Kasumi‐1. Our results add new information on the transcriptional machinery that regulates miR‐29b expression in AML‐derived cells and may help in identifying drugs useful in upmodulation of this miRNA in pre‐treatment of patients with non‐APL leukaemia who can take advantage from hypomethylating agent‐based therapies.

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.

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.

Specific PI-PLCs expression patterns as prognostic factors in breast cancer

In industrialized countries, breast cancer is, in incidence and mortality, in first place among malignant tumors of the female population. In Italy, this neoplasia is diagnosed in about 32,000 women annually and causes approximately 11,000 deaths overall. Like many cancers, breast cancer presents alterations in intracellular signal transduction pathways leading to increased proliferative potential, sustained angiogenesis, apoptosis inhibition, invasiveness and metastatic dissemination. The molecular changes that determine the onset of breast cancer are still largely unknown and, due to the high heterogeneity of this neoplasia, no specific protein pattern allows, at present, to fully characterize and proficiently monitor its progression. Among the molecules involved in intracellular signal transduction pathways, the phosphoinositide dependent phospholipase C (PI-PLC) is involved in tumorigenesis of a variety of tissues, including breast epithelium. Altered expression of phospholipase C-beta 2, -gamma 1 and -delta 4 occur in invasive breast cancers and correlate with tumor-related features, such as cell proliferation and invasiveness, suggesting that these isoforms play an important role in the progression of the disease. Aim of this study was to identify a correlation between the expression levels of different PI-PLC isozymes within single tissue samples and the clinical profile of patients to whom the primary tumor had been removed. This was achieved by performing an immunohistochemical study followed by a retrospective multivariate analysis of invasive breast tumors with different malignant features, in terms of final grade of malignancy, size, proliferation index and receptor pattern, for which a follow-up of at least 5 years was available. Even if preliminary, the obtained results allowed to identify specific expression profiles of the proteins under investigation which showed, independently from the tumor final grade, a significant correlation with the clinical outcome of the patients. Given the heterogeneity that characterizes breast tumors, the obtained results demonstrate that, regardless the histopathological classification, each invasive breast tumor shows a specific pattern of expression of PI-PLC isozymes, that may help to characterize more precisely the neoplasia and contribute to predict the clinical course of individual patients. Acknowledgments. This research was supported by grants from MIUR COFIN and local funds from University of Ferrara (Italy).