Giulia Pozzi

PKCε Controls Mitotic Progression by Regulating Centrosome Migration and Mitotic Spindle Assembly

To form a proper mitotic spindle, centrosomes must be duplicated and driven poleward in a timely and controlled fashion. Improper timing of centrosome separation and errors in mitotic spindle assembly may lead to chromosome instability, a hallmark of cancer. Protein kinase C epsilon (PKCϵ) has recently emerged as a regulator of several cell-cycle processes associated with the resolution of mitotic catenation during the metaphase–anaphase transition and in regulating the abscission checkpoint. However, an engagement of PKCϵ in earlier (pre)mitotic events has not been addressed. Here, we now establish that PKCϵ controls prophase-to-metaphase progression by coordinating centrosome migration and mitotic spindle assembly in transformed cells. This control is exerted through cytoplasmic dynein function. Importantly, it is also demonstrated that the PKCϵ dependency of mitotic spindle organization is correlated with the nonfunctionality of the TOPO2A-dependent G2 checkpoint, a characteristic of many transformed cells. Thus, PKCϵ appears to become specifically engaged in a programme of controls that are required to support cell-cycle progression in transformed cells, advocating for PKCϵ as a potential cancer therapeutic target. Implications: The close relationship between PKCϵ dependency for mitotic spindle organization and the nonfunctionality of the TOPO2A-dependent G2 checkpoint, a hallmark of transformed cells, strongly suggests PKCϵ as a therapeutic target in cancer. Mol Cancer Res; 16(1); 3–15. ©2017 AACR.

The -2518 A/G polymorphism of the monocyte chemoattractant protein-1 as a candidate genetic predisposition factor for secondary myelofibrosis and biomarker of disease severity

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PKCε promotes human Th17 differentiation: Implications in the pathophysiology of psoriasis

PKCε is implicated in T cell activation and proliferation and is overexpressed in CD4+‐T cells from patients with autoimmune Hashimotos thyroiditis. Although this might induce the suspicion that PKCε takes part in autoimmunity, its role in the molecular pathophysiology of immune‐mediated disorders is still largely unknown. We studied PKCε expression in circulating CD4+‐T cells from patients with psoriasis, a skin disorder characterized by an increased amount of Th17 cells, a CD4+ subset that is critical in the development of autoimmunity. Although the mechanisms that underlie Th17 differentiation in humans are still unclear, we here show that: (i) PKCε is overexpressed in CD4+‐T cells from psoriatic patients, and its expression positively correlates with the severity of the disease, being reduced by effective phototherapy; (ii) PKCε interacts with Stat3 during Th17 differentiation and its overexpression results in an enhanced expression of Stat3 and pStat3(Ser727); iii) conversely, when PKCε is forcibly downregulated, CD4+‐T cells show lower levels of pStat3(Ser727) expression and defective in vitro expansion into the Th17‐lineage. These data provide a novel insight into the molecular mechanisms of Th17 cell polarization that is known to play a crucial role in autoimmunity, pinpointing PKCε as a potential target in Th17‐mediated diseases.

Platelet gene expression profile in acute myocardial infarction

Acute myocardial infarction is a sudden event that is fatal in around one-third of patients. It is primarily due to coronary atherosclerotic plaque rupture with subsequent platelet (PLT) activation/aggregation and thrombus formation. PLTs have a key role in the genesis and progression of atherosclerosis and in thrombus formation1. PLTs are anucleated cells which retain mRNA from their megakaryocyte precursor, therefore PLT mRNA is unique in representing a nearly fixed transcriptome2. We tested the hypothesis that platelet transcriptome acts as a fingerprint indicating the development of a future myocardial infarction, with the final goal of identifying a specific STEMI gene-signature, able to discriminate patients with acute event from healthy donors (HD) and from patients affected by stable coronary artery disease (sCAD), the phenotypically closest clinical condition to STEMI. Peripheral blood samples (50mL) were collected in Na-citrate tubes from 20 myocardial infarction patients (MI), 20 sCAD and 20 HD. Highly purified platelets were obtained by leukocyte depletion as previously described3. Platelet RNA extraction were performed by TRIzolTM reagent according to the manufacturer’s protocol. Gene expression profile was analyzed using an Affymetrix GeneChip system (Cancer Genomics and Bioinformatics Laboratory Facility, Kimmel Cancer Center, Jefferson University. Philadelphia, US). The exploratory analysis of PLT transcriptome confirmed differences in gene expression between STEMI, sCAD and HD. Hence, the common differentially expressed genes (DEGs) derived from the STEMI vs sCAD and STEMI vs HD comparisons were obtained and tested by k-nearest neighbor classification and bootstrap. A set of 17 STEMI-related DEGs was identified, showing good sensitivity and specificity for the discrimination of STEMI patients. Overall, we described a STEMI-specific gene expression patterns, suggesting that PLT transcriptome allows to characterize a powerful fingerprint of STEMI theoretically able to predict a future acute event.

PKC epsilon involvement in Th17 in vitro differentiation: implications in psoriasis pathogenesis

Psoriasis is a noncontagious, arytematous-squamose dermatitits affecting both sexes and all races. Although its exact etiology is largely unknown, it is now recognized as one of the most common immune-mediated disorders and several studies demonstrate an impairment of regulatory T-cells (Tregs) function and an up-regulation of IL-17 levels produced by T-helper 17 lymphocytes (Th17)(1,2). Protein kinase C epsilon (PKCe) is a serine/threonine kinase which plays a key role in the proliferation and differentiation of epidermal cells. We have previously demonstrated a role for PKCe in the pathogenesis of the autoimmune disease Hashimoto’s thyroiditis (3). PKCe is over-expressed in CD4+ T lymphocytes isolated from PBMC fraction in patients affected by this pathology and its forced down-modulation primed the TGF-mediated in vitro Treg polarization of human T CD4+ cells. Since it has been demonstrated that PKC-signalling is altered in psoriatic keratinocytes (4), we investigated the involvement of PKCe in Th17 in vitro differentiation and its potentially implication in immune response correlated to psoriasis. Using western blot and real time PCR, we have observed that PKCe protein levels and mRNA increase during Th17-lineage in vitro differentiation from naive CD4+ T cells with a similar trend of Th17 markers of differentiation STAT3 and RoRyT. Moreover, PKCe overexpression significantly increases STAT3 and phosphorylated STAT3 levels, suggesting that PKCe boosts Th17 polarization. Thereafter, we sought to investigate PKCe expression in CD4+ lymphocytes obtained from peripheral blood of psoriatic patients and we observed that PKCe expression levels are significantly higher compared with healthy donors. Intriguingly, we observed a closely correlation of PKCe expression with PASI index, suggesting an involvement of the kinase with the severity of the disease. Collectively these data suggest that PKCe might be involved in Th17 differentiation, that it could be a key factor to regulate Th17 pathological expansion and therefore a potential psoriatic pharmacological target.

Correlation between Protein Kinase Cε expression and thrombotic risk in Primary Myelofibrosis (PMFs)

Myelofibrosis (MF) - either primary (PMF) or arising from a previous PV or ET - is a Philadelphia-negative MPNs characterized by aberrant platelet production and consequent variable platelet count with altered hemostatic function (1). It has already been demonstrated that the risk of thrombotic events is one of the most common co-morbidities associated with PV and ET (2-5). However, risk of thrombotic events in PMF has not been investigated yet. We previously demonstrated that PKCepsilon (PKCe) is over-expressed in platelets from patients with acute myocardial infarction and accounts for their increased reactivity (6). Additionally, we recently showed that PKCe overexpression plays a crucial role in PMF MK impaired differentiation and that its levels correlated with the disease severity (expressed by the IPSS/DIPPS risk category) (7,8). On these bases, we analyzed PKCe expression in platelets from PMF patients, investigating a potential correlation with thrombotic risk and the aggressiveness of the disease. For this study, peripheral blood samples from 6 PMF patients and 3 healthy donors (HD) were collected in Na-citrate tubes. PKCe mRNA and protein levels were determined in platelets purified as described by Carubbi C, 2012. Finally, patients are stratified according to the history of cardio-vascular events and the IPSS/DIPSS risk category. PMF platelets showed significantly higher mRNA levels of PKCe as compared to HD. Protein analysis confirm PKCe over-expression in PMF platelets, almost reaching statistical significance. We then found that platelet from PMF patients who suffered from cardiovascular events display significantly higher levels of PKCe as compared to the one with a negative history. Finally, similarly to what observed in PMF magakaryocytes, we showed a positive correlation between PKCe platelets levels and IPSS/DIPSS risk category, with the lowest levels in low-risk patients and higher levels in high-risk patients. Collectively, our preliminary results indicate that PMF platelets show an aberrant expression of PKCe which correlates with the disease burden and a history of cardiovascular events. This suggests that the over-expression of PKCe may account for PMF platelet altered reactivity and function.

Novel role of PKC ɛ in mitotic spindle stability

Mitosis is a highly regulated process characterized by dramatic and coordinated morphological changes to ensure the fidelity of chromosome segregation. Misseg- regation of mitotic chromosomes leads to a condition that underlies chromosomal instability(1), which is a hallmark of cancer. In order to assure symmetry and bipo- larity of the cell division process, mitotic spindle microtubules properly segregate mitotic chromosomes (2). Among the several isoforms of serine/threonine kinases, PKCe is one of the best understood for its role as a transforming oncogene, and it has been found overexpressed in different types of tumors. In 2008, Saurin and col- leagues demonstrated the involvement of PKCe in the regulation of the late stage of mitosis (3). Through its association with 14-3-3 at the midbody, PKCe is essential for the successful completion of cytokinesis, and the inhibition of functional PKCe-14-3- 3 complex leads to abscission failure and multinucleated phenotype in cells. In this study, we found that PKCe is involved in mitotic spindle stability. Using fluorescence microscopy, we found that the active form of PKCe (phosphorylated at Ser-729), co- localizes to the centrosome in cells in metaphase, where the mitotic spindle nuclea- tion occurs. Furthermore, experiments of co-immunoprecipitation revealed that, when cells are synchronized in metaphase, PKCɛ is associated to ɣ-tubulin, a member of the tubulin superfamily localized to the microtubule organizing centers and is essen- tial for microtubule nucleation from centrosomes. Consequently modulation of PKCɛ expression affects spindle stability: PKCɛ downregulation by specific shRNA results in mitotic spindle disorganization with a reduction of the amount of centrosomal and mitotic ɣ-Tubulin and αβ-tubulin fluorescence. Mitotic spindle formation assays using Nocodazole, known to interfere with the polymerization of microtubules, revealed that cells lacking PKCɛ were unable to regrow microtubules after depolymerization. These results reveal a novel role of PKCɛ in mitotic spindle stability, which likely determinant for genome stability.