Jessika Bertacchini

Pharmacological inhibition of protein kinase CK2 reverts the multidrug resistance phenotype of a CEM cell line characterized by high CK2 level

Protein kinase CK2 is an ubiquitous and constitutively active kinase, which phosphorylates many cellular proteins and is implicated in the regulation of cell survival, proliferation and transformation. We investigated its possible involvement in the multidrug resistance phenotype (MDR) by analysing its level in two variants of CEM cells, namely S-CEM and R-CEM, normally sensitive or resistant to chemical apoptosis, respectively. We found that, while the CK2 regulatory subunit β was equally expressed in the two cell variants, CK2α catalytic subunit was higher in R-CEM and this was accompanied by a higher phosphorylation of endogenous protein substrates. Pharmacological downregulation of CK2 activity by a panel of specific inhibitors, or knockdown of CK2α expression by RNA interference, were able to induce cell death in R-CEM. CK2 inhibitors could promote an increased uptake of chemotherapeutic drugs inside the cells and sensitize them to drug-induced apoptosis in a co-operative manner. CK2 blockade was also effective in inducing cell death of a different MDR line (U2OS). We therefore conclude that inhibition of CK2 can be considered as a promising tool to revert the MDR phenotype.

Lamin A Ser404 Is a nuclear target of akt phosphorylation in C2C12 cells

Akt/PKB is a central activator of multiple signaling pathways coupled with a large number of stimuli. Although both localization and activity of Akt in the nuclear compartment are well-documented, most Akt substrates identified so far are located in the cytoplasm, while nuclear substrates have remained elusive. A proteomic-based search for nuclear substrates of Akt was undertaken, exploiting 2D-electrophoresis/MS in combination with an anti-Akt phosphosubstrate antibody. This analysis indicated lamin A/C as a putative substrate of Akt in C2C12 cells. In vitro phosphorylation of endogenous lamin A/C by recombinant Akt further validated this result. Moreover, by phosphopeptide analysis and point mutation, we established that lamin A/C is phosphorylated by Akt at Ser404, in an evolutionary conserved Akt motif. To delve deeper into this, we raised an antibody against the lamin A Ser404 phosphopeptide which allowed us to determine that phosphorylation of lamin A Ser404 is triggered by the well-known Akt activator insulin, and is therefore to be regarded as a physiological response. Remarkably, expression of S404A lamin A in primary cells from healthy tissue caused the nuclear abnormalities that are a hallmark of Emery-Dreifuss muscular dystrophy (EDMD) cells. Indeed, it is known that mutations at several sites in lamin A/C cause autosomal dominant EDMD. Very importantly, we show here that Akt failed to phosphorylate lamin A/C in primary cells from an EDMD-2 patient with lamin A/C mutated in the Akt consensus motif. Together, our data demonstrate that lamin A/C is a novel signaling target of Akt, and implicate Akt phosphorylation of lamin A/C in the correct function of the nuclear lamina.

The protein kinase Akt/PKB regulates both prelamin A degradation and Lmna gene expression

The serine/threonine kinase Akt/PKB is a major signaling hub integrating metabolic, survival, growth, and cell cycle regulatory signals. The definition of the phospho‐motif cipher driving phosphorylation by Akt led to the identification of hundreds of putative substrates, and it is therefore pivotal to identify those whose phosphorylation by Akt is of consequence to biological processes. The Lmna gene products lamin A/C and the lamin A precursor prelamin A are type V intermediate filament proteins forming a filamentous meshwork, the lamina, underneath the inner nuclear membrane, for nuclear envelope structures organization and interphase chromatin anchoring. In our previous work, we reported that A‐type lamins are phosphorylated by Akt at S301 and S404 in physiological conditions and are therefore bona fide substrates of Akt. We report here that Akt phosphorylation at S404 targets the precursor prelamin A for degradation. We further demonstrate that Akt also regulates Lmna transcription. Our study unveils a previously unknown function of Akt in the control of prelamin A stability and expression. Moreover, given the large number of diseases related to prelamin A, our findings represent a further important step bridging basic A‐type lamin physiology to therapeutic approaches for lamin A‐linked disorders.—Bertacchini, J., Beretti, F., Cenni, V., Guida, M., Gibellini, F., Mediani, L., Marin, O., Maraldi, N. M., de Pol, A., Lattanzi, G., Cocco, L., Marmiroli, S. The protein kinase Akt/PKB regulates both prelamin A degradation and Lmna gene expression. FASEB J. 27, 2145–2155 (2013). www.fasebj.org

A‐type lamins and signaling: The PI 3‐kinase/Akt pathway moves forward

Lamin A/C is a nuclear lamina constituent mutated in a number of human inherited disorders collectively referred to as laminopathies. The occurrence and significance of lamin A/C interplay with signaling molecules is an old question, suggested by pioneer studies performed in vitro. However, this relevant question has remained substantially unanswered, until data obtained in cellular and organismal models of laminopathies have indicated two main aspects of lamin A function. The first aspect is that lamins establish functional interactions with different protein platforms, the second aspect is that lamin A/C activity and altered function may elicit different effects in different cells and tissue types and even in different districts of the same tissue. Both these observations strongly suggest that signaling mechanisms targeting lamin A/C or its binding partners may regulate such a plastic behavior. A number of very recent data show involvement of kinases, as Akt and Erk, or phosphatases, as PP1 and PP2, in lamin A‐linked cellular mechanisms. Moreover, altered activation of signaling in laminopathies and rescue of the pathological phenotype in animal models by inhibitors of signaling pathways, strongly suggest that signaling effectors related to lamin A/C may be implicated in the pathogenesis of laminopathies and may represent targets of therapeutic intervention. In face of such an open perspective of basic and applied research, we review current evidence of lamin A/C interplay with signaling molecules, with particular emphasis on the lamin A‐Akt interaction and on the biological significance of their relationship. J. Cell. Physiol. 220: 553–561, 2009.

Targeting PI3K/AKT/mTOR network for treatment of leukemia

ObjectiveIncreased activity of PI3K/AKT/mTOR pathway has been observed in a huge number of malignancies. This pathway can function as a prosurvival factor in leukemia stem cells and early committed leukemic precursors and its inhibition is regarded as a therapeutic approach. Accordingly, the aim of this review is to evaluate the PI3K/Akt/mTOR inhibitors used in leukemia models.DiscussionInhibition of the PI3K/AKT/mTOR pathway has been reported to have beneficial therapeutic effects in leukemias, both in vitro in leukemia cell lines and in vivo in animal models. Overall, the use of dual PI3K/mTOR inhibitor, dual Akt/RTK inhibitor, Akt inhibitor, selective inhibitor of PI3K, mTOR inhibitor and dual PI3K/PDK1 inhibitor in CML, AML, APL, CLL, B-ALL and T-ALL has a better therapeutic effect than conventional treatments.ConclusionsTargeting the PI3K/Akt/mTOR pathway may have pro-apoptotic and antiproliferative effects on hematological malignancies. Furthermore, modulation of miRNA can be used as a novel therapeutic approach to regulate the PI3K/Akt/mTOR pathway. However, both aspects require further clinical studies.

Feedbacks and adaptive capabilities of the PI3K/Akt/mTOR axis in acute myeloid leukemia revealed by pathway selective inhibition and phosphoproteome analysis.

Acute myeloid leukemia (AML) primary cells express high levels of phosphorylated Akt, a master regulator of cellular functions regarded as a promising drug target. By means of reverse phase protein arrays, we examined the response of 80 samples of primary cells from AML patients to selective inhibitors of the phosphatidylinositol 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) axis. We confirm that >60% of the samples analyzed are characterized by high pathway phosphorylation. Unexpectedly, however, we show here that targeting Akt and mTOR with the specific inhibitors Akti 1/2 and Torin1, alone or in combination, result in paradoxical Akt phosphorylation and activation of downstream signaling in 70% of the samples. Indeed, we demonstrate that cropping Akt or mTOR activity can stabilize the Akt/mTOR downstream effectors Forkhead box O and insulin receptor substrate-1, which in turn potentiate signaling through upregulation of the expression/phosphorylation of selected growth factor receptor tyrosine kinases (RTKs). Activation of RTKs in turn reactivates PI3K and downstream signaling, thus overruling the action of the drugs. We finally demonstrate that dual inhibition of Akt and RTKs displays strong synergistic cytotoxic effects in AML cells and downmodulates Akt signaling to a much greater extent than either drug alone, and should therefore be explored in AML clinical setting.

The oral protein-kinase C β inhibitor enzastaurin (LY317615) suppresses signalling through the AKT pathway, inhibits proliferation and induces apoptosis in multiple myeloma cell lines

Deregulation of the protein kinase C (PKC) signalling pathway has been implicated in tumor progression. Here we investigated the PKC inhibitor enzastaurin for its activity against multiple myeloma (MM) cells. Enzastaurin suppresses cell proliferation in a large panel of human myeloma cell lines (HMCLs), with IC50 values ranging from 1.3 to 12.5 µM and induces apoptosis, which is prevented by the ZVAD-fmk broad caspase inhibitor. These results are consistent with decreased phosphorylation of AKT and GSK3-β, a downstream target of the AKT pathway and a pharmacodynamic marker for enzastaurin. Furthermore, enzastaurin cytotoxicity is retained when HMCLs were cocultured with multipotent mesenchymal stromal cells. Enzastaurin has additive or synergistic cytotoxic effects with bortezomib or thalidomide. Considering the strong anti-myeloma activity of enzastaurin in vitro and in animal models and its safe toxicity profile, phase II studies in MM patients of enzastaurin alone or in combination with other drugs are warranted.

MATER protein as substrate of PKCε in human cumulus cells

High activity of the phosphoinositide 3-kinase/Akt pathway in cumulus cells plays an important role in FSH regulation of cell function and Protein Kinase C epsilon (PKCepsilon) collaborates with these signalling pathways to regulate cell proliferation. Relevant roles in follicular development are played by Maternal Antigen That Embryos Require (MATER) that is a cumulus cell- and oocyte-specific protein dependent on the maternal genome. We recently demonstrated that human MATER localizes at specific domains of oocytes and, for the first time, also in cumulus cells. MATER contains a carboxy-terminal leucine-rich repeat domain involved in protein-protein interactions regulating different cellular functions. Here we investigated the functional role of MATER. Thus, we performed coimmunoprecipitation experiments using HEK293T cells expressing human MATER; a similar approach was then followed in human cumulus/follicular cells. In MATER(+)HEK293T cells, we observed that this protein acts as a phosphorylation substrate of PKCepsilon. Western blot experiments indicate that, unlike oocytes, human cumulus cells express PKCepsilon. Immunoprecipitation and confocal analysis suggest for the first time that MATER protein interacts with this protein kinase in cumulus cells under physiological conditions. Since PKCepsilon is known to collaborate with antiapoptotic signalling pathways, this suggests a novel mechanism for the function of MATER in follicular maturation.

Reverse-phase protein microarrays (RPPA) as a diagnostic and therapeutic guide in multidrug resistant leukemia.

Reverse-phase microarray assays using phospho-specific antibodies (RPPA) can directly measure levels of phosphorylated protein isoforms. In the current study, lysates from parental and multidrug resistant (MDR) CEM leukemia cells were spotted onto reverse-phase protein microarrays and probed with a panel of phospho-antibodies to ERK, PCK and Akt pathways. In particular, the Akt pathway is considered to play significant roles in leukemia and Akt inhibitor therapy has been proposed as a potential tool in the treatment of this disease. The RPPA data prompted us to investigate deeper this pathway. Here, we found that whereas total Akt1 protein level is higher in parental CEM cells, the activated isoform content, p-Akt1, increases in doxorubicin-selected CEM cells (MDR-CEM). This was backed up by Western blot analysis, confirming that Akt1 activity/phosphorylation may be up-regulated in MDR-CEM cells. Further exploration of inhibitory therapy in this system was evaluated. The TNF-related apoptosis-inducing ligand, TRAIL, has been shown to selectively kill tumor cells. Herein, we describe that in MDR-CEM cells TRAIL responsiveness correlates with a reduced expression of endogenous Akt1, suggesting that the MDR phenotype associated to P-gp sensitizes cells to TRAIL therapy.

Molecular targeting of the PKC‐β inhibitor enzastaurin (LY317615) in multiple myeloma involves a coordinated downregulation of MYC and IRF4 expression

The protein kinase C (PKC) pathway has been shown to play a role in the regulation of cell proliferation in several haematological malignancies, including multiple myeloma (MM). Recent data have shown that a PKC inhibitor, enzastaurin, has antiproliferative and proapoptotic activity in a large panel of human myeloma cell lines (HMCLs). In order to further characterise the effect of enzastaurin in MM, we performed gene expression profiling of enzastaurin‐treated KMS‐26 cell line. We identified 62 upregulated and 32 downregulated genes that are mainly involved in cellular adhesion (CXCL12, CXCR4), apoptosis (CTSB, TRAF5, BCL2L1), cell proliferation (IGF1, GADD45A, BCMA (B‐cell maturation antigen), CDC20), transcription regulation (MYC, MX11, IRF4), immune and defence responses. Subsequent validation by Western blotting of selected genes in four enzastaurin‐treated HMCLs was consistent with our microarray analysis. Our data indicate that enzastaurin may affect important processes involved in the proliferation and survival of malignant plasma cells as well as in their interactions with the bone marrow microenvironment and provide a preclinical rationale for the potential role of this drug in the treatment of MM. Copyright