Functions of p120ctn in development and disease

p120 catenin (p120ctn), a component of the cadherin-catenin complex, was the first member to be identified in a most interesting subfamily of the Armadillo family. Several p120ctn isoforms are generated by alternative splicing. These isoforms fulfill pleiotropic functions according to their subcellular localization: modulating the turnover rate of membrane-bound cadherins, regulating the activation of small Rho GTPases in the cytoplasm, and modulating nuclear transcription. Over the last two decades, knowledge of p120ctn has grown remarkably, and this has been achieved in part by using different animal models. At least in frog and mammals, p120ctn is essential for normal development and homeostasis. Here we will discuss the effects of different p120ctn isoforms on cadherin turnover and on signaling in the cytoplasm and the nucleus. We will also elaborate on the structure and function of other members of the p120ctn subfamily: ARVCF, p0071 and delta-catenin. Finally, we will overview the respective roles of p120ctn family members in pathological processes, and particularly in cancer as p120ctn is frequently downregulated or mislocalized in various human tumors

Targeting the Anti-Apoptotic Protein c-FLIP for Cancer Therapy

Cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) is a major resistance factor and critical anti-apoptotic regulator that inhibits tumor necrosis factor-alpha (TNF-alpha), Fas-L, and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis as well as chemotherapy-triggered apoptosis in malignant cells. c-FLIP is expressed as long (c-FLIPL), short (c-FLIPS), and c-FLIPR splice variants in human cells. c-FLIP binds to FADD and/or caspase-8 or -10 in a ligand-dependent and-independent fashion, which in turn prevents death-inducing signaling complex (DISC) formation and subsequent activation of the caspase cascade. Moreover, c-FLIPL and c-FLIPS are known to have multifunctional roles in various signaling pathways, as well as activating and/or upregulating several cytoprotective signaling molecules. Upregulation of c-FLIP has been found in various tumor types, and its downregulation has been shown to restore apoptosis triggered by cytokines and various chemotherapeutic agents. Hence, c-FLIP is an important target for cancer therapy. For example, small interfering RNAs (siRNAs) that specifically knockdown the expression of c-FLIPL in diverse human cancer cell lines augmented TRAIL-induced DISC recruitment and increased the efficacy of chemotherapeutic agents, thereby enhancing effector caspase stimulation and apoptosis. Moreover, small molecules causing degradation of c-FLIP as well as decreasing mRNA and protein levels of c-FLIPL and c-FLIPS splice variants have been found, and efforts are underway to develop other c-FLIP-targeted cancer therapies. This review focuses on (1) the functional role of c-FLIP splice variants in preventing apoptosis and inducing cytokine and drug resistance; (2) the molecular mechanisms that regulate c-FLIP expression; and (3) strategies to inhibit c-FLIP expression and function

Antioxidants and Regulation of Antioxidant Enzymes by Cellular Redox Status

It has been well known that free radicals have numerous detrimental effects in both in vitro and in vivo in biological systems because of their unavoidable and tremendous reactivity against biological macromolecules especially proteins, carbohydrates and nucleic acids. They are produced in the biological systems continuously by various mechanisms and eliminated by cellular antioxidant systems. If not eliminated sufficiently, or there is a disturbance in their neutralization, inevitable cellular damage may occur. Activities or the presence of antioxidant enzymes in the cells are under strong regulation with transcriptional, translational and post translational mechanisms as a consequence of changes in cellular redox potential. Redox sensitive metabolic enzymes or proteins; such as protein phosphatases, NF-?B and AP-1, are capable of sensing the oxidant signals by reversible oxidation of their regulatory units which cause them to transduce the signal and adjust the cellular antioxidant metabolism. Redox sensitive proteins execute their functions via kinases, phosphatases, and transcription factors influencing the steady state levels of antioxidant enzymes. Therefore, cells may sense, transduce, and translate the oxidant signals into appropriate cellular responses depending on the cellular redox state. This rewiev focused on the basic definitions for the free radicals and the tissue defense mechanism and the possible regulation mechanisms of antioxidant enzymes in the situations undergoing reversible changes in cellular redox status

Sorafenib inhibits therapeutic induction of necroptosis in acute leukemia cells

Induction of necroptosis has emerged as an alternative approach to trigger programmed cell death, in particular in apoptosis-resistant cancer cells. Recent evidence suggests that kinase inhibitors targeting oncogenic B-RAF can also affect Receptor-interacting serine/threonine-protein kinase (RIP) 1 and RIP3. Sorafenib, a multi-targeting kinase inhibitor with activity against B-RAF, is used for the treatment of acute leukemia. In the present study, we therefore investigated whether Sorafenib interferes with therapeutic induction of necroptosis in acute leukemia. Here, we report that Sorafenib inhibits necroptotic signaling and cell death in two models of necroptosis in acute leukemia. Sorafenib significantly reduces Second mitochondria-derived activator of caspases (Smac) mimetic-induced necroptosis in apoptosis-resistant acute myeloid leukemia (AML) cells as well as Smac mimetic/Tumor Necrosis Factor (TNF)alpha-induced necroptosis in FADD-deficient acute lymphoblastic leukemia (ALL) cells. Sub- to low micromolar concentrations of Sorafenib corresponding to its plasma levels reported in cancer patients are sufficient to inhibit necroptosis, emphasizing the clinical relevance of our findings. Furthermore, Sorafenib blocks Smac mimetic-mediated phosphorylation of mixed-lineage kinase domain-like protein (MLKL) that marks its activation, indicating that Sorafenib targets components upstream of MLKL such as RIP1 and RIP3. Intriguingly, Sorafenib reduces the Smac mimetic/TNF alpha-stimulated interaction of RIP1 with RIP3 and MLKL, demonstrating that it interferes with the assembly of the necrosome complex. Importantly, Sorafenib significantly protects primary, patient-derived AML blasts from Smac mimetic-induced necroptosis. By demonstrating that Sorafenib limits the anti-leukemic activity of necroptosisinducing drugs in acute leukemia cells, our study has important implications for the use of Sorafenib in the treatment of acute leukemia

Fulvestrant-Induced Cell Death and Proteasomal Degradation of Estrogen Receptor α Protein in MCF-7 Cells Require the CSK c-Src Tyrosine Kinase

Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant

Studies of b-AP15 : a novel inhibitor of proteasome deubiquitinase activity

Bortezomib was the first FDA approved proteasome inhibitor that was initially very successful in treatment of multiple myeloma patients but acquired resistance and adverse side-effect highly decreased patients’ quality of life. Development of 2nd generation proteasome inhibitors that could overcome these shortcomings is thus of prime medical importance. Our group has developed b-AP15 as such a candidate, which targets a different subunit of the proteasome than does bortezomib. In study I, we determined that use of CpdA as a co- translational translocation inhibitor in a co-treatment protocol greatly enhanced proteasome inhibition by b-AP15. Aggresome formation is a resistance mechanism evident after bortezomib treatment. In study II, we demonstrated that b-AP15 did not induce aggresome formation under the same conditions and interestingly we observed less aggresome formation with co-treatment of b- AP15 and bortezomib compared to single treatment with bortezomib. In study III, we demonstrated that lymphoma cell lines were as sensitive to b-AP15 as other cancer cell lines previously reported. The apoptosis induced by b-AP15 correlated to accumulation of polyubiquitination and the ER stress response. In study IV, we observed that the gene expression patterns and apoptosis induction mechanisms of b-AP15 and bortezomib were similar, but not identical. Both induced the expression of Hmox-1 but only b-AP15 could induce ER stress. This study also revealed that ROS scavengers could reduce the apoptosis induced by b-AP15, which was due to activation of AP-1 In study V, the gene expression pattern following Piperlongumine treatment was similar to that of other proteasome inhibitors and the drug could block the ubiquitin–proteasome system in cancer cells. However, Piperlongumine was determined not to be a classic but instead interfered upstream of UPS system. The overall conclusion is that further development of proteasome inhibitors such as b-AP15 should be continued, as increased efficacy is expected following clinical translation

IDENTIFICATION OF THREE NOVEL REGULATORY PATHWAYS INVOLVED IN THE DOWN-REGULATION OF P63 PROTEIN LEVELS

One way to regulate protein functions is by post-translational modification. Post-translational modifications have an important role in the regulation of biological activity of the protein because they allow both to extend the range of functions of a protein and to monitor the activity and determine the activation or inactivation of a protein. The most common protein post-translational modifications include ubiquitylation, phosphorylation and acetylation play an essential role in cellular functions such as cellular differentiation, apoptosis, DNA repair, antigen processing, and stress response. Under particular conditions abnormal post-translational modifications were found in many diseases like: Alzheimer\u2019s disease, Parkinson\u2019s disease, induction of different cancer and others. These abnormal post-translational modifications are permanent and can cause loss or alteration of protein function by changing enzyme activities or capacity aggregation (Stadtman and Levine 2000; Shacter 2000). p63 protein stability is regulated by different protein modifications such phosphorylation, ubiquitylation and sumoylation. p63 is known to be degraded by ubiquitin-mediated proteasomal degradation, the E3 ubiquitin ligase NEDD4-like, ubiquitin protein ligase Itch and ubiquitin-like protein SUMO-1 have been shown to directly interact with p63 and regulate p63 protein stability (Ghioni et al. 2005; Rossi at al. 2006; Rossi et al. 2006) suggest the importance of regulating p63 to tune its biological activity. During my PhD thesis we found three novel and distinct mechanisms that are involved in the regulation of the p63 protein levels; all these mechanisms induce p63 degradation. We demonstrated that these mechanisms are relevant in different physiological contexts and that they are involved in the regulation of p63 biological function. 1. MDM2-Fbw7 pathway contribute to reduce \u394Np63\u3b1 protein levels during keratinocytes differentiation and upon DNA-damage induced by UV exposure and adriamycin treatment. 2. TRIM8 plays a role in enhancing p53 anti-oncogenic activity and at the same time down-modulate oncogenic \u394Np63\u3b1 activity. 3. Hipk2 phosphorylates and promotes proteasomal degradation of \u394Np63\u3b1 to enable an effective DNA-damage response induced by genotoxic drugs. All these evidences indicate that regulation of p63 protein stability is a key mechanism to control p63 activities, in particular during epithelia differentiation and in response to genotoxic agents. The knowledge and the identification of the molecular mechanisms governing p63 regulation under physiological context might be fundamental for understanding the pathogenesis of human syndromes associated to p63 mutations and the mechanism by which p63 promotes disease development. We hope that future studies focusing on the mechanisms involved in p63 protein regulation might increase our knowledge on the p63 role in tumorigenicity and in response to anti-cancer therapy to improve anti-cancer therapies

Multifaceted roles of GSK-3 and Wnt/β-catenin in hematopoiesis and leukemogenesis: opportunities for therapeutic intervention

Glycogen synthase kinase-3 (GSK-3) is well documented to participate in a complex array of critical cellular processes. It was initially identified in rat skeletal muscle as a serine/threonine kinase that phosphorylated and inactivated glycogen synthase. This versatile protein is involved in numerous signaling pathways that influence metabolism, embryogenesis, differentiation, migration, cell cycle progression and survival. Recently, GSK-3 has been implicated in leukemia stem cell pathophysiology and may be an appropriate target for its eradication. In this review, we will discuss the roles that GSK-3 plays in hematopoiesis and leukemogenesis as how this pivotal kinase can interact with multiple signaling pathways such as: Wnt/β-catenin, phosphoinositide 3-kinase (PI3K)/phosphatase and tensin homolog (PTEN)/Akt/mammalian target of rapamycin (mTOR), Ras/Raf/MEK/extracellular signal-regulated kinase (ERK), Notch and others. Moreover, we will discuss how targeting GSK-3 and these other pathways can improve leukemia therapy and may overcome therapeutic resistance. In summary, GSK-3 is a crucial regulatory kinase interacting with multiple pathways to control various physiological processes, as well as leukemia stem cells, leukemia progression and therapeutic resistance. GSK-3 and Wnt are clearly intriguing therapeutic targets

The Mechanisms of Telomere and Telomerase Regulation in Hematologic Malignancies

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The Mechanisms of Telomere and Telomerase Regulation in Hematologic Malignancies

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