Annexin Proteins: Novel Promising Targets for Anticancer Drug Development

Intracellular Ca2+ signaling and Ca2+ homeostasis have long been an important subject area of cell biology. Several intracellular Ca2+ binding proteins have been demonstrated until now, and among these, annexins are characterized by their ability to interact with membrane phospholipids and they form an evolutionary conserved multigene family with the members being expressed throughout animal and plant kingdoms. Annexin proteins are defined by different structural and biochemical criteria, and this multigene family has several biological features. In certain clinical conditions, the alterations on the localization or expression levels of annexin proteins are considered as the causes of pathological results and/or sequelae of disease. So, annexin proteins are indirectly linked to severe human diseases such as cardiovascular disease and cancer. Since annexin proteins are known to play roles in cancer, the researches are focused on defining the clinical significance of certain annexin proteins in cancer development and by the way anticancer treatments in the last decades. This chapter presents detailed information about annexin proteins and the studies on anticancer drug development targeting certain annexins. The studies denominate that targeting of certain annexin proteins reduces tumorigenesis and therapeutic resistance. So, annexin proteins have growing importance for anticancer drug development

Proteomic Characterization of the E3 Ubiquitin-Ligase Hakai: Biological Insights and New Therapeutic Strategies

Programa Oficial de Doutoramento en Bioloxía Celular e Molecular . 5004V01[Resumen] El carcinoma es el tipo más común de cáncer y surge de las células epiteliales. La transición del adenoma al carcinoma se asocia con la pérdida de E-cadherina y, en consecuencia, de los contactos intercelulares. La E-cadherina es un supresor tumoral que está regulado negativamente durante la transición epitelio-mesénquima (EMT) y su pérdida es un marcador de mal pronóstico durante la progresión tumoral. Hakai es una E3 ubiquitina-ligasa que media en la ubiquitinación de la E-cadherina, su endocitosis y consecuente degradación. Aunque la E-cadherina es el sustrato más conocido de Hakai, otras dianas moleculares reguladas por Hakai pueden estar involucradas en la plasticidad celular durante la progresión tumoral. En este trabajo, empleamos la técnica iTRAQ para explorar nuevas rutas moleculares involucradas en la EMT inducida por Hakai. Nuestros resultados muestran que Hakai puede tener una influencia importante sobre proteínas relacionadas con el citoesqueleto, proteínas extracelulares asociadas con el exosoma, proteínas relacionadas con el ARN y proteínas involucradas en metabolismo. Entre las proteínas reguladas por Hakai, describimos la Anexina A2 como un nuevo posible sustrato de Hakai. Además, nuestros resultados revelan que la inhibición farmacológica de Hsp90 con geldanamicina resulta en la degradación de Hakai vía lisosoma. Así, proponemos a Hakai como una nueva proteína cliente de la chaperona Hsp90, destacando un mecanismo novedoso por el cual los inhibidores de Hsp90 pueden influir en el proceso EMT mediado por Hakai y el tratamiento del cáncer.[Resumo] O carcinoma é o tipo de cancro máis común e xorde das células epiteliais. A transición do adenoma ao carcinoma está asociada á perda de E-cadherina e, en consecuencia, aos contactos intercelulares. A E-cadherina é un supresor tumoral que se encontra regulado negativamente durante a transición epitelio-mesénquima (EMT), e a súa perda é un marcador de mala prognose durante a progresión do tumor. Hakai é unha E3 ubiquitina-ligasa que media a ubiquitinización da E-cadherina, a súa endocitose e a súa conseguinte degradación. Aínda que a E-cadherina é o substrato máis coñecido de Hakai, outras dianas moleculares reguladas por Hakai poden estar implicadas na plasticidade celular durante a progresión tumoral. Neste traballo empregamos a técnica iTRAQ para explorar novas vías moleculares implicadas na EMT inducida por Hakai. Os nosos resultados mostran que Hakai pode ter unha influencia importante sobre proteínas relacionadas co citoesqueleto, proteínas extracelulares asociadas co exosoma, proteínas relacionadas co ARN e proteínas implicadas no metabolismo. Entre as proteínas reguladas por Hakai, describimos a Anexina A2 coma un novo posible substrato de Hakai. Ademáis, describimos una relación entre Hakai e o complexo chaperona da proteína Heat shock protein 90 (Hsp90). Tamén, os nosos resultados revelan que a inhibición farmacolóxica de Hsp90 con geldanamicina resulta na degradación de Hakai vía lisosoma. Así, propoñemos a Hakai como unha nova proteína cliente da chaperona Hsp90, destacando un novo mecanismo polo cal os inhibidores de Hsp90 poden influir no proceso de EMT mediado por Hakai e no tratamento do cancro.[Abstract] Carcinoma is the most common type of cancer and arises from epithelial cells. Transition from adenoma to carcinoma is associated with the loss of E-cadherin and, in consequence, the disruption of cell−cell contacts. E-cadherin is a tumor suppressor which is down-regulated during epithelial-to-mesenchymal transition (EMT), and its loss is a predictor of poor prognosis during tumor progression. Hakai is an E3 ubiquitin-ligase that mediates E-cadherin ubiquitination, endocytosis and consequent degradation. Although E-cadherin is the most established substrate for Hakai activity, other regulated molecular targets for Hakai may be involved in cancer cell plasticity during tumor progression. In this work we employed an iTRAQ approach to explore novel molecular pathways involved in Hakai-driven EMT. Our results show that Hakai may have an important influence on cytoskeleton-related proteins, extracellular exosome-associated proteins, RNA-related proteins and proteins involved in metabolism. Among Hakai-down-regulated proteins, we describe Annexin A2 as a new possible susbtrate for Hakai. Moreover, we also report an interaction between Hakai and the heat shock protein 90 (Hsp90) chaperone complex. Besides, our results reveal that the pharmacological inhibition of Hsp90 with geldanamycin results in the degradation of Hakai in a lysosome-dependent manner. Based on that, we propose Hakai as a new client protein of Hsp90 chaperone highlighting a new mechanism by which Hsp90 inhibitors may influence Hakai-mediated EMT process and cancer treatment

Investigation of the intrinsic mechanism of drug resistance in multiple myeloma

The focus of this thesis was to evaluate the mechanisms whereby myeloma cells develop intrinsic resistance with a focus on resistance in the context of bortezomib treatment. The aims of this thesis were to examine multidrug resistance pumps as a mechanism of resistance in MM, to investigate the contribution of p53 signalling perturbations in resistance mechanism in MM, to study the AMPK pathway as an alternative target to overcome MM resistance and finally to characterise myeloma resistance to bortezomib treatment using 2D-DIGE analysis. Focussing on bortezomib resistance models, we found that that overexpression of P-gp attenuates bortezomib activity. Bortezomib is a P-gp substrate and a combination of P-gp inhibitor and bortezomib is able to overcome resistance. Bortezomib is also able to downregulate the expression and function of P-gp. Our findings therefore suggest that combination of a P-gp inhibitor and bortezomib in P-gp positive myeloma would be a reasonable treatment combination to extend use of the drug. We have shown that p53 apoptotic signalling pathways can be accentuated when bortezomib is combined with a Mdm2 inhibitor. In p53 WT cells, nutlin-3 in combination with bortezomib generates additive toxicity in MM cells but is highly synergistic in epithelial models and p53-mutated cell lines. This synergy persists in the presence of BMSCs. This observation has implications more so in epithelial cancers and p53 mutated cancers where single agent bortezomib activity is mild. We have also shown that bortezomib-treated patients who had high expression of nutlin-3-suppressed genes had significantly shorter progression-free (p=0.001, log-rank test) and overall survival (p=0.002, log-rank test) compared to those with low expression levels. AMPK activation is promising as an anticancer pathway and may also be a chemoprevention target. Metformin and AICAR, which activate this pathway, both have demonstrated useful preclinical anticancer properties and have a good therapeutic index in patients. We explored mechanism of cell death and showed that AICAR was able to activate the apoptotic pathway. These agents also synergise with glycolysis inhibitors to further increase cytotoxicity in cancer cells. Identification of proteins whose expression is altered in differing states of sensitivity and resistance provides candidates for better understanding of resistance mechanisms so we also investigated bortezomib resistance in cellular models using proteomic techniques and isolated and identified several novel proteins which may play a role in this phenomenon. Our findings are mechanistically consistent since two of the identified proteins Hsp70 and caspase-3 are known in the literature to be affected by bortezomib treatment

The Role and Regulation of p21 in Myelopoiesis

Elevated levels of the molecular adaptor protein p21waf1/cip1 (p21) and of the IL-3 receptor alpha chain are correlated with chemoresistance and poor prognosis in acute myeloid leukemia (AML). p21 is a core regulator of many biological functions including cell cycle control, apoptosis and differentiation. Our laboratory has demonstrated a decrease in p21 expression levels during cytokine-induced granulocytic differentiation, leading us to hypothesize that p21 antagonizes granulopoiesis. The proliferative cytokine IL-3 has been shown to prevent granulocytic differentiation of murine and human myeloid progenitor cells. We also hypothesized that IL-3 inhibition of differentiation is mediated in part by p21, and tested this in murine 32Dcl3 myeloblasts that are used to model granulopoiesis. Our findings demonstrated that p21 antagonized differentiation by promoting apoptosis of cells exposed to the differentiation inducer G-CSF. We also showed that p21 prevented premature expression of primary granule proteins and contributed to maintenance of the myeloblast phenotype. Furthermore, p21 knockdown accelerated morphologic differentiation of 32Dcl3 cells stimulated to differentiate with G-CSF. We then determined how IL-3 maintains p21 expression in myeloblast cells. We showed that IL-3 stabilized p21 mRNA in myeloblasts leading to high levels of p21 protein. This effect mapped to the 3' untranslated region (UTR) of the p21 transcript. p21 transcript stabilization by IL-3 was independent of PI3-kinase and ERK pathway signaling. In vitro binding assays provided evidence that distinct sets of RNA:protein interactions occur within the proximal 303 nucleotides of the p21 3' UTR and are regulated by IL-3 and G-CSF signaling. Association of a 60-65 kDa protein with p21 riboprobes correlated with IL-3 mediated p21 mRNA stabilization, whereas binding by a 40-42 kDa protein was associated with destabilization of p21 transcripts in 32Dcl3 cells undergoing G-CSF-induced differentiation. These findings provide the first evidence for IL-3-mediated stabilization of mRNA transcripts in myeloid progenitor cells. The finding that p21 antagonized granulopoiesis is also novel. Because high levels of the IL-3 receptor and high p21 expression have separately been linked to poor outcomes in AML, IL-3 mediated p21 mRNA stabilization may contribute to differentiation blockade during AML pathogenesis

HDM2 antagonist MI-219 (spiro-oxindole), but not Nutlin-3 (cis-imidazoline), regulates p53 through enhanced HDM2 autoubiquitination and degradation in human malignant B-cell lymphomas

Abstract Background Lymphomas frequently retain wild-type (wt) p53 function but overexpress HDM2, thereby compromising p53 activity. Therefore, lymphoma is a suitable model for studying the therapeutic value of disrupting the HDM2-p53 interaction by small-molecule inhibitors (SMIs). HDM2 have been developed and are under various stages of preclinical and clinical investigation. Previously, we examined the anti-lymphoma activity of MI-319, the laboratory grade of a new class of HDM2 SMI, the spiro-oxindole, in follicular lymphoma. Since then, MI-219, the clinical grade has become readily available. This study further examines the preclinical effects and mechanisms of MI-219 in a panel of human lymphoma cell lines as well as a cohort of patient-derived B-lymphcytes for its potential clinical use. Results Preclinical assessment of MI-219 was evaluated by means of an in vitro and ex vivo approach and compared to Nutlin-3, the gold standard. Characterization of p53 activity and stability were assessed by quantitative PCR, Western blot, and immunoprecipitation. Biological outcome was measured using Trypan blue exclusion assay, Annexin V/PI, PARP and caspase-3 cleavage. Surprisingly, the overall biological effects of Nutlin-3 were more delayed (48 h) while MI-219 triggered an earlier response (12-24 h), predominantly in the form of apoptotic cell death. Using a cell free autoubiquitination assay, neither agent interfered with HDM2 E3 ligase function. MI-219 was more effective in upregulating wt-p53 stabilization compared to Nutlin-3. MI-219, but not Nutlin-3, enhanced the autoubiquitination and degradation of HDM2. Conclusions Our data reveals unexpected differences between MI-219 and the well-studied Nutlin-3 in lymphoma cell lines and patient samples. We suggest a novel mechanism for MI-219 that alters the functional activity of HDM2 through enhanced autoubiquitination and degradation. Additionally, this mechanism appears to correspond to biological outcome. Our results provide evidence that different classes of HDM2 SMIs elicit molecular events that extend beyond HDM2-p53 dissociation which may be of biological and potentially therapeutic importance

The c-Jun n-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway in paclitaxel-induced apoptosis of cancer cells

The essential cellular functions associated with microtubules have led to a wide use of microtubule-interfering agents in cancer chemotherapy with promising results. Although the most well studied effect of microtubule-interfering agents is an arrest of cells at the G2/M phase of the cell cycle,other effects may also exist. I have observed that paclitaxel (Taxol), docetaxel (Taxotere), vinblastine,vincristine, nocodazole and colchicine activate the c-Jun N-terminal kinase/stress-activated protein kinase(JNK/SAPK) signaling pathway in a variety of human cells. Activation of JNK/SAPK by microtubule interfering agents is dose-dependent and time-dependent and requires interactions with microtubules.Functional activation of the JNKK/SEKl-JNK/SAPK-c Jun cascade was demonstrated by cotransfection with a TPA-response element reporter construct and dominant negative (dn) signal transducers followed by chloramphenicol acetyl-transferase assays. Microtubule-interfering agents also activate both Ras and apoptosis signal-regulating kinase (ASKl), and coexpression of dn Ras and dn ASKl exerted individual and additive inhibition of JNK/SAPK activation by microtubule-interfering agents. These findings suggest that multiple signal transduction pathways are involved with cellular detection of microtubular disarray and subsequent activation of JNK/SAPK.To further examine the role of JNK/SAPK signaling cascades in apoptosis resulting from microtubule dysfunction induced by paclitaxel, I have coexpressed dn signaling proteins of theJNK/SAPK pathway (Ras, ASKl, Rac, JNKK, JNK) in human ovarian cancer cells with a selectable marker to analyze the apoptotic characteristics of cells expressing dn-vectors following exposure to paclitaxel. Expression of these dn signaling proteins had no effect on Bcl-2 phosphorylation, yet inhibited apoptotic changes induced by paclitaxel up to 16 h after treatment. Coexpression of these dna-signaling proteins had no protective effect after 48 h of paclitaxel treatment. These data indicate that: (i) activatedJNK/SAPK acts upstream of membrane changes and caspase-3 activation in paclitaxel-initiated apoptotic pathways, independently of cell cycle stage, (ii) activated JNK/SAPK is not responsible for paclitaxelinducedphosphorylation of Bcl-2, and (iii) apoptosis resulting from microtubule damage may comprise multiple mechanisms, including a JNK/SAPK-dependent early phase and a JNK/SAPK-independent latephase