Sudharsana R. Ande

Apoptosis and cancer: mutations within caspase genes

The inactivation of programmed cell death has profound effects not only on the development but also on the overall integrity of multicellular organisms. Beside developmental abnormalities, it may lead to tumorigenesis, autoimmunity, and other serious health problems. Deregulated apoptosis may also be the leading cause of cancer therapy chemoresistance. Caspase family of cysteinyl-proteases plays the key role in the initiation and execution of programmed cell death. This review gives an overview of the role of caspases, their natural modulators like IAPs, FLIPs, and Smac/Diablo in apoptosis and upon inactivation, and also in cancer development. Besides describing the basic mechanisms governing programmed cell death, a large part of this review is dedicated to previous studies that were focused on screening tumours for mutations within caspase genes as well as their regulators. The last part of this review discusses several emerging treatments that involve modulation of caspases and their regulators. Thus, we also highlight caspase cascade modulating experimental anticancer drugs like cFLIP-antagonist CDDO-Me; cIAP1 antagonists OSU-03012 and ME-BS; and XIAP small molecule antagonists 1396–11, 1396–12, 1396–28, triptolide, AEG35156, survivin/Hsp90 antagonist shephedrin, and some of the direct activators of procaspase-3.

Autophagy and apoptosis dysfunction in neurodegenerative disorders

Autophagy and apoptosis are basic physiologic processes contributing to the maintenance of cellular homeostasis. Autophagy encompasses pathways that target long-lived cytosolic proteins and damaged organelles. It involves a sequential set of events including double membrane formation, elongation, vesicle maturation and finally delivery of the targeted materials to the lysosome. Apoptotic cell death is best described through its morphology. It is characterized by cell rounding, membrane blebbing, cytoskeletal collapse, cytoplasmic condensation, and fragmentation, nuclear pyknosis, chromatin condensation/fragmentation, and formation of membrane-enveloped apoptotic bodies, that are rapidly phagocytosed by macrophages or neighboring cells. Neurodegenerative disorders are becoming increasingly prevalent, especially in the Western societies, with larger percentage of members living to an older age. They have to be seen not only as a health problem, but since they are care-intensive, they also carry a significant economic burden. Deregulation of autophagy plays a pivotal role in the etiology and/or progress of many of these diseases. Herein, we briefly review the latest findings that indicate the involvement of autophagy in neurodegenerative diseases. We provide a brief introduction to autophagy and apoptosis pathways focusing on the role of mitochondria and lysosomes. We then briefly highlight pathophysiology of common neurodegenerative disorders like Alzheimers diseases, Parkinsons disease, Huntingtons disease and Amyotrophic lateral sclerosis. Then, we describe functions of autophagy and apoptosis in brain homeostasis, especially in the context of the aforementioned disorders. Finally, we discuss different ways that autophagy and apoptosis modulation may be employed for therapeutic intervention during the maintenance of neurodegenerative disorders.

Cancer stem cell markers in common cancers – therapeutic implications

Rapid advances in the cancer stem cell (CSC) field have provided cause for optimism for the development of more reliable cancer therapies in the future. Strategies aimed at efficient targeting of CSCs are becoming important for monitoring the progress of cancer therapy and for evaluating new therapeutic approaches. Here, we characterize and compare the specific markers that have been found to be present on stem cells, cancer cells and CSCs in selected tissues (colon, breast, liver, pancreas and prostate). We then discuss future directions of this intriguing new research field in the context of new diagnostic and therapeutic opportunities.

S100A8/A9 induces autophagy and apoptosis via ROS-mediated cross-talk between mitochondria and lysosomes that involves BNIP3

The complex formed by two members of the S100 calcium-binding protein family, S100A8/A9, exerts apoptosis-inducing activity in various cells of different origins. Here, we present evidence that the underlying molecular mechanisms involve both programmed cell death I (PCD I, apoptosis) and PCD II (autophagy)-like death. Treatment of cells with S100A8/A9 caused the increase of Beclin-1 expression as well as Atg12-Atg5 formation. S100A8/A9-induced cell death was partially inhibited by the specific PI3-kinase class III inhibitor, 3-methyladenine (3-MA), and by the vacuole H+-ATPase inhibitor, bafilomycin-A1 (Baf-A1). S100A8/A9 provoked the translocation of BNIP3, a BH3 only pro-apoptotic Bcl2 family member, to mitochondria. Consistent with this finding, ΔTM-BNIP3 overexpression partially inhibited S100A8/A9-induced cell death, decreased reactive oxygen species (ROS) generation, and partially protected against the decrease in mitochondrial transmembrane potential in S100A8/A9-treated cells. In addition, either ΔTM-BNIP3 overexpression or N-acetyl-L-cysteine co-treatment decreased lysosomal activation in cells treated with S100A8/A9. Our data indicate that S100A8/A9-promoted cell death occurs through the cross-talk of mitochondria and lysosomes via ROS and the process involves BNIP3.

The role of prohibitin in cell signaling.

Prohibitin‐1 (PHB, also known as PHB1), a member of the Band‐7 family of proteins, is highly conserved evolutionarily, widely expressed, and present in different cellular compartments. Genetic studies with different organism models have provided strong evidence for an important biological role of PHB in mitochondrial function, cell proliferation, and development. Recent discoveries regarding the involvement of PHB in phophatidylinositol‐3‐kinase (PI3K)/protein kinase B (Akt) and transforming growth factor‐β (TGF‐β)/signal transducers and activators of transcription signaling pathways, and earlier reports on the interaction of PHB with Raf and its critical role in Ras/mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) signaling opened up the possibility that PHB has functions outside of the mitochondria (extramitochondrial) and may be a multifunctional protein. The PI3K/Akt and Ras/MAPK/ERK signaling cascades are versatile signaling processes that diverge from the same receptor tyrosine kinase root, and are involved in cell metabolism, proliferation, and development. Here, we review the emerging role of PHB and its post‐translational modifications in signal transduction pathways, especially in PI3K/Akt and Ras/MAPK/ERK signaling. A recent discovery of opposing effects of PHB on longevity under different metabolic states and its potential connection with insulin/insulin‐like growth factor‐I signaling is also discussed.

Akt-mediated phosphorylation of CDK2 regulates its dual role in cell cycle progression and apoptosis.

Here, we show that CDK2, an S-phase cyclin-dependent kinase, is a novel target for Akt during cell cycle progression and apoptosis. Akt phosphorylates CDK2 at threonine 39 residue both in vitro and in vivo. Although CDK2 threonine 39 phosphorylation mediated by Akt enhances cyclin-A binding, it is dispensable for its basal binding and the kinase activity. In addition, for the first time, we report a transient nucleo-cytoplasmic shuttling of Akt during specific stages of the cell cycle, in particular during the late S and G2 phases. The Akt that is re-localized to the nucleus phosphorylates CDK2 and causes the temporary cytoplasmic localization of the CDK2–cyclin-A complex. The CDK2 cytoplasmic redistribution is required for cell progression from S to G2-M phase, because the CDK2 T39A mutant, which lacks the phosphorylation site and is defective in cytoplasmic localization, severely affects cell cycle progression at the transition from S to G2-M. Interestingly, we also show that the Akt/CDK2 pathway is constitutively activated by some anticancer drugs, such as methotrexate and docetaxel, and under these conditions it promotes, rather than represses, cell death. Thus, the constitutive activation of the Akt/CDK2 pathway and changed subcellular localization promotes apoptosis. By contrast, the transient, physiological Akt/CDK2 activation is necessary for cell cycle progression.

O-GlcNAc modification: why so intimately associated with phosphorylation?

Post-translational modification of proteins at serine and threonine side chains by β-N-acetylglucosamine (O-GlcNAc) mediated by the enzyme β-N-acetylglucosamine transferase has been emerging as a fundamental regulatory mechanism encompassing a wide range of proteins involved in cell division, metabolism, transcription and cell signaling. Furthermore, an extensive interplay between O-GlcNAc modification and serine/threonine phosphorylation in a variety of proteins has been reported to exist. However, our understanding of the regulatory mechanisms involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins is still elusive. Recent success in the mapping of O-GlcNAc modification sites in proteins as a result of technological advancement in mass spectrometry have revealed two important clues which may be inherently connected to the regulation of O-GlcNAc modification and its interplay with phosphorylation in proteins. First, almost all O-GlcNAc modified proteins are known phospho proteins. Second, the prevalence of tyrosine phosphorylation among O-GlcNAc modified proteins is exceptionally higher (~68%) than its normal occurrence (~2%) alone. We hypothesize that phosphorylation may be a requisite for O-GlcNAc modification and tyrosine phosphorylation plays a role in the interplay between O-GlcNAc modification and serine/threonine phosphorylation in proteins. In other words, the interplay between O-GlcNAc modification and phosphorylation is not limited to serine/threonine phosphorylation but also includes tyrosine phosphorylation. Our hypothesis provides an opportunity to understand the underlying mechanism involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins. Furthermore, implication of our hypothesis extends to tyrosine kinase signaling.

The ubiquitin pathway: an emerging drug target in cancer therapy.

Ubiquitination is a highly ordered multistep enzymatic process, carried out by a well-defined systematic ubiquitin pathway, which is required for maintaining appropriate levels and functional activities of various cellular proteins. Targeted regulation of proteins by ubiquitin pathway controls numerous cellular processes including cell proliferation, signal transduction, apoptosis, transcriptional regulation, receptor modulation as well as endocytosis. Significant progress has been made in recent years in not only understanding the structure, function and important regulatory roles of ubiquitin network but also the alterations of ubiquitin pathway in various human diseases including cancer. Based on the progress made, it is now possible to target specifically various components involved in the ubiquitin pathway such as E3 ubiquitin ligases, deubiquitinases and proteosome for potential anticancer therapies. Here we review the potential drug targets available in the ubiquitin system and the small molecule inhibitors that can target these components in the pathway, which can be developed into novel anticancer therapeutics in the near future.

Prohibitin interacts with phosphatidylinositol 3,4,5-triphosphate (PIP3) and modulates insulin signaling

Mitochondrial protein prohibitin (PHB) is known to associate with the plasma membrane of various cell types. However, biological function of plasma membrane associated PHB is not known. Recently we have shown that PHB undergoes tyrosine phosphorylation in response to insulin and here we report that PHB interacts with phosphatidylinositol 3,4,5-triphosphate (PIP3). Furthermore, we demonstrate that over expression of PHB attenuates insulin signaling downstream of phosphatidylinositol 3 (PI3) kinase. This effect was not observed with over expression of tyrosine phosphorylation site mutant-PHB suggesting a role for tyrosine phosphorylation of PHB in this process. Interestingly phosphorylation of PHB by Akt attenuates its interaction with PIP3 and enhances insulin signaling. Thus, phosphorylation of PHB and its interaction with PIP3 may be a part of the regulatory mechanisms that is involved in the modulation of insulin signaling. We speculate that phosphorylation of PHB may serve as a general mechanism in the regulation of PI3 kinase signaling including growth factors and immune receptor signaling.

Interaction between O-GlcNAc modification and tyrosine phosphorylation of prohibitin: implication for a novel binary switch.

Prohibitin (PHB or PHB1) is an evolutionarily conserved, multifunctional protein which is present in various cellular compartments including the plasma membrane. However, mechanisms involved in various functions of PHB are not fully explored yet. Here we report for the first time that PHB interacts with O-linked β-N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and is O-GlcNAc modified; and also undergoes tyrosine phosphorylation in response to insulin. Tyrosine 114 (Tyr114) and tyrosine 259 (Tyr259) in PHB are in the close proximity of potential O-GlcNAc sites serine 121 (Ser121) and threonine 258 (Thr258) respectively. Substitution of Tyr114 and Tyr259 residues in PHB with phenylalanine by site-directed mutagenesis results in reduced tyrosine phosphorylation as well as reduced O-GlcNAc modification of PHB. Surprisingly, this also resulted in enhanced tyrosine phosphorylation and activity of OGT. This is attributed to the presence of similar tyrosine motifs in PHB and OGT. Substitution of Ser121 and Thr258 with alanine and isoleucine respectively resulted in attenuation of O-GlcNAc modification and increased tyrosine phosphorylation of PHB suggesting an association between these two dynamic modifications. Sequence analysis of O-GlcNAc modified proteins having known O-GlcNAc modification site(s) or known tyrosine phosphorylation site(s) revealed a strong potential association between these two posttranslational modifications in various proteins. We speculate that O-GlcNAc modification and tyrosine phosphorylation of PHB play an important role in tyrosine kinase signaling pathways including insulin, growth factors and immune receptors signaling. In addition, we propose that O-GlcNAc modification and tyrosine phosphorylation is a novel previously unidentified binary switch which may provide new mechanistic insights into cell signaling pathways and is open for direct experimental examination.