BBA - Molecular Cell Research (v.1843, #9)

In breast cancer the presence of cells undergoing the epithelial-to-mesenchymal transition is indicative of metastasis progression. Since metabolic features of breast tumour cells are critical in cancer progression and drug resistance, we hypothesized that the lipid content of malignant cells might be a useful indirect measure of cancer progression. In this study Multivariate Curve Resolution was applied to cellular Raman spectra to assess the metabolic composition of breast cancer cells undergoing the epithelial to mesenchymal transition. Multivariate Curve Resolution analysis led to the conclusion that this transition affects the lipid profile of cells, increasing tryptophan but maintaining a low fatty acid content in comparison with highly metastatic cells. Supporting those results, a Partial Least Square-Discriminant analysis was performed to test the ability of Raman spectroscopy to discriminate the initial steps of epithelial to mesenchymal transition in breast cancer cells. We achieved a high level of sensitivity and specificity, 94% and 100%, respectively. In conclusion, Raman microspectroscopy coupled with Multivariate Curve Resolution enables deconvolution and tracking of the molecular content of cancer cells during a biochemical process, being a powerful, rapid, reagent-free and non-invasive tool for identifying metabolic features of breast cancer cell aggressiveness at first stages of malignancy.Display Omitted
Keywords: Biomarker; Breast cancer; Epithelial-to-mesenchymal transition (EMT); Lipid; Metabolism; Raman spectroscopy;

Cell migration to CXCL12 requires simultaneous IKKα and IKKβ-dependent NF-κB signaling by Marianna Penzo; David M. Habiel; Mahalakshmi Ramadass; Richard R. Kew; Kenneth B. Marcu (1796-1804).
CXCL12 and its unique receptor CXCR4, is critical for the homing of a variety of cell lineages during both development and tissue repair. CXCL12 is particularly important for the recruitment of hemato/lymphopoietic cells to their target organs. In conjunction with the damage-associated alarmin molecule HMGB1, CXCL12 mediates immune effector and stem/progenitor cell migration towards damaged tissues for subsequent repair. Previously, we showed that cell migration to HMGB1 simultaneously requires both IKKβ and IKKα-dependent NF-κB activation. IKKβ-mediated activation maintains sufficient expression of HMGB1's receptor RAGE, while IKKα-dependent NF-κB activation ensures continuous production of CXCL12, which complexes with HMGB1 to engage CXCR4. Here using fibroblasts and primary mature macrophages, we show that IKKβ and IKKα are simultaneously essential for cell migration in response to CXCL12 alone. Non-canonical NF-κB pathway subunits RelB and p52 are also both essential for cell migration towards CXCL12, suggesting that IKKα is required to drive non-canonical NF-κB signaling. Flow cytometric analyses of CXCR4 expression show that IKKβ, but not IKKα, is required to maintain a critical threshold level of this CXCL12 receptor. Time-lapse video microscopy experiments in primary MEFs reveal that IKKα is required both for polarization of cells towards a CXCL12 gradient and to establish a basal level of velocity towards CXCL12. In addition, CXCL12 modestly up-regulates IKKα-dependent p52 nuclear translocation and IKKα-dependent expression of the CXCL12 gene. On the basis of our collective results we posit that IKKα is needed to maintain the basal expression of a critical protein co-factor required for cell migration to CXCL12.
Keywords: CXCL12; CXCR4; Cell migration; NF-κB; IKKα; IKKβ;

SUMOylation and deimination of proteins: Two epigenetic modifications involved in Giardia encystation by Cecilia V. Vranych; María R. Rivero; María C. Merino; Gonzalo F. Mayol; Nahuel Zamponi; Belkys A. Maletto; María C. Pistoresi-Palencia; María C. Touz; Andrea S. Rópolo (1805-1817).
SUMOylation, a posttranslational modification of proteins, has been recently described as vital in eukaryotic cells. In a previous work, we analyzed the role of SUMO protein and the genes encoding the putative enzymes of the SUMOylation pathway in the parasite Giardia lamblia. Although we observed several SUMOylated proteins, only the enzyme Arginine Deiminase (ADI) was confirmed as a SUMOylated substrate. ADI is involved in the survival of the parasite and, besides its role in ATP production, it also catalyzes the modification of arginine residues to citrulline in the cytoplasmic tail of surface proteins. During encystation, however, ADI translocates to the nuclei and downregulates the expression of the Cyst Wall Protein 2 (CWP2). In this work, we made site-specific mutation of the ADI SUMOylation site (Lys101) and observed that transgenic trophozoites did not translocate to the nuclei at the first steps of encystation but shuttled in the nuclei late during this process through classic nuclear localization signals. Inside the nuclei, ADI acts as a peptidyl arginine deiminase, being probably involved in the downregulation of CWPs expression and cyst wall formation. Our results strongly indicate that ADI plays a regulatory role during encystation in which posttranslational modifications of proteins are key players.
Keywords: Arginine deiminase; SUMOylation; Nuclear localization; Encystation; Parasites;

Inhibition of endogenous MTF-1 signaling in zebrafish embryos identifies novel roles for MTF-1 in development by Britton O'Shields; Andrew G. McArthur; Andrew Holowiecki; Martin Kamper; Jeffrey Tapley; Matthew J. Jenny (1818-1833).
The metal responsive element-binding transcription factor-1 (MTF-1) responds to changes in cellular zinc levels caused by zinc exposure or disruption of endogenous zinc homeostasis by heavy metals or oxygen-related stress. Here we report the functional characterization of a complete zebrafish MTF-1 in comparison with the previously identified isoform lacking the highly conserved cysteine-rich motif (Cys-X-Cys-Cys-X-Cys) found in all other vertebrate MTF-1 orthologs. In an effort to develop novel molecular tools, a constitutively nuclear dominant-negative MTF-1 (dnMTF-1) was generated as tool for inhibiting endogenous MTF-1 signaling. The in vivo efficacy of the dnMTF-1 was determined by microinjecting in vitro transcribed dnMTF-1 mRNA into zebrafish embryos (1–2 cell stage) followed by transcriptomic profiling using an Agilent 4x44K array on 28- and 36-hpf embryos. A total of 594 and 560 probes were identified as differentially expressed at 28 hpf and 36 hpf, respectively, with interesting overlaps between timepoints. The main categories of genes affected by the inhibition of MTF-1 signaling were: nuclear receptors and genes involved in stress signaling, neurogenesis, muscle development and contraction, eye development, and metal homeostasis, including novel observations in iron and heme homeostasis. Finally, we investigate both the transcriptional activator and transcriptional repressor role of MTF-1 in potential novel target genes identified by transcriptomic profiling during early zebrafish development.
Keywords: MTF-1; Zebrafish; Zinc homeostasis; Metal; Embryonic development;

We studied the regulation of RANKL expression in myeloma by promoter DNA methylation. Methylation-specific polymerase chain reaction showed complete methylation of RANKL promoter in WL-2 myeloma cells but partial methylation in eight other lines. 5-AzadC treatment of WL-2 cells led to demethylation and re-expression of RANKL. Transwell and contact co-culture of WL-2 cells with normal bone marrow-derived mesenchymal stromal cells (BMSCs) resulted in comparable repression of DNA methyltransferase-1 (DNMT1) and re-expression of RANKL in WL-2 cells. Moreover, treatment of WL-2 cells with TNFα led to repression of DNMT1 and re-expression of RANKL in association with upregulation of miR-140-3p and miR-126, which are partially offset by addition of anti-TNFα antibody to transwell-coculture of WL2 with BMSC. Taken together, our results showed that TNFα in the marrow microenvironment led to RANKL demethylation and re-expression in myeloma cells through DNMT1 repression and upregulation of miR-126-3p and miR-140, both known to repress DNMT1 translation.
Keywords: RANKL; Myeloma microenvironment; DNA methylation; DNMT1; MicroRNA; TNFα;

Cholesterol-induced activation of TRPM7 regulates cell proliferation, migration, and viability of human prostate cells by Yuyang Sun; Pramod Sukumaran; Archana Varma; Susan Derry; Abe E. Sahmoun; Brij B. Singh (1839-1850).
Cholesterol has been shown to promote cell proliferation/migration in many cells; however the mechanism(s) have not yet been fully identified. Here we demonstrate that cholesterol increases Ca2 + entry via the TRPM7 channel, which promoted proliferation of prostate cells by inducing the activation of the AKT and/or the ERK pathway. Additionally, cholesterol mediated Ca2 + entry induced calpain activity that showed a decrease in E-cadherin expression, which together could lead to migration of prostate cancer cells. An overexpression of TRPM7 significantly facilitated cholesterol dependent Ca2 + entry, cell proliferation and tumor growth. Whereas, TRPM7 silencing or inhibition of cholesterol synthesis by statin showed a significant decrease in cholesterol-mediated activation of TRPM7, cell proliferation, and migration of prostate cancer cells. Consistent with these results, statin intake was inversely correlated with prostate cancer patients and increase in TRPM7 expression was observed in samples obtained from prostate cancer patients. Altogether, we provide evidence that cholesterol-mediated activation of TRPM7 is important for prostate cancer and have identified that TRPM7 could be essential for initiation and/or progression of prostate cancer.
Keywords: TRPM7; Cholesterol; Calcium and signal transduction; Statin; Cell proliferation and migration; Prostate cancer;

TEIF associated centrosome activity is regulated by EGF/PI3K/Akt signaling by Jing Zhao; Yongxin Zou; Haijing Liu; Huali Wang; Hong Zhang; Wei Hou; Xin Li; Xinying Jia; Jing Zhang; Lin Hou; Bo Zhang (1851-1864).
Centrosome amplification, which is a characteristic of cancer cells, has been understood as a driving force of genetic instability in the development of cancer. In previous work, we demonstrated that TEIF (transcriptional element-interacting factor) distributes in the centrosomes and regulates centrosome status under both physiologic and pathologic conditions. Here we identify TEIF as a downstream effector in EGF/PI3K/Akt signaling. The addition of EGF or transfection of active Akt stimulates centrosome TEIF distribution, resulting in an increase of centrosome splitting and amplification, while inhibitors of either PI3K or Akt attenuate these changes in TEIF and the associated centrosome status. A consensus motif for Akt phosphorylation (RHRVLT) proved to be involved in centrosomal TEIF localization, and the 469-threonine of this motif may be phosphorylated by Akt both in vitro and in vivo. Elimination of this phosphorylated site on TEIF caused reduced centrosome distribution and centrosome splitting or amplification. Moreover, TEIF closely co-localized with C-NAP1 at the proximal ends of centrioles, and centriolar loading of TEIF stimulated by EGF/Akt could displace C-NAP1, resulting in centrosome splitting. These findings reveal linkage of the EGF/PI3K/Akt signaling pathway to regulation of centrosome status which may act as an oncogenic pathway and induce genetic instability in carcinogenesis.
Keywords: Centrosome amplification; EGF; Akt; C-NAP1;

Differential phosphorylation of Akt1 and Akt2 by protein kinase CK2 may account for isoform specific functions by Cristina Girardi; Peter James; Sofia Zanin; Lorenzo A. Pinna; Maria Ruzzene (1865-1874).
Akt (also known as PKB) is a survival kinase frequently up-regulated in cancer; three isoforms of Akt exist, and among them Akt1 and Akt2 are the most widely and highly expressed. They share the same structure and activation mechanism and have many overlapping functions; nevertheless isoform-specific roles and substrates have been reported, which are expected to rely on sequence diversities. In particular, a special role in differentiating Akt1 and Akt2 isoforms has been assigned to the linker region, a short segment between the PH and the catalytic domains. We have previously found that a residue in the linker region (Ser129) is directly phosphorylated by protein kinase CK2 in Akt1; the phosphorylation of the homologous residue in Akt2 (Ser131) has never been analyzed. Here we show that Akt2, endogenously or ectopically expressed in different cell lines, is not phosphorylated on Ser131 by CK2, while in vitro recombinant Akt2 is a CK2 substrate. These data support the hypothesis that in vivo a steric hindrance occurs which prevents the access to the CK2 site. Additionally, we have found that Ser129 phosphorylation is involved in the recognition of the Akt1-specific substrate palladin; this observation provides an explanation of why Akt2, lacking Ser131 phosphorylation in the linker region, has a low efficiency in targeting palladin. CK2-dependent phosphorylation is therefore a crucial event which, discriminating between Akt1 and Akt2, can account for different substrate specificities, and, more in general, for fine tuning of Akt activity in the control of isoform-dependent processes.
Keywords: CK2; Casein kinase 2; CKII; Akt; PKB; Isoform specificity;

Modulation of NRF2 signaling pathway by nuclear receptors: Implications for cancer by Akhileshwar Namani; Yulong Li; Xiu Jun Wang; Xiuwen Tang (1875-1885).
Nuclear factor-erythroid 2 p45-related factor 2 (NRF2, also known as Nfe2l2) plays a critical role in regulating cellular defense against electrophilic and oxidative stress by activating the expression of an array of antioxidant response element-dependent genes. On one hand, NRF2 activators have been used in clinical trials for cancer prevention and the treatment of diseases associated with oxidative stress; on the other hand, constitutive activation of NRF2 in many types of tumors contributes to the survival and growth of cancer cells, as well as resistance to anticancer therapy. In this review, we provide an overview of the NRF2 signaling pathway and discuss its role in carcinogenesis. We also introduce the inhibition of NRF2 by nuclear receptors. Further, we address the biological significance of regulation of the NRF2 signaling pathway by nuclear receptors in health and disease. Finally, we discuss the possible impact of NRF2 inhibition by nuclear receptors on cancer therapy.
Keywords: NRF2; KEAP1; Nuclear receptor; Neh7; Cancer therapy;

Neuroprotection elicited by P2Y13 receptors against genotoxic stress by inducing DUSP2 expression and MAPK signaling recovery by Verónica Morente; Raquel Pérez-Sen; Felipe Ortega; Jaime Huerta-Cepas; Esmerilda G. Delicado; Mª Teresa Miras-Portugal (1886-1898).
Nucleotides activating P2Y13 receptors display neuroprotective actions against different apoptotic stimuli in cerebellar granule neurons. In the present study, P2Y13 neuroprotection was analyzed in conditions of genotoxic stress. Exposure to cisplatin and UV radiation induced caspase-3-dependent apoptotic cell death, and p38 MAPK signaling de-regulation. Pre-treatment with P2Y13 nucleotide agonist, 2methyl-thio-ADP (2MeSADP), restored granule neuron survival and prevented p38 long-lasting activation induced by cytotoxic treatments. Microarray gene expression analysis in 2MeSADP-stimulated cells revealed over-representation of genes related to protein phosphatase activity. Among them, dual-specificity phosphatase-2, DUSP2, was validated as a transcriptional target for P2Y13 receptors by QPCR. This effect could explain 2MeSADP ability to dephosphorylate a DUSP2 substrate, p38, reestablishing the inactive form. In addition, cisplatin-induced p38 sustained activation correlated perfectly with progressive reduction in DUSP2 expression. In conclusion, P2Y13 receptors regulate DUSP2 expression and contribute to p38 signaling homeostasis and survival in granule neurons.
Keywords: P2Y13 receptor; Nucleotide receptor; DUSP; MAPK protein phosphatase; p38; Neuroprotection;

Functional expression of adrenoreceptors in mesenchymal stromal cells derived from the human adipose tissue by Polina D. Kotova; Veronika Yu. Sysoeva; Olga A. Rogachevskaja; Marina F. Bystrova; Alisa S. Kolesnikova; Pyotr A. Tyurin-Kuzmin; Julia I. Fadeeva; Vsevolod A. Tkachuk; Stanislav S. Kolesnikov (1899-1908).
Cultured mesenchymal stromal cells (MSCs) from different sources represent a heterogeneous population of proliferating non-differentiated cells that contains multipotent stem cells capable of originating a variety of mesenchymal cell lineages. Despite tremendous progress in MSC biology spurred by their therapeutic potential, current knowledge on receptor and signaling systems of MSCs is mediocre. Here we isolated MSCs from the human adipose tissue and assayed their responsivity to GPCR agonists with Ca2 + imaging. As a whole, a MSC population exhibited functional heterogeneity. Although a variety of first messengers was capable of stimulating Ca2+ signaling in MSCs, only a relatively small group of cells was specifically responsive to the particular GPCR agonist, including noradrenaline. RT-PCR and immunocytochemistry revealed expression of α1B-, α2A-, and β2-adrenoreceptors in MSCs. Their sensitivity to subtype-specific adrenergic agonists/antagonists and certain inhibitors of Ca2+ signaling indicated that largely the α2A-isoform coupled to PLC endowed MSCs with sensitivity to noradrenaline. The all-or-nothing dose-dependence was characteristic of responsivity of robust adrenergic MSCs. Noradrenaline never elicited small or intermediate responses but initiated large and quite similar Ca2+ transients at all concentrations above the threshold. The inhibitory analysis and Ca2+ uncaging implicated Ca2+-induced Ca2+ release (CICR) in shaping Ca2+ signals elicited by noradrenaline. Evidence favored IP3 receptors as predominantly responsible for CICR. Based on the overall findings, we inferred that adrenergic transduction in MSCs includes two fundamentally different stages: noradrenaline initially triggers a local and relatively small Ca2+ signal, which next stimulates CICR, thereby being converted into a global Ca2+ signal.
Keywords: Mesenchymal stromal cell; Adrenergic receptor; Calcium-induced calcium release; Ca2 + uncaging; IP3 receptor;

Subcellular compartmentation of ascorbate and its variation in disease states by Gábor Bánhegyi; Angelo Benedetti; Éva Margittai; Paola Marcolongo; Rosella Fulceri; Csilla E. Németh; András Szarka (1909-1916).
Beyond its general role as antioxidant, specific functions of ascorbate are compartmentalized within the eukaryotic cell. The list of organelle-specific functions of ascorbate has been recently expanded with the epigenetic role exerted as a cofactor for DNA and histone demethylases in the nucleus. Compartmentation necessitates the transport through intracellular membranes; members of the GLUT family and sodium-vitamin C cotransporters mediate the permeation of dehydroascorbic acid and ascorbate, respectively. Recent observations show that increased consumption and/or hindered entrance of ascorbate in/to a compartment results in pathological alterations partially resembling to scurvy, thus diseases of ascorbate compartmentation can exist. The review focuses on the reactions and transporters that can modulate ascorbate concentration and redox state in three compartments: endoplasmic reticulum, mitochondria and nucleus. By introducing the relevant experimental and clinical findings we make an attempt to coin the term of ascorbate compartmentation disease.Display Omitted
Keywords: Ascorbate; Dehydroascorbic acid; Compartmentation; Scurvy; Arterial tortuosity syndrome; GLUT;

Oligodendrocytes are neuroglial cells responsible, within the central nervous system, for myelin sheath formation that provides an electric insulation of axons and accelerate the transmission of electrical signals. In order to be able to produce myelin, oligodendrocytes progress through a series of differentiation steps from oligodendrocyte precursor cells to mature oligodendrocytes (migration, increase in morphologic complexity and expression pattern of specific markers), which are modulated by cross talk with other nerve cells. If during the developmental stage any of these mechanisms is affected by toxic or external stimuli it may result into impaired myelination leading to neurological deficits. Such being the case, several approaches have been developed to evaluate how oligodendrocyte development and myelination may be impaired. The present review aims to summarize changes that oligodendrocytes suffer from precursor cells to mature ones, and to describe and discuss the different in vitro models used to evaluate not only oligodendrocyte development (proliferation, migration, differentiation and ability to myelinate), but also their interaction with neurons and other glial cells. First we discuss the temporal oligodendrocyte lineage progression, highlighting the differences between human and rodent, usually used as tissue supply for in vitro cultures. Second we describe how to perform and characterize the different in vitro cultures, as well as the methodologies to evaluate oligodendrocyte functionality in each culture system, discussing their advantages and disadvantages. Finally, we briefly discuss the current status of in vivo models for oligodendrocyte development and myelination.
Keywords: Myelinating co-cultures; Ex vivo cultures; In vitro primary cultures; Myelination; Oligodendrocytes;

Myogenesis defect due to Toca-1 knockdown can be suppressed by expression of N-WASP by Bhawana George; Neeraj Jain; Pei Fen Chong; Jun Hou Tan; Thirumaran Thanabalu (1930-1941).
Skeletal muscle formation is a multistep process involving proliferation, differentiation, alignment and fusion of myoblasts to form myotubes which fuse with additional myoblast to form myofibers. Toca-1 (Transducer of Cdc42-dependent actin assembly), is an adaptor protein which activates N-WASP in conjunction with Cdc42 to facilitate membrane invagination, endocytosis and actin cytoskeleton remodeling. Expression of Toca-1 in mouse primary myoblasts and C2C12 myoblasts was up-regulated on day 1 of differentiation and subsequently down-regulated during differentiation. Knocking down Toca-1 expression in C2C12 cells (Toca-1KD cells) resulted in a significant decrease in myotube formation and expression of shRNA-resistant Toca-1 in Toca-1KD cells rescued the myogenic defect, suggesting that the knockdown was specific and Toca-1 is essential for myotube formation. Toca-1KD cells exhibited elongated spindle-like morphology, expressed myogenic markers (MyoD and MyHC) and localized N-Cadherin at cell periphery similar to control cells suggesting that Toca-1 is not essential for morphological changes or expression of proteins critical for differentiation. Toca-1KD cells displayed prominent actin fibers suggesting a defect in actin cytoskeleton turnover necessary for cell–cell fusion. Toca-1KD cells migrated faster than control cells and had a reduced number of vinculin patches similar to N-WASPKO MEF cells. Transfection of N-WASP-expressing plasmid into Toca-1KD cells restored myotube formation of Toca-1KD cells. Thus, our results suggest that Toca-1KD cells have defects in formation of myotubes probably due to reduced activity of actin cytoskeleton regulators such as N-WASP. This is the first study to identify and characterize the role of Toca-1 in myogenesis.
Keywords: Cell adhesion; Arp2/3 complex; Actin cytoskeleton; Membrane fusion; Cell migration; Myogenic differentiation;

Enhanced rate of degradation of basic proteins by 26S immunoproteasomes by Mary Raule; Fulvia Cerruti; Paolo Cascio (1942-1947).
Immunoproteasomes are alternative forms of proteasomes specialized in the generation of MHC class I antigenic peptides and important for efficient cytokine production. We have identified a new biochemical property of 26S immunoproteasomes, namely the ability to hydrolyze basic proteins at greatly increased rates compared to constitutive proteasomes. This enhanced degradative capacity is specific for basic polypeptides, since substrates with a lower content in lysine and arginine residues are hydrolyzed at comparable rates by constitutive and immunoproteasomes. Crucially, selective inhibition of the immunoproteasome tryptic subunit β2i strongly reduces degradation of basic proteins. Therefore, our data demonstrate the rate limiting function of the proteasomal trypsin-like activity in controlling turnover rates of basic protein substrates and suggest new biological roles for immunoproteasomes in maintaining cellular homeostasis by rapidly removing a potentially harmful excess of free histones that can build up under different pathophysiological conditions.Display Omitted
Keywords: Proteasome; Immunoproteasome; Histones; MBP; Proteolysis; Trypsin-like activity;

Keeping the eIF2 alpha kinase Gcn2 in check by Beatriz A. Castilho; Renuka Shanmugam; Richard C. Silva; Rashmi Ramesh; Benjamin M. Himme; Evelyn Sattlegger (1948-1968).
The protein kinase Gcn2 is present in virtually all eukaryotes and is of increasing interest due to its involvement in a large array of crucial biological processes. Some of these are universally conserved from yeast to humans, such as coping with nutrient starvation and oxidative stress. In mammals, Gcn2 is important for e.g. long-term memory formation, feeding behaviour and immune system regulation. Gcn2 has been also implicated in diseases such as cancer and Alzheimer's disease. Studies on Gcn2 have been conducted most extensively in Saccharomyces cerevisiae, where the mechanism of its activation by amino acid starvation has been revealed in most detail. Uncharged tRNAs stimulate Gcn2 which subsequently phosphorylates its substrate, eIF2α, leading to reduced global protein synthesis and simultaneously to increased translation of specific mRNAs, e.g. those coding for Gcn4 in yeast and ATF4 in mammals. Both proteins are transcription factors that regulate the expression of a myriad of genes, thereby enabling the cell to initiate a survival response to the initial activating cue. Given that Gcn2 participates in many diverse processes, Gcn2 itself must be tightly controlled. Indeed, Gcn2 is regulated by a vast network of proteins and RNAs, the list of which is still growing. Deciphering molecular mechanisms underlying Gcn2 regulation by effectors and inhibitors is fundamental for understanding how the cell keeps Gcn2 in check ensuring normal organismal function, and how Gcn2-associated diseases may develop or may be treated. This review provides a critical evaluation of the current knowledge on mechanisms controlling Gcn2 activation or activity.Display Omitted
Keywords: Translational regulation; Gcn2; Gcn1; Uncharged tRNA; Ribosome; eIF2;

The protein phosphatase 2A regulatory subunit B55α is a modulator of signaling and microRNA expression in acute myeloid leukemia cells by Peter P. Ruvolo; Vivian R. Ruvolo; Rodrigo Jacamo; Jared K. Burks; Zhihong Zeng; Seshagiri R. Duvvuri; Liran Zhou; Yihua Qiu; Kevin R. Coombes; Nianxiang Zhang; Suk Y. Yoo; Rongqing Pan; Numsen Hail; Marina Konopleva; George Calin; Steven M. Kornblau; Michael Andreeff (1969-1977).
We recently discovered that the protein phosphatase 2A (PP2A) B55α subunit (PPP2R2A) is under-expressed in primary blast cells and is unfavorable for remission duration in AML patients. In this study, reverse phase protein analysis (RPPA) of 230 proteins in 511 AML patient samples revealed a strong correlation of B55α with a number of proteins including MYC, PKC α, and SRC. B55α suppression in OCI-AML3 cells by shRNA demonstrated that the B subunit is a PKCα phosphatase. B55α does not target SRC, but rather the kinase suppresses protein expression of the B subunit. Finally, the correlation between B55α and MYC levels reflected a complex stoichiometric competition between B subunits. Loss of B55α in OCI-AML3 cells did not change global PP2A activity and the only isoform that is induced is the one containing B56α. In cells containing B55α shRNA, MYC was suppressed with concomitant induction of the competing B subunit B56α (PPP2R5A). A recent study determined that FTY-720, a drug whose action involves the activation of PP2A, resulted in the induction of B55α In AML cells, and a reduction of the B subunit rendered these cells resistant to FTY-720. Finally, reduction of the B subunit resulted in an increase in the expression of miR-191-5p and a suppression of miR-142-3p. B55α regulation of these miRs was intriguing as high levels of miR-191 portend poor survival in AML, and miR-142-3p is mutated in 2% of AML patient samples. In summary, the suppression of B55α activates signaling pathways that could support leukemia cell survival.
Keywords: B55α; AML; Relapse; Cell signaling; miR-142; miR-191;

A mutation leading to super-assembly of twin-arginine translocase (Tat) protein complexes by Roshani Patel; Cvetelin Vasilev; Daniel Beck; Carmine G. Monteferrante; Jan Maarten van Dijl; C. Neil Hunter; Corinne Smith; Colin Robinson (1978-1986).
The Tat system transports folded proteins across the bacterial plasma membrane. The mechanism is believed to involve coalescence of a TatC-containing unit with a separate TatA complex, but the full translocation complex has never been visualised and the assembly process is poorly defined. We report the analysis of the Bacillus subtilis TatAyCy system, which occurs as separate TatAyCy and TatAy complexes at steady state, using single-particle electron microscopy (EM) and advanced atomic force microscopy (AFM) approaches. We show that a P2A mutation in the TatAy subunit leads to apparent super-assembly of Tat complexes. Purification of TatCy-containing complexes leads to a large increase in the TatA:TatC ratio, suggesting that TatAyP2A complexes may have attached to the TatAyCy complex. EM and AFM analyses show that the wild-type TatAyCy complex purifies as roughly spherical complexes of 9–16 nm diameter, whereas the P2A mutation leads to accumulation of large (up to 500 nm long) fibrils that are chains of numerous complexes. Time lapsed AFM imaging, recorded on fibrils under liquid, shows that they adopt a variety of tightly curved conformations, with radii of curvature of 10–12 nm comparable to the size of single TatAyP2A complexes. The combined data indicate that the mutation leads to super-assembly of TatAyP2A complexes and we propose that an individual TatAyP2A complex assembles initially with a TatAyP2ACy complex, after which further TatAyP2A complexes attach to each other. The data further suggest that the N-terminal extracytoplasmic domain of TatAy plays an essential role in Tat complex interactions.
Keywords: Tat; Twin-arginine translocation; Signal peptide; Atomic force microscopy; AFM;

Flotillin depletion affects ErbB protein levels in different human breast cancer cells by Nagham Asp; Sascha Pust; Kirsten Sandvig (1987-1996).
The ErbB3 receptor is an important regulator of cell growth and carcinogenesis. Among breast cancer patients, up to 50–70% have ErbB3 overexpression and 20–30% show overexpressed or amplified ErbB2. ErbB3 has also been implicated in the development of resistance to several drugs used against cancers driven by ErbB1 or ErbB2. One of the main challenges in ErbB-targeting therapy is to inactivate signaling mediated by ErbB2–ErbB3 oncogenic receptor complexes. We analyzed the regulatory role of flotillins on ErbB3 levels and ErbB2–ErbB3 complexes in SKBR3, MCF7 and MDA-MB-134-VI human breast cancer cells. Recently, we described a mechanism for interfering with ErbB2 signaling in breast cancer and demonstrated a molecular complex of flotillin scaffolding proteins with ErbB2 and Hsp90. In the present study, flotillins were found to be in a molecular complex with ErbB3, even in cells without the presence of ErbB2 or other ErbB receptors. Depletion of either flotillin-1 or flotillin-2 resulted in downregulation of ErbB3 and a selective reduction of ErbB2–ErbB3 receptor complexes. Moreover, flotillin-2 depletion resulted in reduced activation of Akt and MAPK signaling cascades, and as a functional consequence of flotillin depletion, breast cancer cells showed an impaired cell migration.Altogether, we provide data demonstrating a novel and functional role of flotillins in the regulation of ErbB protein levels and stabilization of ErbB2–ErbB3 receptor complexes. Thus, flotillins are crucial regulators for oncogenic ErbB function and potential targets for cancer treatment.Display Omitted
Keywords: ErbB3 receptor tyrosine kinase; Flotillin; Breast cancer;

Prostaglandin E2 induces retinoic acid receptor-β up-regulation through MSK1 by Ana B. Fernández-Martínez; Francisco J. Lucio Cazaña (1997-2004).
The pharmacological modulation of putative renoprotective factors hypoxia-inducible factor-1α (HIF-1α) and HIF-1α-regulated vascular endothelial growth factor-A (VEGF-A) in the kidney has therapeutic interest. In human renal proximal tubular HK2 cells, prostaglandin E2 (PGE2) up-regulates HIF-1α and VEGF-A through epidermal growth factor receptor (EGFR)-dependent up-regulation of retinoic acid receptor-β (RARβ). Here we studied the role of mitogen-activated protein kinases (MAPKs) ERK1/2 and p38 and their target kinase, mitogen- and stress activated kinase-1 (MSK1), in the signaling cascade. Treatment of HK2 cells with PGE2 resulted in increased phosphorylation of EGFR, the three studied kinases and the histone H3 (Ser10) at the RARβ gene promoter (the latter has been proposed as a molecular signature of the activated RARβ gene promoter). Prevention of the phosphorylation of EGFR, ERK1/2, p38 MAPK or MSK1 is by incubating, respectively, with AG1478, PD98059, SB203580 or H89 allowed to elucidate the precise phosphorylation order in the signaling cascade triggered by PGE2: first, EGFR; then, ERK1/2 and p38 MAPK and, finally, MSK1. Phosphorylation of MSK1 led to that of Ser10 in histone H3 and to activation of RARβ gene transcription (and the consequent increase in the expression of HIF-1α and VEGF-A), which was suppressed by H89 or by transfecting cells with a vector encoding for a dominant-negative mutant of MSK1. These results highlight the relevance of MSK1 in the up-regulation of RARβ by PGE2. They also may contribute to new therapeutic approaches based upon the pharmacological control of HIF-1α/VEGF-A in the proximal tubule through the modulation of the PGE2/EGFR/MAPK/MSK1/RARβ pathway.
Keywords: Prostaglandin E2; MSK1; EGFR; Retinoic acid receptor; HIF-1α;

Smurf2 regulates the degradation of YY1 by Hyung Min Jeong; Sung Ho Lee; Jinah Yum; Chang-Yeol Yeo; Kwang Youl Lee (2005-2011).
Transcription factor YY1 plays important roles in cell proliferation and differentiation. For example, YY1 represses the expression of muscle-specific genes and the degradation of YY1 is required for myocyte differentiation. The activity of YY1 can be regulated by various post-translational modifications; however, little is known about the regulatory mechanisms for YY1 degradation. In this report, we attempted to identify potential E3 ubiquitin ligases for YY1, and found that Smurf2 E3 ubiquitin ligase can negatively regulate YY1 protein level, but not mRNA level. Smurf2 interacted with YY1, induced the poly-ubiquitination of YY1 and shortened the half-life of YY1 protein. Conversely, an E3 ubiquitin ligase-defective mutant form of Smurf2 or knockdown of Smurf2 increased YY1 protein level. PPxY motif is a typical target recognition site for Smurf2, and the PPxY motif in YY1 was important for Smurf2 interaction and Smurf2-induced degradation of YY1 protein. In addition, Smurf2 reduced the YY1-mediated activation of a YY1-responsive reporter whereas Smurf2 knockdown increased it. Finally, Smurf2 relieved the suppression of p53 activity by YY1. Taken together, our results suggest a novel regulatory mechanism for YY1 function by Smurf2 in which the protein stability and transcriptional activity of YY1 are regulated by Smurf2 through the ubiquitin-proteasome-mediated degradation of YY1.
Keywords: YY1; Smurf2; Ubiquitination; Protein stability;

Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance by Lori Buhlman; Maria Damiano; Giulia Bertolin; Rosa Ferrando-Miguel; Anne Lombès; Alexis Brice; Olga Corti (2012-2026).
Autosomal recessive early-onset Parkinson's disease is most often caused by mutations in the genes encoding the cytosolic E3 ubiquitin ligase Parkin and the mitochondrial serine/threonine kinase PINK1. Studies in Drosophila models and mammalian cells have demonstrated that these proteins regulate various aspects of mitochondrial physiology, including organelle transport, dynamics and turnover. How PINK1 and Parkin orchestrate these processes, and whether they always do so within a common pathway remain to be clarified.We have revisited the role of PINK1 and Parkin in mitochondrial dynamics, and explored its relation to the mitochondrial clearance program controlled by these proteins. We show that PINK1 and Parkin promote Drp1-dependent mitochondrial fission by mechanisms that are at least in part independent. Parkin-mediated mitochondrial fragmentation was abolished by treatments interfering with the calcium/calmodulin/calcineurin signaling pathway, suggesting that it requires dephosphorylation of serine 637 of Drp1. Parkinson's disease-causing mutations with differential impact on mitochondrial morphology and organelle degradation demonstrated that the pro-fission effect of Parkin is not required for efficient mitochondrial clearance. In contrast, the use of Förster energy transfer imaging microscopy revealed that Drp1 and Parkin are co-recruited to mitochondria in proximity of PINK1 following mitochondrial depolarization, indicating spatial coordination between these events in mitochondrial degradation. Our results also hint at a major role of the outer mitochondrial adaptor MiD51 in Drp1 recruitment and Parkin-dependent mitophagy. Altogether, our observations provide new insight into the mechanisms underlying the regulation of mitochondrial dynamics by Parkin and its relation to the mitochondrial clearance program mediated by the PINK1/Parkin pathway.
Keywords: PINK1/Parkin pathway; Mitochondrial dynamics; Calcium/calmodulin/calcineurin signaling; Mitochondrial clearance; Drp1; MiD51;

c-MYC is an oncogenic transcription factor that is degraded by the proteasome pathway. However, the mechanism that regulates delivery of c-MYC to the proteasome for degradation is not well characterized. Here, the results show that the motor protein complex Kinesin-1 transports c-MYC to the cytoplasm for proteasomal degradation. Inhibition of Kinesin-1 function enhanced ubiquitination of c-MYC and induced aggregation of c-MYC in the cytoplasm. Transport studies showed that the c-MYC aggregates moved from the nucleus to the cytoplasm and KIF5B is responsible for the transport in the cytoplasm. Furthermore, inhibition of the proteasomal degradation process also resulted in an accumulation of c-MYC aggregates in the cytoplasm. Moreover, Kinesin-1 was shown to interact with c-MYC and the proteasome subunit S6a. Inhibition of Kinesin-1 function also reduced c-MYC-dependent transformation activities. Taken together, the results strongly suggest that Kinesin-1 transports c-MYC for proteasomal degradation in the cytoplasm and the proper degradation of c-MYC mediated by Kinesin-1 transport is important for transformation activities of c-MYC. In addition, the results indicate that Kinesin-1 transport mechanism is important for degradation of a number of other proteins as well.
Keywords: c-MYC; Kinesin-1; Trafficking; Proteasomal degradation;

Snail1 induced in breast cancer cells in 3D collagen I gel environment suppresses cortactin and impairs effective invadopodia formation by Mi-Sook Lee; Sudong Kim; Baek Gil Kim; Cheolhee Won; Seo Hee Nam; Suki Kang; Hye-Jin Kim; Minkyung Kang; Jihye Ryu; Haeng Eun Song; Doohyung Lee; Sang-Kyu Ye; Noo Li Jeon; Tai Young Kim; Nam Hoon Cho; Jung Weon Lee (2037-2054).
Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFβ1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents.
Keywords: 3D collagen; JNK; Cortactin; Invasion; Snail1;

MicroRNA-296-5p (miR-296-5p) functions as a tumor suppressor in prostate cancer by directly targeting Pin1 by Kuen-Haur Lee; Forn-Chia Lin; Tai-I Hsu; Jen-Tai Lin; Jing-Hong Guo; Chen-Hsun Tsai; Yu-Cheng Lee; Yu-Chieh Lee; Chi-Long Chen; Michael Hsiao; Pei-Jung Lu (2055-2066).
Upregulation of Pin1 was shown to advance the functioning of several oncogenic pathways. It was recently shown that Pin1 is potentially an excellent prognostic marker and can also serve as a novel therapeutic target for prostate cancer. However, the molecular mechanism of Pin1 overexpression in prostate cancer is still unclear. In the present study, we showed that the mRNA expression levels of Pin1 were not correlated with Pin1 protein levels in prostate cell lines which indicated that Pin1 may be regulated at the post-transcriptional level. A key player in post-transcriptional regulation is represented by microRNAs (miRNAs) that negatively regulate expressions of protein-coding genes at the post-transcriptional level. A bioinformatics analysis revealed that miR-296-5p has a conserved binding site in the Pin1 3′-untranslated region (UTR). A luciferase reporter assay demonstrated that the seed region of miR-296-5p directly interacts with the 3′-UTR of Pin1 mRNA. Moreover, miR-296-5p expression was found to be inversely correlated with Pin1 expression in prostate cancer cell lines and prostate cancer tissues. Furthermore, restoration of miR-296-5p or the knockdown of Pin1 had the same effect on the inhibition of the ability of cell proliferation and anchorage-independent growth of prostate cancer cell lines. Our results support miR-296-5p playing a tumor-suppressive role by targeting Pin1 and implicate potential effects of miR-296-5p on the prognosis and clinical application to prostate cancer therapy.
Keywords: miR-296-5p; Pin1; Prostate cancer;

Recent studies reported that protein arginine methyltransferase 6 (PRMT6) enhances estrogen-induced activity of estrogen receptor α (ERα) and dysfunction of PRMT6 is associated with overall better survival for ERα-positive breast cancer patients. However, it is unclear how PRMT6 promotes ERα activity. Here we report that PRMT6 specifically interacts with ERα at its ligand-binding domain. PRMT6 also methylates ERα both in vitro and in vivo. In addition to enhancing estrogen-induced ERα activity, PRMT6 over-expression up-regulates estrogen-independent activity of ERα and PRMT6 gene silencing in MCF7 cells inhibits ligand-independent ERα activation. More interestingly, the effect of PRMT6 on the ligand-independent ERα activity does not require its methyltransferase activity. Instead, PRMT6 competes with Hsp90 for ERα binding: PRMT6 and Hsp90 bindings to ERα are mutually exclusive and PRMT6 over-expression reduces ERα interaction with Hsp90. In conclusion, PRMT6 requires its methyltransferase activity to enhance ERα's ligand-induced activity, but its effect on ligand-independent activity is likely mediated through competing with Hsp90 for binding to the C-terminal domain of ERα. PRMT6-ERα interaction would prevent ERα-Hsp90 association. Since Hsp90 and associated chaperones serve to maintain ERα conformation for ligand-binding yet functionally inactive, inhibition of ERα-Hsp90 interaction would relieve ERα from the constraint of chaperone complex.
Keywords: Steroid hormone receptor; Hsp90; Chaperone binding; Breast cancer;

Nuclear distribution of claudin-2 increases cell proliferation in human lung adenocarcinoma cells by Akira Ikari; Ryo Watanabe; Tomonari Sato; Saeko Taga; Shun Shimobaba; Masahiko Yamaguchi; Yasuhiro Yamazaki; Satoshi Endo; Toshiyuki Matsunaga; Junko Sugatani (2079-2088).
Claudin-2 is expressed in human lung adenocarcinoma tissue and cell lines, although it is absent in normal lung tissue. However, the role of claudin-2 in cell proliferation and the regulatory mechanism of intracellular distribution remain undefined. Proliferation of human adenocarcinoma A549 cells was decreased by claudin-2 knockdown together with a decrease in the percentage of S phase cells. This knockdown decreased the expression levels of ZONAB and cell cycle regulators. Claudin-2 was distributed in the nucleus in human adenocarcinoma tissues and proliferating A549 cells. The nuclear distribution of ZONAB and percentage of S phase cells were higher in cells exogenously expressing claudin-2 with a nuclear localization signal than in cells expressing claudin-2 with a nuclear export signal. Nuclear claudin-2 formed a complex with ZO-1, ZONAB, and cyclin D1. Nuclear distribution of S208A mutant, a dephosphorylated form of claudin-2, was higher than that of wild type. We suggest that nuclear distribution of claudin-2 is up-regulated by dephosphorylation and claudin-2 serves to retain ZONAB and cyclin D1 in the nucleus, resulting in the enhancement of cell proliferation in lung adenocarcinoma cells.
Keywords: Adenocarcinoma; Claudin-2; Cell proliferation; Nuclear distribution;

Gain in toxic function of stefin B EPM1 mutants aggregates: Correlation between cell death, aggregate number/size and oxidative stress by Mira Polajnar; Tina Zavašnik-Bergant; Nataša Kopitar-Jerala; Magda Tušek-Žnidarič; Eva Žerovnik (2089-2099).
EPM1 is a rare progressive myoclonus epilepsy accompanied by apoptosis in the cerebellum of patients. Mutations in the gene of stefin B (cystatin B) are responsible for the primary defect underlying EPM1. Taking stefin B aggregates as a model we asked what comes first, protein aggregation or oxidative stress, and how these two processes correlate with cell death.We studied the aggregation in cells of the stefin B wild type, G4R mutant, and R68X fragment before (Ceru et al., 2010, Biol. Cell). The present study was performed on two more missense mutants of human stefin B, G50E and Q71P, and they similarly showed numerous aggregates upon overexpression. Mutant- and oligomer-dependent increase in oxidative stress and cell death in cells bearing aggregates was shown. On the other hand, there was no correlation between the size and number of the aggregates and cell death. We suggest that differences in toxicity of the aggregates depend on whether they are in oligomeric/protofibrillar or fibrillar form. This in turn likely depends on the mutant's 3D structure where unfolded proteins show lower toxicity. Imaging by transmission electron microscopy showed that the aggregates in cells are of different types: bigger perinuclear, surrounded by membranes and sometimes showing vesicle-like invaginations, or smaller, punctual and dispersed throughout the cytoplasm. All EPM1 mutants studied were inactive as cysteine proteases inhibitors and in this way contribute to loss of stefin B functions. Relevance to EPM1 disease by gain in toxic function is discussed.Display Omitted
Keywords: Protein aggregate; Amyloid; Toxic oligomer; Oxidative stress; Cell death; Cystatin B;

Regulating cell death at, on, and in membranes by Xiaoke Chi; Justin Kale; Brian Leber; David W. Andrews (2100-2113).
Bcl-2 family proteins are central regulators of apoptosis. Various family members are located in the cytoplasm, endoplasmic reticulum, and mitochondrial outer membrane in healthy cells. However during apoptosis most of the interactions between family members that determine the fate of the cell occur at the membranes of intracellular organelles. It has become evident that interactions with membranes play an active role in the regulation of Bcl-2 family protein interactions. Here we provide an overview of various models proposed to explain how the Bcl-2 family regulates apoptosis and discuss how membrane binding affects the structure and function of each of the three categories of Bcl-2 proteins (pro-apoptotic, pore-forming, and anti-apoptotic). We also examine how the Bcl-2 family regulates other aspects of mitochondrial and ER physiology relevant to cell death.
Keywords: Apoptosis; Bcl-2 family; Endoplasmic reticulum; Mitochondrion; Membrane; Embedded Together;

Wnt signaling determines human stromal (mesenchymal) stem cell (hMSC) differentiation fate into the osteoblast or adipocyte lineage. microRNAs (miRNAs) are small RNA molecules of 21–25 nucleotides that regulate many aspects of osteoblast biology. Thus, we examined miRNAs regulated by Wnt signaling in hMSC. We identified miRNA (miR)-141-3p as a Wnt target which in turn inhibited Wnt signaling. Moreover, miR-141-3p inhibited hMSC proliferation by arresting cells at the G1 phase of the cell cycle. miR-141-3p inhibited osteoblast differentiation of hMSC as evidenced by reduced alkaline phosphatase activity, gene expression and in vitro mineralized matrix formation. Bioinformatic studies, Western blot analysis and 3’UTR reporter assay demonstrated that cell division cycle 25A (CDC25A) is a direct target of miR-141-3p. siRNA-mediated knock-down of CDC25A inhibited hMSC proliferation and osteoblast differentiation. In summary, miR-141-3p acts as a negative regulator of hMSC proliferation and osteoblast differentiation. Targeting miR-141-3p could be used as an anabolic therapy of low bone mass diseases, e.g. osteoporosis.
Keywords: Human stromal (mesenchymal) stem cell; miRNA; Cell proliferation and differentiation; Wnt signaling; CDC25A;

Heparanase is a key player in renal fibrosis by regulating TGF-β expression and activity by Valentina Masola; Gianluigi Zaza; Maria Francesca Secchi; Giovanni Gambaro; Antonio Lupo; Maurizio Onisto (2122-2128).
Epithelial–mesenchymal transition (EMT) of tubular cells is one of the mechanisms which contribute to renal fibrosis and transforming growth factor-β (TGF-β) is one of the main triggers. Heparanase (HPSE) is an endo-β-D-glucuronidase that cleaves heparan-sulfate thus regulating the bioavailability of growth factors (FGF-2, TGF-β). HPSE controls FGF-2-induced EMT in tubular cells and is necessary for the development of diabetic nephropathy in mice.The aim of this study was to investigate whether HPSE can modulate the expression and the effects of TGF-β in tubular cells.First we proved that the lack of HPSE or its inhibition prevents the increased synthesis of TGF-β by tubular cells in response to pro-fibrotic stimuli such as FGF-2, advanced glycosylation end products (AGE) and albumin overload.Second, since TGF-β may derive from sources different from tubular cells, we investigated whether HPSE modulates tubular cell response to exogenous TGF-β. HPSE does not prevent EMT induced by TGF-β although it slows its onset; indeed in HPSE-silenced cells the acquisition of a mesenchymal phenotype does not develop as quickly as in wt cells. Additionally, TGF-β induces an autocrine loop to sustain its signal, whereas the lack of HPSE partially interferes with this autocrine loop.Overall these data confirm that HPSE is a key player in renal fibrosis since it interacts with the regulation and the effects of TGF-β. HPSE is needed for pathological TGF-β overexpression in response to pro-fibrotic factors. Furthermore, HPSE modulates TGF-β-induced EMT: the lack of HPSE delays tubular cell transdifferentiation, and impairs the TGF-β autocrine loop.
Keywords: Epithelial–mesenchymal transition; Heparanase; Renal fibrosis; TGF-β;

Recent studies revealed that the interstitial fluid flow in and around tumor tissue not only played an important role in delivering anticancer agents, but also affected the microenvironment, mostly hypoxia, in modulating tumor radio-sensitivity. The current study investigated the hypoxia-independent mechanisms of flow-induced shear stress in sensitizing tumors to radiation.Colon cancer cells were seeded onto glass slides pre-coated with fibronectin. A parallel-plate flow chamber system was used to impose fluid shear stress. Cell proliferation, apoptosis and colony assays were measured after shear stress and/or radiation. Cell cycle analysis and immunoblots of cell adhesion signal molecules were evaluated. The effect of shear stress was reversed by modulating integrin β1 or FAK.Shear stress of 12 dyne/cm2 for 24 h, but not 3 h, enhanced the radiation induced cytotoxicity to colon cancer cells. Protein expression of FAK was significantly down-regulated but not transcriptionally suppressed. By modulating integrin β1 and FAK expression, we demonstrated that shear stress enhanced tumor radiosensitivity by regulating integrin β1/FAK/Akt as well as integrin β1/FAK/cortactin pathways. Shear stress in combination with radiation might regulate integrins signaling by recruiting and activating caspases 3/8 for FAK cleavage followed by ubiquitin-mediated proteasomal degradation.Shear stress enhanced the radiation toxicity to colon cancer cells through suppression of integrin signaling and protein degradation of FAK. The results of our study provide a strong rationale for cancer treatment that combines between radiation and strategy in modulating tumor interstitial fluid flow.
Keywords: Radiation; Shear stress; Integrin; FAK;