Rnd3 interacts with TAO kinases and contributes to mitotic cell rounding and spindle positioning.

Rnd3 is an atypical Rho family protein that is constitutively GTP bound, and acts on membranes to induce loss of actin stress fibers and cell rounding. Phosphorylation of Rnd3 promotes 14-3-3 binding and its relocation to the cytosol. Here, we show that Rnd3 binds to the thousand-and-one amino acid kinases TAOK1 and TAOK2 in vitro and in cells. TAOK1 and TAOK2 can phosphorylate serine residues 210, 218 and 240 near the C-terminus of Rnd3, and induce Rnd3 translocation from the plasma membrane to the cytosol. TAOKs are activated catalytically during mitosis and Rnd3 phosphorylation on serine 210 increases in dividing cells. Rnd3 depletion by RNAi inhibits mitotic cell rounding and spindle centralization, and delays breakdown of the intercellular bridge between two daughter cells. Our results show that TAOKs bind, phosphorylate and relocate Rnd3 to the cytosol and that Rnd3 contributes to mitotic cell rounding, spindle positioning and cytokinesis. Rnd3 can therefore participate in the regulation of early and late mitosis and may also act downstream of TAOKs to affect the cytoskeleton.

A new TAO kinase inhibitor reduces tau phosphorylation at sites associated with neurodegeneration in human tauopathies.

In Alzheimer's disease (AD) and related tauopathies, the microtubule-associated protein tau is highly phosphorylated and aggregates to form neurofibrillary tangles that are characteristic of these neurodegenerative diseases. Our previous work has demonstrated that the thousand-and-one amino acid kinases (TAOKs) 1 and 2 phosphorylate tau on more than 40 residues in vitro. Here we show that TAOKs are phosphorylated and active in AD brain sections displaying mild (Braak stage II), intermediate (Braak stage IV) and advanced (Braak stage VI) tau pathology and that active TAOKs co-localise with both pre-tangle and tangle structures. TAOK activity is also enriched in pathological tau containing sarkosyl-insoluble extracts prepared from AD brain. Two new phosphorylated tau residues (T123 and T427) were identified in AD brain, which appear to be targeted specifically by TAOKs. A new small molecule TAOK inhibitor (Compound 43) reduced tau phosphorylation on T123 and T427 and also on additional pathological sites (S262/S356 and S202/T205/S208) in vitro and in cell models. The TAOK inhibitor also decreased tau phosphorylation in differentiated primary cortical neurons without affecting markers of synapse and neuron health. Notably, TAOK activity also co-localised with tangles in post-mortem frontotemporal lobar degeneration (FTLD) brain tissue. Furthermore, the TAOK inhibitor decreased tau phosphorylation in induced pluripotent stem cell derived neurons from FTLD patients, as well as cortical neurons from a transgenic mouse model of tauopathy (Tau35 mice). Our results demonstrate that abnormal TAOK activity is present at pre-tangles and tangles in tauopathies and that TAOK inhibition effectively decreases tau phosphorylation on pathological sites. Thus, TAOKs may represent a novel target to reduce or prevent tau-associated neurodegeneration in tauopathies.

Targeting TAO Kinases Using a New Inhibitor Compound Delays Mitosis and Induces Mitotic Cell Death in Centrosome Amplified Breast Cancer Cells.

Thousand-and-one amino acid kinases (TAOK) 1 and 2 are activated catalytically during mitosis and can contribute to mitotic cell rounding and spindle positioning. Here, we characterize a compound that inhibits TAOK1 and TAOK2 activity with IC50 values of 11 to 15 nmol/L, is ATP-competitive, and targets these kinases selectively. TAOK inhibition or depletion in centrosome-amplified SKBR3 or BT549 breast cancer cell models increases the mitotic population, the percentages of mitotic cells displaying amplified centrosomes and multipolar spindles, induces cell death, and inhibits cell growth. In contrast, nontumorigenic and dividing bipolar MCF-10A breast cells appear less dependent on TAOK activity and can complete mitosis and proliferate in the presence of the TAOK inhibitor. We demonstrate that TAOK1 and TAOK2 localize to the cytoplasm and centrosomes respectively during mitosis. Live cell imaging shows that the TAOK inhibitor prolongs the duration of mitosis in SKBR3 cells, increases mitotic cell death, and reduces the percentages of cells exiting mitosis, whereas MCF-10A cells continue to divide and proliferate. Over 80% of breast cancer tissues display supernumerary centrosomes, and tumor cells frequently cluster extra centrosomes to avoid multipolar mitoses and associated cell death. Consequently, drugs that stimulate centrosome declustering and induce multipolarity are likely to target dividing centrosome-amplified cancer cells preferentially, while sparing normal bipolar cells. Our results demonstrate that TAOK inhibition can enhance centrosome declustering and mitotic catastrophe in cancer cells, and these proteins may therefore offer novel therapeutic targets suitable for drug inhibition and the potential treatment of breast cancers, where supernumerary centrosomes occur. Mol Cancer Ther; 16(11); 2410-21. ©2017 AACR.

Engineering a vascular endothelial growth factor 165-binding heparan sulfate for vascular therapy.

The therapeutic use of VEGF165 to stimulate blood vessel formation for the treatment of peripheral arterial disease or cardiovascular-related disease has met with limited success. Here we describe an affinity-isolated heparan sulfate glycotherapeutic (HS7(+ve)) that binds to, and enhances the bioactivity of, VEGF165. Application of HS7(+ve) complexed with VEGF165 results in enhanced VEGF165-VEGFR2 interaction, prolonged downstream pErk1/2 signalling, and increased cell proliferation and tube formation in HUVECs, compared with VEGF165 alone. The pro-angiogenic potential of HS7(+ve) was further assessed in vivo using the chick embryo chorioallantoic membrane (CAM) assay. Exogenous dosing with HS7(+ve) alone significantly enhanced the formation of new blood vessels with potencies comparable to VEGF165. These results demonstrate the potential for vascular therapy of glycotherapeutic agents targeted at augmenting the bioactivity of VEGF165.

The dual function of KSR1: a pseudokinase and beyond.

Protein kinases play a pivotal role in regulating many aspects of biological processes, including development, differentiation and cell death. Within the kinome, 48 kinases (~10%) are classified as pseudokinases owing to the fact that they lack at least one conserved catalytic residue in their kinase domain. However, emerging evidence suggest that some pseudokinases, even without the ability to phosphorylate substrates, are regulators of multiple cellular signalling pathways. Among these is KSR1 (kinase suppressor of Ras 1), which was initially identified as a novel kinase in the Ras/Raf pathway. Subsequent studies showed that KSR1 mainly functions as a platform to assemble different cellular components thereby facilitating signal transduction. In the present article, we discuss recent findings regarding KSR1, indicating that it has dual activity as an active kinase as well as a pseudokinase/scaffolding protein. Moreover, the biological functions of KSR1 in human disorders, notably in malignancies, are also reviewed.

Forkhead box proteins: tuning forks for transcriptional harmony.

Forkhead box (FOX) proteins are multifaceted transcription factors that are responsible for fine-tuning the spatial and temporal expression of a broad range of genes both during development and in adult tissues. This function is engrained in their ability to integrate a multitude of cellular and environmental signals and to act with remarkable fidelity. Several key members of the FOXA, FOXC, FOXM, FOXO and FOXP subfamilies are strongly implicated in cancer, driving initiation, maintenance, progression and drug resistance. The functional complexities of FOX proteins are coming to light and have established these transcription factors as possible therapeutic targets and putative biomarkers for specific cancers.

SIRT6 modulates paclitaxel and epirubicin resistance and survival in breast cancer.

In this study, we report the identification of a novel role of SIRT6 in both epirubicin and paclitaxel resistance in breast cancer. We found that SIRT6 protein levels are elevated in paclitaxel- and epirubicin-resistant MCF-7 cells compared with the parental sensitive cells. SIRT6 knockout and depletion sensitized cells to both paclitaxel and epirubicin treatment, whereas SIRT6 ectopic overexpression led to increased resistance to paclitaxel and epirubicin. Moreover, our data suggest that SIRT6 could be mediating epirubicin resistance through enhancing the DNA repair response to epirubicin-induced DNA damage. Clonogenic assays also revealed that mouse embryonic fibroblasts (MEFs) lacking SIRT6 have decreased long-term viability in response to epirubicin. The tumour suppressor FOXO3a increases its levels of acetylation in MEFs depleted of SIRT6, whereas its induction by epirubicin is attenuated in breast cancer cells overexpressing SIRT6. Further cell viability studies demonstrate that deletion of FOXO1/3/4 in MEFs can confer sensitivity to both paclitaxel and epirubicin, suggesting that SIRT6 reduces paclitaxel and epirubicin sensitivity, at least in part, through modulating FOXO acetylation and expression. Consistently, immunohistochemical analysis of 118 breast cancer patient samples revealed that high SIRT6 nuclear staining is significantly associated with poorer overall survival (P = 0.018; Kaplan-Meier analysis). Multivariate Cox analysis demonstrated that nuclear SIRT6 staining remained associated with death after correcting for tumour stage and lymph-node involvement (P = 0.033). Collectively, our data suggest that SIRT6 has a role in paclitaxel and epirubicin sensitivity via targeting FOXO proteins and that SIRT6 could be a useful biomarker and therapeutic target for paclitaxel- and epirubicin-resistant cancer.

The p38 MAPK-MK2 axis regulates E2F1 and FOXM1 expression after epirubicin treatment.

E2F1 is responsible for the regulation of FOXM1 expression, which plays a key role in epirubicin resistance. Here, we examined the role and regulation of E2F1 in response to epirubicin in cancer cells. We first showed that E2F1 plays a key role in promoting FOXM1 expression, cell survival, and epirubicin resistance as its depletion by siRNA attenuated FOXM1 induction and cell viability in response to epirubicin. We also found that the p38-MAPK activity mirrors the expression patterns of E2F1 and FOXM1 in both epirubicin-sensitive and -resistant MCF-7 breast cancer cells, suggesting that p38 has a role in regulating E2F1 expression and epirubicin resistance. Consistently, studies using pharmacologic inhibitors, siRNA knockdown, and knockout mouse embryonic fibroblasts (MEF) revealed that p38 mediates the E2F1 induction by epirubicin and that the induction of E2F1 by p38 is, in turn, mediated through its downstream kinase MK2 [mitogen-activated protein kinase (MAPK)-activated protein kinase 2; MAPKAPK2]. In agreement, in vitro phosphorylation assays showed that MK2 can directly phosphorylate E2F1 at Ser-364. Transfection assays also showed that E2F1 phosphorylation at Ser-364 participates in its induction by epirubicin but also suggests that other phosphorylation events are also involved. In addition, the p38-MK2 axis can also limit c-jun-NH(2)-kinase (JNK) induction by epirubicin and, notably, JNK represses FOXM1 expression. Collectively, these findings underscore the importance of p38-MK2 signaling in the control of E2F1 and FOXM1 expression as well as epirubicin sensitivity.

Targeting of syndecan-1 by microRNA miR-10b promotes breast cancer cell motility and invasiveness via a Rho-GTPase- and E-cadherin-dependent mechanism.

microRNAs are small endogenous noncoding RNAs, which post-transcriptionally regulate gene expression. In breast cancer, overexpression of the transmembrane heparan sulfate proteoglycan syndecan-1, a predicted target of the oncomiR miR-10b, correlates with poor clinical outcome. To investigate the potential functional relationship of miR-10b and syndecan-1, MDA-MB-231 and MCF-7 breast cancer cells were transiently transfected with pre-miR-10b, syndecan-1 siRNA or control reagents, respectively. Altered cell behavior was monitored by proliferation, migration and invasion chamber assays, and time-lapse video microscopy. miR-10b overexpression induced post-transcriptional downregulation of syndecan-1, as demonstrated by quantitative real-time PCR (qPCR), flow cytometry, and 3'UTR luciferase assays, resulting in increased cancer cell migration and matrigel invasiveness. Syndecan-1 silencing generated a copy of this phenotype. Adhesion to fibronectin and laminin and basal cell proliferation was increased. Syndecan-1 coimmunoprecipitated with focal adhesion kinase, which showed increased activation upon syndecan-1 depletion. Affymetrix screening and confirmatory qPCR and Western blotting analysis of syndecan-1-deficient cells revealed upregulation of ATF-2, COX-2, cadherin-11, vinculin, actin γ 2, MYL9, transgelin-1, RhoA/C, matrix metalloproteinase 2 (MMP2) and heparanase, and downregulation of AML1/RUNX1, E-cadherin, CLDN1, p21WAF/CIP, cyclin-dependent kinase 6, TLR-4, PAI1/2, Collagen1alpha1, JHDM1D, Mpp4, MMP9, matrilin-2 and ANXA3/A10. Video microscopy demonstrated massively increased Rho kinase-dependent motility of syndecan-1-depleted cells, which displayed increased filopodia formation. We conclude that syndecan-1 is a novel target of the oncomiR miR-10b. Rho-GTPase-dependent modulation of cytoskeletal function and downregulation of E-cadherin expression are identified as relevant effectors of the miR-10b-syndecan-1 axis, which emerges as a promising target for the development of new therapeutic approaches for breast cancer.

FOXM1: From cancer initiation to progression and treatment.

The Forkhead box protein M1 (FOXM1) transcription factor is a regulator of myriad biological processes, including cell proliferation, cell cycle progression, cell differentiation, DNA damage repair, tissue homeostasis, angiogenesis and apoptosis. Elevated FOXM1 expression is found in cancers of the liver, prostate, brain, breast, lung, colon, pancreas, skin, cervix, ovary, mouth, blood and nervous system, suggesting it has an integral role in tumorigenesis. Recent research findings also place FOXM1 at the centre of cancer progression and drug sensitivity. In this review the involvement of FOXM1 in various aspects of cancer, in particular its role and regulation within the context of cancer initiation, progression, and cancer drug response, will be summarised and discussed.

Phosphorylation of FOXO3a on Ser-7 by p38 promotes its nuclear localization in response to doxorubicin.

FOXO3a is a forkhead transcription factor that regulates a multitude of important cellular processes, including proliferation, apoptosis, differentiation, and metabolism. Doxorubicin treatment of MCF-7 breast carcinoma cells results in FOXO3a nuclear relocation and the induction of the stress-activated kinase p38 MAPK. Here, we studied the potential regulation of FOXO3a by p38 in response to doxorubicin. Co-immunoprecipitation studies in MCF-7 cells demonstrated a direct interaction between p38 and FOXO3a. We also showed that p38 can bind and phosphorylate a recombinant FOXO3a directly in vitro. HPLC-coupled phosphopeptide mapping and mass spectrometric analyses identified serine 7 as a major site for p38 phosphorylation. Using a phosphorylated Ser-7 FOXO3a antibody, we demonstrated that FOXO3a is phosphorylated on Ser-7 in response to doxorubicin. Immunofluorescence staining studies showed that upon doxorubicin treatment, the wild-type FOXO3a relocalized to the nucleus, whereas the phosphorylation-defective FOXO3a (Ala-7) mutant remained largely in the cytoplasm. Treatment with SB202190 also inhibits the doxorubicin-induced FOXO3a Ser-7 phosphorylation and nuclear accumulation in MCF-7 cells. In addition, doxorubicin caused the nuclear translocation of FOXO3a in wild-type but not p38-depleted mouse fibroblasts. Together, our results suggest that p38 phosphorylation of FOXO3a on Ser-7 is essential for its nuclear relocalization in response to doxorubicin.

Differential expression of metallothionein in gastrointestinal stromal tumors and gastric carcinomas.

Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors that account for about 2% of gastric tumors. Metallothioneins (MTs) are multifunctional proteins associated with carcinogenesis and known to be coded by 10 functional MT genes. This study evaluated MT mRNA and protein expression in GISTs and compared the expression levels with gastric carcinomas. An immunohistochemical study of MT protein expression was performed in 15 GISTs (specifically located in the stomach) and 38 early stage gastric carcinomas. The percentage of cells stained and intensity of staining were determined. MT-2A mRNA expression was investigated in 6 GISTs and 6 early stage gastric carcinoma patients. All GISTs displayed positive nuclear immunostaining, with most GISTs having predominantly mildly stained nuclei (93.3%). On the other hand, 37 out of 38 gastric carcinoma cases were positively stained for nuclear MT with 24 cases (63.2%) exhibiting predominantly mild nuclear staining, 7 cases (18.4%) moderate nuclear staining, and 6 cases (15.8%) strong nuclear staining. Nuclear MT expression was found to be significantly lower in GIST samples when compared with gastric carcinoma tissues based on the percentage stained and immunoreactive score. We then established that the MT-2A gene transcript was the most abundant MT isoform in MKN28 gastric cancer cells and analyzed its expression in GIST and gastric carcinoma tissues. We found that GISTs had significantly lower MT-2A mRNA levels than gastric carcinoma tissues. Lower MT-2A gene expression and nuclear MT protein expression in GISTs when compared with gastric carcinomas may reflect their different underlying biology and divergent histogenesis.

Differential roles for membrane-bound and soluble syndecan-1 (CD138) in breast cancer progression.

The heparan sulfate proteoglycan syndecan-1 (Sdc1) modulates cell proliferation, adhesion, migration and angiogenesis. Proteinase-mediated shedding converts Sdc1 from a membrane-bound coreceptor into a soluble effector capable of binding the same ligands. In breast carcinomas, Sdc1 overexpression correlates with poor prognosis and an aggressive phenotype. To distinguish between the roles of membrane-bound and shed forms of Sdc1 in breast cancer progression, human MCF-7 breast cancer cells were stably transfected with plasmids overexpressing wild-type (WT), constitutively shed and uncleavable forms of Sdc1. Overexpression of WT Sdc1 increased cell proliferation, whereas overexpression of constitutively shed Sdc1 decreased proliferation. Fibroblast growth factor-2-mediated mitogen-activated protein kinase signaling was reduced following small-interfering RNA (siRNA)-mediated knockdown of Sdc1 expression. Constitutively, membrane-bound Sdc1 inhibited invasiveness, whereas soluble Sdc1 promoted invasion of MCF-7 cells into matrigel matrices. The latter effect was reversed by the matrix metalloproteinase inhibitors N-isobutyl-N-(4-methoxyphenylsufonyl) glycyl hydroxamic acid and tissue inhibitor of metalloproteinase (TIMP)-1. Affymetrix microarray analysis identified TIMP-1, Furin and urokinase-type plasminogen activator receptor as genes differentially regulated in soluble Sdc1-overexpressing cells. Endogenous TIMP-1 expression was reduced in cells overexpressing soluble Sdc1 and increased in those overexpressing the constitutively membrane-bound Sdc1. Moreover, E-cadherin protein expression was downregulated in cells overexpressing soluble Sdc1. Our results suggest that the soluble and membrane-bound forms of Sdc1 play different roles at different stages of breast cancer progression. Proteolytic conversion of Sdc1 from a membrane-bound into a soluble molecule marks a switch from a proliferative to an invasive phenotype, with implications for breast cancer diagnostics and potential glycosaminoglycan-based therapies.

Targeting heparan sulfate proteoglycans in breast cancer treatment.

Breast carcinoma is one of the leading causes of mortality among female cancers globally. Heparan sulfate proteoglycans, found predominantly on cell surfaces and in the extracellular matrix, are known to regulate breast cancer cellular behavior. Many studies have shown that these molecules serve as potential biomarkers for breast cancer. In addition, they have aberrant expression patterns and participate in various molecular signaling pathways in tumor progression. There is substantial interest in targeting heparan sulfate proteoglycans for cancer treatment, which needs to be tailored according to the roles that each proteoglycan plays in cancer biology. Current clinical trials using phosphomannopentaose sulfate, a heparan sulfate mimic, and various forms of heparin have produced promising results in breast cancer patients. Besides heparan sulfate chains, novel therapeutic agents could potentially be developed to regulate the proteoglycan core proteins as well as enzymes that modify heparan sulfation patterns. This review discusses the current use and future prospective applications of heparan sulfate proteoglycans, which have been recently patented, as therapeutic targets in breast cancer treatment.

Comparison of the effects of differentially sulphated bovine kidney- and porcine intestine-derived heparan sulphate on breast carcinoma cellular behaviour.

Heparan sulphate is a sulphated glycosaminoglycan and is able to bind to and regulate the activity of many growth and signalling factors. We have previously shown that its expression is correlated with tumour grade and cell proliferation in breast phyllodes tumours. In this study, we examined the use of heparan sulphate as a biomarker of invasive ductal carcinoma and the effects of differentially sulphated heparan species on breast cancer cell behaviour. Immunohistochemistry using the 10E4 monoclonal antibody was carried out on 32 paraffin-embedded breast cancer specimens and paired non-cancerous breast tissues to compare the expression patterns of heparan sulphate. Upregulated expression of the sulphated 10E4 epitope in heparan sulphate was detected in both epithelial and stromal compartments of breast cancer compared with normal mammary tissues, with a 2.8X increase in immunoreactivity score. To determine the effects of differentially sulphated heparan sulphate molecules on breast cancer behaviour, cultured breast carcinoma cells were treated with chlorate, a competitive inhibitor of glycosaminoglycan sulphation, and two different heparan sulphate species. Inhibition of glycosaminoglycan sulphation resulted in a significant increase in cancer cell adhesion and a reduction in cell migration, together with upregulated expression of focal adhesion kinase and paxillin. Both porcine intestine- and bovine kidney-derived heparan sulphate species could block the change in cell adhesion. However, the former heparan sulphate species completely abolished, while the latter exacerbated, the chlorate-induced decrease in cell migration. The results show that heparan sulphate is a useful biomarker of breast invasive ductal carcinoma. Different sulphation patterns of heparan sulphate residues have differential effects in regulating breast cancer cellular behaviour, and this may be exploited to develop heparan sulphate into a useful target for treatment of breast carcinoma.

Immunohistochemical expression of heparan sulfate correlates with stromal cell proliferation in breast phyllodes tumors.

Phyllodes tumors are fibroepithelial neoplasms typified by stromal proliferation. We have previously shown the role of pathologic parameters and the prognostic significance of p53 and CD117 protein expression in these tumors. In this study, we evaluated the expression of heparan sulfate, which has been implicated in many biological processes such as cell adhesion, embryogenesis, and tumorigenesis (including malignant transformation of mammary cells) in 232 breast phyllodes tumors. We used a monoclonal antibody, 10E4, to examine the localization of heparan sulfate in phyllodes tumors by immunohistochemistry. The immunoreactivity of both epithelial and stromal components was examined and analyzed with pathological parameters and other immunohistochemical markers, including p53, MIB1, bcl2, and CD117. Stromal 10E4 expression was significantly associated with tumor grade, stromal p53, and MIB1 expression in proliferating cells, suggesting that heparan sulfate may participate in malignant tumor growth.

Systematic analysis of snake neurotoxins' functional classification using a data warehousing approach.

MOTIVATION: Sequence annotations, functional and structural data on snake venom neurotoxins (svNTXs) are scattered across multiple databases and literature sources. Sequence annotations and structural data are available in the public molecular databases, while functional data are almost exclusively available in the published articles. There is a need for a specialized svNTXs database that contains NTX entries, which are organized, well annotated and classified in a systematic manner. RESULTS: We have systematically analyzed svNTXs and classified them using structure-function groups based on their structural, functional and phylogenetic properties. Using conserved motifs in each phylogenetic group, we built an intelligent module for the prediction of structural and functional properties of unknown NTXs. We also developed an annotation tool to aid the functional prediction of newly identified NTXs as an additional resource for the venom research community. AVAILABILITY: We created a searchable online database of NTX proteins sequences (http://research.i2r.a-star.edu.sg/Templar/DB/snake_neurotoxin). This database can also be found under Swiss-Prot Toxin Annotation Project website (http://www.expasy.org/sprot/).