Fibroblast growth factor signalling influences homologous recombination-mediated DNA damage repair to promote drug resistance in ovarian cancer.

BACKGROUND: Ovarian cancer patients frequently develop chemotherapy resistance, limiting treatment options. We have previously shown that individuality in fibroblast growth factor 1 (FGF1) expression influences survival and chemotherapy response. METHODS: We used MTT assays to assess chemosensitivity to cisplatin and carboplatin following shRNA-mediated knockdown or heterologous over-expression of FGF1 (quantified by qRT-PCR and immunoblot analysis), and in combination with the FGFR inhibitors AZD4547 and SU5402, the ATM inhibitor KU55933 and DNA-PK inhibitor NU7026. Immunofluorescence microscopy was used to quantify the FGF1-dependent timecourse of replication protein A (RPA) and γH2AX foci formation. RESULTS: Pharmacological inhibition of FGF signalling reversed drug resistance in immortalised cell lines and in primary cell lines from drug-resistant ovarian cancer patients, while FGF1 over-expression induced resistance. Ataxia telangiectasia mutated (ATM) phosphorylation, but not DNA adduct formation was FGF1 dependent, following cisplatin or carboplatin challenge. Combining platinum drugs with the ATM inhibitor KU55933, but not with the DNA-PK inhibitor NU7026 re-sensitised resistant cells. FGF1 expression influenced the timecourse of damage-induced RPA and γH2AX nuclear foci formation. CONCLUSION: Drug resistance arises from FGF1-mediated differential activation of high-fidelity homologous recombination DNA damage repair. FGFR and ATM inhibitors reverse platinum drug resistance, highlighting novel combination chemotherapy approaches for future clinical trial evaluation.

Quantitative proteome analysis of temporally resolved phagosomes following uptake via key phagocytic receptors.

Macrophages operate at the forefront of innate immunity and their discrimination of foreign versus "self" particles is critical for a number of responses including efficient pathogen killing, antigen presentation, and cytokine induction. In order to efficiently destroy the particles and detect potential threats, macrophages express an array of receptors to sense and phagocytose prey particles. In this study, we accurately quantified a proteomic time-course of isolated phagosomes from murine bone marrow-derived macrophages induced by particles conjugated to seven different ligands representing pathogen-associated molecular patterns, immune opsonins or apoptotic cell markers. We identified a clear functional differentiation over the three timepoints and detected subtle differences between certain ligand-phagosomes, indicating that triggering of receptors through a single ligand type has mild, but distinct, effects on phagosome proteome and function. Moreover, our data shows that uptake of phosphatidylserine-coated beads induces an active repression of NF-κB immune responses upon Toll-like receptor (TLR)-activation by recruitment of anti-inflammatory regulators to the phagosome. This data shows for the first time a systematic time-course analysis of bone marrow-derived macrophages phagosomes and how phagosome fate is regulated by the receptors triggered for phagocytosis.

Mapping the substrate landscape of protein phosphatase 2A catalytic subunit PPP2CA.

Protein phosphatase 2A (PP2A) is an essential Ser/Thr phosphatase. The PP2A holoenzyme complex comprises a scaffolding (A), regulatory (B), and catalytic (C) subunit, with PPP2CA being the principal catalytic subunit. The full scope of PP2A substrates in cells remains to be defined. To address this, we employed dTAG proteolysis-targeting chimeras to efficiently and selectively degrade dTAG-PPP2CA in homozygous knock-in HEK293 cells. Unbiased global phospho-proteomics identified 2,204 proteins with significantly increased phosphorylation upon dTAG-PPP2CA degradation, implicating them as potential PPP2CA substrates. A vast majority of these are novel. Bioinformatic analyses revealed involvement of the potential PPP2CA substrates in spliceosome function, cell cycle, RNA transport, and ubiquitin-mediated proteolysis. We identify a pSP/pTP motif as a predominant target for PPP2CA and confirm some of our phospho-proteomic data with immunoblotting. We provide an in-depth atlas of potential PPP2CA substrates and establish targeted degradation as a robust tool to unveil phosphatase substrates in cells.

Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes.

The adenosine monophosphate (AMP)-activated protein kinase (AMPK) has a crucial role in maintaining cellular energy homeostasis. This study shows that human and mouse T lymphocytes express AMPKalpha1 and that this is rapidly activated in response to triggering of the T cell antigen receptor (TCR). TCR stimulation of AMPK was dependent on the adaptors LAT and SLP76 and could be mimicked by the elevation of intracellular Ca(2+) with Ca(2+) ionophores or thapsigargin. AMPK activation was also induced by energy stress and depletion of cellular adenosine triphosphate (ATP). However, TCR and Ca(2+) stimulation of AMPK required the activity of Ca(2+)-calmodulin-dependent protein kinase kinases (CaMKKs), whereas AMPK activation induced by increased AMP/ATP ratios did not. These experiments reveal two distinct pathways for the regulation of AMPK in T lymphocytes. The role of AMPK is to promote ATP conservation and production. The rapid activation of AMPK in response to Ca(2+) signaling in T lymphocytes thus reveals that TCR triggering is linked to an evolutionally conserved serine kinase that regulates energy metabolism. Moreover, AMPK does not just react to cellular energy depletion but also anticipates it.

Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR).

mTOR (mammalian target of rapamycin) stimulates cell growth by phosphorylating and promoting activation of AGC (protein kinase A/protein kinase G/protein kinase C) family kinases such as Akt (protein kinase B), S6K (p70 ribosomal S6 kinase) and SGK (serum and glucocorticoid protein kinase). mTORC1 (mTOR complex-1) phosphorylates the hydrophobic motif of S6K, whereas mTORC2 phosphorylates the hydrophobic motif of Akt and SGK. In the present paper we describe the small molecule Ku-0063794, which inhibits both mTORC1 and mTORC2 with an IC50 of approximately 10 nM, but does not suppress the activity of 76 other protein kinases or seven lipid kinases, including Class 1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher concentrations. Ku-0063794 is cell permeant, suppresses activation and hydrophobic motif phosphorylation of Akt, S6K and SGK, but not RSK (ribosomal S6 kinase), an AGC kinase not regulated by mTOR. Ku-0063794 also inhibited phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3-phosphoinositide-dependent protein kinase-1). We interpret this as implying phosphorylation of Ser473 promotes phosphorylation of Thr308 and/or induces a conformational change that protects Thr308 from dephosphorylation. In contrast, Ku-0063794 does not affect Thr308 phosphorylation in fibroblasts lacking essential mTORC2 subunits, suggesting that signalling processes have adapted to enable Thr308 phosphorylation to occur in the absence of Ser473 phosphorylation. We found that Ku-0063794 induced a much greater dephosphorylation of the mTORC1 substrate 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) than rapamycin, even in mTORC2-deficient cells, suggesting a form of mTOR distinct from mTORC1, or mTORC2 phosphorylates 4E-BP1. Ku-0063794 also suppressed cell growth and induced a G1-cell-cycle arrest. Our results indicate that Ku-0063794 will be useful in delineating the physiological roles of mTOR and may have utility in treatment of cancers in which this pathway is inappropriately activated.

Antigen receptor regulation of phosphoinositide-dependent kinase 1 pathways during thymocyte development.

Phosphoinositide-dependent kinase 1 (PDK1) is essential for T cell development but little is know about the stimuli that regulate PDK1 signaling in vivo. The thymus contains a heterogeneous mixture of cells at different stages of development making it difficult to use biochemical techniques to examine the activity of PDK1 pathways as thymocytes develop in situ. Herein, we use a single cell assay to quantify activation of the PDK1 target kinase ribosomal S6 kinase 1 (S6K1) in different murine thymocyte subsets immediately ex vivo. This technique allows an assessment of S6K1 activation as thymocytes respond to developmental stimuli in vivo. These studies reveal that only a small percentage of thymocytes show evidence for activation of PDK1 mediated signaling in situ. The thymic subpopulations that contain active PDK1/S6K1 are those known to be responding to signaling by the pre T cell receptor and the mature alpha/beta T cell antigen receptor (TCR). Moreover, loss of antigen receptor signaling in T cell progenitors that cannot rearrange their TCR beta locus prevents in vivo activation of S6K1. The present data identifying antigen receptor signaling as a key activator of PDK1 mediated signaling afford a molecular explanation for the important role of this molecule in T cells.

Phosphoinositide-dependent kinase l (PDK1) haplo-insufficiency inhibits production of alpha/beta (alpha/beta) but not gamma delta (gamma/delta) T lymphocytes.

In the present study, we have explored the impact of deleting a single allele of PDK1 in T cell progenitors on alpha/beta and gamma/delta T cell development. The data show that deleting a single allele of PDK1 allows differentiation of alpha/beta T cells but prevents their proliferative expansion in the thymus. Accordingly, mice with T cells that are haplo-insufficient for PDK1 have reduced numbers of thymocytes and alpha/beta peripheral T cells. T cell progenitors also give rise to gamma/delta T cells but in contrast to the loss of alpha/beta T cells in T-PDK1 null and haplo-insufficient mice, there were increased numbers of gamma/delta T cells. The production of alpha/beta T cells is dependent on the proliferative expansion of thymocytes and is determined by a balance between the frequency with which cells enter the proliferative phase of the cell cycle and rates of cell death. Herein, we show that PDK1 haplo-insufficient thymocytes have no defects in their ability to enter the cell cycle but show increased apoptosis. PDK1 thus plays a determining role in the development of alpha/beta T lymphocytes but does not limit gamma/delta T cell development.

Effects of Brussels sprout juice on the cell cycle and adhesion of human colorectal carcinoma cells (HT29) in vitro.

Consumption of Brassica vegetables is associated with a reduced risk of cancer of the alimentary tract in animal models and human populations. We used raw juice extracted from Brussels sprouts rich in the glucosinolate sinigrin to explore the effect of naturally occurring glucosinolate breakdown products on cell cycle progression and apoptosis in human colorectal carcinoma cells (HT29). Juice was prepared from sprout tissue immediately before use, and the glucosinolate breakdown products were determined by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. The cell cycle was analyzed by flow cytometry on detached and adherent cells, and apoptosis was measured in the detached population by annexin V staining. Twenty-four hours after challenge with juice (10 microL/mL), 7-13% of adherent cells had detached from the substratum but the majority (82%) of these cells had not entered apoptosis, whereas only 33% of detached control cells were not apoptotic (p < 0.05). The main glucosinolate breakdown products were as follows: the sinigrin breakdown product, 1-cyano-2,3-epithiopropane (ca. 38 mM); the gluconapin hydrolysis product, 3-butenyl isothiocyanate (ca. 2.2.mM); the glucobrassicin metabolite, ascorbigen (ca. 8 mM); and low concentrations of other indole glucosinolate-derived hydrolysis products such as neoascorbigen and 3,3'-diindolylmethane. A variety of biologically active glucosinolate breakdown products are released by mechanical disruption of raw Brussels sprout tissue, but contrary to previous assumptions, allyl isothiocyanate is not the main compound responsible for the inhibition of cell proliferation.

Discovery of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amine aurora kinase inhibitors.

Through cell-based screening of our kinase-directed compound collection, we discovered that a subset of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amines were potent cytotoxic agents against cancer cell lines, suppressed mitotic histone H3 phosphorylation, and caused aberrant mitotic phenotypes. It was subsequently established that these compounds were in fact potent inhibitors of aurora A and B kinases. It was shown that potency and selectivity of aurora kinase inhibition correlated with the presence of a substituent at the aniline para-position in these compounds. The anticancer effects of lead compound 4-methyl-5-(2-(4-morpholinophenylamino)pyrimidin-4-yl)thiazol-2-amine (18; K(i) values of 8.0 and 9.2 nM for aurora A and B, respectively) were shown to emanate from cell death following mitotic failure and increased polyploidy as a consequence of cellular inhibition of aurora A and B kinases. Preliminary in vivo assessment showed that compound 18 was orally bioavailable and possessed anticancer activity. Compound 18 (CYC116) is currently undergoing phase I clinical evaluation in cancer patients.

Siglec-5 (CD170) can mediate inhibitory signaling in the absence of immunoreceptor tyrosine-based inhibitory motif phosphorylation.

Siglec-5 (CD170) is a member of the recently described human CD33-related siglec subgroup of sialic acid binding Ig-like lectins and is expressed on myeloid cells of the hemopoietic system. Similar to other CD33-related siglecs, Siglec-5 contains two tyrosine-based motifs in its cytoplasmic tail implicated in signaling functions. To investigate the role of these motifs in Siglec-5-dependent signaling, we used transfected rat basophil leukemia cells as a model system. Tyrosine phosphorylation of Siglec-5 led to recruitment of the tyrosine phosphatases SHP-1 and SHP-2, as seen in both pull-down assays and microscopy. Siglec-5 could efficiently inhibit FcepsilonRI-mediated calcium fluxing and serotonin release after co-cross-linking. Surprisingly, a double tyrosine to alanine mutant of Siglec-5 could still mediate strong inhibition of serotonin release in the absence of detectable tyrosine phosphorylation, whereas a double tyrosine to phenylalanine mutant lost all inhibitory activity. In comparison, suppression of Siglec-5-dependent adhesion to red blood cells was reversed by either tyrosine to alanine or tyrosine to phenylalanine mutations of the membrane proximal tyrosine-based motif. Using an in vitro phosphatase assay with synthetic and recombinant forms of the cytoplasmic tail, it was shown that a double alanine mutant of Siglec-5 had weak, but significant SHP-1 activating properties similar to those of wild type, non-phosphorylated cytoplasmic tail, whereas a double phenylalanine mutant was inactive. These findings establish that Siglec-5 can be classified as an inhibitory receptor with the potential to mediate SHP-1 and/or SHP-2-dependent signaling in the absence of tyrosine phosphorylation.

Allyl-isothiocyanate causes mitotic block, loss of cell adhesion and disrupted cytoskeletal structure in HT29 cells.

Epidemiological evidence indicates that Brassica vegetables protect against colorectal cancer. Brassicas contain glucosinolates, the breakdown products of which exert antiproliferative effects against cancer cells. We have examined the effects of allyl-isothiocyanate (AITC), a major breakdown product of the glucosinolate sinigrin, on proliferation and death of colorectal cancer cells. HT-29 colorectal cells were exposed to AITC for 24 h and the number of adherent and detached cells determined. Both populations were analysed for cell-cycle characteristics and examined by light and electron microscopy for features of apoptosis and mitosis. Evidence of apoptosis was also determined by flow cytometric analysis of Annexin V staining in the detached population of cells. AITC-treated cells were also stained for alpha-tubulin. Treatment caused cells to round up after 7 h of exposure and subsequently detach. At 24 h these cells were blocked in mitosis. Detached AITC-treated cells showed no signs of apoptosis as assessed by morphological features or by Annexin V staining but they did show evidence of disrupted tubulin. AITC inhibits proliferation of cancer cells by causing mitotic block associated with disruption of alpha-tubulin in a manner analogous to a number of chemotherapeutic agents.

PTEN M-CBR3, a versatile and selective regulator of inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5). Evidence for Ins(1,3,4,5,6)P5 as a proliferative signal.

The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumor suppressor is a phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) 3-phosphatase that plays a crucial role in regulating many cellular processes by antagonizing the phosphoinositide 3-kinase signaling pathway. Although able to metabolize soluble inositol phosphates in vitro, the question of their significance as physiological substrates is unresolved. We show that inositol phosphates are not regulated by wild type PTEN, but that a synthetic mutant, PTEN M-CBR3, previously thought to be inactive toward inositides, can selectively regulate inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5). Transfection of U87-MG cells with PTEN M-CBR3 lowered Ins(1,3,4,5,6)P5 levels by 60% without detectable effect on PtdInsP3. Although PTEN M-CBR3 is a 3-phosphatase, levels of myo-inositol 1,4,5,6-tetrakisphosphate were not increased, whereas myo-inositol 1,3,4,6-tetrakisphospate levels increased by 80%. We have used PTEN M-CBR3 to study the physiological function of Ins(1,3,4,5,6)P5 and have found that Ins(1,3,4,5,6)P5 does not modulate PKB phosphorylation, nor does it regulate clathrin-mediated epidermal growth factor receptor internalization. By contrast, PTEN M-CBR3 expression, and the subsequent lowering of Ins(1,3,4,5,6)P5, are associated with reduced anchorage-independent colony formation and anchorage-dependent proliferation in U87-MG cells. Our results, together with previously published data, suggest that Ins(1,3,4,5,6)P5 has a role in proliferation.