Better Understanding of Phosphoinositide 3-Kinase (PI3K) Pathways in Vasculature: Towards Precision Therapy Targeting Angiogenesis and Tumor Blood Supply.

The intracellular PI3K-AKT-mTOR pathway is involved in regulation of numerous important cell processes including cell growth, differentiation, and metabolism. The PI3Kα isoform has received particular attention as a novel molecular target in gene therapy, since this isoform plays critical roles in tumor progression and tumor blood flow and angiogenesis. However, the role of PI3Kα and other class I isoforms, i.e. PI3Kß, γ, δ, in the regulation of vascular tone and regional blood flow are largely unknown. We used novel isoform-specific PI3K inhibitors and mice deficient in both PI3Kγ and PI3Kδ (Pik3cg(-/-)/Pik3cd(-/-)) to define the putative contribution of PI3K isoform(s) to arterial vasoconstriction. Wire myography was used to measure isometric contractions of isolated murine mesenteric arterial rings. Phenylephrine-dependent contractions were inhibited by the pan PI3K inhibitors wortmannin (100 nM) and LY294002 (10 µM). These vasoconstrictions were also inhibited by the PI3Kα isoform inhibitors A66 (10 µM) and PI-103 (1 µM), but not by the PI3Kß isoform inhibitor TGX 221 (100 nM). Pik3cg(-/-)/Pik3cd(-/-)-arteries showed normal vasoconstriction. We conclude that PI3Kα is an important downstream element in vasoconstrictor GPCR signaling, which contributes to arterial vasocontraction via α1-adrenergic receptors. Our results highlight a regulatory role of PI3Kα in the cardiovascular system, which widens the spectrum of gene therapy approaches targeting PI3Kα in cancer cells and tumor angiogenesis and regional blood flow.

The centrosomal protein TACC3 controls paclitaxel sensitivity by modulating a premature senescence program.

Microtubule-interfering cancer drugs such as paclitaxel (PTX) often cause chemoresistance and severe side effects, including neurotoxicity. To explore potentially novel antineoplastic molecular targets, we investigated the cellular response of breast carcinoma cells to short hairpin(sh)RNA-mediated depletion of the centrosomal protein transforming acidic coiled coil (TACC) 3, an Aurora A kinase target expressed during mitosis. Unlike PTX, knockdown of TACC3 did not trigger a cell death response, but instead resulted in a progressive loss of the pro-apoptotic Bcl-2 protein Bim that links microtubule integrity to spindle poison-induced cell death. Interestingly, TACC3-depleted cells arrested in G1 through a cellular senescence program characterized by the upregulation of nuclear p21(WAF), downregulation of the retinoblastoma protein and extracellular signal-regulated kinase 1/2, formation of HP1γ (phospho-Ser83)-positive senescence-associated heterochromatic foci and increased senescence-associated ß-galactosidase activity. Remarkably, the onset of senescence following TACC3 knockdown was strongly accelerated in the presence of non-toxic PTX concentrations. Thus, we conclude that mitotic spindle stress is a major trigger of premature senescence and propose that the combined targeting of the centrosomal Aurora A-TACC3 axis together with drugs interfering with microtubule dynamics may efficiently improve the chemosensitivity of cancer cells.

TACC3 depletion sensitizes to paclitaxel-induced cell death and overrides p21WAF-mediated cell cycle arrest.

Regulators of the mitotic spindle apparatus are attractive cellular targets for antitumor therapy. The centrosomal protein transforming acidic coiled coil (TACC) 3 is required for spindle assembly and proper chromosome segregation. In this study, we employed an inducible RNA interference approach to downregulate TACC3 expression. We show that TACC3 knock-down in NIH3T3 fibroblasts caused aneuploidy, but failed to overtly impair mitotic progression. TACC3 depletion rather triggered a postmitotic p53-p21(WAF) pathway and led to a reversible cell cycle arrest. Similar effects were induced by low concentrations of paclitaxel, a spindle poison used in antitumor therapy. Interestingly, however, and unlike in TACC3-proficient cells, paclitaxel was able to induce strong polyploidy and subsequent apoptosis in TACC3-depleted cells. Even though paclitaxel treatment was associated with the activation of the survival kinase Akt and an antiapoptotic expression of cytoplasmic p21(WAF) and cyclin D1, this inhibition of cell death was abrogated by depletion of TACC3. Thus, our data identify TACC3 as a potential target to overcome p21(WAF)-associated protection of transformed cells against paclitaxel-induced cell death.

Gadd45gamma is dispensable for normal mouse development and T-cell proliferation.

Gadd45gamma, a family member of the growth arrest and DNA damage-inducible gene family 45 (Gadd45), is strongly induced by interleukin-2 (IL-2) in peripheral T cells. While in most tissues all Gadd45 family members are expressed, Gadd45gamma is the only member that is induced by IL-2. Here we show that the IL-2-induced expression of Gadd45gamma is dependent on a signaling pathway mediated by the tyrosine kinase Jak3 and the transcription factors Stat5a and Stat5b (signal transducer and activator of transcription). Previous studies with ectopically overexpressed Gadd45gamma in various cell lines implicated its function in negative growth control. To analyze the physiological role of Gadd45gamma we used homologous recombination to generate mice lacking Gadd45gamma. Gadd45gamma-deficient mice develop normally, are indistinguishable from their littermates, and are fertile. Furthermore, hematopoiesis in mice lacking Gadd45gamma is not impaired and Gadd45gamma-deficient T lymphocytes show normal responses to IL-2. These data demonstrate that Gadd45gamma is not essential for normal mouse development and hematopoiesis, possibly due to functional redundancy among the Gadd45 family members. Gadd45gamma is also dispensable for IL-2-induced T-cell proliferation.

Stat5a/b contribute to interleukin 7-induced B-cell precursor expansion, but abl- and bcr/abl-induced transformation are independent of stat5.

The cytokines interleukin 7 (IL-7) and interleukin 4 (IL-4) regulate lymphoid differentiation and function and activate the transcription factor Stat5. Using mice deficient for the 2 highly related transcription factors, Stat5a and Stat5b (Stat5a/b(-/-)), we investigated the role of Stat5 for B-cell differentiation, expansion, and function. Peripheral blood B cells of Stat5-deficient mice are significantly reduced, but no proliferation defects in response to various mitogenic stimuli are found. Also, IgM and IgG1 antibody production and immunoglobulin class switching are not affected. Pre- and pro-B cells of Stat5-deficient animals were found to have reduced responses to IL-7. Pro- and pre-B cells are the target cells of the abl oncogene and numerous studies have suggested that Stat5a/b is essential for transformation by derivatives of the Abelson (abl) gene. To assess the role of Stat5a/b in transformation, we have evaluated the ability of various abl derivatives to transform cells from Stat5a/b-deficient mice in vitro or in vivo. We demonstrate that the absence of Stat5a/b is not essential for the induction of lymphoid or myeloid tumors in vivo or on the ability to transform bone marrow cells in vitro.

Stat5 activation is uniquely associated with cytokine signaling in peripheral T cells.

The activation and subsequent proliferation of peripheral T cells requires the engagement of the T cell and a cytokine receptor, typically the IL-2 or IL-4 receptors. Critical to understanding the regulation of peripheral T cells is the knowledge of the unique contributions of each receptor to full T cell activation and cell cycle progression. Mice deficient in Stat5a and Stat5b have demonstrated the essential role that these highly related proteins play in cell cycle progression following peripheral T cell activation. Here we demonstrate that activation of the Stat5 proteins by tyrosine phosphorylation is uniquely contributed by cytokine receptor signaling and specifically does not occur through the T cell receptor complex.

Reconstitution of IL6-inducible differentiation of a myeloid leukemia cell line by activated Stat factors.

Differentiation of the myeloid leukemia cell line M1 by treatment with IL6-type cytokines depends on activation of the Jak/Stat (Janus kinase/signal transducer and activator of transcription) pathway. Defects in this cascade are correlated with an impaired cytokine-inducible differentiation of various other myeloid cell lines. Although treatment with IL-6 increased the amount of activated transcription factor Stat3 in the myeloid leukemia line C, differentiation was not induced. However, after cotransfection with expression constructs for the tyrosine kinase Jak2 and Stat factors 3 or 5a, treatment of the cells with IL-6 caused a decrease in the number of viable cells. In parallel, an increase in the percentage of differentiated cells occurred. These findings are consistent with the hypothesis that the Jak/Stat signaling cascade plays an important role in cytokine-induced differentiation of myeloid leukemia cells.

Modulation of the activation status of Stat5a during LIF-induced differentiation of M1 myeloid leukemia cells.

Treatment of M1 myeloid leukemia cells with leukemia inhibitory factor (LIF) causes activation of transcription factors Stat1, Stat3 and Stat5a (signal transducers and activators of transcription). DNA-binding of Stat proteins was detectable for extended periods of time in LIF-treated M1 cells, which simultaneously underwent terminal differentiation. The relative composition of Stat factors in the protein-DNA complexes changed during time. Whereas Stat3 was activated up to 36 h during treatment with LIF, Stat5a was activated only short-termed. Similarly, high expression of the immediate early gene CIS (cytokine-inducible SH2-containing protein), a known target gene of Stat5 in hematopoietic cells, occurred only during the onset of differentiation. This suggests a role of Stat5a in the early phase of LIF-induced differentiation and growth arrest of M1 cells.

Induction of differentiation by IL-6-type cytokines is impaired in myeloid leukaemia cells unable to activate Stat5a.

The block of differentiation in myeloid leukaemia can be overcome by treatment with a variety of agents including cytokines. Interleukin 6 (IL-6) and leukaemia inhibitory factor (LIF) induce macrophage differentiation and growth arrest through activation of the Janus kinase (Jak)/signal transducers and activators of transcription (Stat) signal pathway in murine M1 myeloid leukaemia cells. Treatment of various other myeloid leukaemia lines with LIF or IL-6 did not lead to induction of differentiation. Several defects in the cytokine triggered Jak/Stat signal pathway were striking in these lines. They expressed a decreased or undetectable amount of at least one of the components of the specific cytokine receptor complexes. Three lines contained a constitutively activated Jak/Stat signal cascade and in two of them, lines C and BMC-63, this cascade was inducible by treatment with IL-6, despite of a very low density of IL-6-receptors. Apart from the cytokine receptors, additional components of the Jak/Stat signal cascade were altered in these lines. Expression and activation of the transcription factor Stat5a and the tyrosine kinase Jak2 were markedly decreased compared to M1 cells, suggesting a role of activated Stat5a in the induction of differentiation. These results demonstrate a direct correlation between alterations in the Jak/Stat signal pathway and the inability to differentiate after cytokine treatment of myeloid leukaemia cells.

Members of the family of IL-6-type cytokines activate Stat5a in various cell types.

Interleukin-6 (IL-6)-type cytokines activate transcription factors Stat1 and Stat3 (signal transducers and activators of transcription). Here we report that leukemia inhibitory factor (LIF) and IL-6 activate Stat5a in M1 myeloid leukemia cells in addition. In murine embryonal stem (ES) cells stably transfected with an expression vector for Stat5a treatment with LIF resulted in tyrosine phosphorylation and DNA-binding of this transcription factor. Transfection of an expression construct for Stat5a in human hepatoma cells caused a dose-dependent increase in LIF-triggered transcriptional activity. Our data demonstrate that Stat5a is activated by IL-6-type cytokines and can mediate transcriptional activity in addition to Stat1 and Stat3.