The transcription factor FOXO3a is a crucial cellular target of gefitinib (Iressa) in breast cancer cells

Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) that causes growth delay in cancer cell lines and human tumor xenografts expressing high levels of EGFR. An understanding of the downstream cellular targets of gefitinib will allow the discovery of biomarkers for predicting outcomes and monitoring anti-EGFR therapies and provide information for key targets for therapeutic intervention. In this study, we investigated the role of FOXO3a in gefitinib action and resistance. Using two gefitinib-sensitive (i.e., BT474 and SKBR3) as well as three other resistant breast carcinoma cell lines (i.e., MCF-7, MDA-MB-231, and MDA-MB-453), we showed that gefitinib targets the transcription factor FOXO3a to mediate cell cycle arrest and cell death in sensitive breast cancer cells. In the sensitive cells, gefitinib treatment causes cell cycle arrest predominantly at the G(0)-G(1) phase and apoptosis, which is associated with FOXO3a dephosphorylation at Akt sites and nuclear translocation, whereas in the resistant cells, FOXO3a stays phosphorylated and remains in the cytoplasm. The nuclear accumulation of FOXO3a in response to gefitinib was confirmed in tumor tissue sections from breast cancer patients presurgically treated with gefitinib as monotherapy. We also showed that knockdown of FOXO3a expression using small interfering RNA (siRNA) can rescue sensitive BT474 cells from gefitinib-induced cell-proliferative arrest, whereas reintroduction of active FOXO3a in resistant MDA-MB-231 cells can at least partially restore cell-proliferative arrest and sensitivity to gefitinib. These results suggest that the FOXO3a dephosphorylation and nuclear localization have a direct role in mediating the gefitinib-induced proliferative arrest and in determining sensitivity to gefitinib.Supported by the German Cancer Aide Foundation (J. Krol)and the Association of International Cancer Research (R. Francis).Andrew Sunters and Andreas Polychronic were fellows funded by CancerResearch UK. Andre Albergaria is a recipient of a grant from Fundação para a Ciência e a Tecnologia, Portugal. This work was sponsored by theBreast Cancer Research Trust and Cancer Research U

Constitutively Nuclear FOXO3a Localization Predicts Poor Survival and Promotes Akt Phosphorylation in Breast Cancer

Background: The PI3K-Akt signal pathway plays a key role in tumorigenesis and the development of drug-resistance. Cytotoxic chemotherapy resistance is linked to limited therapeutic options and poor prognosis. Methodology/Principal Findings: Examination of FOXO3a and phosphorylated-Akt (P-Akt) expression in breast cancer tissue microarrays showed nuclear FOXO3a was associated with lymph node positivity (p = 0.052), poor prognosis (p = 0.014), and P-Akt expression in invasive ductal carcinoma. Using tamoxifen and doxorubicin-sensitive and -resistant breast cancer cell lines as models, we found that doxorubicin- but not tamoxifen-resistance is associated with nuclear accumulation of FOXO3a, consistent with the finding that sustained nuclear FOXO3a is associated with poor prognosis. We also established that doxorubicin treatment induces proliferation arrest and FOXO3a nuclear relocation in sensitive breast cancer cells. Induction of FOXO3a activity in doxorubicin-sensitive MCF-7 cells was sufficient to promote Akt phosphorylation and arrest cell proliferation. Conversely, knockdown of endogenous FOXO3a expression reduced PI3K/Akt activity. Using MDA-MB-231 cells, in which FOXO3a activity can be induced by 4-hydroxytamoxifen, we showed that FOXO3a induction up-regulates PI3K-Akt activity and enhanced doxorubicin resistance. However FOXO3a induction has little effect on cell proliferation, indicating that FOXO3a or its downstream activity is deregulated in the cytotoxic drug resistant breast cancer cells. Thus, our results suggest that sustained FOXO3a activation can enhance hyperactivation of the PI3K/Akt pathway. Conclusions/Significance: Together these data suggest that lymph node metastasis and poor survival in invasive ductal breast carcinoma are linked to an uncoupling of the Akt-FOXO3a signaling axis. In these breast cancers activated Akt fails to inactivate and re-localize FOXO3a to the cytoplasm, and nuclear-targeted FOXO3a does not induce cell death or cell cycle arrest. As such, sustained nuclear FOXO3a expression in breast cancer may culminate in cancer progression and the development of an aggressive phenotype similar to that observed in cytotoxic chemotherapy resistant breast cancer cell models. © 2010 Chen et al.published_or_final_versio

Homologous and heterologous desensitization of guanylyl cyclase-B signaling in GH3 somatolactotropes

The guanylyl cyclases, GC-A and GC-B, are selective receptors for atrial and C-type natriuretic peptides (ANP and CNP, respectively). In the anterior pituitary, CNP and GC-B are major regulators of cGMP production in gonadotropes and yet mouse models of disrupted CNP and GC-B indicate a potential role in growth hormone secretion. In the current study, we investigate the molecular and pharmacological properties of the CNP/GC-B system in somatotrope lineage cells. Primary rat pituitary and GH3 somatolactotropes expressed functional GC-A and GC-B receptors that had similar EC50 properties in terms of cGMP production. Interestingly, GC-B signaling underwent rapid homologous desensitization in a protein phosphatase 2A (PP2A)-dependent manner. Chronic exposure to either CNP or ANP caused a significant down-regulation of both GC-A- and GC-B-dependent cGMP accumulation in a ligand-specific manner. However, this down-regulation was not accompanied by alterations in the sub-cellular localization of these receptors. Heterologous desensitization of GC-B signaling occurred in GH3 cells following exposure to either sphingosine-1-phosphate or thyrotrophin-releasing hormone (TRH). This heterologous desensitization was protein kinase C (PKC)-dependent, as pre-treatment with GF109203X prevented the effect of TRH on CNP/GC-B signaling. Collectively, these data indicate common and distinct properties of particulate guanylyl cyclase receptors in somatotropes and reveal that independent mechanisms of homologous and heterologous desensitization occur involving either PP2A or PKC. Guanylyl cyclase receptors thus represent potential novel therapeutic targets for treating growth-hormone-associated disorders

Lrp5 Is Not Required for the Proliferative Response of Osteoblasts to Strain but Regulates Proliferation and Apoptosis in a Cell Autonomous Manner

Although Lrp5 is known to be an important contributor to the mechanisms regulating bone mass, its precise role remains unclear. The aim of this study was to establish whether mutations in Lrp5 are associated with differences in the growth and/or apoptosis of osteoblast-like cells and their proliferative response to mechanical strain in vitro. Primary osteoblast-like cells were derived from cortical bone of adult mice lacking functional Lrp5 (Lrp5−/−), those heterozygous for the human G171V High Bone Mass (HBM) mutation (LRP5G171V) and their WT littermates (WTLrp5, WTHBM). Osteoblast proliferation over time was significantly higher in cultures of cells from LRP5G171V mice compared to their WTHBM littermates, and lower in Lrp5−/− cells. Cells from female LRP5G171V mice grew more rapidly than those from males, whereas cells from female Lrp5−/− mice grew more slowly than those from males. Apoptosis induced by serum withdrawal was significantly higher in cultures from Lrp5−/− mice than in those from WTHBM or LRP5G171V mice. Exposure to a single short period of dynamic mechanical strain was associated with a significant increase in cell number but this response was unaffected by genotype which also did not change the ‘threshold’ at which cells responded to strain. In conclusion, the data presented here suggest that Lrp5 loss and gain of function mutations result in cell-autonomous alterations in osteoblast proliferation and apoptosis but do not alter the proliferative response of osteoblasts to mechanical strain in vitro

Association of Tat with Promoters of PTEN and PP2A Subunits Is Key to Transcriptional Activation of Apoptotic Pathways in HIV-Infected CD4+ T Cells

Apoptosis in HIV-1-infected CD4+ primary T cells is triggered by the alteration of the PI3K and p53 pathways, which converge on the FOXO3a transcriptional activator. Tat alone can cause activation of FOXO3a and of its proapoptotic target genes. To understand how Tat affects this pathway, we carried out ChIP-Chip experiments with Tat. Tat associates with the promoters of PTEN and two PP2A subunit genes, but not with the FOXO3a promoter. PTEN and PP2A encode phosphatases, whose levels and activity are increased when Tat is expressed. They counteract phosphorylation of Akt1 and FOXO3a, and so activate transcriptional activity of FOXO3a. FOXO3a promotes increased transcription of Egr-1, which can further stimulate the transcription of PTEN, thereby reinforcing the pathway that leads to FOXO3a transcriptional activation. RNAi experiments support the role of PTEN and PP2A in the initiation of the Tat-mediated cascade, which is critical to apoptosis. The increased accumulation of PTEN and PP2A subunit mRNAs during Tat expression is more likely to be the result of increased transcription initiation and not relief of promoter-proximal pausing of RNAPII. The Tat-PTEN and -PP2A promoter interactions provide a mechanistic explanation of Tat-mediated apoptosis in CD4+ T cells

Role of endocrine and paracrine factors in the adaptation of bone to mechanical loading

There appears to be no unique mechanically sensitive pathway by which changes in bone loading regulate bone mass and architecture to ensure adequate structural strength. Rather, strain-derived changes in bone cells activate a number of nonspecific strain-sensitive pathways (including calcium fluxes, prostanoids, nitric oxide, extracellular signal-regulated kinase, and sclerostin), the activities of which are modified by a number of factors (including estrogen receptors) for which this contribution is subsidiary to other purposes. The strain-sensitive pathways modified by these factors interact with a number of other pathways, some of which appear to have specific osteoregulatory potential (eg, the parathyroid hormone pathway), whereas others such as the Wnt pathway appear to be associated primarily with the response mechanisms of proliferation, differentiation, and apoptosis. The outcome of these multiple interactions are stimuli for local bone formation, resorption, or maintenance of the status quo, to maintain existing bone architecture or adapt it to a new mechanical regimen

The mouse fibula as a suitable bone for the study of functional adaptation to mechanical loading

Bones' functionally adaptive responses to mechanical loading can usefully be studied in the tibia by the application of loads between the knee and ankle in normal and genetically modified mice. Such loading also deforms the fibula. Our present study was designed to ascertain whether the fibula adapts to loading in a similar way to the tibia and could thus provide an additional bone in which to study functional adaptation. The right tibiae/fibulae in C57BL/6 mice were subjected to a single period of axial loading (40 cycles at 10 Hz with 10-second intervals between each cycle; approximately 7 min/day, 3 alternate days/week, 2 weeks). The left tibiae/fibulae were used as non-loaded, internal controls. Both left and right fibulae and tibiae were analyzed by micro-computed tomography at the levels of the mid-shaft of the fibula and 25% from its proximal and distal ends. We also investigated the effects of intermittent parathyroid hormone (iPTH) on the (re)modelling response to 2-weeks of loading and the effect of 2-consecutive days of loading on osteocytes' sclerostin expression. These in vivo experiments confirmed that the fibula showed similar loading-related (re)modelling responses to those previously documented in the tibia and similar synergistic increases in osteogenesis between loading and iPTH. The numbers of sclerostin-positive osteocytes at the proximal and middle fibulae were markedly decreased by loading. Collectively, these data suggest that the mouse fibula, as well as the tibia and ulna, is a useful bone in which to assess bone cells' early responses to mechanical loading and the adaptive (re)modelling that this engenders

Effects of 2500, 2900 and 3400 με on proliferation of primary osteoblast-like cells derived from female and male WT (A), <i>Lrp5</i>^−/− (B) and <i>LRP5</i>^G171V (C) mice.

<p>Changes in absolute number of cells between static and strain of both genotypes and genders are shown. Results are mean ± SEM of three independent experiments. Experiments were repeated 3 times. No significant differences at 2500 and 2900 με were observed. ***p<0.001 and *p<0.05 compared with the static control within the gender. (D) The effects of 3400 με on proliferation of primary osteoblast-like cells derived from female and male <i>LRP5</i>^G171V and <i>Lrp5</i>^−/− mice and their WT littermates. Percentage differences between static and strain of both genotypes and genders are shown. Results are the mean ± SEM of three independent experiments. Experiments were repeated 3 times. There were no significant differences between groups.</p