Activation Of FOXO3a Research Articles

Tyrosine nitrations impaired intracellular trafficking of FSHR to the cell surface and FSH-induced Akt-FoxO3a signaling in human granulosa cells.

Many infertile women suffered from poor ovarian response, and increased reactive oxygen species with age might mediate the poor ovarian response to FSH. In this study, we collected follicular fluids and isolated granulosa cells from female patients. Increased levels of peroxynitrite, tyrosine nitrations of FSH receptor (FSHR) and apoptosis were obviously detectable with decreased FSHR protein expressions in granulosa cells of the poor ovarian responders. In KGN (a human ovarian granulosa cell line) cells, exogenous peroxynitrite could sequester FSHR in the cytoplasm, and these dislocated FSHR might suffer from proteasome-mediated degradations. Here, we identified four peroxynitrite-mediated nitrated tyrosine residues of FSHR. Site-directed mutagenesis of FSHR revealed that Y626 was pivotal for intracellular trafficking of FSHR to the cell surface. Akt-induced inactivation of FoxO3a was required for the repression of FSH on granulosa cell apoptosis. However, peroxynitrite impaired FSH-induced Akt-FoxO3a signaling, while FSHR-Y626A mutant took similar effects. In addition, FoxO3a knockdown indeed impaired FSH-mediated cell survival, while FoxO3a-S253A mutant reversed that significantly.

Defect of SIRT1-FoxO3a axis is associated with the production of reactive oxygen species during protein kinase CK2 downregulation-mediated cellular senescence and nematode aging

We investigated whether SIRT1 is associated with reactive oxygen species (ROS) accumulation during CK2 downregulation-mediated senescence. SIRT1 overexpression suppressed ROS accumulation, reduced transcription of FoxO3a target genes, and nuclear export and acetylation of FoxO3a, which were induced by CK2 downregulation in HCT116 and MCF-7 cells. Conversely, overexpression of a dominant-negative mutant SIRT1 (H363Y) counteracted decreased ROS levels, increased transcriptional activity of FoxO3a, and increased nuclear import and decreased acetylation of FoxO3a, which were induced by CK2 upregulation. CK2 downregulation destabilized SIRT1 protein via an ubiquitin-proteasome pathway in human cells, whereas CK2 overexpression reduced ubiquitination of SIRT1. Finally, the SIRT1 activator resveratrol attenuated the accumulation of ROS and lipofuscin as well as lifespan shortening, and reduced expression of the DAF-16 target gene sod-3, which were induced by CK2 downregulation in nematodes. Altogether, this study demonstrates that inactivation of the SIRT1–FoxO3a axis, at least in part, is involved in ROS generation during CK2 downregulation-mediated cellular senescence and nematode aging.

The tumor suppressor FOXO3a mediates the response to EGFR inhibition in glioblastoma cells.

Although EGFR activation is a hallmark of glioblastoma (GBM), anti-EGFR therapy has so far not yielded the desired effects. Targeting PI3K/Akt has been proposed as a strategy to increase the cellular sensitivity to EGFR inhibitors. Here we evaluated the contribution of FOXO3a, a key Akt target, in the response of GBM cells to EGFR inhibition. FOXO3a activation was assessed by immunofluorescence and gene reporter assays, and by evaluating target gene expression using Western blotting and qRT-PCR. Cellular effects were evaluated using cell viability and apoptosis assays, i.e., Annexin V/PI staining and caspase 3/7 activity measurements. Drug synergism was evaluated by performing isobolographic analyses. Gene silencing experiments were performed using stable shRNA transfections. We found that EGFR inhibition in GBM cells led to FOXO3a activation and to transcriptional modulation of its key targets, including repression of the oncogene FOXM1. In addition, we found that specific FOXO3a activation recapitulated the molecular effects of EGFR inhibition, and that the FOXO3a activator trifluoperazine, a FDA-approved antipsychotic agent, reduced GBM cell growth. Subsequent isobolographic analyses of combination experiments indicated that trifluoperazine and erlotinib cooperated synergistically and that their concomitant treatment induced a robust activation of FOXO3a, leading to apoptosis in GBM cells. Using gene silencing, we found that FOXO3a is essential for the response of GBM cells to EGFR inhibition. Our data indicate that FOXO3a activation is a crucial event in the response of GBM cells to EGFR inhibition, suggesting that FOXO3a may serve as an actionable therapeutic target that can be modulated using FDA-approved drugs.

Estrogen Signaling in ERα-Negative Breast Cancer: ERβ and GPER.

Estrogen receptors are important regulators of the growth of breast tumors. Three different receptors for estrogens have been identified in breast tumors, two nuclear receptors, ERα and ERβ, and a G-protein coupled estrogen receptor 1 (GPER) that initiates non-genomic effects of estrogens in the cytosol. Recent findings show that the stimulation of cytoplasmic ERα and ERβ also triggers non-genomic signaling pathways. The treatment of breast cancer with anti-estrogens depends on the presence of ERα. About 40% of all breast cancers, however, do not express ERα. One subgroup of these tumors overexpress Her-2, another important group is designated as triple-negative breast cancer, as they neither express ERα, nor progesterone receptors, nor do they overexpress Her-2. This review addresses the signaling of ERβ and GPER in ERα-negative breast tumors. In addition to the well-established EGF-receptor transactivation pathways of GPER, more recent findings of GPER-dependent activation of FOXO3a, the Hippo-pathway, and HOTAIR-activation are summarized.

Perfluorooctane sulfonate induces apoptosis via activation of FoxO3a and upregulation of proapoptotic Bcl-2 proteins in PC12 cells.

Perfluorooctane sulfonate (PFOS), a kind of organic pollutant widely found in the environment and biota, could alter normal brain development and produce cognitive dysfunction. For the past years, the neurotoxic effects of PFOS have been shown. Recent studies have proven that PFOS can induce neuronal apoptosis and cause neurotoxicity, but the regulatory proteins referred to the process have not been clarified. In this study, PC12 cells were used to investigate the changes of the expression of apoptosis-related proteins, forkhead box O3 (FoxO3a) and pro-apoptotic Bcl-2 proteins. We detected that the levels of cleaved caspase-3 and cleaved PARP were up-regulated obviously in PFOS-treated PC12 cells by using Western blotting, and that the apoptotic rate of PC12 cells was increased significantly by using flow cytometry, verifying that PFOS could induce neuronal apoptosis. Western blot analysis and immunofluorescence revealed obvious up-regulation of the expression of FoxO3a and proapoptotic Bcl-2 proteins. In addition, knockdown of FoxO3a gene inhibited Bim expression and apoptosis. According to the data, we believe that FoxO3a may play a crucial role in PFOS-induced neurotoxicity.

ING4 suppresses hepatocellular carcinoma via a NF-κB/miR-155/FOXO3a signaling axis.

The tumor suppressor ING4 has been shown to be reduced in human HCC. The alteration of ING4 contributes to HCC progression. However, its effect in HCC and the potential mechanism is largely unclear. Herein, we found that downregulation of ING4 in HCC tumor tissues was closely associated with cancer staging, tumor size and vascular invasion. Lentivirus-mediated ING4 overexpression significantly inhibited proliferation, migration and invasion, and induced cell cycle G1 phase arrest and apoptosis in MHCC97H human HCC cells. Moreover, overexpression of ING4 dramatically suppressed MHCC97H tumor cell growth and metastasis to lung in vivo in athymic BALB/c nude mice. Mechanistic studies revealed that overexpression of ING4 markedly increased expression of FOXO3a both at the mRNA and protein level as well as enhanced nuclear level and transcriptional activity of FOXO3a in MHCC97H tumor cells. In addition, ING4 repressed transcriptional activity of NF-κB and expression of miR-155 targeting FOXO3a. Knockdown of ING4 exhibited opposing effects in MHCC97L human HCC cells. Interestingly, knockdown of FOXO3a attenuated not only ING4-elicited tumor suppression but also ING4-mediated regulatory effect on FOXO3a downstream targets, confirming that FOXO3a is involved in ING4-directed tumor-inhibitory effect in HCC. Overexpression of miR-155 attenuated ING4-induced upregulation of FOXO3a, whereas inhibition of miR-155 blunted ING4 knockdown-induced reduction of FOXO3a. Furthermore, inhibition of NF-κB markedly impaired ING4 knockdown-induced upregulation of miR-155 and downregulation of FOXO3a. Taken together, our study provided the first compelling evidence that ING4 can suppress human HCC growth and metastasis to a great extent via a NF-κB/miR-155/FOXO3a pathway.

SIRT6, a novel direct transcriptional target of FoxO3a, mediates colon cancer therapy.

SIRT6, NAD+-dependent deacetylase sirtuin 6, has recently shown to suppress tumor growth in several types of cancer. Colon cancer is a challenging carcinoma associated with high morbidity and death. However, whether SIRT6 play a direct role in colon tumorigenesis and the underlying mechanism are not understood.Methods: To investigate the role of SIRT6 in colon cancer, we firstly analyzed the specimens from 50 colorectal cancer (CRC) patients. We generated shSIRT6 LoVo cells and xenograft mouse to reveal the essential role of SIRT6 in cell apoptosis and tumor growth. To explore the underlying mechanism of SIRT6 regulation, we performed FRET and real-time fluorescence imaging in living cells, real-time PCR, immunoprecipitaion, immunohistochemistry, flow cytometry and luciferase reporter assay.Results: The expression level of SIRT6 in patients' specimens is lower than that of normal controls, and patients with higher SIRT6 level have a better prognosis. Here, we identified that transcriptional factor FoxO3a is a direct up-stream of SIRT6 and positively regulated SIRT6 expression, which in turn, promotes apoptosis by activating Bax and mitochondrial pathway. Functional studies reveal that Akt inactivation increases FoxO3a activity and augment its binding to SIRT6 promoter, leading to elevated SIRT6 expression. Knocking down SIRT6 abolished apoptotic responses and conferred resistance to the treatment of BKM120. Combinational therapies with conventional drugs showed synergistic chemosensitization, which was SIRT6-dependent both in vitro and in vivo.Conclusion: The results uncover SIRT6 as a new potential biomarker for colon cancer, and its unappreciated mechanism about transcription and expression via Akt/FoxO3a pathway.

Estradiol ameliorates antioxidant axis SIRT1-FoxO3a-MnSOD/catalase in the heart of fructose-fed ovariectomized rats

Harmful effects of fructose-rich diet (FRD) were predominantly observed in males, suggesting protective effects of estrogens. Little is known about AMPK/sirtuin-1 (SIRT1)/forkhead box O3 (FoxO3a)/manganese superoxide dismutase (MnSOD)/catalase signaling in the heart in state of metabolic syndrome and oxidative stress induced by fructose over-consumption. We investigated the effect of 10% FRD on expression of AMPK-SIRT1-FoxO3a-MnSOD/catalase axis in myocardium and potentially beneficial effect of 17β-estradiol replacement. The expression of NADPH oxidase 4 (Nox4) and miRNA-155, unfavorable regulators of this axis, were also investigated. FRD significantly increased AMPK and decreased FoxO3a activity, decreased SIRT1, MnSOD and Nox4 protein expression while E2 reverted these changes, except for Nox4, and increased catalase protein level. E2 diminished Nox4 and MnSOD mRNA level in FRD ovariectomized rats. These results suggest independent response of AMPK and SIRT to FRD treatment. The proposed signaling in the heart should be further investigated in the prooxidative and antioxidative milieu.

Protection of dilator function of coronary arteries from homocysteine by tetramethylpyrazine: Role of ER stress in modulation of BKCa channels

ObjectivesWe recently reported the involvement of ER stress-mediated BKCa channel inhibition in homocysteine-induced coronary dilator dysfunction. In another study, we demonstrated that tetramethylpyrazine (TMP), an active ingredient of the Chinese herb Chuanxiong, possesses potent anti-ER stress capacity. The present study investigated whether TMP protects BKCa channels from homocysteine-induced inhibition and whether suppression of ER stress is a mechanism contributing to the protection. Furthermore, we explored the signaling transduction involved in TMP-conferred protection on BKCa channels. MethodsBKCa channel-mediated relaxation was studied in porcine small coronary arteries. Expressions of BKCa channel subunits, ER stress molecules, and E3 ubiquitin ligases, as well as BKCa ubiquitination were determined in porcine coronary arterial smooth muscle cells (PCASMCs). Whole-cell BKCa currents were recorded. ResultsExposure of PCASMCs to homocysteine or the chemical ER stressor tunicamycin increased the expression of ER stress molecules, which was significantly inhibited by TMP. Suppression of ER stress by TMP preserved the BKCa β1 protein level and restored the BKCa current in PCASMCs, concomitant with an improved BKCa-mediated dilatation in coronary arteries. TMP attenuated homocysteine-induced BKCa β1 protein ubiquitination, in which inhibition of ER stress-mediated FoxO3a activation and FoxO3a-dependent atrogin-1 and Murf-1 was involved. ConclusionsReversal of BKCa channel inhibition via suppressing ER stress-mediated loss of β1 subunits contributes to the protective effect of TMP against homocysteine on coronary dilator function. Inhibition of FoxO3a-dependent ubiquitin ligases is involved in TMP-conferred normalization of BKCa β1 protein level. These results provide new mechanistic insights into the cardiovascular benefits of TMP.

The increased activity of a transcription factor inhibits autophagy in diabetic embryopathy

Maternal diabetes induces neural tube defects and stimulates the activity of the forkhead box O3 (Fox)O3a in the embryonic neuroepithelium. We previously demonstrated that deleting the FOXO3a gene ameliorates maternal diabetes-induced neural tube defects. Macroautophagy (hereafter referred to as "autophagy") is essential for neurulation. Rescuing autophagy suppressed by maternal diabetes in the developing neuroepithelium inhibits neural tube defect formation in diabetic pregnancy. This evidence suggests a possible link between FoxO3a and impaired autophagy in diabetic embryopathy. We aimed to determine whether maternal diabetes suppresses autophagy through FoxO3a, and if the transcriptional activity of FoxO3a is required for the induction of diabetic embryopathy. We used a well-established type 1 diabetic embryopathy mouse model, in which diabetes was induced by streptozotocin, for our invivo studies. To determine if FoxO3a mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium, we induced diabetic embryopathy in FOXO3a gene knockout mice and FoxO3a dominant negative transgenic mice. Embryos were harvested at embryonic day 8.5 to determine FoxO3a and autophagy activity and at embryonic day 10.5 for the presence of neural tube defects. We also examined the expression of autophagy-related genes. C17.2 neural stem cells were used for invitro examination of the potential effects of FoxO3a on autophagy. Deletion of the FOXO3a gene restored the autophagy markers, lipidation of microtubule-associated protein 1A/1B-light chain 3I to light chain 3II, in neurulation stage embryos. Maternal diabetes decreased light chain 3I-positive puncta number in the neuroepithelium, which was restored by deleting FoxO3a. Maternal diabetes also decreased the expression of positive regulators of autophagy (Unc-51 like autophagy activating kinase 1, Coiled-coil myosin-like BCL2-interacting protein, and autophagy-related gene 5) and the negative regulator of autophagy, p62. FOXO3a gene deletion abrogated the dysregulation of autophagy genes. Invitro data showed that the constitutively active form of FoxO3a mimicked high glucose in repressing autophagy. In cells cultured under high-glucose conditions, overexpression of the dominant negative FoxO3a mutant blocked autophagy impairment. Dominant negative FoxO3a overexpression in the developing neuroepithelium restored autophagy and significantly reduced maternal diabetes-induced apoptosis and neural tube defects. Our study revealed that diabetes-induced FoxO3a activation inhibited autophagy in the embryonic neuroepithelium. We also observed that FoxO3a transcriptional activity mediated the teratogenic effect of maternal diabetes because dominant negative FoxO3a prevents maternal diabetes-induced autophagy impairment and neural tube defect formation. Our findings suggest that autophagy activators could be therapeutically effective in treating maternal diabetes-induced neural tube defects.

Akt2 mediates glucocorticoid resistance in lymphoid malignancies through FoxO3a/Bim axis and serves as a direct target for resistance reversal

Glucocorticoids (GCs) are widely used drugs in the treatment of lymphoid malignancies; resistance of GCs in lymphocytes confers poor prognosis and the mechanisms are poorly understood. Here, we found T-acute lymphoblastic leukemia (T-ALL) cells acquire resistance to dexamethasone (DEX)-mediated killing through abnormal activation of Akt, resulting in inhibition of the FoxO3a/Bim pathway. The resistant state was reported to be associated with increased glycolysis, NOTCH1 activating mutations and activated PI3K/ serum GS regulated kinases (SGK) pathway. Use of aforementioned pathway inhibitors blocked FoxO3a-phosphorylation and partially improved DEX-mediated killing of GC-resistant T-ALL cells, further revealing the essential role of the FoxO3a/Bim pathway in the development of GC resistance. Inhibition of Akt is most effective at restoring sensitivity to DEX of GC-resistant lymphocytes in vitro and in vivo, but shows significant hepatotoxicity in vivo. A significantly elevated expression of Akt2 not Akt1 in intrinsically, secondarily GC-resistant lymphocytes and relapsed/refractory ALL patients implicates a more specific target for GC resistance. Mechanistically, Akt2 has a stronger binding capacity with FoxO3a compared to Akt1, and acts as a direct and major negative regulator of FoxO3a activity driving GC resistance. Pharmacologic inhibition of Akt2 more effectively restores sensitivity to GCs than inhibition of Akt1 in vitro, shows higher synergistic effect acting with DEX, and reverses GC resistance in GC-resistant T- or B- lymphoid tumors in vivo with reduced liver toxicity. In summary, these results suggest that Akt2 might serve as a more direct and specific kinase mediating GC resistance through FoxO3a/Bim signaling pathway, and Akt2 inhibition may be explored as a promising target for treating GC-resistant hematopoietic malignancies.

Effect of insulin on thyroid cell proliferation, tumor cell migration, and potentially related mechanisms

ABSTRACTBackground: Diabetes has recently been identified as a risk factor for a variety of cancers, possibly due to hyperinsulinemia or exogenous insulin use. Thyroid cancer is the most common endocrine malignancy, and its incidence has been exponentially increasing worldwide at an alarming rate. The aim of this study was to establish whether insulin use affects thyroid cancer development and progression, specifically cell proliferation and migration in vitro.Methods: In this study, we investigated the effects of the insulin agents most commonly used in the clinic, regular human insulin (HI) and insulin glargine (IG), on the proliferation and migration of thyroid cells.Results: Both HI and IG affected the thyroid cells in a dose-dependent manner and at high concentrations significantly promoted thyroid cell proliferation and tumor cell migration. The promoting effect might be elicited by activation of the insulin receptor and insulin-like growth factor-1 receptor and through the downstream Akt-signaling pathway, which inhibits the activity of the tumor-suppressor FoxO3a. In particular, MAPK-signaling cascades were activated in papillary thyroid carcinoma cell-1 cells but not in follicular rat thyroid-5 cells.Conclusion: The in vitro evidence demonstrated that HI and IG can promote thyroid cell proliferation and tumor cell migration at supraphysiological concentrations, but the effect was not significant at low concentrations. Whether high-dose insulins could affect diabetic patients with thyroid cancer or undetected (pre)cancerous lesions needs further in vivo study.Abbreviations: HI: human regular insulin; IG: insulin glargine; IR: insulin receptor; IGF-1R: insulin-like growth factor-1 receptor; Akt: protein kinase B (PKB); MAPK: mitogen-activated protein kinase; FoxO3a: the forkhead box-containing protein: class O 3a

Inhibition of mTORC2 enhances UVB-induced apoptosis in keratinocytes through a mechanism dependent on the FOXO3a transcriptional target NOXA but independent of TRAIL

The primary cause of non-melanoma skin cancer (NMSC) is ultraviolet B (UVB) radiation. We have shown previously that mTORC2 inhibition sensitizes keratinocytes to UVB-induced apoptosis mediated by the transcription factor FOXO3a. FOXO3a is a key regulator of apoptosis and a tumor suppressor in several cancer types. Activation of FOXO3a promotes apoptosis through the coordinated expression of a variety of target genes, including TRAIL and NOXA. We hypothesized that in the setting of mTORC2 inhibition, the UVB-induced expression of these factors would lead to apoptosis in a FOXO3a-dependent manner. Using spontaneously immortalized human keratinocytes (HaCaT cells), we observed that both TRAIL and NOXA expression increased in cells exposed to UVB and the TOR kinase inhibitor Torin 2. Similar to knockdown of FOXO3a, NOXA knockdown reversed the sensitization to UVB-induced apoptosis caused by mTORC2 inhibition. In contrast, loss of TRAIL by either knockdown or knockout actually enhanced expression of nuclear FOXO3a, which maintained apoptosis. These surprising results are not due to faulty death receptor signaling in HaCaT cells, as we found that the cells undergo extrinsic apoptosis in response to treatment with recombinant TRAIL. Even more striking, TRAIL knockout cells were sensitized to recombinant TRAIL-induced apoptosis compared to wild-type HaCaT cells, with the largest increase occurring in the presence of mTORC2 inhibition. Taken together, these studies provide strong evidence that mTORC2 controls UVB-induced apoptosis by regulating NOXA expression downstream of FOXO3a. Moreover, FOXO3a transcriptional activation by mTORC2 inhibitors may be a valuable target for prevention or therapy of NMSC, especially in cases with low endogenous TRAIL.

SIRT1 downregulation mediated Manganese-induced neuronal apoptosis through activation of FOXO3a-Bim/PUMA axis

Manganese (Mn) is an essential trace element. Excessive exposure to Mn may lead to neuronal death and neurodegenerative disorders. Accumulating evidence has shown that silent mating type information regulation 2 homolog 1 (SIRT1) plays a vital role in brain damage. However, whether aberrant SIRT1 levels contribute to Mn-induced neurotoxicity remains unknown. In this study, we report the important role of SIRT1 downregulation during Mn-induced neuronal apoptosis. Mn was found to downregulate SIRT1 protein levels in the rat pheochromocytoma (PC12) cells and mouse brain tissues. Mn enhanced SIRT1 protein degradation and downregulated its gene expression. Furthermore, Mn induced cell apoptosis in a dose-dependent manner both in vitro and in vivo, and resulted in an increase in forkhead box O (FOXO) 3a expression and acetylation. SIRT1 activation by resveratrol clearly attenuated Mn-triggered apoptosis and FOXO3a activation. Mn markedly increased the expression of Bcl-2 interacting mediator of cell death (Bim) and p53-up-regulated modulator of apoptosis (PUMA), whereas downregulation of FOXO3a significantly inhibited their upregulation and subsequent apoptosis. In summary, we determined that Mn downregulated SIRT1 by multiple mechanisms, thus led to apoptosis via activation of the FOXO3a–Bim/PUMA axis in PC12 cells. These findings on the impact of Mn on SIRT1 may lead to an improved understanding of Mn-induced neurotoxicity and provide a molecular target to antagonise Mn-associated neuronal damage.

Lipopolysaccharide induced the proliferation of mouse lung fibroblasts by suppressing FoxO3a/p27 pathway.

Aberrant aggregation and activation of lung fibroblasts is a key process in pulmonary fibrosis, but the underlying mechanism remains enigmatic. Forkhead Box O3a (FoxO3a) is considered to be an important transcription factor that could regulate both cell cycle and cell viability. To investigate the role of FoxO3a on LPS-induced lung fibroblast proliferation, we transfected FoxO3a-SiRNA or FoxO3a-OE lentivirus into cultured mouse lung fibroblasts to knockdown or overexpress FoxO3a and pretreated mouse lung fibroblasts with gefitinib to enhance FoxO3a activity. The proliferation of lung fibroblasts was evaluated by CCK8 assay, the expression of FoxO3a, phosphorylated FoxO3a (p-FoxO3a) and p27 were measured by Western blot. We found that the proliferation of mouse lung fibroblasts mediated by LPS is accompanied by the inactivation of FoxO3a. The knockdown of FoxO3a could further decreased the expression of p27 mediated by LPS, while the overexpression of FoxO3a significantly increased the expression of p27 and suppressed LPS-induced lung fibroblast proliferation. Upon treating fibroblasts with gefitinib, the phosphorylation of FoxO3a was reduced and FoxO3a translocated into the nucleus, the expression of p27 was significantly increased and the proliferation of lung fibroblasts mediated by LPS could also be inhibited effectively. The results indicate that overexpression and reduced phosphatase activity of FoxO3a inhibit LPS-induced lung fibroblast proliferation through the activation of FoxO3a/p27 signaling pathways. Thus, to enhance FoxO3a activity could be a potential therapeutic target for LPS-induced pulmonary fibrosis.

Abstract 5888: Disruption of FOXO3a-miRNAs mediated feedback inhibition of IGF2-IRS1 signaling contributes to Herceptin resistance in HER2 positive breast cancer cells

Abstract Resistance to HER2-target therapy remains a clinical challenge for HER2+ breast cancer subtype. The molecular mechanisms for Herceptin resistance remain elusive. Here, we demonstrated that the IGF2 and IRS1 protein levels were increased in Herceptin resistant breast cancer cells and that the IGF2/IGF1R/IRS1/Akt/mTOR signaling was involved in Herceptin resistance. In Herceptin sensitive HER2+ breast cancer cells, IRS1 protein level resulted from mTOR2 mediated FOXO3a status determined the feedback regulation of IGF2. Upon high dose rhIGF2 treatment, the mTOR1 translationally upregulated PPP3CB protein level that recovered the function of FOXO3a inactivated by mTOR2. FOXO3a positively transcriptionally regulated miR-128-3p and miR-30-5p expression that targeted IRS1 mRNA to control the feedback regulation of IGF2 signaling. In agreement, in Herceptin resistant cells, the decrease of miR-128-3p and miR-30-5p resulted from constitutively inactivation of FOXO3a by mTOR2, led to increase of IRS1 protein level, even in response to high endogenous IGF2 protein level in culture medium. High IGF2 protein level was resulted from decrease of miR-193a-5p that was also regulated by FOXO3a. Unexpectedly, in Herceptin resistant cells, Src/p-STAT5 signaling cooperating with HDAC1 transcriptionally repressed PPP3CB expression resulting into FOXO3a inactivation. Moreover, we confirmed that IRS1 knockdown reversed the Herceptin resistance in vivo using a xenograft mouse model. Serums form patients with poor response to Herceptin, had higher IGF2 protein level. Tissues from patients with poor response had higher IRS1, p-Akts473, p-FOXO3a and p-Src levels, but had lower PPP3CB, miR-128-3p, miR-30-5p and miR-193a-5p levels. In conclusions, disruption of PPP3CB-FOXO3a-miRNAs mediated feedback inhibition of IGF2-IRS1 signaling plays pivotal role in Herceptin resistance of HER2+ breast cancer. Related molecules in this signaling may represent potential biomarkers and therapeutic targets for HER2+ breast cancer treatment. Keywords: Breast cancer, Herceptin resistance, IGF2, IRS1, FOXO3a This study was supported by grants from the National Natural Science Foundation of China: No.81672616, No.81402196 and No.81272450, supported by grants from Guangdong Natural Science Funds for Distinguished Young Scholars No.2016A030306003 (G Zheng). Citation Format: Zhimin He. Disruption of FOXO3a-miRNAs mediated feedback inhibition of IGF2-IRS1 signaling contributes to Herceptin resistance in HER2 positive breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5888.

PO-203 A novel member in the β-catenin destruction complex: may MAPK14/P38α foster new therapeutic approaches in colorectal cancer?

IntroductionOne of the most commonly deregulated signalling pathways in colorectal cancer (CRC) is the Wnt cascade, which is controlled by APC. APC regulates β-catenin levels, thereby modulating the transcriptional activity of the TCF/LEF transcription factors. High levels of nuclear β-catenin lead to constitutive activation of the Wnt pathway, loss of normal cellular architecture and neoplastic transformation. Previous reports indicate that Wnts are capable of activating p38 MAPKs. A few older studies carried out in other tissues provided evidence that Wnt3a could activate p38 suggesting that the p38 pathway may feed into the canonical Wnt/β-catenin pathway at least at the level of GSK3β. We recently showed that p38α is required to maintain CRC metabolism and survival, as its inhibition leads to activation of FoxO3A, autophagy, cell death and tumour growth reduction both in vitro and in vivo.Material and methodsWe performed extensive characterisation of the functional interaction between p38 and the APC/β-catenin/GSK3β complex (co-localization analysis by confocal microscopy and co-immunoprecipitation studies) in several cell lines in vitro and in the APCMin/+ mouse preclinical model in vivo.Results and discussionsOur data showed that CRC cells have higher levels of activated p38 than their normal counterparts, and experiments using kinase-specific inhibitors revealed that these cells are ‘addicted’ to p38 activity. Interestingly, p38α blockade reduced the size and number of adenomas in the small bowel of APCMin/+ mice. Significant results were obtained in vivo by co-immunoprecipitation analysis of tissues from normal mice and APCMin/+ mice treated or not with AOM. Our findings confirmed the presence of p38α in APC/β-catenin/GSK3β complexes in CRC cells. Importantly, p38α co-localised with β-catenin in both normal and cancer cells; however, these proteins were confined to the cytoplasm in colonocytes, while they occupied discrete nuclear regions in CRC cells. These data were further corroborated by the inhibitory effect of p38α blockade on β-catenin-responsive genes (i.e. c-Myc, cyclin D1/2). Characterisation of this novel functional interaction was also extended with chromatin immunoprecipitation experiments.ConclusionIdentification of p38α as a novel member of the APC/β-catenin/GSK3β complex could help elucidate mechanisms contributing to human colon tumour pathogenesis and allow for the development of new strategies for CRC treatment.

Dysregulation of DAF-16/FOXO3A-mediated stress responses accelerates oxidative DNA damage induced aging

DNA damage is presumed to be one type of stochastic macromolecular damage that contributes to aging, yet little is known about the precise mechanism by which DNA damage drives aging. Here, we attempt to address this gap in knowledge using DNA repair-deficient C. elegans and mice. ERCC1-XPF is a nuclear endonuclease required for genomic stability and loss of ERCC1 in humans and mice accelerates the incidence of age-related pathologies. Like mice, ercc-1 worms are UV sensitive, shorter lived, display premature functional decline and they accumulate spontaneous oxidative DNA lesions (cyclopurines) more rapidly than wild-type worms. We found that ercc-1 worms displayed early activation of DAF-16 relative to wild-type worms, which conferred resistance to multiple stressors and was important for maximal longevity of the mutant worms. However, DAF-16 activity was not maintained over the lifespan of ercc-1 animals and this decline in DAF-16 activation corresponded with a loss of stress resistance, a rise in oxidant levels and increased morbidity, all of which were cep-1/ p53 dependent. A similar early activation of FOXO3A (the mammalian homolog of DAF-16), with increased resistance to oxidative stress, followed by a decline in FOXO3A activity and an increase in oxidant abundance was observed in Ercc1-/- primary mouse embryonic fibroblasts. Likewise, in vivo, ERCC1-deficient mice had transient activation of FOXO3A in early adulthood as did middle-aged wild-type mice, followed by a late life decline. The healthspan and mean lifespan of ERCC1 deficient mice was rescued by inactivation of p53. These data indicate that activation of DAF-16/FOXO3A is a highly conserved response to genotoxic stress that is important for suppressing consequent oxidative stress. Correspondingly, dysregulation of DAF-16/FOXO3A appears to underpin shortened healthspan and lifespan, rather than the increased DNA damage burden itself.

Activation of FOXO3a reverses 5-Fluorouracil resistance in human breast cancer cells

Breast cancer is the most frequently diagnosed tumor type and the primary leading cause of cancer deaths in women worldwide. Drug resistance is the major obstacle for breast cancer treatment improvement. TRAIL-inducing compound 10 (Tic10), a novel activator of FOXO3, exhibits potent antitumor efficacy both in vitro and in vivo. In the present study, we investigated the resistance reversal effect of Tic10 on multidrug-resistant breast cancer cells T47D/5Fu derived from T47D breast cancer cells. We found that FOXO3a was significantly decreased in T47D/5-Fu cells, whereas treatment of Tic10 enhances FOXO3a expression and nuclear translocation. Moreover, treatment of Tic10 could reverses 5-Fluorouracil resistance of T47D/5-Fu cells via induction of G0/G1 cell cycle arrest and apoptosis. Furthermore, we found that Tic10 decreased the expression of CDK4 via FOXO3a-dependment mechanism. In addition, our data showed that Tic10 could sensitize drug resistant T47D/5-Fu cells to 5-Fu in vivo. Taken together, these data suggested Tic10 as capable of restoring sensitivity for drug-resistant breast cancer cells.

β-arrestin1-medieated inhibition of FOXO3a contributes to prostate cancer cell growth invitro and invivo.

Recently, β‐arrestin1 has been indicated as a prostate cancer promoter through promoting cell proliferation and epithelial to mesenchymal transition, but its underlying mechanism remains unclear. Here, our data revealed that β‐arrestin1 could promote cell growth through inhibiting the transcriptional activity and expression of FOXO3a in prostate cancer cells in vitro and in vivo. We found that β‐arrestin1 could promote the cell and tumor growth of prostate cancer, and β‐arrestin1 expression represented a negative correlation with FOXO3a expression but not FOXO1 expression in prostate cancer cell lines and tissues. In addition, forced expression of β‐arrestin1 induced a significant decrease of FOXO3a expression but had no clear effect on FOXO1 expression. Mechanistically, β‐arrestin1 could interact with FOXO3a and MDM2, respectively, and promote the interaction between FOXO3a and MDM2, whereas it had no obvious interaction with FOXO1. Furthermore, β‐arrestin1 could inhibit the transcriptional activity of FOXO3a via Akt and ERK1/2 pathways. Together, our results revealed a novel mechanism for β‐arrestin1 in the regulation of the prostate cancer procession through inhibiting FOXO3a.