Regulation Of FOXO3a Research Articles

SIRT3 inhibits prostate cancer metastasis through regulation of FOXO3A by suppressing Wnt/β-catenin pathway

SIRT3, a mitochondrial NAD+-dependent deacetylase, has been reported to restrain prostate cancer growth both in vitro and in vivo, however, its role in metastatic prostate cancer has not been revealed. In this study, we reported that SIRT3 inhibited the epithelial-mesenchymal transition (EMT) and migration of prostatic cancer cells in vitro and their metastasis in vivo. Consistently, based on analyses of tissue microarray and microarray datasets, lower SIRT3 expression level was correlated with higher prostate cancer Gleason scores, and SIRT3 expression were significantly decreased in metastatic tissues compared with prostate tumor tissues. Mechanistically, SIRT3 promoted FOXO3A expression by attenuating Wnt/β-catenin pathway, thereby inhibiting EMT and migration of prostate cancer cells. Indeed, SIRT3's inhibitory effect on EMT and migration of prostate cancer cells can be rescued after applying Wnt/β-catenin pathway activator LiCl, or boosted by wnt inhibitor XAV939. Together, this study revealed a novel mechanism for prostate cancer metastasis that involves SIRT3/ Wnt/β-catenin/ FOXO3A signaling to modulate EMT and cell migration.

Abstract 2323: The inhibitory role of Kaempferol-3-O-rutinoside induced AMPK activation on the growth of human breast cancer cell lines

Abstract Solidago virga-aurea, herbaceous perennial plant of the family Asteraceae, has astringent, diuretic, and antiseptic assets. In our current study, we found that kaempferol-3- O -rutinoside (KR) from Solidago virga-aurea extract prompted cellular apoptosis in breast cancer cells. The present study was conducted to investigate its effects on MCF-7 cells with respect to the induction of inhibitory effect on cell viability against MDA-MB-231 and MCF-7 breast cancer cell lines. In addition, apoptotic protein, especially Bim, was also increased in KR treated MDA-MB-231 and MCF-7 cell line. However, an anti-apoptotic protein, Bcl-xL, was not changed by KR. To find out positive regulator of Bim expression, FOXO (forkhead-box transcription factor, group O) 3a and AMPK (5' AMP-activated protein kinase) protein expressions were examined in KR treated cells. Immunoblot analysis showed dramatically increased expressions of FOXO3a and AMPK in KR treated MDA-MB-231 and MCF-7 cells. In addition, cell cycle analysis indicated that G0/G1 arrest was intensely increased in KR treated MDA-MB-231 and MCF-7 cells. Moreover, phospho-AMPK (pT172) and phospho-FOXO3a (S472) expression were also increased in KR treated MDA-MB-231 and MCF-7 cells. Our results suggest that KR induces apoptosis by AMPK-FOXO3a mediated Bim expression. The mechanisms of KR mediated AMPK and FOXO3a regulation is not fully elucidated yet, but further studies about these molecular mechanisms between two proteins will give some ideas for cancer therapies for certain breast cancer patients. Citation Format: Chea Ha Kim. The inhibitory role of Kaempferol-3-O-rutinoside induced AMPK activation on the growth of human breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2323. doi:10.1158/1538-7445.AM2017-2323

Abstract 4308: Cordycepin induces apoptosis by caveolin-1-mediated JNK regulation of Foxo3a in human lung adenocarcinoma cells

Abstract Forkhead transcription factor (Foxo3a) is a critical effector of JNK-mediated tumor suppression. However, it is not clear whether the caveolin-1 (CAV1)-mediated JNK/Foxo3a pathway is involved in cancer cell apoptosis. We found that cordycepin upregulates CAV1 expression, which was accompanied by JNK phosphorylation (p-JNK), and, subsequently Foxo3a translocation into the nucleus, resulting in the upregulation of Bax protein expression. Furthermore, we found that CAV1 overexpression upregulated p-JNK whereas CAV1 siRNA downregulated p-JNK. Additionally, SP600125, a specific JNK inhibitor, significantly increased Foxo3a phosphorylation, which attenuated Foxo3a translocation into the nucleus, indicating that CAV1 mediates JNK regulation of Foxo3a. Foxo3a siRNA downregulated Bax protein and attenuated A549 apoptosis, indicating that the CAV1-mediated JNK/Foxo3a pathway induces the apoptosis of A549 lung cancer cells. Cordycepin significantly decreased tumor volume in nude mice. Taken together, these results indicate that cordycepin promotes CAV1 upregulation to enhance JNK/Foxo3a signaling pathway activation inducing apoptosis in lung cancer cells and support its potential as a therapeutic agent for lung cancer. Citation Format: Eunbi Jo, Hyun Jin Jang, Ik-Soon Jang. Cordycepin induces apoptosis by caveolin-1-mediated JNK regulation of Foxo3a in human lung adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4308. doi:10.1158/1538-7445.AM2017-4308

Abstract 1480: Mir-182 is involved in sulindac anticancer activity in colon cancer

Abstract Nonsteroidal anti-inflammatory drugs (NSAIDs) display promising antineoplastic activity in many human solid tumors including colorectal cancer. Previous studies reported that sulindac sulfide (SS) can inhibit the growth of tumor cells through cyclooxygenase-2 (COX-2) dependent or independent pathways. Obviously, COX-2 independent pathway involves a low toxic property to support the clinical potential for using sulindac as a chemoprevention drug. However, the molecular mechanisms responsible for COX-2 independent pathway have not been completely elucidated. In this project, we employed two human colon cancer cell lines, referred to HCT116 and HT29. HCT116 cells are characterized for low COX-2 expression, while HT29 cells show relatively high COX-2 expression. We found that SS could unbiasedly inhibit the growth of HCT116 and HT29 cells by arresting cells in G1/G2 phases. CyclinG2 was found to be upregulated in response to SS treatment. FOXO3a has been reported to regulate CyclinG2 expression at the transcriptional level. Our results demonstrate that SS could also upregulate FOXO3a. By using the loss-of-function strategy, we found that SS could not efficiently upregulate CyclinG2 and lead to cell cycle arrest in cells with FOXO3a knockdown. In addition, we studied the mechanism that could be involved in SS regulation of FOXO3a. Our previous studies reported that a panel of miRNAs could be altered by SS treatment in both colon and breast cancer cells. In the downregulated miRNA list, we found that miR-182 could potentially target FOXO3a. By using luciferase assay, we validated the direct regulation of miR-182 on the expression of FOXO3a. When miR-182 was downregulated, SS could neither efficiently upregulate the expression of FOXO3a nor inhibit cell growth as it did in control cells. We gained highly consistent results in HT29 and HT29 cells with COX-2 knockdown. Therefore, our study demonstrates a pathway consisting of miR-182/ FOXO3a/ CyclinG2 as a novel mechanism responsible for SS anticancer activity in colon cancer, which may imply a COX-2 independent pathway. Citation Format: Hongyou Zhao, Bin Yi, Zhipin Liang, Ruixia Ma, Yaguang Xi. Mir-182 is involved in sulindac anticancer activity in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1480. doi:10.1158/1538-7445.AM2017-1480

Lycopene Protects Keratinocytes Against UVB Radiation-Induced Carcinogenesis via Negative Regulation of FOXO3a Through the mTORC2/AKT Signaling Pathway.

Lycopene, one of the most potent anti-oxidants, has been reported to exhibit potent anti-proliferative properties in a wide range of cancer cells through modulation of the cell cycle and apoptosis. Forkhead box O3 (FOXO3a) plays a pivotal role in modulating the expression of genes involved in cell death. Herein, we investigated the role of FOXO3a signaling in the anti-cancer effects of lycopene. Results showed that lycopene pretreatment attenuated UVB-induced cell hyper-proliferation and promoted apoptosis, accompanied by decreased cyclin-dependent kinase 2 (CDK2) and CDK4 complex in both human keratinocytes and SKH-1 hairless mice. FOXO3a is phosphorylated in response to UVB irradiation and sequestered in the cytoplasm, while lycopene pretreatment rescued this sensitization. Gene ablation of FOXO3a attenuated lycopene-induced decrease in cell hyper-proliferation, CDK2, and CDK4 complex, indicating a critical role of FOXO3a in the lycopene-induced anti-proliferative effect of keratinocytes during UVB irradiation. Transfection with FOXO3a siRNA inhibited the lycopene-induced increase in cell apoptosis, BAX and cleaved PARP expression. Moreover, loss of AKT induced further accelerated lycopene-induced FOXO3a dephosphorylation, while loss of mechanistic target of rapamycin complex 2 (mTORC2) by transfection with RICTOR siRNA induced levels of AKT phosphorylation comparable to those obtained with lycopene. In contrast, overexpression of AKT or mTORC2 decreased the effects of lycopene on the expression of FOXO3a as well as AKT phosphorylation, suggesting that lycopene depends on the negative modulation of mTORC2/AKT signaling. Taken together, our findings demonstrate that the mTORC2/AKT/FOXO3a axis plays a critical role in the anti-proliferative and pro-apoptotic effects of lycopene in UVB-induced photocarcinogenesis. J. Cell. Biochem. 119: 366-377, 2018. © 2017 Wiley Periodicals, Inc.

Overexpression of FoxO3a is associated with glioblastoma progression and predicts poor patient prognosis.

Forkhead transcription factor FoxO3a has been reported to have ambiguous functions and distinct mechanisms in various solid tumors, including glioblastoma (GBM). Although a preliminary analysis of a small sample of patients indicated that FoxO3a aberrations in glioma might be related to aggressive clinical behavior, the clinical significance of FoxO3a in glioblastoma remains unclear. We investigated the expression of FoxO3a in a cohort of 91 glioblastoma specimens and analyzed the correlations of protein expression with patient prognosis. Furthermore, the functional impact of FoxO3a on GBM progression and the underlying mechanisms of FoxO3a regulation were explored in a series of in vitro and in vivo assays. FoxO3a expression was elevated in glioblastoma tissues, and high nuclear FoxO3a expression in human GBM tissues was associated with poor prognosis. Moreover, knockdown of FoxO3a significantly reduced the colony formation and invasion ability of GBM cells, whereas overexpression of FoxO3a promoted the colony formation and invasion ability. The results of in vivo GBM models further confirmed that FoxO3a knockdown inhibited GBM progression. More, the pro-oncogenic effects of FoxO3a in GBM were mediated by the activation of c-Myc, microtubule-associated protein 1 light chain 3 beta (LC3B) and Beclin1 in a mixed-lineage leukemia 2 (MLL2)-dependent manner. These findings suggest that high FoxO3a expression is associated with glioblastoma progression and that FoxO3a independently indicates poor prognosis in patients. FoxO3a might be a novel prognostic biomarker or a potential therapeutic target in glioblastoma.

Cordycepin induces apoptosis by caveolin-1-mediated JNK regulation of Foxo3a in human lung adenocarcinoma

Forkhead transcription factor (Foxo3a) is a downstream effector of JNK-induced tumor suppression. However, it is not clear whether the caveolin-1 (CAV1)-mediated JNK/Foxo3a pathway is involved in cancer cell apoptosis. We found that cordycepin upregulates CAV1 expression, which was accompanied by JNK phosphorylation (p-JNK) and subsequent Foxo3a translocation into the nucleus, resulting in the upregulation of Bax protein expression. Furthermore, we found that CAV1 overexpression upregulated p-JNK, whereas CAV1 siRNA downregulated p-JNK. Additionally, SP600125, a specific JNK inhibitor, significantly increased Foxo3a phosphorylation, which downregulated Foxo3a translocation into the nucleus, indicating that CAV1 mediates JNK regulation of Foxo3a. Foxo3a siRNA downregulated Bax protein and attenuated A549 apoptosis, indicating that the CAV1-mediated JNK/Foxo3a pathway induces the apoptosis of A549 lung cancer cells. Cordycepin significantly decreased tumor volume in nude mice. Taken together, these results indicate that cordycepin promotes CAV1 upregulation to enhance JNK/Foxo3a signaling pathway activation, inducing apoptosis in lung cancer cells, and support its potential as a therapeutic agent for lung cancer.

Abstract 3520: Solidago virga-aurea extract induced apoptosis of breast cancer cells through FOXO3 mediated bim expression

Abstract Solidago virga-aurea (European goldenrod or woundwort) is an herbaceous perennial plant of the family Asteraceae. It is used for medical system with astringent, diuretic, antiseptic and homeopathic properties. And the studies about Solidago virga-aurea are in progress for the other functions. Recently, we found that Solidago virga-aurea extract induces apoptosis of breast cancer cells. MTT assay showed that cellular viability was decreased in Solidago virga-aurea extract treated MDA-MB-231 and MCF-7 breast cancer cell lines by dose and time dependent manner. And apoptotic protein, especially bim, was increased in Solidago virga-aurea extract treated MCF-7 cell line. However, anti-apoptotic protein, Bcl-xL, does not changed by Solidago virga-aurea extract. In order to find out positive regulator of bim expression, we tested whether FOXO (forkhead-box transcription factor, group O) 3a and AMPK (5’ AMP-activated protein kinase) protein expression. In immunoblot analysis showed that FOXO3a expression level was dramatically increased in Solidago virga-aurea treated MCF-7 cells without changes of AMPK expression. Moreover, immunostaining showed that Solidago virga-aurea induces co-localization of FOXO3a and bim proteins in MCF-7 and MB-231 cells. Taken together, Solidago virga-aurea induces apoptosis by FOXO3a mediated up-regulation of bim protein. The mechanisms of Solidago virga-aurea mediated FOXO3a regulation is not fully elucidated yet, but further studies about this molecular mechanism between two proteins will give some ideas for cancer therapies for certain breast cancer patients. Citation Format: Haesung Kim, Chea Ha Kim, Jeong-Hyeon Kim, Jeong-Min Lee, Jeong-Ho Jeon, Jae-Yong Lee. Solidago virga-aurea extract induced apoptosis of breast cancer cells through FOXO3 mediated bim expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3520.

Protein kinase C dependent development of dendritic cells.

Abstract Dendritic cells (DC) are professional antigen presenting cells that are important activators of adaptive immune responses and play a role in the activation of CD8+ T cells that target tumor cells. Our lab has also previously shown that protein kinase C (PKC) βII is essential for DC differentiation and that tumor derived factors, particularly IL-6, inhibit DC differentiation by repressing the expression of PKCβII through a Stat3 dependent mechanism. However, the pathways activated downstream of PKCβII during DC differentiation have yet to be fully characterized. To study this, we used the K562 cell line, which differentiates into DCs with addition of PMA, a known chemical activator of conventional PKC isoenzymes. We have also shown previously that PMA specifically activates PKCβII in K562 cells. Addition of PMA to K562 cells showed the activation of the ERK1/2 and NFκB pathways, including both the canonical and non-canonical NFκB pathways. Chemical inhibition of either the ERK1/2 or NFκB pathway yielded immature DCs with a reduced capacity to stimulate T-cell proliferation when incubated with PMA. Inhibition of ERK1/2 also showed a change in levels of RelB, a NFκB transcription factor, with PMA treatment and during PMA induced DC differentiation. Our lab has also shown that matured DCs reduce Foxo3a protein levels with the stimulation of CD80/CD86 through an Akt dependent mechanism. Inhibition of either the ERK1/2 or NFκB pathways showed an increase in Foxo3a levels, indicating a role for the ERK1/2 and NFκB pathways in Foxo3a regulation during differentiation into mature DCs outside of Akt. Taken together, these results show an integral role for both the ERK and NFκB pathways downstream of PKCβII activation during DC differentiation.

FOXO3a and Posttranslational Modifications Mediate Glucocorticoid Sensitivity in B-ALL.

Glucocorticoids are widely used to treat B acute lymphoblastic leukemia (B-ALL); however, the molecular mechanism underlying glucocorticoid response and resistance is unclear. In this study, the role and regulation of FOXO3a in mediating the dexamethasone response in B-ALL were investigated. The results show that FOXO3a mediates the cytotoxic function of dexamethasone. In response to dexamethasone, it was found that FOXO3a translocates into the nucleus, where it induces the expression of downstream targets, including p27Kip1 and Bim, important for proliferative arrest and cell death in the sensitive RS4;11 and SUP-B15 B-ALL cells. FOXO3a activation by dexamethasone is mediated partially through the suppression of the PI3K/Akt signaling cascade. Furthermore, two posttranslational modifications were uncovered, phosphorylation on Ser-7 and acetylation on Lys-242/5, that associated with FOXO3a activation by dexamethasone. Immunoblot analysis showed that the phosphorylation on Ser-7 of FOXO3a is associated with p38/JNK activation, whereas the acetylation on Lys-242/5 is correlated with the downregulation of SIRT1/2/6 and the induction of the acetyltransferase CBP/p300. Collectively, these results indicate that FOXO3a is essential for dexamethasone response in B-ALL cells, and its nuclear translocation and activation is associated with its phosphorylation on Ser-7 and acetylation on Lys-242/245. These posttranslational events can be exploited as biomarkers for B-ALL diagnosis and as drug targets for B-ALL treatment, particularly for overcoming the glucocorticoid resistance. FOXO3a and its posttranslational regulation are essential for dexamethasone response, and targeting FOXO3a and sirtuins may enhance the dexamethasone-induced cytotoxicity in B-ALL cells.

Simvastatin prevents triple-negative breast cancer metastasis in pre-clinical models through regulation of FOXO3a.

We previously reported using statins was correlated with improved metastasis-free survival in aggressive breast cancer. The purpose of this study was to examine the effect of statins on metastatic colonization by triple-negative breast cancer (TNBC) cells. TNBC cell lines were treated with simvastatin and then studied for cell cycle progression and proliferation in vitro, and metastasis formation in vivo, following injection of statin-treated cells. Reverse-phase protein assay (RPPA) analysis was performed on statin-treated and control breast cancer cells. RNA interference targeting FOXO3a was used to measure the impact of simvastatin on FOXO3a-expressing cells. The prognostic value of FOXO3a mRNA expression was examined in eight public breast cancer gene expression datasets including 1479 patients. Simvastatin increased G1/S-phase arrest of the cell cycle and inhibited both proliferation and migration of TNBC cells in vitro. In vitro pre-treatment and in vivo treatment with simvastatin reduced metastases. Phosphorylated FOXO3a was downregulated after simvastatin treatment in (RPPA) analysis. Ectopic expression of FOXO3a enhanced mammosphere formation and migratory capacity in vitro. Knockdown of FOXO3a attenuated the effect of simvastatin on mammosphere formation and migration. Analysis of public gene expression data demonstrates FOXO3a mRNA downregulation was independently associated with shorter metastasis-free survival in all breast cancers, as well as in TNBC breast cancers. Simvastatin inhibits in vitro endpoints associated with metastasis through a FOXO3a mechanism and reduced metastasis formation in vivo. FOXO3a expression is prognostic for metastasis formation in patient data. Further investigation of simvastatin as a cancer therapy is warranted.

Forkhead box K2 modulates epirubicin and paclitaxel sensitivity through FOXO3a in breast cancer.

The forkhead transcription factor FOXK2 has recently been implicated in cancer cell proliferation and survival, but a role in cancer chemotherapeutic drug resistance has hitherto not been explored. Here we demonstrate that FOXK2 has a central role in mediating the cytotoxic drug response in breast cancer. Clonogenic and cell viability assays showed that enhanced FOXK2 expression sensitizes MCF-7 breast cancer cells to paclitaxel or epirubicin treatment, whereas FOXK2 depletion by small interfering RNAs (siRNAs) confers drug resistance. Our data also showed that the activation of the tumour suppressor FOXO3a by paclitaxel and epirubicin is mediated through the induction of FOXK2, as depletion of FOXK2 by siRNA limits the induction of FOXO3a by these drugs in MCF-7 cells. Chromatin immunoprecipitation (ChIP) analysis showed that in response to drug treatment, FOXK2 accumulates and binds to the proximal FOXO3a promoter region in MCF-7 cells. Furthermore, we also uncovered that FOXK2 is deregulated and, therefore, can express at high levels in the nucleus of both the paclitaxel and epirubicin drug-resistant MCF-7 cells. Our results showed that ectopically overexpressed FOXK2 accumulates in the nuclei of drug-resistant MCF-7 cells but failed to be recruited to target genes, including FOXO3a. Crucially, we found that FOXO3a is required for the anti-proliferative and epirubicin-induced cytotoxic function of FOXK2 in MCF-7 cells by sulphorhodamine and clonogenic assays. The physiological importance of the regulation of FOXO3a by FOXK2 is further confirmed by the significant correlations between FOXO3a and FOXK2 expression in breast carcinoma patient samples. Further survival analysis also reveals that high nuclear FOXK2 expression significantly associates with poorer clinical outcome, particularly in patients who have received conventional chemotherapy, consistent with our finding that FOXK2 is deregulated in drug-resistant cells. In summary, our results suggest that paclitaxel and epirubicin target the FOXK2 to modulate their cytotoxicity and deregulated FOXK2 confers drug resistance.

Abstract 27: The role of the mTORC2-dependent regulation of FOXO3a in UVB-induced apoptosis

Abstract The greatest environmental risk factor for developing non-melanoma skin cancer (NMSC) is ultraviolet B (UVB) radiation. Both complexes of the mammalian target of rapamycin (mTOR) signaling pathway, the rapamycin-sensitive mTOR complex 1 (mTORC1) and the rapamycin-resistant mTOR complex 2 (mTORC2), are stimulated in response to UVB. Cell survival in UVB-induced keratinocyte carcinogenesis has been shown to be mTORC2-depedent. The tumor suppressor FOXO3a, a member of the Forkhead box family of proteins, is an important transcription factor involved in regulating cell survival. FOXO3a activity is attenuated as a result of AKT activation, which is downstream of mTORC2. UVB has been shown to activate AKT through the mTORC2 pathway; however, the role of FOXO3a in UVB-induced apoptosis in keratinocytes has yet to be studied. To investigate the role of mTORC2 and FOXO3a in UVB-induced apoptosis, we utilized both human keratinocytes (HaCaT cells) as well as inducible Rictor-deficient mouse embryonic fibroblasts (iRictKO cells) following treatment with 4-hydroxy-tamoxifen (4OHT). We also employed the use of inhibitors and shRNA targeting Rictor or mSIN1. The Rictor and mSIN1 proteins are key structural components unique to mTORC2 and their removal dramatically lowers mTORC2 activity. Finally, we compared the effects of shRNA targeting FOXO3a with the expression of a constitutively active mutant FOXO3a. For all the experiments described, cells were exposed to an apoptotic dose of UVB and allowed to incubate for defined experimentally established intervals. Our results show that there is an mTORC2-dependent regulation of FOXO3a expression, localization, and activity upon UVB-irradiation. Through western analysis we demonstrate that UVB exposure promotes the phosphorylation of FOXO3a by AKT in cells with intact mTORC2 signaling, which is repressed when mTORC2 is inhibited. This phosphorylation has been shown in other systems to reduce FOXO3a activity by causing a cytoplasmic localization of the protein. In agreement with this, we observed a rapid cytoplasmic localization of FOXO3a and decreased FOXO3a promoter activation in response to UVB, which is rescued following disruption of mTORC2 signaling. Additionally, we find that FOXO3a protein expression is reduced upon UVB-irradiation when mTORC2-signaling is reduced. These data support previous findings that suggest the mTORC2-driven FOXO3a cytoplasmic sequestration protects FOXO3a from dephosphorylation and degradation. Taken together, these studies provide strong evidence that mTORC2 plays a critical role in regulating FOXO3a expression, localization and activity following UVB-irradiation. In addition, our results support the hypothesis that the mTORC2-dependent UVB-induced apoptosis is due at least in part to FOXO3a. Supported by ES19242 (LMS) Citation Format: Robert P. Feehan, Lisa M. Shantz. The role of the mTORC2-dependent regulation of FOXO3a in UVB-induced apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 27. doi:10.1158/1538-7445.AM2015-27

CCN5/WISP-2 promotes growth arrest of triple-negative breast cancer cells through accumulation and trafficking of p27(Kip1) via Skp2 and FOXO3a regulation.

The matricellular protein CCN5/WISP-2 represents a promising target in triple-negative breast cancer (TNBC) because treatment or induced activation of CCN5 in TNBC cells promotes cell growth arrest at the G0/G1 phase, reduces cell proliferation and delays tumor growth in the xenograft model. Our studies found that the p27(Kip1) tumor suppressor protein is upregulated and relocalized to the nucleus from cytoplasm by CCN5 in these cells and that these two events (upregulation and relocalization of p27(Kip1)) are critical for CCN5-induced growth inhibition of TNBC cells. In the absence of CCN5, p27(Kip1) resides mostly in the cytoplasm, which is associated with the aggressive nature of cancer cells. Mechanistically, CCN5 inhibits Skp2 expression, which seems to stabilize the p27(Kip1) protein in these cells. On the other hand, CCN5 also recruits FOXO3a to mediate the transcriptional regulation of p27(Kip1). The recruitment of FOXO3a is achieved by the induction of its expression and activity through shifting from cytoplasm to the nucleus. Our data indicate that CCN5 blocks PI3K/AKT signaling to dephosphorylate at S318, S253 and Thr32 in FOXO3a for nuclear relocalization and activation of FOXO3a. Moreover, inhibition of α6β1 receptors diminishes CCN5 action on p27(Kip1) in TNBC cells. Collectively, these data suggest that CCN5 effectively inhibits TNBC growth through the accumulation and trafficking of p27(Kip1) via Skp2 and FOXO3a regulation, and thus, activation of CCN5 may have the therapeutic potential to kill TNBC.

Estrogen receptor β upregulates FOXO3a and causes induction of apoptosis through PUMA in prostate cancer.

Estrogen receptor β (ERβ) is emerging as a critical factor in understanding prostate cancer biology. Although reduced in prostate cancer above Gleason grade 3, ERβ is a potential drug target at the initial stage of the disease. In human prostate cancer cells, we found that ERβ causes apoptosis by increasing the expression of pro-apoptotic factor p53-upregulated modulator of apoptosis (PUMA), independent of p53, but dependent on the forkhead transcription factor class-O family member, FOXO3a. FOXO3a has previously been shown to induce PUMA after growth factor withdrawal and inhibition of the Akt pathway. Surprisingly, the phosphorylation of FOXO3a remained unchanged, while the mRNA and total protein levels of FOXO3a were increased in response to ERβ expression or treatment of PC3, 22Rv1 and LNCaP cells with the ERβ-specific ligands 3β-Adiol (5α-androstane-3β,17β-diol), DPN (diarylpropionitrile) or 8β-VE2 (8-vinylestra-1,3,5 (10)-triene-3,17β-diol). Knockdown of FOXO3a or ERβ expression abolished the increase of PUMA in response to 3β-Adiol in LNCaP and PC3 cells, suggesting that FOXO3a mediates the apoptotic effect of 3β-Adiol-activated ERβ. Moreover, the ventral prostate of ERβ-/- mice had decreased expression of FOXO3a and PUMA compared with the ERβ+/+ mice, indicating a relationship between ERβ and FOXO3a expression. The regulation of FOXO3a by ERβ in normal basal epithelial cells indicates a function of ERβ in cell differentiation and maintenance of cells in a quiescent state. In addition, the expression of ERβ, FOXO3a and PUMA is comparable and higher in benign prostatic hyperplasia than in prostate cancer Gleason grade 4 or higher, where there is substantial loss of ERβ, FOXO3a and PUMA. We conclude that ERβ induces apoptosis of prostate cancer cells by increasing transcription of FOXO3a, leading to an increase of PUMA and subsequent triggering of apoptosis via the intrinsic pathway involving caspase-9. Furthermore, we conclude that ligands specifically activating ERβ could be useful pharmaceuticals in the treatment of prostate cancer.

IKBKE phosphorylation and inhibition of FOXO3a: a mechanism of IKBKE oncogenic function.

Forkhead box O (FOXO) transcription factors are emerging as key regulators of cell survival and growth. The transcriptional activity and subcellular localization of FOXO are tightly regulated by post-translational modifications. Here we report that IKBKE regulates FOXO3a through phosphorylation of FOXO3a-Ser644. The phosphorylation of FOXO3a resulted in its degradation and nuclear-cytoplasmic translocation. Previous studies have shown that IKBKE directly activates Akt and that Akt inhibits FOXO3a by phosphorylation of Ser32, Ser253 and Ser315. However, the activity of Akt-nonphosphorytable FOXO3a-A3 (i.e., converting 3 serine residues to alanine) was inhibited by IKBKE. Furthermore, overexpression of IKBKE correlates with elevated levels of pFOXO3a-S644 in primary lung and breast tumors. IKBKE inhibits cellular function of FOXO3a and FOXO3a-A3 but, to a much less extent, of FOXO3a-S644A. These findings suggest that IKBKE regulates FOXO3a primarily through phosphorylation of SerS644 and that IKBKE exerts its cellular function, at least to some extent, through regulation of FOXO3a.

Abstract 843: Inhibition of AKT signaling promotes FOXO3a-dependent apoptosis in castration-resistant prostate cancer cells.

Abstract Treatment for aggressive prostate cancers involves hormonal ablation therapy, radical prostatectomy, radiation therapy, chemotherapy or in combination. Despite their initial sensitivity to these treatments, prostate cancer eventually progresses to castration -resistant prostate cancer (CRPC), which is both aggressive and refractory to current therapeutic modalities. Hence, CRPC demands the identification of novel targeted therapies with minimal side effects. We identified Withaferin-A (WA) a major bioactive compound derived from the medicinal plant, Withania somnifera, is extensively used in Asian herbal medicines to treat a variety of ailments, including cancer. Earlier, we reported that WA induced the pro-apoptotic gene, prostate apoptosis response-4 (Par-4) and apoptosis in CRPC cells. These results encouraged us to determine the mechanism of action of WA on CRPC cells. It is known that activated AKT negatively regulates forkhead transcription factor class O family protein (FOXO3a) that regulates genes involved with tumorigenesis, cell cycle arrest or apoptosis. In the present study, we found that FOXO3a is an upstream of Par-4 signaling which transactivates Par-4 function in CRPC cells. Inhibition of AKT activation by WA facilitates nuclear translocation of FOXO3a which in turn activates Par-4 mediated apoptosis in CRPC cells. Our confocal and immuno-precipitation studies confirmed that upon activation. FOXO3a and Par-4 were co-localized in the nucleus. Similar results were found with overexpression of FOXO3a showing transactivation of Par-4 and growth arrest in CRPC cells. Treatment of actinomycin-D or cyclohexmide along with WA, confirmed that FOXO3a regulation on Par-4 function in CRPC cells. On contrary, inhibition of FOXO3a by siRNA resulted in down regulation of Par-4 and apoptosis by WA in CRPC cells suggesting FOXO3a activation is necessary for the induction of Par-4 mediated apoptosis. Promoter bashing studies suggest a direct interaction of FOXO3a with Par-4 promoter; a sequential deletion of FOXO3a DNA binding sites in Par-4 promoter fails to induce Par-4 activation upon WA treatment in CRPC cells. Overall, our studies conclude that Par-4 is a target of FOXO3a, and Par-4 activation is required for induction of apoptosis in CRPC cells. Activation of FOXO3a is an attractive target for the treatment of CRPC and molecules such as WA can be explored further for the treatment of CRPC. Citation Format: Trinath P. Das, Chendil Damodaran. Inhibition of AKT signaling promotes FOXO3a-dependent apoptosis in castration-resistant prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 843. doi:10.1158/1538-7445.AM2013-843

Abstract 1715: Estrogen receptor β induces apoptosis through PUMA independent of p53 in prostate cancer cells.

Abstract Estrogen receptor β1’s (ERβ1) expression is reduced during the progression of prostate cancer. In agreement with several studies indicating a tumor suppressive effect of ERβ, we find that expression of ERβ increases apoptosis in the prostate cancer cells. Cells expressing ERβ showed an increase in apoptotic cell population by TUNEL assay and flow cytometric analysis. In addition, cells expressing ERβ showed an increase in sub G1 cell population compared to the control cells. Also, ERβ decreases the expression of anti-apoptotic factor Bcl-2 and increases the expression of the pro-apoptotic factor PUMA. Regulation of PUMA was found to be independent of p53. Although phosphorylation of AKT was decreased by ERβ expression we could not see any reduction of phosphorylated FOXO3A, a known regulator of PUMA. On the other hand total mRNA and protein of FOXO3A increased indicating a direct regulation of FOXO3A by ERβ. Using non-transfected PC3 and LNCaP cells we could observe induction of FOXO3A and PUMA as well as apoptosis in response to added 3β-adiol, DPN or 8β-VE2 indicating that the small amount of ERβ expressed in these cell lines are sufficient to induce apoptosis in response to ERβ specific ligands. Overall, our study indicates an apoptotic action of ERβ and suggests that it can be advantageous to treat prostate cancer with ERβ agonist. Citation Format: Prasenjit Dey, Jan-Ake Gustafsson, Anders M. Strom. Estrogen receptor β induces apoptosis through PUMA independent of p53 in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1715. doi:10.1158/1538-7445.AM2013-1715

Nuclear Localization Marker of FOXO3a: Can it be Used to Predict Doxorubicin Response?

Nuclear Localization Marker of FOXO3a: Can it be Used to Predict Doxorubicin Response?

Aberrant microRNA-182 expression is associated with glucocorticoid resistance in lymphoblastic malignancies

Glucocorticoid (GC) resistance in lymphoblastic malignancies is related to treatment failure and is a marker of poor prognosis. Previous studies have suggested that microRNA-182 (miR-182) functions as an oncogene and plays a role in tumorigenesis, through regulation of FOXO3A. FOXO3A has been implicated in tumor suppression and GC-induced apoptosis, suggesting that FOXO3A has potential as a therapeutic target. Herein we investigated the role of miR-182 in GC sensitivity in lymphoblastic malignancies. Expression of miR-182 was consistently higher in human and mouse GC-resistant cell lines than in GC-sensitive cell lines. Furthermore, increased expression of miR-182 reduced total FOXO3A expression but had no significant effect on phospho-FOXO3A. Additionally Bim, as a downstream target of FOXO3A, was reduced by overexpression of miR-182, and increased by down-regulation of miR-182. These results demonstrate that miR-182 is involved in glucocorticoid resistance, via targeting of FOXO3A, and that restoration of miR-182 is a potentially promising therapeutic strategy in lymphoblastic malignancies.