Nuclear Accumulation Of FOXO3a Research Articles

Abstract 5330: Nuclear protein export inhibitor selinexor chemotherapy combination for pancreatic cancer therapy

Abstract Background: The majority of pancreatic ductal adenocarcinoma (PDAC) patients experience disease progression after Gemcitabine-nab-Paclitaxel (GemPac) treatment indicating the need for more effective combination therapies for this highly recalcitrant disease. Earlier we showed that nuclear exporter protein exportin 1 (XPO1) is a valid therapeutic target in PDAC and selective inhibitor of nuclear export (SINE) selinexor (Sel), synergistically enhances the efficacy of GemPac in pancreatic cancer cells, spheroids, patient derived tumors and showed evaluable response in PDAC patients in a Phase I study. Here we investigated the mechanisms of this synergy utilizing PDAC cellular models in vitro and LSL-Kras G12D/+; Trp53 fl/+; Pdx1-Cre (KPC) mouse model. Methods: We used single-nuclei RNA sequencing in KPC tumors, and poly A RNA sequencing in PDAC cells post sel-Gem-Pac treatment. Prioritized sn-seq and RNA-seq identified molecules were validated in in vitro or in the PDAC patient samples through siRNA mediated silencing, quantitative gene expression, cytotoxicity and related assays. Results: In KPC model, selinexor-GemPac caused statistically significant enhancement in survival compared to controls (p less than 0.05). Molecular analysis of residual KPC tumors showed re-organization of tumor stromal architecture, suppression of proliferation and nuclear retention of tumor suppressors. Histopathological analysis for Ki67 suggests comparatively lower rate of proliferation and enhanced nuclear accumulation of FOXO3a in the treated mice. Loosening of stroma and lowering of collagen fiber is also evident in treated mice from Picrosirius and Masson’s trichrome staining. Additionally, the KPC derived primary tumor cells showed reduced colony formation upon combination treatment. Sn-seq showed reduction in tumor cell heterogeneity (less cell clusters based on gene expression), CDK8, CD44 and Camk1d stem cell and EMT related genes alongside significant alterations in mitochondrial signaling after combination treatment. In cellular models, GO and GSEA analyses of RNA-seq data predicted DNA templated regulation of transcription and DNA replication associated genes in Sel or Sel-GemPac mediated growth inhibition. The top downregulated genes including drug resistant related gene PFN1 and HSP90 identified in Sel or Sel-GemPac treated PDAC cells from RNA-seq data were validated using RNAi and confirmed their role in synergy. Conclusions: Taken together Selinexor in combination with Gemcitabine-nab-Paclitaxel enhanced the survival of KPC mice through inhibition of well-connected tumor promoting molecules resulting in broad penetration in PDAC supporting signaling. A phase II study involving selinexor-gemcitabine combination in patients with metastatic PDAC is ongoing. Citation Format: Md. Hafiz Uddin, Sahar Bannoura, Yiwei Li, Husain Y. Khan, Amro Aboukameel, Mohammed N. Al-Hallak, Rafic Beydoun, Steve Kim, Yosef Landesman, Ramzi M. Mohammad, Anthony Shields, Asfar S. Azmi. Nuclear protein export inhibitor selinexor chemotherapy combination for pancreatic cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5330.

Resistance to BTK inhibition by ibrutinib can be overcome by preventing FOXO3a nuclear export and PI3K/AKT activation in B-cell lymphoid malignancies

Chronic activation of the Bruton’s tyrosine kinase (BTK)-mediated B-cell receptor (BCR) signaling is a hallmark of many B-cell lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL). Ibrutinib, an FDA approved, orally administered BTK inhibitor, has demonstrated high response rates, however, complete responses are infrequent and acquired resistance to BTK inhibition can emerge. In this study, we generated ibrutinib-resistant (IB-R) cell lines by chronic exposure of CLL and activated B-cell (ABC)-DLBCL cells to ibrutinib in order to investigate the mechanism of acquired resistance to ibrutinib. IB-R cell lines demonstrated downregulation of FOXO3a and PTEN levels and activation of AKT, with their levels being low in the nuclei of resistant cells in comparison to the sensitive counterparts. Inhibition of PI3K and AKT using idelalisib and MK2206, respectively increased ibrutinib-induced apoptosis in IB-R cells by downregulation of pAKT473 and restoring FOXO3a levels, demonstrating the importance of these cell survival factors for ibrutinib-resistance. Notably, the exportin 1 inhibitor, selinexor synergized with ibrutinib in IB-R cells and restored nuclear abundance of FOXO3a and PTEN, suggesting that nuclear accumulation of FOXO3a and PTEN facilitates increase in ibrutinib-induced apoptosis in IB-R cells. These data demonstrate that reactivation of FOXO3a nuclear function enhances the efficacy of ibrutinib and overcomes acquired resistance to ibrutinib. Together, these findings reveal a novel mechanism that confers ibrutinib resistance via aberrant nuclear/cytoplasmic subcellular localization of FOXO3a and could be exploited by rational therapeutic combination regimens for effectively treating lymphoid malignancies.

Hippo kinases regulate cell junctions to inhibit tumor metastasis in response to oxidative stress

Reactive oxygen species (ROS) are key regulators in cell proliferation, survival, tumor initiation and development. However, the role of ROS in tumor metastasis is less clear. Here, we show that oxidative stress inhibited tumor metastasis via activation of Hippo kinase MST1/2, which led to the phosphorylation and nuclear accumulation of FoxO3a, resulting in upregulation of ΔNp63α expression and suppression of cell migration independent of YAP. Strikingly, while loss of MST1 led to and disruption of cell-cell junction exemplified by reduced E-cadherin expression, resulting in scattered cell growth, loss of MST2 led to disruption of cell-matrix adhesion as evidenced by reduced integrin β4, resulting in increased cell migration and tumor metastasis. Furthermore, expression of MST1 and MST2 was down-regulated in human breast carcinoma. Furthermore, oxidative stress inhibited HER2-or PI3K-mediated tumor metastasis via the MST2-FoxO3a-ΔNp63α pathway. Together, these results that this noncanonical Hippo MST2-FoxO3a-ΔNp63α pathway may play a critical role in ROS-mediated regulation of cell migration and tumor metastasis.

Suppression of FoxO3a attenuates neurobehavioral deficits after traumatic brain injury through inhibiting neuronal autophagy

Traumatic brain injury (TBI) is a serious insult that frequently leads to neurological impairments. Forkhead box O (FoxO) 3a, as transcription factor, has been confirmed to modulate autophagic process. Moreover, FoxO3a is expressed throughout the brain including the hippocampus. However, the role of FoxO3a in the pathophysiology of TBI is unclear. The present study is designed to investigate whether FoxO3a has the neuroprotective effects on rats subjected to TBI, and further to explore the potential molecular mechanisms. Thus, a rat model of TBI was created by using a modified weight-drop device to mimic the insults of TBI. The results showed that FoxO3a was significantly increased in the serum of patients with TBI as well as in experimental animals. Furthermore, our data also demonstrated that TBI stimulated the translocation of FoxO3a from the cytosol to the nucleus. Additionally, we found that knockdown of FoxO3a by siRNA silencing significantly improved neurobehavioral dysfunctions and conferred a better neuroprotective effects after TBI, evidenced by promoting motor behavioral recovery, attenuating learning and memory impairments, and partially reversing neuronal damage in the hippocampus. To further investigate the molecular mechanisms underlying this neuroprotection, we identified that nuclear accumulation of Foxo3a could induce highly expression of autophagy pathway genes including LC-3, Beclin-1, p62, ATG12, and ATG14, and finally initiate neurological impairments. Interestingly, silencing FoxO3a by siRNA remarkably inhibited the induction of neuronal autophagy after TBI, and activated autophagy was closely related to TBI-induced neurological deficits. Taken together, these findings indicated that FoxO3a knockdown conferred neuroprotective effects after TBI through inhibiting the activation of neuronal autophagy.

Inactivation of the FoxO3a transcription factor is associated with the production of reactive oxygen species during protein kinase CK2 downregulation-mediated senescence in human colon cancer and breast cancer cells

We previously showed that protein kinase CK2 downregulation mediates senescence through the reactive oxygen species (ROS)–p53–p21Cip1/WAF1 pathway in various human cells. In the present study, we investigated whether the FoxO3a transcription factor is associated with ROS production during CK2 downregulation-induced senescence in human colon cancer HCT116 and breast cancer MCF-7 cells. FoxO3a overexpression suppressed ROS production and p53 stabilization induced by a CK2α knockdown. CK2α downregulation induced nuclear export of FoxO3a through stimulation of AKT-mediated phosphorylation of FoxO3a and decreased transcription of its target genes (Cu/ZnSOD, MnSOD, and catalase). In contrast, CK2α overexpression inhibited AKT-mediated FoxO3a phosphorylation. This resulted in nuclear accumulation of FoxO3a, and elevated expression of its target genes. Therefore, these data indicate for the first time that CK2 downregulation stimulates ROS generation by inhibiting FoxO3a during premature senescence in human colon and breast cancer cells.

PTEN-regulated AKT/FoxO3a/Bim signaling contributes to Human cell glioblastoma apoptosis by platinum-maurocalcin conjugate

A previous report has shown that a chimera between a platinum complexing agent (1) and the cell penetrating peptide maurocalcin, synthesized with D-amino acids, (DMCa), termed Pt-1-DMCa, is a highly successful anticancer compound that works by targeting the intracellular redox system in glioblastoma (GBM) cells. However, the detailed cellular mechanism whereby the conjugate specifically kills tumor cells remains unclear. Herein, we show that Pt-1-DMCa induces apoptosis in Human U87 GBM cells through reactive oxygen species (ROS)-dependent modulation of the PI3K/AKT/FoxO3a signalling pathway. First, we found that Pt-1-DMCa treatment of these cells induces inhibition of AKT and nuclear accumulation of FoxO3a thereby facilitating transcription of the target genes Bim and PTEN. Modulation of the AKT/FoxO3a/Bim signaling pathway by RNA interference confirms that these signaling events are critical for Pt-1-DMCa-induced apoptosis of U87 GBM cells. Furthermore, we reveal that FoxO3a-mediated up-regulation of PTEN exerts an additional inhibitory effect on the AKT survival pathway. Thus, our results demonstrate that the conjugate can induce ROS-dependent FoxO3a-mediated apoptosis in U87 cells through PTEN-mediated inhibition of the PI3K/AKT survival axis. Our results help elucidate the molecular mechanisms underlying Pt-1-DMCa-induced cell death in U87 GBM cells and support a theoretical basis for future applications of the MCa peptide.

Foxo3a Transcriptionally Upregulates AQP4 and Induces Cerebral Edema Following Traumatic Brain Injury

Increased cranial pressure due to development of edema contributes significantly to the pathology of traumatic brain injury (TBI). Induction of an astrocytic water channel protein, Aquaporin 4 (AQP4), is known to predominantly contribute to cytotoxic edema following TBI. However, the mechanism for the increase in AQP4 following 24 h of TBI is poorly understood. Here we show that transcriptional activation of a ubiquitously expressed mammalian forkhead transcription factor, Foxo3a, induces cerebral edema by increasing the AQP4 level in the controlled cortical impact model of TBI in mice. TBI stimulates nuclear translocation of Foxo3a in astrocytes and subsequently augments its binding to AQP4 promoter in pericontusional cortex. Nuclear accumulation of Foxo3a is augmented by a decrease in phosphorylation at its Ser256 residue due to inactivation of Akt after TBI. Depletion of Foxo3a in mice rescues cytotoxic edema by preventing induction of AQP4 as well as attenuates memory impairment after TBI in mice.

PTEN-regulated AKT/FoxO3a/Bim signaling contributes to reactive oxygen species-mediated apoptosis in selenite-treated colorectal cancer cells

Mounting evidence shows that selenium possesses chemotherapeutic potential against tumor cells, including leukemia, prostate cancer and colorectal cancer (CRC) cells. However, the detailed mechanism by which sodium selenite specifically kills tumor cells remains unclear. Herein, we demonstrated that supranutritional doses of selenite-induced apoptosis in CRC cells through reactive oxygen species (ROS)-dependent modulation of the PI3K/AKT/FoxO3a signaling pathway. First, we found that selenite treatment in HCT116 and SW480 CRC cells caused inhibition of AKT and the nuclear accumulation of FoxO3a by western blot and immunofluorescence analyses, respectively, thereby facilitating transcription of the target genes bim and PTEN. Modulation of the AKT/FoxO3a/Bim signaling pathway by chemical inhibitors or RNA interference revealed that these events were critical for selenite-induced apoptosis in CRC cells. Additionally, we discovered that FoxO3a-mediated upregulation of PTEN exerted a further inhibitory effect on the AKT survival pathway. We also corroborated our findings in vivo by performing immunohistochemistry experiments. In summary, our results show that selenite could induce ROS-dependent FoxO3a-mediated apoptosis in CRC cells and xenograft tumors through PTEN-mediated inhibition of the PI3K/AKT survival axis. These results help to elucidate the molecular mechanisms underlying selenite-induced cell death in tumor cells and provide a theoretical basis for translational applications of selenium.

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?

Serotonin promotes the proliferation of serum-deprived hepatocellular carcinoma cells via upregulation of FOXO3a

BackgroundPeripheral serotonin is involved in tumorigenesis and induces a pro-proliferative effect in hepatocellular carcinoma (HCC) cells; however, the intracellular mechanisms by which serotonin exerts a mitogenic effect remain unclear. In this research, we examined whether FOXO3a, a transcription factor at the interface of crucial cellular processes, plays a role downstream of serotonin in HCC cells.ResultsThe cell viability and expression of FOXO3a was assessed in three HCC cell lines (Huh7, HepG2 and Hep3B) during serum deprivation in the presence or absence of serotonin. Serum free media significantly inhibited HCC proliferation and led to reduced expression and nuclear accumulation of FOXO3a. Knockdown of FOXO3a enhanced the ability of serum deprivation to inhibit HCC cells proliferation. And overexpression of non-phosphorylated FOXO3a in HCC cells reversed serum-deprivation-induced growth inhibition. Serotonin reversed the serum-deprivation-induced inhibition of cell proliferation and upregulated FOXO3a in Huh7 cells; however, serotonin had no effect on the proliferation of serum-deprived HepG2 or Hep3B cells. In addition to proliferation, serotonin also induced phosphorylation of AKT and FOXO3a in serum-deprived Huh7 cells but not in HepG2 and Hep3B cells. However, the phosphorylation of FOXO3a induced by serotonin did not export FOXO3a from nucleus to cytoplasm in serum-deprived Huh7 cells. Consequently, we demonstrated that serotonin promoted the proliferation of Huh7 cells by increasing the expression of FOXO3a. We also provide preliminary evidence that different expression levels of the 5-HT2B receptor (5-HT2BR) may contribute to the distinct effects of serotonin in different serum-deprived HCC cells.ConclusionsThis study demonstrates that FOXO3a functions as a growth factor in serum-deprived HCC cells and serotonin promotes the proliferation of serum-deprived HCC cells via upregulation of FOXO3a, in the presence of sufficient levels of the serotonin receptor 5-HT2BR. Drugs targeting the serotonin-5-HT2BR-FOXO3a pathway may provide a novel target for anticancer therapy.

Constitutively nuclear FOXO3a localization predicts poor survival and promotes Akt phosphorylation in breast cancer.

BackgroundThe 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 FindingsExamination 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/SignificanceTogether 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.

The Forkhead Transcription Factor FOXO3a Increases Phosphoinositide-3 Kinase/Akt Activity in Drug-Resistant Leukemic Cells through Induction of PIK3CA Expression

The phosphoinositide-3 kinase (PI3K)/Akt signal pathway plays a key role in the tumorigenesis of many cancers and in the subsequent development of drug resistance. Using the K562 chronic myelogenous leukemia (CML) cell line and the doxorubicin-resistant derivative lines KD30 and KD225 as models, we observed that enhanced PI3K/Akt activity and the acquisition of chemoresistance correlated unexpectedly with the increased expression and nuclear accumulation of FOXO3a. Moreover, we found that the induction of FOXO3a activity in naïve K562 cells was sufficient to enhance PI3K/Akt activity and to confer resistance to the cytotoxic effects of doxorubicin. Conversely, the knockdown of endogenous FOXO3a expression reduced PI3K/Akt activity and sensitized these cells to doxorubicin. Further chromatin immunoprecipitation and promoter mutation analyses demonstrated that FOXO3a regulates the expression of the PI3K catalytic subunit p110alpha through the activation of a promoter region proximal to a novel untranslated exon upstream from the reported transcription start site of the p110alpha gene PIK3CA. As was the case for FOXO3a, the expression or knockdown of p110alpha was sufficient to amplify or reduce PI3K/Akt activity, respectively. Thus, our results suggest that the chronic activation of FOXO3a by doxorubicin in CML cells can enhance survival through a feedback mechanism that involves enhanced p110alpha expression and hyperactivation of the PI3K/Akt pathway.

Significance of Nuclear Accumulation of Foxo3a in Esophageal Squamous Cell Carcinoma

To investigate the value of Foxo3a in predicting the response to neoadjuvant treatment of, and prognosis for, esophageal squamous cell carcinoma. Immunohistochemical staining was performed in a retrospective series of 60 biopsied esophageal squamous cell carcinomas, and the correlation between nuclear accumulation of Foxo3a and clinicopathologic features was analyzed, including patient survival. In addition, in vitro biologic changes, radiosensitivity, and in vivo tumorigenicity of esophageal carcinoma cells after experimental manipulation of Foxo3a expression levels were determined. Clinical findings point to a significant correlation between the nuclear accumulation of Foxo3a and the survival rate of esophageal cancer patients. In addition, Foxo3a is a significant predictor for the response to neoadjuvant therapy. In cell culture, irradiation and oxidative stress seemed to result in nuclear accumulation of Foxo3a. Down-regulation of Foxo3a significantly decreased radiosensitivity but had no obvious effect on tumor growth, as measured by a clonogenic assay in vitro and growth delay in vivo. Nuclear accumulation of Foxo3a in tumor cells was correlated with increased radiosensitivity and with improved patient survival. Thus, it is suggested that Foxo3a may be a potential marker for esophageal cancer.

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.

Regulation of FOXO3a by brain-derived neurotrophic factor in differentiated human SH-SY5Y neuroblastoma cells

FOXO3a is a ubiquitously expressed mammalian forkhead transcription factor with a high expression level in adult brain. The activity of FOXO3a is inhibited by growth factors through activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling, which phosphorylates FOXO3a and decreases the level of FOXO3a in the nucleus. In the present study, we examined the regulation of FOXO3a by brain-derived neurotrophic factor (BDNF) in retinoic acid (RA)-differentiated human SH-SY5Y neuroblastoma cells. BDNF caused a rapid and time-dependent decrease of nuclear FOXO3a with a corresponding increase of cytosolic FOXO3a. The rate of the BDNF-induced nuclear/cytosolic redistribution was consistent with the time course of BDNF-induced threonine 32-phosphorylation of FOXO3a, and was mediated by the PI3K/Akt signaling pathway. Active FOXO3a rapidly increased the level of Bcl-2-interacting mediator (bim) in differentiated SH-SY5Y cells, and BDNF decreased the FOXO3a-induced increase of bim through activation of both PI3K/Akt and Erk signaling pathways. Thapsigargin, an endoplasmic reticulum (ER) stress-inducing agent, significantly decreased threonine 32-phosphorylation of FOXO3a, and increased nuclear and decreased cytosolic FOXO3a, suggesting that thapsigargin activates FOXO3a. Treatment with BDNF completely reversed and blocked the thapsigargin-induced dephosphorylation and nuclear accumulation of FOXO3a. In addition, protein phosphatase 1/2A inhibitors increased threonine 32-phosphorylation of FOXO3a, decreased nuclear FOXO3a, and blocked thapsigargin-induced activity of FOXO3a. The regulatory effect of BDNF on FOXO3a and its target genes may play a significant role in the BDNF-mediated neuronal survival, differentiation, and plasticity.