Chuan He Yang

Interferon α/β Promotes Cell Survival by Activating Nuclear Factor κB through Phosphatidylinositol 3-Kinase and Akt

Interferons (IFNs) play critical roles in host defense by modulating gene expression via activation of signal transducer and activator of transcription (STAT) factors. IFN-α/β also activates another transcription factor, nuclear factor κB (NF-κB), which protects cells against apoptotic stimuli. NF-κB activation requires the IFN-dependent association of STAT3 with the IFNAR1 chain of the IFN receptor. IFN-dependent NF-κB activation involves the sequential activation of a serine kinase cascade involving phosphatidylinositol 3-kinase (PI-3K) and Akt. Whereas constitutively active PI-3K and Akt induce NF-κB activation, Ly294002 (a PI-3K inhibitor), dominant-negative PI-3K, and kinase-dead Akt block IFN-dependent NF-κB activation. Moreover, dominant-negative PI-3K blocks IFN-promoted degradation of κBox α. Ly294002, a dominant-negative PI-3K construct, and kinase-dead Akt block IFN-promoted cell survival, enhancing apoptotic cell death. Therefore, STAT3, PI-3K, and Akt are components of an IFN signaling pathway that promotes cell survival through NF-κB activation.

IFN INDUCES MIR-21 THROUGH A SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 3-DEPENDENT PATHWAY AS A SUPPRESSIVE NEGATIVE FEEDBACK ON IFN-INDUCED APOPTOSIS

The microRNA miR-21 is overexpressed in many human cancers, wherein accumulating evidence indicates that it functions as an oncogene. Here, we report that the cytokine IFN rapidly induces miR-21 expression in human and mouse cells. Signal transducer and activator of transcription 3 (STAT3) was implicated in this pathway based on the lack of IFN effect on miR-21 expression in prostate cancer cells with a deletion in the STAT3 gene. STAT3 ablation abrogated IFN induction of miR-21, confirming the important role of STAT3 in regulating miR-21. Chromatin immunoprecipitation analysis showed that STAT3 directly bound the miR-21 promoter in response to IFN. Experiments in mouse embryo fibroblasts with a genetic deletion of the p65 NF-κB subunit showed that IFN-induced miR-21 expression was also dependent on NF-κB. STAT3 silencing blocked both IFN-induced p65 binding to the miR-21 promoter and p65 nuclear translocation. Thus, IFN-induced miR-21 expression is coregulated by STAT3 and NF-κB at the level of the miR-21 promoter. Several cell death regulators were identified as downstream targets of miR-21, including PTEN and Akt. Functional experiments in prostate cancer cells directly showed that miR-21 plays a critical role in suppressing IFN-induced apoptosis. Our results identify miR-21 as a novel IFN target gene that functions as a key feedback regulator of IFN-induced apoptosis.

MicroRNA miR-21 Regulates the Metastatic Behavior of B16 Melanoma Cells

Background: miRNAs are involved in many critical biological processes. Results: miR-21 induction is STAT3-dependent, and miR-21 knockdown inhibited melanoma cell proliferation and migration and enhanced apoptosis. Although B16 cells produced large lung metastases in mice, miR-21 knockdown cells only formed small lung lesions. Conclusion: miR-21 regulates the metastatic behavior of melanoma. Significance: miR-21 is identified as a potential drug target in melanoma. MicroRNA-21 (miR-21) is overexpressed in many human tumors and has been linked to various cellular processes altered in cancer. miR-21 is also up-regulated by a number of inflammatory agents, including IFN, which is of particular interest considering the close relationship between inflammation and cancer. Because miR-21 appears to be overexpressed in human melanoma, we examined the role of miR-21 in cancer development and metastasis in B16 mouse melanoma cells. We found that miR-21 is a member of an IFN-induced miRNA subset that requires STAT3 activation. To characterize the role of miR-21 in melanoma behavior, we transduced B16 cells with lentivirus encoding a miR-21 antagomir and isolated miR-21 knockdown B16 cells. miR-21 knockdown or IFN treatment alone inhibited B16 cell proliferation and migration in vitro, and in combination they had an enhanced effect. Moreover, miR-21 knockdown sensitized B16 cells to IFN-induced apoptosis. In B16 cells miR-21 targeted tumor suppressor (PTEN and PDCD4) and antiproliferative (BTG2) proteins. To characterize the role of miR-21 in vivo, empty vector- and antagomiR-21-transduced B16 melanoma cells were injected via tail vein into syngeneic C57BL/6 mice. Although empty vector-transduced B16 cells produced large lung metastases, miR-21 knockdown cells only formed small lung lesions. Importantly, miR-21 knockdown tumor-bearing mice exhibited prolonged survival compared with empty vector tumor-bearing mice. Thus, miR-21 regulates the metastatic behavior of B16 melanoma cells by promoting cell proliferation, survival, and migration/invasion as well as by suppressing IFN action, providing important new insights into the role of miR-21 in melanoma.

DNA Damage Induces NF-κB-dependent MicroRNA-21 Up-regulation and Promotes Breast Cancer Cell Invasion

Background: DNA damage response and miRNAs have been linked to cancer progression. Results: Genotoxic drug induces up-regulation of miR-21 in a NF-κB- and STAT3-dependent manner, which correlates with enhanced breast cancer cell invasion. Conclusion: Genotoxic NF-κB activation promotes breast cancer invasion via miR-21 induction. Significance: Genotoxic NF-κB signaling pathway may serve as a drug target to reduce therapeutic resistance and metastasis in breast cancer. NF-κB activation induced by genotoxic treatment in cancer cells has been associated with therapeutic resistance in multiple human malignancies. Therapeutic resistance also correlates with high metastatic potential in human cancers, including breast cancer. Whether genotoxic treatment-activated NF-κB also contributes to cancer metastasis following radiation and chemotherapy is unclear. Here, we show that chemotherapeutic drug-induced NF-κB activation promotes breast cancer cell migration and invasion. The increased metastatic potential is dependent on IL-6 induction mediated by genotoxic NF-κB activation. Moreover, genotoxic treatment also up-regulates oncogenic microRNA-21 (miR-21) expression through eliciting NF-κB recruitment to the miR-21 promoter region, where it cooperates with signal transducer and activator of transcription 3 (STAT3) to activate miR-21 transcription. DNA damage-induced histone H3 phosphorylation via activated MSK1 creates an open chromatin structure for NF-κB/STAT3-driven transactivation of miR-21. NF-κB-dependent IL-6 up-regulation is responsible for STAT3 activation and recruitment to the miR-21 promoter upon genotoxic stress. Induction of miR-21 may enable cancer cells to elude DNA damage-induced apoptosis and enhance the metastatic potential of breast cancer cells through repressing expression of PTEN and PDCD4. Our data support a critical role of DNA damage-induced NF-κB activation in promoting cancer metastasis following genotoxic treatment, and NF-κB-dependent miR-21 induction may contribute to both therapeutic resistance and metastasis in breast cancer.

Constitutive Activation of Signal Transducer and Activator of Transcription 3 (STAT3) and Nuclear Factor κB Signaling in Glioblastoma Cancer Stem Cells Regulates the Notch Pathway

Background: Glioma cancer stem cells (CSCs) are believed to drive tumorigenesis. Results: Glioma CSCs show constitutive activation of the STAT3/NF-κB signaling pathway and the Notch pathway. Conclusion: A novel relationship between glioma CSCs and the Notch pathway is defined, involving the constitutive activation of STAT3 and NF-κB signaling. Significance: The STAT3, NF-κB, and Notch pathways provide novel therapeutic targets to treat glioma. Malignant gliomas are locally aggressive, highly vascular tumors that have a dismal prognosis, and present therapies provide little improvement in the disease course and outcome. Many types of malignancies, including glioblastoma, originate from a population of cancer stem cells (CSCs) that are able to initiate and maintain tumors. Although CSCs only represent a small fraction of cells within a tumor, their high tumor-initiating capacity and therapeutic resistance drives tumorigenesis. Therefore, it is imperative to identify pathways associated with CSCs to devise strategies to selectively target them. In this study, we describe a novel relationship between glioblastoma CSCs and the Notch pathway, which involves the constitutive activation of STAT3 and NF-κB signaling. Glioma CSCs were isolated and maintained in vitro using an adherent culture system, and the biological properties were compared with the traditional cultures of CSCs grown as multicellular spheres under nonadherent culture conditions. Interestingly, both adherent and spheroid glioma CSCs show constitutive activation of the STAT3/NF-κB signaling pathway and up-regulation of STAT3- and NF-κB-dependent genes. Gene expression profiling also identified components of the Notch pathway as being deregulated in glioma CSCs, and the deregulated expression of these genes was sensitive to treatment with STAT3 and NF-κB inhibitors. This finding is particularly important because Notch signaling appears to play a key role in CSCs in a variety of cancers and controls cell fate determination, survival, proliferation, and the maintenance of stem cells. The constitutive activation of STAT3 and NF-κB signaling pathways that leads to the regulation of Notch pathway genes in glioma CSCs identifies novel therapeutic targets for the treatment of glioma.

The Curcumin Analog EF24 Targets NF-κB and miRNA-21, and Has Potent Anticancer Activity In Vitro and In Vivo

EF24 is a curcumin analog that has improved anticancer activity over curcumin, but its therapeutic potential and mechanism of action is unknown, which is important to address as curcumin targets multiple signaling pathways. EF24 inhibits the NF-κB but not the JAK-STAT signaling pathway in DU145 human prostate cancer cells and B16 murine melanoma cells. EF24 induces apoptosis in these cells apparently by inhibiting miR-21 expression, and also enhances the expression of several miR-21 target genes, PTEN and PDCD4. EF24 treatment significantly suppressed the growth of DU145 prostate cancer xenografts in immunocompromised mice and resulted in tumor regression. EF24 enhanced the expression of the miR-21 target PTEN in DU145 tumor tissue, but suppressed the expression of markers of proliferating cells (cyclin D1 and Ki67). In syngeneic mice injected with B16 cells, EF24 treatment inhibited the formation of lung metastasis, prolonged animal survival, inhibited miR-21 expression and increased the expression of miR-21 target genes. Expression profiling of miRNAs regulated by EF24 in vitro and in vivo showed that the antitumor activity of EF24 reflected the enhanced expression of potential tumor suppressor miRNAs as well as the suppressed expression of oncogenic miRNAs, including miR-21. Taken together, our data suggest that EF24 is a potent anticancer agent and selectively targets NF-κB signaling and miRNA expression, indicating that EF24 has significant potential as a therapeutic agent in various cancers.

Non‐conventional signal transduction by type 1 interferons: The NF‐κB pathway

Type I interferons (IFNs) regulate diverse cellular functions by modulating the expression of IFN‐stimulated genes (ISGs) through the activation of the well established signal transduction pathway of the Janus Kinase (JAK) and signal transducers and activators of transcription (STAT) proteins. Although the JAK–STAT signal transduction pathway is critical in mediating IFNs antiviral and antiproliferative activities, other signaling pathways are activated by IFNs and regulate cellular response to IFN. The NF‐κB transcription factor regulates the expression of genes involved in cell survival and immune responses. We have identified a novel IFN mediated signal pathway that leads to NF‐κB activation and demonstrate that a subset of ISGs that play key roles in cellular response to IFN is regulated by NF‐κB. This review focuses on the IFN‐induced NF‐κB activation pathway and the role of NF‐κB in ISG expression, antiviral activity and apoptosis, and the therapeutic application of IFN in cancer and infectious disease. J. Cell. Biochem. 102: 1087–1094, 2007.

The Short Form of the Interferon α/β Receptor Chain 2 Acts as a Dominant Negative for Type I Interferon Action

We have characterized the functional properties of the short form of the human interferon α/β receptor chain 2 (IFNAR2), denoted IFNAR2.1. IFNAR2.1 contains a shortened cytoplasmic domain when compared with the recently cloned full-length IFNAR2 chain (IFNAR2.2). We show that IFNα8 and IFNβ1b induce antiviral and antiproliferative activity in mouse cell transfectants expressing the human IFNAR1 chain of the receptor and induce the formation of STAT1/STAT2 dimers in IFN-stimulated response element (ISRE)-dependent gel shift assays. In contrast, coexpression of IFNAR2.1 with IFNAR1 reduces the IFN-induced antiviral, antiproliferative and ISRE-dependent gel shift binding activity conferred by IFNAR1 alone. No antiviral or antiproliferative response to IFN, nor IFN-induced ISRE-dependent gel shift binding activity, was observed when IFNAR2.1 was expressed alone in murine cells. Therefore, IFNAR2.1 acts as a dominant negative for these IFN-induced activities. Our results suggest that IFNAR2.1 represents a nonfunctional version of the full-length chain (IFNAR2.2).

MicroRNA-21 Promotes Glioblastoma Tumorigenesis by Down-regulating Insulin-like Growth Factor-binding Protein-3 (IGFBP3)

Background: miR-21 is overexpressed in many human cancers, including glioblastoma. Results: Insulin-like growth factor (IGF)-binding protein-3 (IGFBP3) is a novel miR-21 target gene and inhibits gliomagenesis in vitro and in vivo. Conclusion: miR-21 down-regulates IGFBP3, which acts as a tumor suppressor in human glioblastoma. Significance: IGFBP3 may have promise as a therapeutic target and prognostic marker for glioblastoma. Despite advances in surgery, imaging, chemotherapy, and radiation, patients with glioblastoma multiforme (GBM), the most common histological subtype of glioma, have an especially dismal prognosis; >70% of GBM patients die within 2 years of diagnosis. In many human cancers, the microRNA miR-21 is overexpressed, and accumulating evidence indicates that it functions as an oncogene. Here, we report that miR-21 is overexpressed in human GBM cell lines and tumor tissue. Moreover, miR-21 expression in GBM patient samples is inversely correlated with patient survival. Knockdown of miR-21 in GBM cells inhibited cell proliferation in vitro and markedly inhibited tumor formation in vivo. A number of known miR-21 targets have been identified previously. By microarray analysis, we identified and validated insulin-like growth factor (IGF)-binding protein-3 (IGFBP3) as a novel miR-21 target gene. Overexpression of IGFBP3 in glioma cells inhibited cell proliferation in vitro and inhibited tumor formation of glioma xenografts in vivo. The critical role that IGFBP3 plays in miR-21-mediated actions was demonstrated by a rescue experiment, in which IGFBP3 knockdown in miR-21KD glioblastoma cells restored tumorigenesis. Examination of tumors from GBM patients showed that there was an inverse relationship between IGFBP3 and miR-21 expression and that increased IGFBP3 expression correlated with better patient survival. Our results identify IGFBP3 as a novel miR-21 target gene in glioblastoma and suggest that the oncogenic miRNA miR-21 down-regulates the expression of IGFBP3, which acts as a tumor suppressor in human glioblastoma.

Induction of miRNA-181a by genotoxic treatments promotes chemotherapeutic resistance and metastasis in breast cancer

Acquired therapeutic resistance is the major drawback to effective systemic therapies for cancers. Aggressive triple-negative breast cancers (TNBC) develop resistance to chemotherapies rapidly, whereas the underlying mechanisms are not completely understood. Here we show that genotoxic treatments significantly increased the expression of miR-181a in TNBC cells, which enhanced TNBC cell survival and metastasis upon Doxorubicin treatment. Consistently, high miR-181a level associated with poor disease free survival and overall survival after treatments in breast cancer patients. The upregulation of miR-181a was orchestrated by transcription factor STAT3 whose activation depended on NF-κB-mediated IL-6 induction in TNBC cells upon genotoxic treatment. Intriguingly, activated STAT3 not only directly bound to MIR181A1 promoter to drive transcription but also facilitated the recruitment of MSK1 to the same region where MSK1 promoted a local active chromatin state by phosphorylating histone H3. We further identified BAX as a direct functional target of miR-181a, whose suppression decreased apoptosis and increased invasion of TNBC cells upon Dox treatment. These results were further confirmed by evidence that suppression of miR-181a significantly enhanced therapeutic response and reduced lung metastasis in a TNBC orthotopic model. Collectively, our data suggested that miR-181a induction had a critical role in promoting therapeutic resistance and aggressive behavior of TNBC cells upon genotoxic treatment. Antagonizing miR-181a may serve as a promising strategy to sensitize TNBC cells to chemotherapy and mitigate metastasis.