STAT3 is required for proliferation and maintenance of multipotency in glioblastoma stem cells.

Signal transducer and activator of transcription 3 (STAT3) regulates diverse cellular processes, including cell growth, differentiation, and apoptosis, and is frequently activated during tumorigenesis. Recently, putative glioblastoma stem cells (GBM-SCs) were isolated and characterized. These cells can self-renew indefinitely in culture, are highly tumorigenic, and retain the ability to differentiate in culture. We have found that treatment of GBM-SCs with two chemically distinct small molecule inhibitors of STAT3 DNA-binding inhibits cell proliferation and the formation of new neurospheres from single cells. Genetic knockdown of STAT3 using a short hairpin RNA also inhibits GBM-SC proliferation and neurosphere formation, confirming that these effects are specific to STAT3. Although STAT3 inhibition can induce apoptosis in serum-derived GBM cell lines, this effect was not observed in GBM-SCs grown in stem cell medium. Markers of neural stem cell multipotency also decrease upon STAT3 inhibition, suggesting that STAT3 is required for maintenance of the stem-like characteristics of these cells. Strikingly, even a transient inhibition of STAT3 leads to irreversible growth arrest and inhibition of neurosphere formation. These data suggest that STAT3 regulates the growth and self-renewal of GBM-SCs and is thus a potential target for cancer stem cell-directed therapy of glioblastoma multiforme.

A Large-Scale RNAi Screen Identifies SGK1 as a Key Survival Kinase for GBM Stem Cells.

Glioblastoma multiforme (GBM) is the most common type of primary malignant brain cancer and has a very poor prognosis. A subpopulation of cells known as GBM stem-like cells (GBM-SC) have the capacity to initiate and sustain tumor growth and possess molecular characteristics similar to the parental tumor. GBM-SCs are known to be enriched in hypoxic niches and may contribute to therapeutic resistance. Therefore, to identify genetic determinants important for the proliferation and survival of GBM stem cells, an unbiased pooled shRNA screen of 10,000 genes was conducted under normoxic as well as hypoxic conditions. A number of essential genes were identified that are required for GBM-SC growth, under either or both oxygen conditions, in two different GBM-SC lines. Interestingly, only about a third of the essential genes were common to both cell lines. The oxygen environment significantly impacts the cellular genetic dependencies as 30% of the genes required under hypoxia were not required under normoxic conditions. In addition to identifying essential genes already implicated in GBM such as CDK4, KIF11, and RAN, the screen also identified new genes that have not been previously implicated in GBM stem cell biology. The importance of the serum and glucocorticoid-regulated kinase 1 (SGK1) for cellular survival was validated in multiple patient-derived GBM stem cell lines using shRNA, CRISPR, and pharmacologic inhibitors. However, SGK1 depletion and inhibition has little effect on traditional serum grown glioma lines and on differentiated GBM-SCs indicating its specific importance in GBM stem cell survival.Implications: This study identifies genes required for the growth and survival of GBM stem cells under both normoxic and hypoxic conditions and finds SGK1 as a novel potential drug target for GBM. Mol Cancer Res; 16(1); 103-14. ©2017 AACR.

Signal transducer and activator of transcription 3 (STAT3) regulates human telomerase reverse transcriptase (hTERT) expression in human cancer and primary cells.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that plays a critical role in cytokine and growth factor signaling and is frequently activated in human tumors. Human telomerase reverse transcriptase (hTERT) is also often overexpressed in tumor cells and mediates cellular immortalization. Here we report that STAT3 directly regulates the expression of hTERT in a variety of human cancer cells. Moreover, STAT3 activity is required for the survival of many human tumors, and hTERT expression contributes to the survival of STAT3-dependent tumor cells. In addition, we find that growth factors and cytokines stimulate hTERT expression in primary human cells in a STAT3-dependent manner. Thus, STAT3 is a key regulator of hTERT expression in both normal and tumor cells.

Regulation of the JMJD3 (KDM6B) histone demethylase in glioblastoma stem cells by STAT3.

The growth factor and cytokine regulated transcription factor STAT3 is required for the self-renewal of several stem cell types including tumor stem cells from glioblastoma. Here we show that STAT3 inhibition leads to the upregulation of the histone H3K27me2/3 demethylase Jmjd3 (KDM6B), which can reverse polycomb complex-mediated repression of tissue specific genes. STAT3 binds to the Jmjd3 promoter, suggesting that Jmjd3 is a direct target of STAT3. Overexpression of Jmjd3 slows glioblastoma stem cell growth and neurosphere formation, whereas knockdown of Jmjd3 rescues the STAT3 inhibitor-induced neurosphere formation defect. Consistent with this observation, STAT3 inhibition leads to histone H3K27 demethylation of neural differentiation genes, such as Myt1, FGF21, and GDF15. These results demonstrate that the regulation of Jmjd3 by STAT3 maintains repression of differentiation specific genes and is therefore important for the maintenance of self-renewal of normal neural and glioblastoma stem cells.

5-HT induction of c-fos gene expression requires reactive oxygen species and Rac1 and Ras GTPases.

Serotonin (5-HT) stimulates superoxide release, phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK), and DNA synthesis in bovine pulmonary artery smooth muscle cells. Both p42/p44 MAPK and reactive oxygen species (ROS) generation are required for 5-HT-induced growth in SMC. Agents that block the production of ROS, or ROS scavengers, block MAPK activation by 5-HT. However, specific signal transduction by 5-HT leading to proteins that control entrance into the cell cycle are not well defined in smooth muscle cells. Here, we show by Western blot that 5-HT upregulates c-Fos, an immediate early gene product known to regulate the entrance of quiescent cells into the cell cycle. Northern blots showed that c-fos mRNA is induced by 5-HT in 30 min. This induction is blocked by PD98059, indicating that activation of MAPK is required. 5-HT-induced expression of a 350 bp c-fos promoter in a luciferase reporter is blocked by PD98059 and diphenyliodonium (DPI). The GTPases Rac1 and Ras have been implicated in growth factor-induced generation of ROS. Overexpression of either dominant negative (DN) Rac1 or DN Ras inhibited 5-HT-mediated c-fos promoter activation. 5-HT also induced expression from a truncated c-fos promoter containing an isolated serum response element. This activation was blocked by DPI and PD98059. Overexpression of activated Ras and Rac1 were additive for activation of the serum response element promoter. Regulation of cyclin D1, a protein shown to be regulated by c-fos and required for entry into the cell cycle, is upregulated by 5-HT and is blocked by DPI and PD98059. Nuclear factor-kappaB, which can also regulate cyclin D1, was not activated. We conclude that 5-HT stimulates c-fos and cyclin D1 expression through a ROS-dependent mechanism that requires Ras, Rac1, and MAPK.

Knockdown of STAT3 expression by RNAi induces apoptosis in astrocytoma cells.

BACKGROUND: Astrocytomas are the most common type of primary central nervous system tumors. They are frequently associated with genetic mutations that deregulate cell cycle and render these tumors resistant to apoptosis. STAT3, signal transducer and activator of transcription 3, participates in several human cancers by inducing cell proliferation and inhibiting apoptosis and is frequently activated in astrocytomas. METHODS: RNA interference was used to knockdown STAT3 expression in human astrocytes and astrocytoma cell lines. The effect of STAT3 knockdown on apoptosis, cell proliferation, and gene expression was then assessed by standard methods. RESULTS: We have found that STAT3 is constitutively activated in several human astrocytoma cell lines. Knockdown of STAT3 expression by siRNA induces morphologic and biochemical changes consistent with apoptosis in several astrocytoma cell lines, but not in primary human astrocytes. Moreover, STAT3 is required for the expression of the antiapoptotic genes survivin and Bcl-xL in the A172 glioblastoma cell line. CONCLUSION: These results show that STAT3 is required for the survival of some astrocytomas. These studies suggest STAT3 siRNA could be a useful therapeutic agent for the treatment of astrocytomas.

Clinically relevant doses of chemotherapy agents reversibly block formation of glioblastoma neurospheres.

Glioblastoma patients have a poor prognosis, even after surgery, radiotherapy, and chemotherapy with temozolomide or 1,3-bis(2-chloroethy)-1-nitrosourea. We developed an in vitro recovery model using neurosphere cultures to analyze the efficacy of chemotherapy treatments, and tested whether glioblastoma neurosphere-initiating cells are resistant. Concentrations of chemotherapy drugs that inhibit neurosphere formation are similar to clinically relevant doses. Some lines underwent a transient cell cycle arrest and a robust recovery of neurosphere formation. These results indicate that glioblastoma neurospheres can regrow after treatment with chemotherapy drugs. This neurosphere recovery assay will facilitate studies of chemo-resistant subpopulations and methods to enhance glioblastoma therapy.

Haploid genetic screens in human cells identify host factors used by pathogens.

Loss-of-function genetic screens in model organisms have elucidated numerous biological processes, but the diploid genome of mammalian cells has precluded large-scale gene disruption. We used insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8. Using this approach, we identified host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects of diphtheria toxin and exotoxin A. We also identified genes needed for the action of cytolethal distending toxin, including a cell-surface protein that interacts with the toxin. This approach has both conceptual and practical parallels with genetic approaches in haploid yeast.

PDGF-induced human airway smooth muscle cell proliferation requires STAT3 and the small GTPase Rac1.

The signal transducers and activators of transcription (STAT) family of transcription factors regulates a variety of biological functions including cellular proliferation, transformation, apoptosis, and differentiation. We have previously determined that PDGF activates the STAT pathway in human airway smooth muscle cells (HASMC) and that the Jak and Src kinases are required for both PDGF-induced STAT activation and HASMC proliferation. As increased airway smooth muscle (ASM) volume is associated with airflow obstruction and disease severity in patients with asthma, it is important to elucidate the cellular and molecular pathways that regulate ASM accumulation. In this paper, we investigated the requirement of STAT3 for PDGF-induced HASMC proliferation. We demonstrate that knockdown of STAT3 expression in HASMC resulted in a significant decrease in mitogen-induced cellular proliferation. Additionally, PDGF-induced activation of STAT3 required the small GTP-binding protein Rac1, and Rac1 was also required for PDGF-induced HASMC proliferation. Furthermore, PDGF treatment induced STAT3 and Rac1 to physically associate and translocate to the nucleus, identifying one mechanism by which STAT3 is regulated in response to PDGF in HASMC. Finally, we determined that STAT3 expression is required for PDGF-mediated regulation of cell cycle targets cyclin D3 and p27. These data define a novel mitogenic signaling pathway in airway smooth muscle cells leading from PDGF to Rac1 and STAT3 and subsequent cell cycle gene regulation. Thus, targeting STAT3 may prove to be a novel therapeutic approach for patients with severe asthma and significant airway wall remodeling, as manifested by ASM accumulation.

Inhibition of the Src and Jak kinases protects against lipopolysaccharide-induced acute lung injury.

The cascade of cellular and molecular pathways mediating acute lung injury is complex and incompletely defined. Although the Src and Jak family of kinases is upregulated in LPS-induced murine lung injury, their role in the development of lung injury is unknown. Here we report that systemic inhibition of these kinases using specific small molecule inhibitors (PP2, SU6656, tyrphostin A1) significantly attenuated LPS-induced lung injury, as determined by histologic and capillary permeability assays. These inhibitors blocked LPS-dependent cytokine and chemokine production in the lung and in the serum. In contrast, lung-targeted inhibition of these kinases in the airway epithelium via adenoviral-mediated gene transfer of dominant negative Src or of suppressor of cytokine signaling (SOCS-1) disrupted lung cytokine production but had no effect on systemic cytokine production or lung vascular permeability. Mice were significantly protected from lethal LPS challenge by the small molecule inhibitors of Jak and Src kinase. Importantly, this protection was still evident even when the inhibitors were administered 6 hours after LPS challenge. Taken together, these observations suggest that Jak and Src kinases participate in acute lung injury and verify the potential of this class of selective tyrosine kinase inhibitors to serve as novel therapeutic agents for this disease.

Role of the JAK-STAT pathway in PDGF-stimulated proliferation of human airway smooth muscle cells.

Airway remodeling, as manifested by an increase in airway smooth muscle mass, mucous gland hyperplasia, and subepithelial fibrosis, contributes to the airway hyperresponsiveness and fixed obstruction seen in some asthmatic patients. Here we investigated whether the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway contributes to platelet-derived growth factor (PDGF)-stimulated mitogenesis of human airway smooth muscle cells (HASMC). PDGF treatment of quiescent HASMC resulted in the rapid tyrosine phosphorylation and DNA binding of STAT1 and STAT3. This phosphorylation was blocked by inhibition of Src and JAK2 kinases. In addition, STAT activation by PDGF was found to be redox dependent. Moreover, PDGF-induced thymidine uptake was completely blocked by pretreatment of HASMC with the STAT kinase inhibitors AG-490, SU-6656, and PP2. Interestingly, the JAK pathway was required for HASMC mitogenesis independently of mitogen-activated protein kinase activation. Inhibition of the Src and JAK kinases blocked PDGF-stimulated gene expression of the STAT target genes cyclin D1 and c-myc. These results indicate that the JAK-STAT pathway contributes to PDGF-induced mitogenesis, and thus this pathway may be important in the airway remodeling seen in some asthmatic patients.

Shiga toxin 1 triggers a ribotoxic stress response leading to p38 and JNK activation and induction of apoptosis in intestinal epithelial cells.

Shiga toxins made by Shiga toxin-producing Escherichia coli (STEC) are associated with hemolytic uremic syndrome. Shiga toxins (Stxs) may access the host systemic circulation by absorption across the intestinal epithelium. The effects of Stxs on this cell layer are not completely understood, although animal models of STEC infection suggest that, in the gut, Stxs may participate in both immune activation and apoptosis. Stxs have one enzymatically active A subunit associated with five identical B subunits. The A subunit inactivates ribosomes by cleaving a specific adenine from the 28S rRNA. We have previously shown that Stxs can induce multiple C-X-C chemokines in intestinal epithelial cells in vitro, including interleukin-8 (IL-8), and that Stx-induced IL-8 expression is linked to induction of c-Jun mRNA and p38 mitogen-activated protein (MAP) kinase pathway activity. We now report Stx1 induction of both primary response genes c-jun and c-fos and activation of the stress-activated protein kinases, JNK/SAPK and p38, in the intestinal epithelial cell line HCT-8. By 1 h of exposure to Stx1, mRNAs for c-jun and c-fos are induced, and both JNK and p38 are activated; activation of both kinases persisted up to 24 h. Stx1 enzymatic activity was required for kinase activation; a catalytically defective mutant toxin did not activate either. Stx1 treatment of HCT-8 cells resulted in cell death that was associated with caspase 3 cleavage and internucleosomal DNA fragmentation; this cytotoxicity also required Stx1 enzymatic activity. Blocking Stx1-induced p38 and JNK activation with the inhibitor SB202190 prevented cell death and diminished Stx1-associated caspase 3 cleavage. In summary, these data link the Stx1-induced ribotoxic stress response with both chemokine expression and apoptosis in the intestinal epithelial cell line HCT-8 and suggest that blocking host cell MAP kinases may prevent these Stx-associated events.

Activation of the STAT pathway in acute lung injury.

Acute lung injury (ALI) is a devastating clinical problem with a mortality as high as 60%. It is now appreciated that ALI represents a cytokine excess state that involves the microvasculature of multiple organs. The signal transducers and activators of transcription (STAT) family of transcription factors activate critical mediators of cytokine responses, but there is limited knowledge about their role in mediating ALI. In the present study, we demonstrate that the STAT transcription factors are activated rapidly in the lungs after intraperitoneal and intranasal LPS administration in mice. We also demonstrated that LPS activates both the STAT kinases, Src and JAK, in the lung with kinetics that are consistent with STAT activation. LPS treatment resulted in STAT3 activation throughout the resident lung cells, as well as in the recruited inflammatory cells. Whereas direct LPS treatment did not lead to STAT activation in cultured epithelial or endothelial cells, IL-6 activated STAT3 in both of these cell types. Furthermore, IL-6 was induced by LPS in serum and in the lung with kinetics consistent with STAT3 activation, suggesting that IL-6 may be one mechanism of STAT activation by LPS. In addition, STAT activation required reactive oxygen species, as the overexpression of catalase in mice prevented LPS-mediated STAT activation in the lung. STATs may be a common pathway for mediating ALI, regardless of the inciting factor, as STAT activation also occurred in both a gastric acid aspiration and acute pancreatitis model of ALI. Finally, STATs are activated in the lung long before signs of ALI are present, suggesting that the STAT transcription factors may play a role in initiating the inflammatory response seen in the lung.