STAT3 is activated by JAK2 independent of key oncogenic driver mutations in non-small cell lung carcinoma

Constitutive activation of STAT3 is a common feature in many solid tumors including non-small cell lung carcinoma (NSCLC). While activation of STAT3 is commonly achieved by somatic mutations to JAK2 in hematologic malignancies, similar mutations are not often found in solid tumors. Previous work has instead suggested that STAT3 activation in solid tumors is more commonly induced by hyperactive growth factor receptors or autocrine cytokine signaling. The interplay between STAT3 activation and other well-characterized oncogenic "driver" mutations in NSCLC has not been fully characterized, though constitutive STAT3 activation has been proposed to play an important role in resistance to various small-molecule therapies that target these oncogenes. In this study we demonstrate that STAT3 is constitutively activated in human NSCLC samples and in a variety of NSCLC lines independent of activating KRAS or tyrosine kinase mutations. We further show that genetic or pharmacologic inhibition of the gp130/JAK2 signaling pathway disrupts activation of STAT3. Interestingly, treatment of NSCLC cells with the JAK1/2 inhibitor ruxolitinib has no effect on cell proliferation and viability in two-dimensional culture, but inhibits growth in soft agar and xenograft assays. These data demonstrate that JAK2/STAT3 signaling operates independent of known driver mutations in NSCLC and plays critical roles in tumor cell behavior that may not be effectively inhibited by drugs that selectively target these driver mutations.     

Inhibition of GM-CSF/IL-3/IL-5 signaling by antisense oligodeoxynucleotides targeting the common beta chain of their receptors.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 play a key role in allergic inflammation. They mediate their effect via receptors that consist of two distinct subunits, a cytokine-specific alpha subunit and a common beta subunit (betac) that transduces cell signaling. We sought to down-regulate the biologic activities of GM-CSF, IL-3, and IL-5 simultaneously by inhibiting betac mRNA expression with antisense technology. Experiments were performed with TF-1 cells (a human erythroleukemia cell line expressing GM-CSF, IL-3, and IL-5 receptors, which proliferates in response to these cytokines), monocytic U937 cells, which require these cytokines for differentiation, and purified human eosinophils. Cells were treated with antisense phosphorothioate oligodeoxynucleotides (ODN) targeting betac mRNA. In contrast to nontreated cells and cells treated by sense or mismatched ODN, antisense ODN inhibited betac mRNA expression and significantly decreased the level of cell surface betac protein expression on TF-1 and U937 cells. Receptor function was also affected. Antisense ODN were able to inhibit TF-1 cell proliferation in vitro in the presence of GM-CSF, IL-3, or IL-5 in the culture medium and eosinophil survival. We suggest that antisense ODN against betac may provide a new therapeutic alternative for the treatment of neoplastic or allergic diseases associated with eosinophilic inflammation.     

Analysis of signals and functions of the chimeric human granulocyte-macrophage colony-stimulating factor receptor in BA/F3 cells and transgenic mice

Receptors for GM-CSF, IL-3, and IL-5 are composed of two subunits: alpha, which is specific for each cytokine, and betac, which is shared by all. Although the role of betac in signal transduction has been extensively studied, the role of the alpha subunit has remained to be clarified. To analyze the role of the human (h) GM-CSF receptor alpha subunit, we constructed a chimeric receptor subunit composed of extracellular and transmembrane regions of alpha fused with the cytoplasmic region of betac, designated alpha/beta. In BA/F3 cells, chimeric receptor composed of alpha/beta,beta can transduce signals for mitogen-activated protein kinase cascade activation and proliferation in response to hGM-CSF. Although phosphorylation of Jak1 but not of Jak2 occurred with stimulation of hGM-CSF, the dominant-negative Jak2 but not the dominant-negative Jak1 suppresses c-fos promoter activation. To determine whether the chimeric receptor alpha/beta,beta is functional in vivo, we developed transgenic mice expressing the chimeric receptor alpha/beta,beta. Bone marrow cells from the transgenic mice expressing the alpha/beta,beta receptor form not only GM colonies but also various lineages of colonies in response to GM-CSF. In addition, mast cells were produced when bone marrow cells of the transgenic mouse were cultured with hGM-CSF. Thus, it appears that the cytoplasmic region of the alpha subunit is not required for hGM-CSF promoting activities, even in bone marrow cells.     

Clarification of the role of N-glycans on the common beta-subunit of the human IL-3, IL-5 and GM-CSF receptors and the murine IL-3 beta-receptor in ligand-binding and receptor activation

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 are related cytokines that play key roles in regulating the differentiation, proliferation, survival and activation of myeloid blood cells. The cell surface receptors for these cytokines are composed of cytokine-specific alpha-subunits and a common beta-receptor (betac), a shared subunit that is essential for receptor signaling in response to GM-CSF, IL-3 and IL-5. Previous studies have reached conflicting conclusions as to whether N-glycosylation of the betac-subunit is necessary for functional GM-CSF, IL-3 and IL-5 receptors. We sought to clarify whether betac N-glycosylation plays a role in receptor function, since all structural studies of human betac to date have utilized recombinant protein lacking N-glycosylation at Asn(328). Here, by eliminating individual N-glycans in human betac and the related murine homolog, beta(IL-3), we demonstrate unequivocally that ligand-binding and receptor activation are not critically dependent on individual N-glycosylation sites within the beta-subunit although the data do not preclude the possibility that N-glycans may exert some sort of fine control. These studies support the biological relevance of the X-ray crystal structures of the human betac domain 4 and the complete ectodomain, both of which lack N-glycosylation at Asn(328).     

Induction of cell shape changes through activation of the interleukin-3 common beta chain receptor by the RON receptor-type tyrosine kinase.

The RON receptor-type tyrosine kinase, a member of the hepatocyte growth factor receptor family, is a receptor for macrophage-stimulating protein (MSP). Recently, we observed that MSP induces morphological changes in interleukin (IL)-3-dependent Ba/F3 cells ectopically expressing RON. We show here that stimulation of those cells with either MSP or IL-3 increases tyrosine phosphorylation of proteins of 130, 110, 90, 62, and 58 kDa and induces similar morphological changes, accompanied by unique nuclear shape and redistribution of F-actin. A tyrosine kinase inhibitor, genistein, blocked both the increase in tyrosine phosphorylation and morphological changes. Upon stimulation with either MSP or IL-3, prominent tyrosine-phosphorylated pp90 was similarly co-immunoprecipitated with the common beta chain of IL-3 receptor (betac). Unlike IL-3, stimulation with MSP increased tyrosine phosphorylation of betac without activation of JAK2, resulting in morphological changes with modest cell growth. Confocal immunofluorescence analyses showed colocalization of RON, betac, and tyrosine-phosphorylated proteins. In vitro kinase assays revealed that autophosphorylated RON phosphorylated betac. These results suggest that the signaling pathway for morphological changes through betac and its associated protein pp90 is distinct from the pathway for cell growth in the IL-3 signal transduction system.     

JAK1 N-terminus binds to conserved Box 1 and Box 2 motifs of cytokine receptor common beta subunit but signal activation requires JAK1 C-terminus

The human interleukin-3 receptor (hIL-3R) consists of a unique alpha subunit (hIL-3Ralpha) and a common beta subunit (betac). Binding of IL-3 to IL-3R activates Janus kinases JAK1 and JAK2. Our previously study showed that JAK2 and JAK1 were constitutively associated with the hIL-3Ralpha and betac subunits, respectively. In this study, we further demonstrate that JAK2 binds to the intracellular domain of hIL-3Ralpha and JAK1 binds to the Box 1 and Box 2 motifs of betac using GST-hIL-3R fusion proteins in pull-down assays. JAK1 mutational analysis revealed that its JH7-3 domains bound directly to the Box 1 and Box 2 motifs of betac. We further examined the role of JAK1 JH7-3 domains in JAK1 and JAK2-mediated signaling using the CDJAKs fusion proteins, which consisted of a CD16 extracellular domain, a CD7 transmembrane domain, and either JAK1 (CDJAK1), JAK2 (CDJAK2), or JAK1-JH7-3 domains (CDJAK1-JH7-3) as intracellular domains. Anti-CD16 antibody crosslinking of wild type fusion proteins CDJAK1 with CDJAK2 could mimic IL-3 signaling, however, the crosslinking of fusion proteins CDJAK1-JH7-3 with CDJAK2 failed to activate downstream proteins. These results suggest that the JAK1-JH7-3 domains are required for betac interaction and abolish wild type JAK1 and JAK2-mediated signaling.     

Downregulation of Bim, a proapoptotic relative of Bcl-2, is a pivotal step in cytokine-initiated survival signaling in murine hematopoietic progenitors

Two distinct signaling pathways regulate the survival of interleukin-3 (IL-3)-dependent hematopoietic progenitors. One originates from the membrane-proximal portion of the cytoplasmic domain of the IL-3 receptor (betac chain), which is shared by IL-3 and granulocyte-macrophage colony-stimulating factor and is involved in the regulation of Bcl-x(L) through activation of STAT5. The other pathway emanates from the distal region of the betac chain and overlaps with downstream signals from constitutively active Ras proteins. Although the latter pathway is indispensable for cell survival, its downstream targets remain largely undefined. Here we show that the expression of Bim, a member of the BH3-only subfamily of cell death activators, is downregulated by IL-3 signaling through either of two major Ras pathways: Raf/mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/mammalian target of rapamycin. Akt/phosphokinase B does not appear to play a significant role in this regulatory cascade. Bim downregulation has important implications for cell survival, since enforced expression of this death activator at levels equivalent to those induced by cytokine withdrawal led to apoptosis even in the presence of IL-3. We conclude that Bim is a pivotal molecule in cytokine regulation of hematopoietic cell survival.     

H2O2-induced activation of transcription factors STAT1 and STAT3: the role of EGF receptor and tyrosine kinase JAK2

Reactive oxygen species initiate multiple signal transduction pathways including tyrosine kinase signaling. Here, we demonstrate tyrosine phosphorylation of EGF receptor, STAT3, and, to a lesser extent, STAT1 upon H2O2 treatment of HER14 cells (NIH3T3 fibroblasts transfected with full-length EGF receptor). Maximum phosphorylation levels were observed in 5 min of stimulation at 1-2 mM H2O2. It has been shown that the intrinsic EGF-receptor tyrosine kinase is responsible for the receptor phosphorylation upon H2O2 stimulation. STAT3 and STAT1 activation in HER14 cells was demonstrated to depend on EGF receptor kinase activity, rather than JAK2 activity, while in both K721A and CD126 cells (NIH3T3 transfected with kinase-dead EGF receptor, and EGF receptor lacking major autophosphorylation sites, respectively) STAT1 and STAT3 tyrosine phosphorylation requires JAK2 kinase activity. Furthermore, STAT3 is constitutively phosphorylated in K721A and CD126 cells, and STAT1 H2O2-stimulated activation in these cells is much more prominent than in HER14. In all the cell lines used, Src-kinase activity was demonstrated to be unnecessary for ROS-initiated phosphorylation of STATs. Herein, we postulate that EGF receptor plays a role in H2O2-induced STAT activation in HER14 cells. Our data also prompted a hypothesis of constitutive inhibition of JAK2-dependent STAT activation in this cell line.     

{beta}1 Integrin and IL-3R coordinately regulate STAT5 activation and anchorage-dependent proliferation

We previously demonstrated that integrin-dependent adhesion activates STAT5A, a well known target of IL-3-mediated signaling. Here, we show that in endothelial cells the active beta1 integrin constitutively associates with the unphosphorylated IL-3 receptor (IL-3R) beta common subunit. This association is not sufficient for activating downstream signals. Indeed, only upon fibronectin adhesion is Janus Kinase 2 (JAK2) recruited to the beta1 integrin-IL-3R complex and triggers IL-3R beta common phosphorylation, leading to the formation of docking sites for activated STAT5A. These events are IL-3 independent but require the integrity of the IL-3R beta common. IL-3 treatment increases JAK2 activation and STAT5A and STAT5B tyrosine and serine phosphorylation and leads to cell cycle progression in adherent cells. Expression of an inactive STAT5A inhibits cell cycle progression upon IL-3 treatment, identifying integrin-dependent STAT5A activation as a priming event for IL-3-mediated S phase entry. Consistently, overexpression of a constitutive active STAT5A leads to anchorage-independent cell cycle progression. Therefore, these data provide strong evidence that integrin-dependent STAT5A activation controls IL-3-mediated proliferation.     

Autocrine-mediated activation of STAT3 correlates with cell proliferation in breast carcinoma lines

The intracellular signals driving the proliferation of breast carcinoma (BC) cells have been widely studied. Both the mitotic and metastatic potential of BC cells have been linked to the frequent overexpression of ErbB family members. Other signaling molecules, including the estrogen receptor, the tyrosine kinases c-Src and Syk, and STAT proteins, especially STAT3, have also been implicated in BC tumor growth. Here we have examined ErbB and STAT protein expression and activation in six BC-derived cell lines. ErbB expression and tyrosine phosphorylation varied considerably among the six cell lines. However, STAT protein expression and activation were more consistent. Two levels of STAT3 activation were distinguished in DNA-binding assays: an epidermal growth factor-inducible, high level that requires both ErbB1 and Janus kinase (JAK) activity and an elevated serum-dependent level that is maintained by autocrine/paracrine signaling and requires JAK activity but is independent of ErbB1 kinase activity. BC cell growth could be inhibited by dominant-negative versions of STAT3 and the JAK inhibitor AG490 but not by PD153035 or PD168393, inhibitors of ErbB1 kinase activity. This indicates that BC cell proliferation may be a consequence of STAT3 activation by autocrine/paracrine signals.     

Interleukin-2 (IL-2) receptor-betagamma signalling is activated by c-Kit in the absence of IL-2, or by exogenous IL-2 via JAK3/STAT5 in human papillomavirus-associated cervical cancer

Activation of the interleukin-2 receptor (IL-2R) induces signalling cascades promoting T cell proliferation. However, signal transduction pathways triggered in IL-2R-expressing solid tumours are unknown. This report shows that human papillomavirus (HPV)-associated cervical cancer cells express an IL-2R composed of beta and gamma chains (IL-2Rbetagamma), and that IL-2-mediated activation increases the phosphorylation of JAK3 and STAT5, stimulating cell proliferation. Interestingly, endogenous IL-2 is not produced by these cells, suggesting the activation of IL-2Rbetagamma by an alternative mechanism. Accordingly, we found that Stem Cell Factor (SCF)-activated c-Kit induces phosphorylation of the IL-2Rbeta chain in the absence of IL-2. Moreover, inhibition of IL-2Rbeta phosphorylation by blocking c-Kit tyrosine kinase activity abolishes both, IL-2 and SCF-mediated proliferation. Thus, these results demonstrate that IL-2 triggers a JAK3/STAT5 cascade in HPV-associated cervical cancer cells expressing IL-2Rbetagamma, and that this receptor can be alternatively activated by SCF-activated c-Kit in the absence of IL-2.     

Simultaneous activation of JAK1 and JAK2 confers IL-3 independent growth on Ba/F3 pro-B cells

JAK1 and JAK2 are tyrosine kinases involved in the regulation of cell proliferation, differentiation, and survival. These proteins may play a key role in mediating the effects of the cytokine IL-3 on hematopoietic cells. IL-3 induces tyrosine phosphorylation of both JAK1 and JAK2. However, it is not clear whether the activation of JAK1, JAK2, or both is sufficient to confer factor-independent growth in IL-3 dependent cells. To address this issue, fusion proteins CD16/CD7/JAK (CDJAK), comprised of a CD16 extracellular domain, a CD7 transmembrane domain, and a JAK cytoplasmic region (either a wild-type JAK or a dominant negative mutant of JAK) were constructed. We established several Ba/F3 derivatives that stably overexpress the conditionally active forms of either CDJAK1, CDJAK2, or both these fusion proteins. In this study, the autophosphorylation of CDJAK1 or CDJAK2 was induced by crosslinking with anti-CD16 antibody. We demonstrated that, like their wild-type counterparts, CDJAK1 and CDJAK2 were preassociated with the IL-3 receptor beta and alpha subunits, respectively. Furthermore, the simultaneous activation of both CDJAK1 and CDJAK2 fusion proteins, but not either one alone, led to the tyrosine phosphorylation of the IL-3 receptor beta subunit, the activation of downstream signaling molecules, including STAT5, Akt, and MAPK, and the conferring of factor-independent growth to IL-3-dependent Ba/F3 cells. Coexpression of dominant negative mutants CDJAK1KE or CDJAK2KE with wild type CDJAK2 or CDJAK1, respectively, inhibited these activation activities. These results suggest that JAK1 and JAK2 must work cooperatively and not independently and that their actions are dependent on having normal kinase activity to trigger downstream signals leading to IL-3 independent proliferation and survival of Ba/F3 cells.     

Interleukin-1beta-induced iNOS expression in human lung carcinoma A549 cells: involvement of STAT and MAPK pathways

For understanding of signaling molecules important in lung cancer growth and progression, IL-1beta effect was analyzed on iNOS expression and key signaling molecules in human lung carcinoma A549 cells and established the role of specific signaling molecules by using specific chemical inhibitors. IL-1beta exposure (10 ng/ml) induced strong iNOS expression in serum starved A549 cells. Detailed molecular analyses showed that IL-1beta increased expression of phosphorylated STAT1 (Tyr701 and Ser727) and STAT3 (Tyr705 and Ser727) both in total cell lysates and nuclear lysates. Further, IL-1beta exposure strongly activated MAPKs (ERK1/2, JNK1/2 and p38) and Akt as well as increased nuclear levels of NF-kappaB and HIF-1alpha in A549 cells. Use of specific chemical inhibitors for JAK1 kinase (piceatannol), JAK2 kinase (AG-490), MEK1/2 (PD98059) and JNK1/2 (SP600125) revealed that IL-1beta-induced iNOS expression involved signaling pathways in addition to JAK-STAT and ERK1/2-JNK1/2 activation. Overall, these results suggested that instead of specific pharmacological inhibitors, use of chemopreventive agents with broad spectrum efficacy to inhibit IL-1beta-induced signaling cascades and iNOS expression would be a better strategy towards lung cancer prevention and/or treatment.