STAT5 deficiency in hepatocytes reduces diethylnitrosamine-induced liver tumorigenesis in mice.

Chronic liver diseases and the development of hepatocellular carcinoma are closely linked and pose a major medical challenge as treatment options are limited. Animal studies have shown that genetic deletion of the signal transducer and activator of transcription (STAT) 5 in liver is associated with higher susceptibility to fatty liver disease, fibrosis and cancer, indicating a protective role of hepatic STAT5 in mouse models of chronic liver disease. To investigate the role of STAT5 in the etiology of liver cancer in more detail, we applied the chemical carcinogen diethylnitrosamine (DEN) to mice harboring a hepatocyte-specific deletion of Stat5 (S5KO). At 8 months after DEN injections, tumor formation in S5KO was significantly reduced. This was associated with diminished tumor frequency and less aggressive liver cancer progression. Apoptosis and inflammation markers were not changed in S5KO livers suggesting that the reduced tumor burden was not due to impaired inflammatory response. Despite reduced mRNA expression of the DEN bio-activator cytochrome P450 2e1 (Cyp2e1) in S5KO livers, protein levels were similar. Yet, delayed tumor formation in S5KO mice coincided with decreased activation of c-Jun N-terminal Kinase (JNK). Taken together, while STAT5 has a protective role in fatty liver-associated liver cancer, it exerts oncogenic functions in DEN-induced liver cancer.

Growth-hormone-induced signal transducer and activator of transcription 5 signaling causes gigantism, inflammation, and premature death but protects mice from aggressive liver cancer.

UNLABELLED: Persistently high levels of growth hormone (GH) can cause liver cancer. GH activates multiple signal-transduction pathways, among them janus kinase (JAK) 2-signal transducer and activator of transcription (STAT) 5 (signal transducer and activator of transcription 5). Both hyperactivation and deletion of STAT5 in hepatocytes have been implicated in the development of hepatocellular carcinoma (HCC); nevertheless, the role of STAT5 in the development of HCC as a result of high GH levels remains enigmatic. Thus, we crossed a mouse model of gigantism and inflammatory liver cancer caused by hyperactivated GH signaling (GH(tg) ) to mice with hepatic deletion of STAT5 (STAT5(Δhep) ). Unlike GH(tg) mice, GH(tg) STAT5(Δhep) animals did not display gigantism. Moreover, the premature mortality, which was associated with chronic inflammation, as well as the pathologic alterations of hepatocytes observed in GH(tg) mice, were not observed in GH(tg) animals lacking STAT5. Strikingly, loss of hepatic STAT5 proteins led to enhanced HCC development in GH(tg) mice. Despite reduced chronic inflammation, GH(tg) STAT5(Δhep) mice displayed earlier and more advanced HCC than GH(tg) animals. This may be attributed to the combination of increased peripheral lipolysis, hepatic lipid synthesis, loss of hepatoprotective mediators accompanied by aberrant activation of tumor-promoting c-JUN and STAT3 signaling cascades, and accumulation of DNA damage secondary to loss of cell-cycle control. Thus, HCC was never observed in STAT5(Δhep) mice. CONCLUSION: As a result of their hepatoprotective functions, STAT5 proteins prevent progressive fatty liver disease and the formation of aggressive HCC in the setting of hyperactivated GH signaling. At the same time, they play a key role in controlling systemic inflammation and regulating organ and body size.

Impairment of hepatic growth hormone and glucocorticoid receptor signaling causes steatosis and hepatocellular carcinoma in mice.

UNLABELLED: Growth hormone (GH)-activated signal transducer and activator of transcription 5 (STAT5) and the glucocorticoid (GC)-responsive glucocorticoid receptor (GR) are important signal integrators in the liver during metabolic and physiologic stress. Their deregulation has been implicated in the development of metabolic liver diseases, such as steatosis and progression to fibrosis. Using liver-specific STAT5 and GR knockout mice, we addressed their role in metabolism and liver cancer onset. STAT5 single and STAT5/GR double mutants developed steatosis, but only double-mutant mice progressed to liver cancer. Mechanistically, STAT5 deficiency led to the up-regulation of prolipogenic sterol regulatory element binding protein 1 (SREBP-1) and peroxisome proliferator activated receptor gamma (PPAR-γ) signaling. Combined loss of STAT5/GR resulted in GH resistance and hypercortisolism. The combination of both induced expression of adipose tissue lipases, adipose tissue lipid mobilization, and lipid flux to the liver, thereby aggravating STAT5-dependent steatosis. The metabolic dysfunctions in STAT5/GR compound knockout animals led to the development of hepatic dysplasia at 9 months of age. At 12 months, 35% of STAT5/GR-deficient livers harbored dysplastic nodules and ∼ 60% hepatocellular carcinomas (HCCs). HCC development was associated with GH and insulin resistance, enhanced tumor necrosis factor alpha (TNF-α) expression, high reactive oxygen species levels, and augmented liver and DNA damage parameters. Moreover, activation of the c-Jun N-terminal kinase 1 (JNK1) and STAT3 was prominent. CONCLUSION: Hepatic STAT5/GR signaling is crucial for the maintenance of systemic lipid homeostasis. Impairment of both signaling cascades causes severe metabolic liver disease and promotes spontaneous hepatic tumorigenesis.

Stat5a serine 725 and 779 phosphorylation is a prerequisite for hematopoietic transformation.

Stat5 transcription factors are essential gene regulators promoting proliferation, survival, and differentiation of all hematopoietic cell types. Mutations or fusions of oncogenic tyrosine kinases often result in constitutive Stat5 activation. We have modeled persistent Stat5 activity by using an oncogenic Stat5a variant (cS5). To analyze the hitherto unrecognized role of Stat5 serine phosphorylation in this context, we have generated cS5 constructs with mutated C-terminal serines 725 and 779, either alone or in combination. Genetic complementation assays in primary Stat5(null/null) mast cells and Stat5(DeltaN) T cells demonstrated reconstitution of proliferation with these mutants. Similarly, an in vivo reconstitution experiment of transduced Stat5(null/null) fetal liver cells transplanted into irradiated wild-type recipients revealed that these mutants exhibit biologic activity in lineage differentiation. By contrast, the leukemogenic potential of cS5 in bone marrow transplants decreased dramatically in cS5 single-serine mutants or was completely absent upon loss of both serine phosphorylation sites. Our data suggest that Stat5a serine phosphorylation is a prerequisite for cS5-mediated leukemogenesis. Hence, interference with Stat5a serine phosphorylation might provide a new therapeutic option for leukemia and myeloid dysplasias without affecting major functions of Stat5 in normal hematopoiesis.

Oncogenic Kit controls neoplastic mast cell growth through a Stat5/PI3-kinase signaling cascade.

The D816V-mutated variant of Kit triggers multiple signaling pathways and is considered essential for malignant transformation in mast cell (MC) neoplasms. We here describe that constitutive activation of the Stat5-PI3K-Akt-cascade controls neoplastic MC development. Retrovirally transduced active Stat5 (cS5(F)) was found to trigger PI3K and Akt activation, and to transform murine bone marrow progenitors into tissue-infiltrating MCs. Primary neoplastic Kit D816V(+) MCs in patients with mastocytosis also displayed activated Stat5, which was found to localize to the cytoplasm and to form a signaling complex with PI3K, with consecutive Akt activation. Finally, the knock-down of either Stat5 or Akt activity resulted in growth inhibition of neoplastic Kit D816V(+) MCs. These data suggest that a downstream Stat5-PI3K-Akt signaling cascade is essential for Kit D816V-mediated growth and survival of neoplastic MCs.

Antitumor Activity of the IGF-1/IGF-2-Neutralizing Antibody Xentuzumab (BI 836845) in Combination with Enzalutamide in Prostate Cancer Models.

Androgen deprivation therapy and second-generation androgen receptor signaling inhibitors such as enzalutamide are standard treatments for advanced/metastatic prostate cancer. Unfortunately, most men develop resistance and relapse; signaling via insulin-like growth factor (IGF) has been implicated in castration-resistant prostate cancer. We evaluated the antitumor activity of xentuzumab (IGF ligand-neutralizing antibody), alone and in combination with enzalutamide, in prostate cancer cell lines (VCaP, DuCaP, MDA PCa 2b, LNCaP, and PC-3) using established in vitro assays, and in vivo, using LuCaP 96CR, a prostate cancer patient-derived xenograft (PDX) model. Xentuzumab + enzalutamide reduced the viability of phosphatase and tensin homolog (PTEN)-expressing VCaP, DuCaP, and MDA PCa 2b cells more than either single agent, and increased antiproliferative activity and apoptosis induction in VCaP. Xentuzumab or xentuzumab + enzalutamide inhibited IGF type 1 receptor and AKT serine/threonine kinase (AKT) phosphorylation in VCaP, DuCaP, and MDA PCa 2b cells; xentuzumab had no effect on AKT phosphorylation and proliferation in PTEN-null LNCaP or PC-3 cells. Knockdown of PTEN led to loss of antiproliferative activity of xentuzumab and reduced activity of xentuzumab + enzalutamide in VCaP cells. Xentuzumab + enzalutamide inhibited the growth of castration-resistant LuCaP 96CR PDX with acquired resistance to enzalutamide, and improved survival in vivo The data suggest that xentuzumab + enzalutamide combination therapy may overcome castration resistance and could be effective in patients who are resistant to enzalutamide alone. PTEN status as a biomarker of responsiveness to combination therapy needs further investigation.

The different functions of Stat5 and chromatin alteration through Stat5 proteins.

Stat5 proteins modulate gene transcription upon cytokine- and growth factor action. Stat5a and Stat5b proteins alone are weak activators of transcription. They can modify chromatin organization through oligomerization and they act predominantly in co-operation and interaction with other proteins. The conservative view of exclusively nuclear functions of Stat5 was challenged by the observation of additional Stat5 effects in the cytoplasm, resulting in activation of the PI3K-Akt pathway. We summarize biological consequences of mutations in conserved domains of Stat5 or of deletions in the N- or C-terminal domains with impact on target gene transcription. Formation of higher-order oligomers is dramatically changed upon amino- or carboxyterminal deletions in Stat5 proteins. Mutations in or deletion of the Stat5 N-terminus leads to diminished leukemogenic potential of oncogenic Stat5, probably due to the inability to form Stat5 tetramers. The Stat5 N-terminal domain prevents persistent activation and can act as a DNA-docking platform for the glucocorticoid receptor (GR). The corresponding protocols should facilitate follow-up studies on Stat5 proteins and their contribution to normal physiological versus pathological processes through differential chromatin binding.

Hepatic Deletion of Janus Kinase 2 Counteracts Oxidative Stress in Mice.

Genetic deletion of the tyrosine kinase JAK2 or the downstream transcription factor STAT5 in liver impairs growth hormone (GH) signalling and thereby promotes fatty liver disease. Hepatic STAT5 deficiency accelerates liver tumourigenesis in presence of high GH levels. To determine whether the upstream kinase JAK2 exerts similar functions, we crossed mice harbouring a hepatocyte-specific deletion of JAK2 (JAK2Δhep) to GH transgenic mice (GHtg) and compared them to GHtgSTAT5Δhep mice. Similar to GHtgSTAT5Δhep mice, JAK2 deficiency resulted in severe steatosis in the GHtg background. However, in contrast to STAT5 deficiency, loss of JAK2 significantly delayed liver tumourigenesis. This was attributed to: (i) activation of STAT3 in STAT5-deficient mice, which was prevented by JAK2 deficiency and (ii) increased detoxification capacity of JAK2-deficient livers, which diminished oxidative damage as compared to GHtgSTAT5Δhep mice, despite equally severe steatosis and reactive oxygen species (ROS) production. The reduced oxidative damage in JAK2-deficient livers was linked to increased expression and activity of glutathione S-transferases (GSTs). Consistent with genetic deletion of Jak2, pharmacological inhibition and siRNA-mediated knockdown of Jak2 led to significant upregulation of Gst isoforms and to reduced hepatic oxidative DNA damage. Therefore, blocking JAK2 function increases detoxifying GSTs in hepatocytes and protects against oxidative liver damage.

Pharmacodynamic and antineoplastic activity of BI 836845, a fully human IGF ligand-neutralizing antibody, and mechanistic rationale for combination with rapamycin.

Insulin-like growth factor (IGF) signaling is thought to play a role in the development and progression of multiple cancer types. To date, therapeutic strategies aimed at disrupting IGF signaling have largely focused on antibodies that target the IGF-I receptor (IGF-IR). Here, we describe the pharmacologic profile of BI 836845, a fully human monoclonal antibody that utilizes an alternative approach to IGF signaling inhibition by selectively neutralizing the bioactivity of IGF ligands. Biochemical analyses of BI 836845 demonstrated high affinity to human IGF-I and IGF-II, resulting in effective inhibition of IGF-induced activation of both IGF-IR and IR-A in vitro. Cross-reactivity to rodent IGFs has enabled rigorous assessment of the pharmacologic activity of BI 836845 in preclinical models. Pharmacodynamic studies in rats showed potent reduction of serum IGF bioactivity in the absence of metabolic adverse effects, leading to growth inhibition as evidenced by reduced body weight gain and tail length. Moreover, BI 836845 reduced the proliferation of human cell lines derived from different cancer types and enhanced the antitumor efficacy of rapamycin by blocking a rapamycin-induced increase in upstream signaling in vitro as well as in human tumor xenograft models in nude mice. Our data suggest that BI 836845 represents a potentially more effective and tolerable approach to the inhibition of IGF signaling compared with agents that target the IGF-I receptor directly, with potential for rational combinations with other targeted agents in clinical studies.

Hepatic growth hormone and glucocorticoid receptor signaling in body growth, steatosis and metabolic liver cancer development.

Growth hormone (GH) and glucocorticoids (GCs) are involved in the control of processes that are essential for the maintenance of vital body functions including energy supply and growth control. GH and GCs have been well characterized to regulate systemic energy homeostasis, particular during certain conditions of physical stress. However, dysfunctional signaling in both pathways is linked to various metabolic disorders associated with aberrant carbohydrate and lipid metabolism. In liver, GH-dependent activation of the transcription factor signal transducer and activator of transcription (STAT) 5 controls a variety of physiologic functions within hepatocytes. Similarly, GCs, through activation of the glucocorticoid receptor (GR), influence many important liver functions such as gluconeogenesis. Studies in hepatic Stat5 or GR knockout mice have revealed that they similarly control liver function on their target gene level and indeed, the GR functions often as a cofactor of STAT5 for GH-induced genes. Gene sets, which require physical STAT5-GR interaction, include those controlling body growth and maturation. More recently, it has become evident that impairment of GH-STAT5 signaling in different experimental models correlates with metabolic liver disease, ranging from hepatic steatosis to hepatocellular carcinoma (HCC). While GH-activated STAT5 has a protective role in chronic liver disease, experimental disruption of GC-GR signaling rather seems to ameliorate metabolic disorders under metabolic challenge. In this review, we focus on the current knowledge about hepatic GH-STAT5 and GC-GR signaling in body growth, metabolism, and protection from fatty liver disease and HCC development.

Serine phosphorylation of the Stat5a C-terminus is a driving force for transformation.

Persistent tyrosine phosphorylation of Stat3 and Stat5 is associated with oncogenic activity. Phosphorylation of the conserved tyrosine residue (pTyr) was long believed to be the only essential prerequisite to promote activation and nuclear translocation of Stat proteins. It has become evident, however, that post-translational protein modifications like serine phosphorylation, acetylation, glycosylation as well as protein splicing and processing constitute further regulatory mechanisms to modulate Stat transcriptional activity and to provide an additional layer of specificity to Jak-Stat signal transduction. Significantly, most vertebrate Stat proteins contain one conserved serine phosphorylation site within their transactivation domains. This phosphorylation motif is located within a P(M)SP sequence. Stat transcription factor activity is negatively influenced by mutation of the serine to alanine. Moreover, it was shown for both Stat3 and Stat5 that their capacity to transform cells was diminished. This review addresses recent advances in understanding the regulation and the biochemical and biological consequences of Stat serine phosphorylation. In particular, we discuss their role in persistently activated Stat proteins for cancer research.

Interleukin-6 and oncostatin M stimulation of proliferation of prostate cancer 22Rv1 cells through the signaling pathways of p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase.

INTRODUCTION: Interleukin-6 (IL-6) is a pleiotropic regulator of prostate cancer cell growth. Oncostatin M (OSM), an IL-6-type cytokine, affects the growth of prostate cancers in a paracrine and autocrine manner. In order to understand better the mechanisms controlling proliferation and intracellular signaling by these cytokines in advanced prostate carcinoma, we performed studies in 22Rv1 cells derived from the relapsed xenograft CWR22R. METHODS: Expression of IL-6 and OSM receptors (OSMR-beta) and elements of signal transduction pathways in 22Rv1 cells were investigated by RT-PCR. Proliferation was assessed by cell counting after treatment with either IL-6 or OSM. IL-6 secretion was measured in conditioned medium from 22Rv1 cells by ELISA. Expression and phosphorylation status of signal transducers and activators of transcription factor (STAT) 3, mitogen-activated protein kinases (MAPK) p44/p42 and p38, and protein kinase B (Akt) was investigated by Western blot. RESULTS: 22Rv1 cells express both subunits of the IL-6 receptor (gp80 and gp130) and leukemia inhibitory factor receptor-beta (LIFR-beta) but not OSMR-beta. Their proliferation was stimulated by IL-6 or OSM and the maximal effect was observed at a concentration of 10 ng/ml of either cytokine. Interestingly, neither IL-6 nor OSM induced phosphorylation of STAT3. OSM modestly increased the phosphorylation of p38 and both cytokines exerted an effect on Akt phosphorylation. CONCLUSIONS: IL-6 and OSM stimulate proliferation of 22Rv1 cells, at least in part through activation of the phosphatidylinositol 3-kinase (PI 3-K) signaling pathway. Our data provide additional evidence for the growth-stimulatory role of IL-6 and related cytokines in advanced prostate cancer and may serve as a basis for the development of novel experimental therapies.