Transcriptional and Physiological Roles for STAT Proteins in Leptin Action.

Molecular Metabolism (2019)

2019 Jan 24

Pan W, Allison MB, Sabatini P, Rupp A, Adams J, Patterson C, Jones JC, Olson DP, Myers MG.
| DOI: doi:10.1016/j.molmet.2019.01.007

Abstract Objectives Leptin acts via its receptor LepRb on specialized neurons in the brain to modulate food intake, energy expenditure, and body weight. LepRb activates signal transducers and activators of transcription (STATs, including STAT1, STAT3, and STAT5) to control gene expression. Methods Because STAT3 is crucial for physiologic leptin action, we used TRAP-seq to examine gene expression in LepRb neurons of mice ablated for Stat3 in LepRb neurons (Stat3LepRbKO mice), revealing the STAT3-dependent transcriptional targets of leptin. To understand roles for STAT proteins in leptin action, we also ablated STAT1 or STAT5 from LepRb neurons and expressed a constitutively-active STAT3 (CASTAT3) in LepRb neurons. Results While we also found increased Stat1 expression and STAT1-mediated transcription of leptin-regulated genes in Stat3LepRbKO mice, ablating Stat1 in LepRb neurons failed to alter energy balance (even on the Stat3LepRbKO background); ablating Stat5 in LepRb neurons also failed to alter energy balance. Importantly, expression of a constitutively-active STAT3 (CASTAT3) in LepRb neurons decreased food intake and body weight and improved metabolic parameters in leptin-deficient (ob/ob) mice, as well as in wild-type animals. Conclusions Thus, STAT3 represents the unique STAT protein required for leptin action and STAT3 suffices to mediate important components of leptin action in the absence of other LepRb signals.

Spatial molecular and cellular determinants of STAT3 activation in liver fibrosis progression in non-alcoholic fatty liver disease

JHEP Reports

2022 Nov 01

Jiao, J;Sanchez, J;Saldarriaga, O;Solis, L;Tweardy, D;Maru, D;Stevenson, H;Beretta, L;
| DOI: 10.1016/j.jhepr.2022.100628

Background & Aims The prevalence of non-alcoholic fatty liver disease (NAFLD) and its severe form, non-alcoholic steatohepatitis (NASH), is increasing. Subjects with NASH often develop liver fibrosis and advanced liver fibrosis is the main determinant of mortality in NASH patients. We and others have reported that STAT3 contributes to liver fibrosis and hepatocellular carcinoma in mice. Methods Here, we explored whether STAT3 activation in hepatocytes and in non-hepatocytes areas, measured by phospho-STAT3 (pSTAT3), is associated with liver fibrosis progression in 133 patients with NAFLD. We further characterized the molecular and cellular determinants of STAT3 activation by integrating spatial distribution and transcriptomic changes in fibrotic NAFLD liver. Results pSTAT3 scores in non-hepatocytes areas progressively increased with fibrosis severity (r=0.53, p

Loss of P53 Function Activates JAK2–STAT3 Signaling to Promote Pancreatic Tumor Growth, Stroma Modification, and Gemcitabine Resistance in Mice and is Associated With Patient Survival

Gastroenterology.

2016 Mar 18

Wörmann SM, Song L, Ai J, Diakopoulos KN, Görgülü K, Ruess D, Campbell A, Doglioni C, Jodrell D, Neesse A, Demir EI, Karpathaki AP, Barenboim M, Hagemann T, Rose-John S, Sansom O, Schmid RM, Protti MP, Lesina M, Algül H
PMID: 27003603 | DOI: 10.1053/j.gastro.2016.03.010.

Abstract

BACKGROUND & AIMS:

One treatment strategy for pancreatic ductal adenocarcinoma is to modify, rather than deplete, the tumor stroma. Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) is associated with progression of pancreatic and other solid tumors. We investigated whether loss of P53 function contributes to persistent activation of STAT3 and modification of the pancreatic tumor stroma in patients and mice.

METHODS:

Stat3, Il6st (encodes gp130), or Trp53 were disrupted, or a mutant form of P53 (P53R172H) or transgenic sgp130 were expressed, in mice that developed pancreatic tumors due to expression of activated KRAS (KrasG12D, KC mice). Pancreata were collected and analyzed by immunohistochemistry, in situ hybridization, quantitative reverse-transcription PCR, or immunoblot assays; fluorescence-activated cell sorting to identify immune cells. We obtained frozen pancreatic tumor specimens from patients and measured levels of phosphorylated STAT3 and P53 by immunohistochemistry; protein levels were associated with survival using Kaplan-Meier analyses. We measured levels of STAT3, P53, ligands for gp130, interleukin-6, cytokines, sonic hedgehog signaling, STAT3 phosphorylation (activation), and accumulation of reactive oxygen species in primary pancreatic cells from mice. Mice with pancreatic tumors were given gemcitabine and a JAK2 inhibitor; tumor growth was monitored by 3-dimensional ultrasound.

RESULTS:

STAT3 was constitutively phosphorylated in pancreatic tumor cells from KC mice with loss or mutation of P53. Tumor cells of these mice accumulated reactive oxygen species and had lower activity of the phosphatase SHP2 and prolonged phosphorylation of JAK2, compared to tumors from KC mice with functional P53. These processes did not require the gp130 receptor. Genetic disruption of Stat3 in mice, or pharmacologic inhibitors of JAK2 or STAT3 activation, reduced fibrosis and the numbers of pancreatic stellate cells in the tumor stroma and altered the types of immune cells that infiltrated tumors. Mice given a combination of gemcitabine and a JAK2 inhibitor formed smaller tumors and survived longer than mice given control agents; the tumor stroma had fewer activated pancreatic stellate cells, lower levels of periostin, and alterations in collagen production and organization. Phosphorylation of STAT3 correlated with P53 mutation and features of infiltrating immune cells in human pancreatic tumors. Patients whose tumors had lower levels of phosphorylated STAT3 and functional P53 had significantly longer survival times than patients with high levels of phosphorylated STAT3 and P53 mutation.

CONCLUSION:

In pancreatic tumors of mice, loss of P53 function activates JAK2-STAT3 signaling, which promotes modification of the tumor stroma and tumor growth and resistance to gemcitabine. In human pancreatic tumors, STAT3 phosphorylation correlated with P53 mutation and patient survival time. Inhibitors of this pathway slow tumor growth and stroma formation, alter immune cell infiltration, and prolong survival of mice.

STAT-3 RNAscope Determination in Human Diffuse Large B-Cell Lymphoma1.

Translational Oncology (2019)

2019 Jan 09

Tamma R, Ingravallo G, Albano F, Gaudio F, Annese T, Ruggieri S, Lorusso L, Errede M, Maiorano E, Specchia G, Ribatti D.
| DOI: 10.1016/j.tranon.2018.12.008

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma. Signal transducer and activator of transcription 3 (STAT3) is a cytoplasmic transcription with many important functions, including regulation of cell proliferation, differentiation, survival, angiogenesis, and immune response. MATERIALS AND METHODS: In this study, we have compared by means of RNAscope technology STAT3 RNA expression in human DLBCL in a selected group of activated B-cell–like DLBCL (ABC-DLBCL) patients with another group of germinal center B-cell–like DLBCL (GBC-DLBCL) patients. RESULTS: The results have shown that ABC DLBCL tissue samples contained a significantly higher number of STAT3-positive cells as compared with GCB tissue samples. Moreover, by means of confocal immunofluorescence analysis, we have observed that tumor vessels in ABC samples appeared lined by endothelial cells expressing both FVIII and STAT3 signals, while in GCB samples, only few vessels coexpressed FVIII and STAT3. CONCLUSIONS: These data confirm other reports showing that STAT3 is highly expressed and activated in ABC-DLBCL and our previously published data demonstrating that, in primary central nervous system lymphoma, tumor vessels appeared lined by endothelial cells expressing both FVIII and STAT3.

Activation of STAT3 signaling pathway in the kidney of COVID-19 patients

Journal of nephrology

2021 Oct 09

Salem, F;Li, XZ;Hindi, J;Casablanca, NM;Zhong, F;El Jamal, SM;Haroon Al Rasheed, MR;Li, L;Lee, K;Chan, L;He, JC;
PMID: 34626364 | DOI: 10.1007/s40620-021-01173-0

Acute kidney injury is common in patients with COVID-19, however mechanisms of kidney injury remain unclear. Since cytokine storm is likely a cause of AKI and glomerular disease, we investigated the two major transcription factors, STAT3 and NF-kB, which are known to be activated by cytokines.This is an observational study of the postmortem kidneys of 50 patients who died with COVID-19 in the Mount Sinai Hospital during the first pandemic surge. All samples were reviewed under light microscopy, electron microscopy, and immunofluorescence by trained renal pathologists. In situ hybridization evaluation for SARS-CoV-2 and immunostaining of transcription factors STAT3 and NF-kB were performed.Consistent with previous findings, acute tubular injury was the major pathological finding, together with global or focal glomerulosclerosis. We were not able to detect SARS-CoV-2 in kidney cells. ACE2 expression was reduced in the tubular cells of patients who died with COVID-19 and did not co-localize with TMPRSS2. SARS-CoV-2 was identified occasionally in the mononuclear cells in the peritubular capillary and interstitium. STAT3 phosphorylation at Tyr705 was increased in 2 cases in the glomeruli and in 3 cases in the tubulointerstitial compartments. Interestingly, STAT3 phosphorylation at Ser727 increased in 9 cases but only in the tubulointerstitial compartment. A significant increase in NF-kB phosphorylation at Ser276 was also found in the tubulointerstitium of the two patients with increased p-STAT3 (Tyr705).Our findings suggest that, instead of tyrosine phosphorylation, serine phosphorylation of STAT3 is commonly activated in the kidney of patients with COVID-19.

Development of resistance to FAK inhibition in pancreatic cancer is linked to stromal depletion.

Gut

2019 May 10

Jiang H, Liu X, Knolhoff BL, Hegde S, Lee KB, Jiang H, Fields RC, Pachter JA, Lim KH, DeNardo DG.
PMID: 31076405 | DOI: 10.1136/gutjnl-2018-317424

Abstract

OBJECTIVE:

We investigated how pancreatic cancer developed resistance to focal adhesion kinase (FAK) inhibition over time.

DESIGN:

Pancreatic ductal adenocarcinoma (PDAC) tumours from KPC mice (p48-CRE; LSL-KRasG12D/wt; p53flox/wt) treated with FAK inhibitor were analysed for the activation of a compensatory survival pathway in resistant tumours. We identified pathways involved in the regulation of signal transducer and activator of transcription 3 (STAT3) signalling on FAK inhibition by gene set enrichment analysis and verified these outcomes by RNA interference studies. We also tested combinatorial approaches targeting FAK and STAT3 in syngeneic transplantable mouse models of PDAC and KPC mice.

RESULTS:

In KPC mice, the expression levels of phosphorylated STAT3 (pSTAT3) were increased in PDAC cells as they progressed on FAK inhibitor therapy. This progression corresponded to decreased collagen density, lowered numbers of SMA+ fibroblasts and downregulation of the transforming growth factor beta (TGF-β)/SMAD signalling pathway in FAK inhibitor-treated PDAC tumours. Furthermore, TGF-β production by fibroblasts in vitro drives repression of STAT3 signalling and enhanced responsiveness to FAK inhibitor therapy. Knockdown of SMAD3 in pancreatic cancer cells abolished the inhibitory effects of TGF-β on pSTAT3. We further found that tumour-intrinsic STAT3 regulates the durability of the antiproliferative activity of FAK inhibitor, and combinatorial targeting of FAK and Janus kinase/STAT3 act synergistically to suppress pancreatic cancer progression in mouse models.

CONCLUSION:

Stromal depletion by FAK inhibitor therapy leads to eventual treatment resistance through the activation of STAT3 signalling. These data suggest that, similar to tumour-targeted therapies, resistance mechanisms to therapies targeting stromal desmoplasia may be critical to treatment durability.

GLI1 activates pro-fibrotic pathways in myelofibrosis fibrocytes

Cell death & disease

2022 May 20

Manshouri, T;Veletic, I;Li, P;Yin, CC;Post, SM;Verstovsek, S;Estrov, Z;
PMID: 35595725 | DOI: 10.1038/s41419-022-04932-4

Bone marrow (BM) fibrosis was thought to be induced exclusively by mesenchymal stromal cells (MSCs). However, we and others found that neoplastic fibrocytes induce BM fibrosis in myelofibrosis (MF). Because glioma-associated oncogene-1 (GLI1), an effector of the Hedgehog pathway, plays a role in the induction of BM fibrosis, we wondered whether GLI1 affects fibrocyte-induced BM fibrosis in MF. Multiplexed fluorescence immunohistochemistry analysis of MF patients' BM detected high levels of GLI1 in MF fibrocytes compared to MSCs or normal fibrocytes. Immunostaining, RNA in situ hybridization, gene expression analysis, and western immunoblotting detected high levels of GLI1 and GLI1-induced matrix metalloproteases (MMP) 2 and 9 in MF patients BM-derived cultured fibrocytes. Similarly, MF patients' BM-derived GLI1+ fibrocytes were found in BMs and spleens of MF xenograft mice. GLI1 silencing reduced the levels of MMP2/9, phosphorylated SMAD2/3, and procollagen-I, and knockdown or inhibition of GLI1 decreased fibrocyte formation and induced apoptosis of both fibrocytes and fibrocyte progenitors. Because Janus kinase (JAK)2-induced STAT3 is constitutively activated in MF and because STAT3 induces GLI1 expression, we sought to determine whether STAT3 activates GLI1 in MF fibrocytes. Imaging analysis detected phosphotyrosine STAT3 in MF patients' BM fibrocytes, and transfection of fibrocytes with STAT3-siRNA or treatment with a JAK1/2 inhibitor ruxolitinib reduced GLI1 and MMP2/9 levels. Chromatin immunoprecipitation and a luciferase assay revealed that STAT3 induced the expression of the GLI1 gene in both MF BM fibrocytes and fibrocyte progenitors. Together, our data suggest that STAT3-activated GLI1 contributes to the induction of BM fibrosis in MF.

E2F and STAT3 provide transcriptional synergy for histone variant H2AZ activation to sustain glioblastoma chromatin accessibility and tumorigenicity

Cell death and differentiation

2022 Jan 20

Yoon, J;Grinchuk, OV;Tirado-Magallanes, R;Ngian, ZK;Tay, EXY;Chuah, YH;Lee, BWL;Feng, J;Crasta, KC;Ong, CT;Benoukraf, T;Ong, DST;
PMID: 35058574 | DOI: 10.1038/s41418-021-00926-5

The histone variant H2AZ is overexpressed in diverse cancer types where it facilitates the accessibility of transcriptional regulators to the promoters of cell cycle genes. However, the molecular basis for its dysregulation in cancer remains unknown. Here, we report that glioblastomas (GBM) and glioma stem cells (GSCs) preferentially overexpress H2AZ for their proliferation, stemness and tumorigenicity. Chromatin accessibility analysis of H2AZ2 depleted GSC revealed that E2F1 occupies the enhancer region within H2AZ2 gene promoter, thereby activating H2AZ2 transcription. Exploration of other H2AZ2 transcriptional activators using a customized "anti-H2AZ2" query signature for connectivity map analysis identified STAT3. Co-targeting E2F and STAT3 synergistically reduced the levels of H2AZ, histone 3 lysine 27 acetylation (H3K27ac) and cell cycle gene transcription, indicating that E2F1 and STAT3 synergize to activate H2AZ gene transcription in GSCs. Remarkably, an E2F/STAT3 inhibitor combination durably suppresses GSC tumorigenicity in an orthotopic GBM xenograft model. In glioma patients, high STAT3 signaling is associated with high E2F1 and H2AZ2 expression. Thus, GBM has uniquely opted the use of E2F1- and STAT3-containing "enhanceosomes" that integrate multiple signaling pathways to achieve H2AZ gene activation, supporting a translational path for the E2F/STAT3 inhibitor combination to be applied in GBM treatment.

Inflammatory Infiltrate and Angiogenesis in Mantle Cell Lymphoma

Transl Oncol.

2020 Mar 13

Annese T, Ingravallo G, Tamma R, De Giorgis M, Maiorano E, Perrone T, Albano F, Specchia G, Ribatti D
PMID: 32120334 | DOI: 10.1016/j.tranon.2020.100744

Mantle cell lymphoma (MCL) is an aggressive and rare B-cell non-Hodgkin lymphoma classified in two clinicopathological subtypes according to SOX11 expression and mutation state of immunoglobulin variable region heavy chain (IgVH) gene. The transcription factor SOX11, overexpressed in 78%-93% of MCL patients, plays a central role in modulating tumor microenvironment prosurvival signals and angiogenic genes. In this work, we have explored the lymph node microenvironment of three subgroups of MCL patients classified according to SOX11 expression as negative, light, and strong. CD34+ microvessels, CD4+ and CD8+ T-lymphocytes, CD68+ and CD163+ macrophages, and the oncogene p53 expression were evaluated by immunohistochemistry. Moreover, STAT3 mRNA expression was analyzed by RNA-scope assay. Our results confirmed increased angiogenesis in the sample of patients positive to SOX11 compared to the negative ones and demonstrated that angiogenesis and SOX11 expression positively correlate to a higher T-lymphocytes inflammatory infiltrate. On the contrary, angiogenesis and SOX11 expression negatively correlate with macrophage's inflammatory infiltrate and p53 expression. STAT3 mRNA expression level was not relevant concerning angiogenesis or SOX11 expression. Overall, our data indicate that, in MCL, SOX11 expression is associated with increased angiogenesis and a high CD4+ and CD8+ T-cell infiltration, which are not sustained by CD163+ macrophages infiltrate and p53 expression

KLHDC7B-DT aggravates pancreatic ductal adenocarcinoma development via inducing cross-talk between cancer cells and macrophages

Clinical science (London, England : 1979)

2021 Feb 26

Li, MX;Wang, HY;Yuan, CH;Ma, ZL;Jiang, B;Li, L;Zhang, L;Xiu, DR;
PMID: 33538300 | DOI: 10.1042/CS20201259

Tumor microenvironment (TME) exerts key roles in pancreatic ductal adenocarcinoma (PDAC) development. However, the factors regulating the cross-talk between PDAC cells and TME are largely unknown. In the present study, we identified a long noncoding RNA (lncRNA) KLHDC7B divergent transcript (KLHDC7B-DT), which was up-regulated in PDAC and correlated with poor survival of PDAC patients. Functional assays demonstrated that KLHDC7B-DT enhanced PDAC cell proliferation, migration, and invasion. Mechanistically, KLHDC7B-DT was found to directly bind IL-6 promoter, induce open chromatin structure at IL-6 promoter region, activate IL-6 transcription, and up-regulate IL-6 expression and secretion. The expression of KLHDC7B-DT was positively correlated with IL-6 in PDAC tissues. Via inducing IL-6 secretion, KLHDC7B-DT activated STAT3 signaling in PDAC cells in an autocrine manner. Furthermore, KLHDC7B-DT also activated STAT3 signaling in macrophages in a paracrine manner, which induced macrophage M2 polarization. KLHDC7B-DT overexpressed PDAC cells-primed macrophages promoted PDAC cell proliferation, migration, and invasion. Blocking IL-6/STAT3 signaling reversed the effects of KLHDC7B-DT on macrophage M2 polarization and PDAC cell proliferation, migration, and invasion. In conclusion, KLHDC7B-DT enhanced malignant behaviors of PDAC cells via IL-6-induced macrophage M2 polarization and IL-6-activated STAT3 signaling in PDAC cells. The cross-talk between PDAC cells and macrophages induced by KLHDC7B-DT represents potential therapeutic target for PDAC.

Euglycemia Restoration by Central Leptin in Type 1 Diabetes Requires STAT3 Signaling but Not Fast-Acting Neurotransmitter Release.

Diabetes.

2016 Jan 28

Xu Y, Chang JT, Myers MG Jr, Xu Y, Tong Q.
PMID: 26822087 | DOI: -

Central leptin action is sufficient to restore euglycemia in insulinopenic type 1 diabetes (T1D); however, the underlying mechanism remains poorly understood. To examine the role of intracellular STAT3 pathways, we used LepRs/s mice with disrupted leptin-pSTAT3 signaling to test the effect of central leptin on euglycemia restoration. These mice developed STZ-induced T1D, which was surprisingly not associated with hyperglucagonemia, a typical manifestation in T1D. Further, leptin action on euglycemia restoration was abrogated in these mice, which was associated with refractory hypercorticosteronemia. To examine the role of fast-acting neurotransmitters glutamate and γ-aminobutyric acid (GABA), two major neurotransmitters in the brain, from LepR neurons, we used mice with disrupted release of glutamate, GABA or both from LepR neurons. Surprisingly, all mice responded normally to leptin-mediated euglycemia restoration, which was associated with expected correction from hyperglucagonemia and hyperphagia. In contrast, mice with loss of glutamate and GABA appeared to develop an additive obesity effect over those with loss of single neurotransmitter release. Thus, our study reveals that STAT3 signaling, but not fast-acting neurotransmitter release, is required for leptin action on euglycemia restoration, and that hyperglucagonemia is not required for T1D.

Long non-coding RNA LEISA promotes progression of lung adenocarcinoma via enhancing interaction between STAT3 and IL-6 promoter

Oncogene

2021 Apr 15

Wu, S;Liu, B;Zhang, Y;Hong, R;Liu, S;Xiang, T;Tao, T;Cai, J;Wu, J;Li, M;Guan, H;
PMID: 33859372 | DOI: 10.1038/s41388-021-01769-7

Long non-coding RNAs (lncRNAs) are emerging as a new class of regulators for a variety of biological processes and have been suggested to play pivotal roles in cancer development and progression. Our current study found that a lncRNA, designated enhancing IL-6/STAT3 signaling activation (LEISA, ENST00000603468), functioned as an oncogenic lncRNA in lung adenocarcinoma (LAD), a major form of non-small cell lung carcinoma, which is one of the most frequently diagnosed malignancies with high morbidity and mortality worldwide, and was involved in the regulation of STAT3 induced IL-6 transcription. Our data showed that LEISA was highly expressed in, and correlated with the clinical progression and prognosis of LAD. Ectopic expression of LEISA promoted the proliferation and suppressed apoptosis of LAD cells in vitro and in vivo. Mechanistically, we demonstrated that LEISA recruited STAT3 to bind the promoter of IL-6 and upregulated IL-6 expression. Taken together, our work identifies LEISA as a potential diagnostic biomarker and therapeutic target for LAD.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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BACKGROUND & AIMS:

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CONCLUSION:

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Advanced Cell Diagnostics

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