BACKGROUND: Neuroendocrine prostate cancer (NEPC) is a lethal subset of prostate cancer which is characterized by neuroendocrine differentiation and loss of androgen receptor (AR) signaling. Growing evidence reveals that cell lineage plasticity is crucial in the failure of NEPC therapies. Although studies suggest the involvement of the neural transcription factor PAX6 in drug resistance, its specific role in NEPC remains unclear.
METHODS: The expression of PAX6 in NEPC was identified via bioinformatics and immunohistochemistry. CCK8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay were used to illustrate the key role of PAX6 in the progression of in vitro. ChIP and Dual-luciferase reporter assays were conducted to confirm the binding sequences of AR in the promoter region of PAX6, as well as the binding sequences of PAX6 in the promoter regions of STAT5A and MET. For in vivo validation, the xenograft model representing NEPC subtype underwent pathological analysis to verify the significant role of PAX6 in disease progression. Complementary diagnoses were established through public clinical datasets and transcriptome sequencing of specific cell lines. ATAC-seq was used to detect the chromatin accessibility of specific cell lines.
RESULTS: PAX6 expression was significantly elevated in NEPC and negatively regulated by AR signaling. Activation of PAX6 in non-NEPC cells led to NE trans-differentiation, while knock-down of PAX6 in NEPC cells inhibited the development and progression of NEPC. Importantly, loss of AR resulted in an enhanced expression of PAX6, which reprogramed the lineage plasticity of prostate cancer cells to develop NE phenotypes through the MET/STAT5A signaling pathway. Through ATAC-seq, we found that a high expression level of PAX6 elicited enhanced chromatin accessibility, mainly through attenuation of H4K20me3, which typically causes chromatin silence in cancer cells.
CONCLUSIONS: This study reveals a novel neural transcription factor PAX6 could drive NEPC progression and suggest that it might serve as a potential therapeutic target for the management of NEPC.

OBJECTIVE: To explore the potential role of signal transducer and activator of transcription 5A (STAT5A) in the metastasis of breast cancer, and its mechanism of regulation underlying.
RESULTS: TCGA datasets were used to evaluate the expression of STAT5A in normal and different cancerous tissues through TIMER2.0, indicating that STAT5A level was decreased in breast cancer tissues compared with normal ones. Gene Set Enrichment Analysis predicted that STAT5A was associated with the activation of immune cells and cell cycle process. We further demonstrated that the infiltration of immune cells was positively associated with STAT5A level. Influorescence staining revealed the expression and distribution of F-actin was regulated by STAT5A, while colony formation assay, wound healing and transwell assays predicted the inhibitory role of STAT5A in the colony formation, migratory and invasive abilities in breast cancer cells. In addition, overexpression of the Notch3 intracellular domain (N3ICD), the active form of Notch3, resulted in the increased expression of STAT5A. Conversely, silencing of Notch3 expression by siNotch3 decreased STAT5A expression, supporting that STAT5A expression is positively associated with Notch3 in human breast cancer cell lines and breast cancer tissues. Mechanistically, chromatin immunoprecipitation showed that Notch3 was directly bound to the STAT5A promoter and induced the expression of STAT5A. Moreover, overexpressing STAT5A partially reversed the enhanced mobility of breast cancer cells following Notch3 silencing. Low expression of Notch3 and STAT5A predicted poorer prognosis of patients with breast cancer.
CONCLUSIONS: The present study demonstrates that Notch3 inhibits metastasis in breast cancer through inducing transcriptionally STAT5A, which was associated with tumor-infiltrating immune cells, providing a novel strategy to treat breast cancer.

Inhibition of STAT5 was recently reported to reduce murine atherosclerosis. However, the role of STAT5 isoforms, and more in particular STAT5A in macrophages in the context of human atherosclerosis remains unknown.
Here, we demonstrate reciprocal expression regulation of STAT5A and STAT5B in human atherosclerotic lesions. The former was highly upregulated in ruptured over stable plaque and correlated with macrophage presence, a finding that was corroborated by the high chromosomal accessibility of STAT5A but not B gene in plaque macrophages. Phosphorylated STAT5 correlated with macrophages confirming its activation status. As macrophage STAT5 is activated by GM-CSF, we studied the effects of its silencing in GM-CSF differentiated human macrophages. STAT5A knockdown blunted the immune response, phagocytosis, cholesterol metabolism, and augmented apoptosis terms on transcriptional levels. These changes could partially be confirmed at functional level, with significant increases in apoptosis and decreases in lipid uptake and IL-6, IL-8, and TNFa cytokine secretion after STAT5A knockdown. Finally, inhibition of general and isoform A specific STAT5 significantly reduced the secretion of TNFa, IL-8 and IL-10 in ex vivo tissue slices of advanced human atherosclerotic plaques.
In summary, we identify STAT5A as an important determinant of macrophage functions and inflammation in the context of atherosclerosis and show its promise as therapeutic target in human atherosclerotic plaque inflammation.

OBJECTIVE: Multiple transcription factors (TFs) have previously been shown to control hypertrophic chondrocyte-specific mouse type X collagen gene (Col10a1) expression via interaction with Col10a1 promoters. This study aims to investigate the role and mechanism of the potential binding factor signal transduction and transcription activator 5a (Stat5a) of Col10a1 cis-enhancer, in controlling Col10a1 gene expression and chondrocyte hypertrophic differentiation.
METHODS: The potential Col10a1 regulator was predicted by the transcription factor affinity prediction (TRAP) analysis of the 150-bp Col10a1 cis enhancer. Stat5a was screened and verified by qRT-PCR, western blot and IHC analyses. Transfection of Stat5a siRNA or expression plasmid into MCT and ATDC5 cells was performed to either knockdown or over-express Stat5a and to investigate the influence of Stat5a on Col10a1 gene expression during the chondrocyte hypertrophy. Dual-luciferase reporter assay was performed to explore the mechanism of Stat5a affecting Col10a1 transcription. Alcian blue, alkaline phosphatase, and alizarin red staining, as well as qRT-PCR analyses of related marker genes were performed to investigate the effect and possible mechanism of Stat5a on chondrocyte differentiation.
RESULTS: The potential binding factor of Col10a1 cis-enhancer Stat5a and Col10a1 were both highly expressed and positively correlated within hypertrophic chondrocytes in vitro and in situ. Knockdown of Stat5a reduced Col10a1 expression, while overexpression of Stat5a enhanced Col10a1 expression in hypertrophic chondrocytes, suggesting Stat5a as a positive Col10a1 regulator. Mechanistically, Stat5a was shown to potentiate the reporter activity mediated by Col10a1 promoter/enhancer. In addition, Stat5a increased the intensity of alkaline phosphatase staining of ATDC5 cells and the expression of relevant hypertrophic marker genes including Runx2, which was consistent with the expression of Stat5a and Col10a1.
CONCLUSIONS: Our results support that Stat5a promoted Col10a1 expression and chondrocyte hypertrophic differentiation, possibly via interaction with the 150-bp Col10a1 cis-enhancer.

Studies examining the role of signal transducer and activator of transcription 5 (STAT5) in various cancers have produced controversial results. To address this controversy, we examined the prognostic role of STAT5a in cancer patients across multiple cancers. Transcription levels of STAT5a between tumors and normal tissues, obtained from public databases, were analyzed for statistical differences using Cox regression analysis with the outcome as overall survival and covariate of interest as high STAT5a expression. Meta-analysis was then conducted to summarize the hazard ratio estimate from the Cox regression analyses. We found that STAT5a was significantly under-expressed in breast, lung, and ovarian cancers, while STAT5a was significantly overexpressed in lymphoid neoplasm diffuse large B-cell lymphoma, glioblastoma, and glioma. High STAT5a expression was significantly associated with favorable survival in bladder cancer (lnHR = -0.8689 [-1.4087, -0.3292], P-value = 0.0016), breast cancer (lnHR = -0.7805 [-1.1394, -0.4215], P-value < 0.0001) and lung cancer (lnHR = -0.3255 [-0.6427, -0.0083], P-value = 0.0443). After adjusting for clinicopathological factors, high STAT5a expression remained significantly associated with favorable survival in breast cancer (lnHR = -0.6091 [-1.0810, -0.1372], P-value = 0.0114). These results suggest that higher STAT5a expression is associated with favorable overall survival in breast cancer, and therefore might have protective effects, and that STAT5a expression could be a potential prognostic biomarker, especially in breast cancer. However, the prognostic role of STAT5a is dependent on cancer type.

Lipopolysaccharide (LPS) has previously been implicated in insulin resistance by generating an innate immune response and activating inflammatory cascades. Many studies have discovered a relationship between high levels of serum LPS and the advancement of diabetic microvascular problems, indicating that LPS may play a role in the control of critical signaling pathways connected to insulin resistance. The current study focused on signaling pathways linked to insulin resistance and explored probable mechanisms of LPS-induced insulin resistance in a murine model. It next looked at the effects of burdock, bee pollen, and -lipoic acid on LPS-induced inflammation and autoimmune defects in rats. LPS intoxication was induced via ip injection for one week in a dose of 10 mg/kg followed by α-lipoic acid, Burdock and bee pollen in an oral treatment for one month. Following that, biochemical and molecular studies were performed. The RNA expression of the regulating genes STAT5A and PTEN was measured. In addition, ATF-4 and CHOP as autophagy biomarkers were also subjected to mRNA quantification. The results demonstrated a considerable improvement in the -lipoic acid, Burdock, and bee pollen treated groups via modifying oxidative stress indicators as well as molecular ones. Furthermore, glucose concentration in serum and α-amylase were also improved upon treatment with the superiority of α-lipoic acid for modulating all estimated parameters. In conclusion: the results declared in the current study suggested that α-lipoic acid could regulate insulin resistance signaling pathways induced by LPS intoxication.

Definitive diagnosis to sudden cardiac death (SCD) is often challenging since the postmortem examination on SCD victims could hardly demonstrate an adequate cause of death. It is therefore important to uncover the inherited risk component to SCD. Signal transducer and activators of transcription 5 A (STAT5A) is a member of the STAT family and a transcription factor that is activated by many cell ligands and associated with various cardiovascular processes. In this study, we performed a systematic variant screening on the STAT5A to filter potential functional genetic variations. Based on the screening results, an insertion/deletion polymorphism (rs3833144) in 3\'UTR of STAT5A was selected as the candidate variant. A total of 159 SCD cases and 668 SCD matched healthy controls was enrolled to perform a case-control study and evaluate the association between rs3833144 and SCD susceptibility in Chinese populations. Logistic regression analysis showed that the deletion allele of rs3833144 had significantly increased the SCD risk (odds ratio (OR) = 1.54; 95% confidence interval (CI) = 1.18-2.01; P = 0.000955). Further genotype-expression eQTL analysis showed that samples with deletion allele appeared to lower expression of STAT5A, and in silico prediction suggested the local 3 D structure changes of STAT5A mRNA caused by the variant. On the other hand, the bioinformatic analysis presented that promoters of RARA and PTGES3L-AARSD1 could interact with rs3833144, and eQTL analysis showed the higher expression of both genes in samples with deletion allele. Dual-luciferase activity assays also suggested the significant regulatory role of rs3833144 in gene transcription. Our current data thus suggested a possible involvement of rs3833144 to SCD predisposition in Chinese populations and rs3833144 with potential function roles may become a candidate marker for SCD diagnosis and prevention.

The conversion of signal transducer and activator of transcription (STAT) proteins from latent to active transcription factors is central to cytokine signaling. Triggered by their signal-induced tyrosine phosphorylation, it is the assembly of a range of cytokine-specific STAT homo- and heterodimers that marks a key step in the transition of hitherto latent proteins to transcription activators. In contrast, the constitutive self-assembly of latent STATs and how it relates to the functioning of activated STATs is understood less well. To provide a more complete picture, we developed a co-localization-based assay and tested all 28 possible combinations of the seven unphosphorylated STAT (U-STAT) proteins in living cells. We identified five U-STAT homodimers-STAT1, STAT3, STAT4, STAT5A, and STAT5B-and two heterodimers-STAT1:STAT2 and STAT5A:STAT5B-and performed semi-quantitative assessments of the forces and characterizations of binding interfaces that support them. One STAT protein-STAT6-was found to be monomeric. This comprehensive analysis of latent STAT self-assembly lays bare considerable structural and functional diversity in the ways that link STAT dimerization before and after activation.

KIF5B-RET gene rearrangement occurs in ~1% of lung adenocarcinomas. Recently, targeted agents that inhibit RET phosphorylation have been evaluated in several clinical studies; however, little is known about the role of this gene fusion in driving lung cancer. Immunohistochemistry was used to evaluate the expression of the FOXA2 protein in tumor tissues of patients with lung adenocarcinoma. KIF5B-RET fusion cells proliferated in a cohesive form and grew tightly packed with variable-sized colonies. The expression of RET and its downstream signaling molecules, including p-BRAF, p-ERK, and p-AKT, increased. In KIF5B-RET fusion cells, the intracellular expression of p-ERK was higher in the cytoplasm than in the nucleus. Two transcription factors, STAT5A and FOXA2, exhibiting significantly different expressions at the mRNA level, were finally selected. p-STAT5A was highly expressed in the nucleus and cytoplasm, whereas the expression of the FOXA2 protein was lower; however, it was much higher in the nucleus than in the cytoplasm. Compared with the expression of FOXA2 in the RET rearrangement-wild NSCLC (45.0%), high expression (3+) were observed in most RET rearrangement NSCLCs (94.4%). Meanwhile, KIF5B-RET fusion cells began to increase belatedly from day 7 and only doubled on day 9 in 2D cell culture. However, tumors in mice injected with KIF5B-RET fusion cells began to rapidly increase from day 26. In cell cycle analyses, the KIF5B-RET fusion cells in G0/G1 were increased on day 4 (50.3 ± 2.6%) compared with the empty cells (39.3 ± 5.2%; P = 0.096). Cyclin D1 and E2 expressions were reduced, whereas CDK2 expression slightly increased. pRb and p21 expression was diminished compared with the empty cells, TGF-β1 mRNA was highly expressed, and the proteins were accumulated mostly in the nucleus. Twist mRNA and protein expression was increased, whereas Snail mRNA and protein expression was decreased. Particularly, in KIF5B-RET fusion cells treated with FOXA2 siRNA, the expression of TGF-β 1 mRNA was remarkably reduced but Twist1 and Snail mRNA were increased. Our data suggest that cell proliferation and invasiveness in KIF5B-RET fusion cells are regulated by the upregulation of STAT5A and FOXA2 through the continuous activation of multiple RET downstream signal cascades, including the ERK and AKT signaling pathways. We found that TGF-β1 mRNA, where significant increments were observed in KIF5B-RET fusion cells, is regulated at the transcriptional level by FOXA2.

Identification of causative genes or genetic variants associated with phenotype traits benefits the genetic improvement of animals. CISH plays a role in immunity and growth, however, the upstream transcriptional factors of porcine CISH and the genetic variations in these factors remain unclear. In this study, we firstly identified the minimal core promoter of porcine CISH and confirmed the existence of STATx binding sites. Overexpression and RT-qPCR demonstrated STAT5A increased CISH transcriptional activity (P < 0.01) and mRNA expression (P < 0.01), while GATA1 inhibited CISH transcriptional activity (P < 0.01) and the following mRNA expression (P < 0.05 or P < 0.01). Then, the putative functional genetic variations of porcine STAT5A were screened and a PCR-SSCP was established for genotype g.508A>C and g.566C>T. Population genetic analysis showed the A allele frequency of g.508A>C and C allele frequency of g.566C>T was 0.61 and 0.94 in Min pigs, respectively, while these two alleles were fixed in the Landrace population. Statistical analysis showed that Min piglets with CC genotype at g.566C>T or Hap1: AC had higher 28-day body weight, 35-day body weight, and ADG than TC or Hap3: CT animals (P < 0.05, P < 0.05). Further luciferase activity assay demonstrated that the activity of g.508A>C in the C allele was lower than the A allele (P < 0.05). Collectively, the present study demonstrated that STAT5A positively regulated porcine CISH transcription, and SNP g.566C>T in the STAT5A was associated with the Min piglet growth trait.