Shan L, etal., Breast Cancer Res Treat. 2004 Dec;88(3):263-72.
Signal transducer and activator of transcription (Stat) 5a is a transcription factor mediating the action of specific cytokines, growth factors and hormones on gene expression. In the mammary gland, Stat5a is well recognized for its function in prolactin signali
ng, lobuloalveolar development, and milk protein expression during pregnancy and lactation. Latent cytoplasmic Stat5a is activated by tyrosine phosphorylation and following dimerization undergoes nuclear import. In the current study, Stat5a expression was examined immunohistochemically in carcinomas induced by the chemical carcinogens 7,12-dimethylbenz[a]anthracene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. A high percentage of carcinomas showed nuclear labeling of Stat5a [44 of 68 (65%)] with Stat5a nuclear labeling index ranging from 18 to 77%. In contrast, control normal mammary gland tissue displayed cytosolic expression. Carcinomas with different Stat5a staining patterns (cytoplasmic or nuclear) showed a statistical difference for the proliferating cell nuclear antigen (PCNA) labeling, tumor differentiation, nuclear grade, mitotic activity, and tumor size. High Stat5a nuclear expression was closely correlated with the higher-grade carcinomas. Stat5a nuclear expression was also detected in intraductal proliferations (10 of 21 lesions) and in ductal carcinomas in situ (13 of 15 lesions). Immunohistochemical analysis was further carried out in human breast cancers. Stat5a nuclear expression was detected in ductal and lobular carcinomas and DCIS at a frequency of 48% (15/31), 33% (2/6), and 40% (2/5), respectively. Nuclear expression of Stat5a in human breast cancers also correlated with the PCNA nuclear labeling index. The findings implicate activated Stat5a in mammary gland cancer development in the rat and human.
Ren S, etal., Oncogene. 2002 Jun 20;21(27):4335-9.
A genetic study was conducted to determine if activated Stat5a contributes to mammary carcinogenesis and to evaluate the mechanism. Similar to human breast cancers, a proportion of mammary adenocarcinomas in the WAP-TAg transgenic mouse model demonstrate constit
utive Stat5a/b and Stat3 activation. Stat5a activation is linked to mammary epithelial cell survival and differentiation, and proliferation in hematopoetic cell lineages. Breeding WAP-TAg mice to mice carrying germ-line deletions of the Stat5a gene generated mice with reduced levels of Stat5a. Hemizygous loss of the Stat5a allele significantly reduced levels of Stat5a expression without altering mammary gland development or transgene expression levels. In comparison to mice carrying two wild-type Stat5a alleles, hemizygous loss of the Stat5a allele reduced the number of mice with palpable tumors at 7 months of age (67% from 100%, P<0.05), resulted in smaller tumors at 7 months of age (3.8 cm3 from 7.6 cm3, P=0.003), delayed first tumor appearance (208 days from 188 days, P=0.01), and increased the apoptotic index in the adenocarcinomas (4.3+/-0.3 from 1.2+/-0.2, P=0.016). Neither cell proliferation nor differentiation in the cancers was altered. Decreasing Stat5a activation levels could be a therapeutic approach for reducing survival of breast cancer cells.
The rat homolog of sheep mammary gland factor (MGF)/Stat5 has been isolated and used to study the regulation of Stat5 during mammary gland development and PRL regulation in COS cells transfected with Stat5a and the PRL receptor. Two alternatively spliced isoform
s, designated Stat5a1 and Stat5a2, were identified, the latter encoding a carboxy-terminal truncated protein. A polyclonal antibody to a carboxy-terminal peptide of Stat5a1 was generated and used to measure the level of this isoform during mammary gland development and after PRL induction in COS cells transiently transfected with Stat5a and the long form of the PRL receptor. Surprisingly, Stat5a mRNA and protein were readily detected both in virgin rats and after mammary gland involution. The levels of Stat5a increased during pregnancy, were highest in late pregnancy, and then, unexpectedly, decreased during lactation, the time at which the highest levels of milk protein gene expression are observed. Electrophoretic mobility shift assays using the specific anti-Stat5a1 antisera demonstrated that Stat5a1 comprises part of the heterogeneous, PRL-inducible, protein-DNA complex associated with the beta-casein GAS site. Immunocytochemical analysis detected considerable cytoplasmic and some nuclear staining for Stat5a1 during late pregnancy and predominantly nuclear staining during early lactation. The lack of correspondence of Stat5a gene expression and beta-casein gene expression suggests that Stat5 activation may facilitate the interaction of other factors binding within composite response elements identified recently in the milk protein gene promoters that are then responsible for the stable expression of milk protein genes in terminally differentiated mammary epithelial cells.
Hu X, etal., Proc Natl Acad Sci U S A. 2013 Jun 18;110(25):10213-8. doi: 10.1073/pnas.1221243110. Epub 2013 Jun 3.
Tumor suppressors known to date impede cancer growth by arresting the cell cycle or promoting apoptosis. Here we show that unphosphorylated human STAT5A functions as a tumor suppressor capable of repressing multiple oncogenes via heterochromatin formation. Unph
osphorylated STAT5A binds to heterochromatin protein 1alpha (HP1alpha) and stabilizes heterochromatin. Expressing unphosphorylated STAT5A or HP1alpha inhibits colon cancer growth in mouse xenograft models. Transcriptome profiling shows that expressing an unphosphorylatable STAT5A has similar effects to overexpressing HP1alpha in global gene expression. Notably, the majority of the genes commonly repressed by unphosphorylated STAT5A and HP1alpha have been implicated in cancer development. Finally, down-regulation, somatic mutations, and deletions of STAT5 genes are found in certain human cancers. These results suggest that unphosphorylated STAT5A may epigenetically suppress tumor growth by promoting heterochromatin formation.
Haddad BR, etal., Am J Pathol. 2013 Jun;182(6):2264-75. doi: 10.1016/j.ajpath.2013.02.044. Epub 2013 May 7.
The molecular mechanisms underlying progression of prostate cancer (PCa) to castrate-resistant (CR) and metastatic disease are poorly understood. Our previous mechanistic work shows that inhibition of transcription factor Stat5 by multiple alternative methods induces extensive rapid apoptotic death
of Stat5-positive PCa cells in vitro and inhibits PCa xenograft tumor growth in nude mice. Furthermore, STAT5A/B induces invasive behavior of PCa cells in vitro and in vivo, suggesting involvement of STAT5A/B in PCa progression. Nuclear STAT5A/B protein levels are increased in high-grade PCas, CR PCas, and distant metastases, and high nuclear STAT5A/B expression predicts early disease recurrence and PCa-specific death in clinical PCas. Based on these findings, STAT5A/B represents a therapeutic target protein for advanced PCa. The mechanisms underlying increased Stat5 protein levels in PCa are unclear. Herein, we demonstrate amplification at the STAT5A/B gene locus in a significant fraction of clinical PCa specimens. STAT5A/B gene amplification was more frequently found in PCas of high histologic grades and in CR distant metastases. Quantitative in situ analysis revealed that STAT5A/B gene amplification was associated with increased STAT5A/B protein expression in PCa. Functional studies showed that increased STAT5A/B copy numbers conferred growth advantage in PCa cells in vitro and as xenograft tumors in vivo. The work presented herein provides the first evidence of somatic STAT5A/B gene amplification in clinical PCas.
Nie ZY, etal., J Exp Clin Cancer Res. 2020 Jan 17;39(1):17. doi: 10.1186/s13046-019-1502-7.
BACKGROUND: STAT5 plays an important role in the transformation of hematopoietic cells by BCR-ABL. However, the downstream target genes activated by STAT5 in chronic myeloid leukemia (CML) cells remain largely unclear. Here, we investigated the mechanistic functional relationship between
STAT5A-regulated microRNA and CML cell apoptosis. METHODS: The expression of USP15, Caspase-6, STAT5A-regulated miR-202-5p and STAT5A was detected by qRT-PCR and Western blotting in CML cell lines and PBMCs of CML patients. Cell apoptosis was evaluated by flow cytometry. Both gain- and loss-of-function experiments were used to investigate the roles of USP15, miR-202-5p and STAT5A in CML. Luciferase reporter assay detected the effect of miR-202-5p on USP15 expression. Xenograft animal model was used to test the effect of anti-miR-202-5p and pimozide on K562 cell xenograft growth. RESULTS: USP15 expression was significantly downregulated in CML cell lines and PBMCs of CML patients. Depletion of USP15 increased, whereas overexpression of USP15 reduced the resistance of CML cells to Imatinib. Further, decreased deubiquitinating activity of USP15 by USP15 downregulation led to reduced caspase-6 level, thus attenuating CML cell apoptosis. Mechanistically, miR-202-5p was upregulated in K562G cells and negatively regulated USP15 expression by directly targeting USP15 3'-UTR. Correspondingly, upregulation of miR-202-5p enhanced the resistance of CML cells to Imatinib by inhibiting cell apoptosis. Importantly, STAT5A was upregulated in CML cells and directly activated miR-202-5p transcription by binding to the pre-miR-202 promoter. Pimozide induced CML cell apoptosis and significantly reduced K562 cell xenograft growth in vivo by blocking STAT5A/miR-202-5p/USP15/Caspase-6 regulatory axis. CONCLUSIONS: we provide the first evidence that de-regulated STAT5A/miR-202-5p/USP15/Caspase-6 regulatory axis suppresses the apoptosis of CML cells, targeting this pathway might be a promising therapeutic approach for the treatment of CML.
Liu HW, etal., Cancers (Basel). 2019 Nov 27;11(12). pii: cancers11121888. doi: 10.3390/cancers11121888.
BACKGROUND: Glioblastoma (GBM), a malignant grade IV tumor, is the most malignant brain tumor due to its hyper-proliferative and apoptosis-evading characteristics. The signal transducer and activators of transcription (STAT) family genes, including STAT3 and STAT5A
>STAT5A, have been indicated to play important roles in GBM progression. Increasing number of reports suggest that garcinol, a polyisoprenylated benzophenone and major bioactive component of Garcinia indica contains potent anti-cancer activities. MATERIAL AND METHODS: The present study investigated the anti-GBM effects of garcinol, focusing on the STAT3/STAT5A activation, using a combination of bioinformatics, in vitro, and ex vivo assays. RESULTS: Our bioinformatics analysis of The Cancer Genome Atlas (TCGA)-GBM cohort (n = 173) showed that STAT3 and STAT5A are preferentially elevated in primary and recurrent GBM, compared to non-tumor brain tissues, and is significantly correlated with reduced overall survival. In support, our immunohistochemical staining of a GBM cohort (n = 45) showed an estimated 5.3-fold (p < 0.001) elevation in STAT3 and STAT5A protein expression in primary and recurrent GBM versus the non-tumor group. In vitro, garcinol treatment significantly suppressed the proliferative, invasive, and migratory potential of U87MG or GBM8401 cells, dose-dependently. In addition, garcinol anticancer effect significantly attenuated the GBM stem cell-like phenotypes, as reflected by diminished ability of U87MG or GBM8401 to form colonies and tumorspheres and suppressed expression of OCT4 and SOX2. Furthermore, analysis on GBM transcriptome revealed an inverse correlation between the level of STAT3/5A and hsa-miR-181d. Garcinol-mediated anti-GBM effects were associated with an increased hsa-miR-181d/STAT3 and hsa-miR-181d/5A ratio. The results were further verified in vivo using U87MG mouse xenograft model where administration of garcinol significantly inhibited tumor growth. CONCLUSIONS: We present evidence of anti-GBM efficacy of garcinol mediated by enhancing the hsa-miR-181d/STAT3 and hsa-miR-181d/5A ratios in GBM cells. Our findings suggest a potential new therapeutic agent for combating aggressive GBM.
Tan D, etal., Chin J Physiol. 2015 Oct 31;58(5):275-84. doi: 10.4077/CJP.2015.BAD322.
As a very important transcription factor, signal transducer and activator of transcription 5a (Stat5a) has been reported to be involved in human reproductive cancers such as breast, prostate and ovarian cancer. However, up to date, the exact role of Stat5a
='font-weight:700;'>Stat5a in breast cancer is still not clear. The data reported are conflicting. D5 Stat5a is a variant of Stat5a we cloned recently. This newly cloned variant behave like its full length counterpart in terms of dimerization, being activated by prolactin and nuclear translocation, however it also behave differently in terms of effect on cell proliferation and interaction with other transcription factors. In the present study, we examined its effect on cell proliferation of cultured breast cancer cells (MCF-10A and MCF-7) by using adenovirus-mediated gene transfer and MTS technology. Also, quantitative real time polymerase chain reaction (qRT-PCR), chromatin immunoprecipitation assay (ChIP) and Western blot were used to probe the possible changes of insulin-like growth factor binding protein-7 (IGFBP-7) expression including mRNA and protein, and the epigenetic changes with overexpression of this newly cloned variant. The results clarified that D5 Stat5a (1) behaves as a promoting factor to the cell proliferation of MCF-10A and MCF-7, (2) induces enhancer of zeste homology 2 (EZH2) expression in breast epithelial cells, as well as histone 3 trimethylation of IGFBP-7 promoter region, and (3) lower IGFBP-7 expression was detected in breast cancer tissue. Taking together, we concluded that the mitogenic effect of D5 Stat5a on breast cells is, at least partly, through up-regulation of histone methyltransferase, EZH2, and therefore inhibiting IGFBP-7 expression by increasing H3K27Me3 of IGFBP-7 promoter region.
Hoang DT, etal., Mol Cancer Ther. 2015 Mar;14(3):713-26. doi: 10.1158/1535-7163.MCT-14-0819. Epub 2014 Dec 31.
Although poorly understood, androgen receptor (AR) signaling is sustained despite treatment of prostate cancer with antiandrogens and potentially underlies development of incurable castrate-resistant prostate cancer. However, therapies targeting the AR signaling axis eventually fail when prostate ca
ncer progresses to the castrate-resistant stage. Stat5a/b, a candidate therapeutic target protein in prostate cancer, synergizes with AR to reciprocally enhance the signaling of both proteins. In this work, we demonstrate that Stat5a/b sequesters antiandrogen-liganded (MDV3100, bicalutamide, flutamide) AR in prostate cancer cells and protects it against proteasomal degradation in prostate cancer. Active Stat5a/b increased nuclear levels of both unliganded and antiandrogen-liganded AR, as demonstrated in prostate cancer cell lines, xenograft tumors, and clinical patient-derived prostate cancer samples. Physical interaction between Stat5a/b and AR in prostate cancer cells was mediated by the DNA-binding domain of Stat5a/b and the N-terminal domain of AR. Moreover, active Stat5a/b increased AR occupancy of the prostate-specific antigen promoter and AR-regulated gene expression in prostate cancer cells. Mechanistically, both Stat5a/b genetic knockdown and antiandrogen treatment induced proteasomal degradation of AR in prostate cancer cells, with combined inhibition of Stat5a/b and AR leading to maximal loss of AR protein and prostate cancer cell viability. Our results indicate that therapeutic targeting of AR in prostate cancer using antiandrogens may be substantially improved by targeting of Stat5a/b.
Talati PG, etal., Am J Pathol. 2015 Sep;185(9):2505-22. doi: 10.1016/j.ajpath.2015.04.026.
Active Stat5a/b predicts early recurrence and disease-specific death in prostate cancer (PC), which both typically are caused by development of metastatic disease. Herein, we demonstrate that Stat5a/b induces epithelial-to-
mesenchymal transition (EMT) of PC cells, as shown by Stat5a/b regulation of EMT marker expression (Twist1, E-cadherin, N-cadherin, vimentin, and fibronectin) in PC cell lines, xenograft tumors in vivo, and patient-derived PCs ex vivo using organ explant cultures. Jak2-Stat5a/b signaling induced functional end points of EMT as well, indicated by disruption of epithelial cell monolayers and increased migration and adhesion of PC cells to fibronectin. Knockdown of Twist1 suppressed Jak2-Stat5a/b-induced EMT properties of PC cells, which were rescued by re-introduction of Twist1, indicating that Twist1 mediates Stat5a/b-induced EMT in PC cells. While promoting EMT, Jak2-Stat5a/b signaling induced stem-like properties in PC cells, such as sphere formation and expression of cancer stem cell markers, including BMI1. Mechanistically, both Twist1 and BMI1 were critical for Stat5a/b induction of stem-like features, because genetic knockdown of Twist1 suppressed Stat5a/b-induced BMI1 expression and sphere formation in stem cell culture conditions, which were rescued by re-introduction of BMI1. By using human prolactin knock-in mice, we demonstrate that prolactin-Stat5a/b signaling promoted metastases formation of PC cells in vivo. In conclusion, our data support the concept that Jak2-Stat5a/b signaling promotes metastatic progression of PC by inducing EMT and stem cell properties in PC cells.
Nanashima N, etal., J Biol Chem. 2005 Dec 30;280(52):43010-6. Epub 2005 Oct 14.
Hirosaki hairless rat (HHR) is a mutant strain spontaneously derived from Sprague-Dawley rats (SDR), and its inheritance is autosomal recessive. In addition to hair loss, female HHRs show involution of the mammary gland at an early stage of lactation. In the present study we investigated the mammary
gland development in HHR. Morphological examinations revealed that HHR mammary glands are underdeveloped in virgins and exhibit distended alveoli on day 1 of lactation (L1), followed by involution. Milk secretion was observed on L1 in HHR. Whey acidic protein and other proteins were increased in milk of HHR and heterozygous rats on SDS-polyacrylamide gel electrophoresis. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay revealed apoptosis induction in HHRs at an early stage of lactation. By Western blotting, signal transducer and activator of transcription (STAT) 5A levels in cytoplasmic and nuclear fractions of the mammary glands were not different between HHR and SDR on L1 and L7. Nuclear localization of STAT5A in HHR and SDR was confirmed by immunohistochemistry. Tyr-phosphorylated STAT5A was not detected in HHR but was detected in SDR nuclear fractions. Several proteins modified with O-linked N-acetylglucosamine (O-GlcNAc) were detected in HHR nuclear extract on L1, although not in SDR or heterozygous rats by Western blotting. When HHR nuclear extract was applied to wheat germ agglutinin-agarose, a part of STAT5A was recovered in bound fractions. STAT5A of SDR or heterozygous rat nuclei were not bound to the lectin. Electrophoretic mobility shift assay revealed that STAT5A modified with O-GlcNAc is bound to the STAT5-responsive element. These results indicate that the mammary glands of HHR showed terminal differentiation for a short period, followed immediately by involution. In HHR, STAT5A is modified with O-GlcNAc but is not Tyr-phosphorylated. This type of glycosylation is suggested to be involved in the transient activation of STAT5A in HHR.
Papaconstantinou AD and Snyderwine EG, FEBS Lett. 2007 Jan 9;581(1):29-33. Epub 2006 Dec 5.
In the present study we addressed whether proliferation and apoptosis in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced rat mammary gland carcinomas were different between carcinomas with high and low expression of phosphotyrosine (pY)-STAT5a. We determined that carcinomas with high pY-STAT
5a were more proliferative (MIB5 immunostaining) and had a higher expression of cyclin D1 and estrogen receptor alpha. Furthermore, carcinomas with elevated pY-STAT5a demonstrated lower apoptosis as measured by the TUNEL assay and the Bcl-2 to Bax ratio, and showed increased expression of the long and short isoforms of the prolactin receptor. The results of this study are consistent with the notion that activated STAT5a may provide a growth advantage in some types of mammary gland cancers.
Deregulated estrogen signaling is evidently linked to breast cancer pathophysiology, although the role of signal transducer and activator of transcription (Stat)5a, integral to normal mammary gland development, is less clear. A mouse model of mammary epithelial cell-targeted deregulated estrogen rec
eptor alpha (ERalpha) expression [conditional ERalpha in mammary epithelium (CERM)] was crossed with mice carrying a germ line deletion of Stat5a [Stat5a-/-] to investigate interactions between ERalpha and Stat5a in mammary tissue. CERM, CERM/Stat5a+/-, CERM/Stat5a-/-, Stat5a+/-, Stat5a-/- and wild-type (WT) mice were generated to test the roles of ERalpha and Stat5a on pubertal differentiation and cancer progression with and without exposure to the chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Only CERM/Stat5a-/- mice demonstrated delayed pubertal terminal end bud differentiation. Without DMBA exposure, Stat5a loss abrogated ERalpha-initiated hyperplastic alveolar nodule (HAN) development and, similarly, Stat5a-/- mice did not develop HANs. However, although Stat5a loss still reduced ERalpha-initiated HAN prevalence following DMBA exposure, Stat5a loss without deregulated ERalpha was associated with an increased HAN prevalence compared with WT. Progression to ERalpha(+) and ERalpha(-) adenocarcinoma was found in all CERM-containing genotypes (CERM, CERM/Stat5a+/-, CERM/Stat5a-/-) and ERalpha(+) adenocarcinoma in the Stat5a-/- genotype. The mammary epithelial cell proliferative index was increased only in CERM mice independent of Stat5a loss. No differences in apoptotic indices were found. In summary, Stat5a cooperated with deregulated ERalpha in retarding pubertal mammary differentiation and contributed to ERalpha-initiated preneoplasia, but its loss did not prevent development of invasive cancer. Moreover, in the absence of deregulated ERalpha, Stat5a loss was associated with development of both HANs and invasive cancer following DMBA exposure.
Signal transducer and activator of transcription 5A (STAT5A) contributes to B-cell responses to cytokines through suppressor of cytokine signaling (Socs) genes in innate immunity. However, its direct roles in B-cell responses to chemokines are poorly understood.
The cytokines interleukin 7 (IL-7) and interleukin 4 (IL-4) regulate lymphoid differentiation and function and activate the transcription factor Stat5. Using mice deficient for the 2 highly related transcription factors, Stat5a and Stat5b (Stat5a
ght:700;'>Stat5a/b(-/-)), we investigated the role of Stat5 for B-cell differentiation, expansion, and function. Peripheral blood B cells of Stat5-deficient mice are significantly reduced, but no proliferation defects in response to various mitogenic stimuli are found. Also, IgM and IgG1 antibody production and immunoglobulin class switching are not affected. Pre- and pro-B cells of Stat5-deficient animals were found to have reduced responses to IL-7. Pro- and pre-B cells are the target cells of the abl oncogene and numerous studies have suggested that Stat5a/b is essential for transformation by derivatives of the Abelson (abl) gene. To assess the role of Stat5a/b in transformation, we have evaluated the ability of various abl derivatives to transform cells from Stat5a/b-deficient mice in vitro or in vivo. We demonstrate that the absence of Stat5a/b is not essential for the induction of lymphoid or myeloid tumors in vivo or on the ability to transform bone marrow cells in vitro.
