Development of a STAT3 reporter prostate cancer cell line for high throughput screening of STAT3 activators and inhibitors

STAT3 is constitutively activated in several cancers, including prostate cancer, and is therefore, a potential target for cancer therapy. DU-145 prostate cancer cells were stably co-transfected with STAT3 reporter and puromycin resistant plasmids to create a stable STAT3 reporter cell line that can be used for high throughput screening of STAT3 modulators. The applicability of this cell line was tested with two known activators and inhibitors of STAT3. As expected, EGF and IL-6 increased STAT3 reporter activity and enhanced the nuclear localization of phosphorylated STAT3 (pSTAT3); whereas Cucurbitacin I and AG490 decreased STAT3 reporter activity dose and time-dependently and reduced the localization of pSTAT3 in the nuclei of prostate cancer cells. Given the importance of STAT3 in cancer initiation and progression, the development of a stable STAT3 reporter cell line in prostate cancer cells provides a rapid, sensitive, and cost effective method for the screening of potential STAT3 modulators.     

PTPN9的表达下调通过抑制STAT3活化促进结直肠癌细胞凋亡

目的:探讨PTPN9对结直肠癌细胞生长和存活的影响及其机制。方法:建立稳定PTPN9高表达的细胞系,使用Real-timePCR检测其内源性变达,使用细胞集落实验及Caspase-3、Caspase-9,检测其细胞活力及凋亡情况。同时我们抑制了细胞中PTPN9的表达,使用实时PCR来验证敲低效率,应用100μM H_2O_2诱导凋亡模型,利用CCK-8测定来确定细胞活力,Caspase-9和Cas-pase-3测定检测其凋亡情况。最后我们应用Western blot技术,检测PTPN9抑制STAT3通路,来调控细胞凋亡。结果:PTPN9的表达在结直肠癌组织中下调。PTPN9的高表达减慢细胞生长和集落的形成,从而诱导结直肠癌细胞凋亡。相反,PTPN9低表达促进细胞生长和存活。此外,PTPN9负责调控STAT3的活化,并在结直肠癌中抑制核易位,并且通过抑制STAT3通路,来抑制PTPN9低表达对细胞凋亡的影响。结论:PTPN9在结直肠癌组织中通过抑制STAT3的活化而抑制细胞生长和存活。     

A role of autophagy in PTP4A3-driven cancer progression

Autophagy, a “self-eating” cellular process, has dual roles in promoting and suppressing tumor growth, depending on cellular context. PTP4A3/PRL-3, a plasma membrane and endosomal phosphatase, promotes multiple oncogenic processes including cell proliferation, invasion, and cancer metastasis. In this study, we demonstrate that PTP4A3 accumulates in autophagosomes upon inhibition of autophagic degradation. Expression of PTP4A3 enhances PIK3C3-BECN1-dependent autophagosome formation and accelerates LC3-I to LC3-II conversion in an ATG5-dependent manner. PTP4A3 overexpression also enhances the degradation of SQSTM1, a key autophagy substrate. These functions of PTP4A3 are dependent on its catalytic activity and prenylation-dependent membrane association. These results suggest that PTP4A3 functions to promote canonical autophagy flux. Unexpectedly, following autophagy activation, PTP4A3 serves as a novel autophagic substrate, thereby establishing a negative feedback-loop that may be required to fine-tune autophagy activity. Functionally, PTP4A3 utilizes the autophagy pathway to promote cell growth, concomitant with the activation of AKT. Clinically, from the largest ovarian cancer data set (GSE 9899, n = 285) available in GEO, high levels of expression of both PTP4A3 and autophagy genes significantly predict poor prognosis of ovarian cancer patients. These studies reveal a critical role of autophagy in PTP4A3-driven cancer progression, suggesting that autophagy could be a potential Achilles heel to block PTP4A3-mediated tumor progression in stratified patients with high expression of both PTP4A3 and autophagy genes.     

A role of autophagy in PTP4A3-driven cancer progression

Autophagy, a “self-eating” cellular process, has dual roles in promoting and suppressing tumor growth, depending on cellular context. PTP4A3/PRL-3, a plasma membrane and endosomal phosphatase, promotes multiple oncogenic processes including cell proliferation, invasion, and cancer metastasis. In this study, we demonstrate that PTP4A3 accumulates in autophagosomes upon inhibition of autophagic degradation. Expression of PTP4A3 enhances PIK3C3-BECN1-dependent autophagosome formation and accelerates LC3-I to LC3-II conversion in an ATG5-dependent manner. PTP4A3 overexpression also enhances the degradation of SQSTM1, a key autophagy substrate. These functions of PTP4A3 are dependent on its catalytic activity and prenylation-dependent membrane association. These results suggest that PTP4A3 functions to promote canonical autophagy flux. Unexpectedly, following autophagy activation, PTP4A3 serves as a novel autophagic substrate, thereby establishing a negative feedback-loop that may be required to fine-tune autophagy activity. Functionally, PTP4A3 utilizes the autophagy pathway to promote cell growth, concomitant with the activation of AKT. Clinically, from the largest ovarian cancer data set (GSE 9899, n = 285) available in GEO, high levels of expression of both PTP4A3 and autophagy genes significantly predict poor prognosis of ovarian cancer patients. These studies reveal a critical role of autophagy in PTP4A3-driven cancer progression, suggesting that autophagy could be a potential Achilles heel to block PTP4A3-mediated tumor progression in stratified patients with high expression of both PTP4A3 and autophagy genes.     

脑缺血大鼠海马信号转导与转录激活子-3的激活及其调控

以往的研究表明,在脑缺血/再灌注的皮层和纹状体组织中信号转导与转录激活子-3(STAT3)被激活。本实验旨在研究SD大鼠四动脉结扎诱导的全脑缺血是否引起海马组织STAT3的快速激活及其调控机制。结果表明,脑缺血导致STAT3快速磷酸化激活及DNA结合活性增加。胞浆STAT3的磷酸化水平从缺血5min起就显著增高,10min达高峰(增加约1．7倍),然后开始下降。核内STAT3的磷酸化水平则逐渐增加,缺血30min时达高峰(增加约2．3倍)。电泳迁移率改变分析法显示,STAT3的DNA结合活性从缺血5min起就显著增加,30min达高峰(增加约3．2倍)。进一步的研究表明,缺血前20min腹腔注射给药,然后缺血30min,发现蛋白酪氨酸激酶抑制剂染料木黄酮和抗氧化剂N-乙酞半胱氨酸能显著地抑制核内STAT3的磷酸化水平及DNA结合活性的增加(磷酸化水平从2．3和2．5倍分别降为1．2和1．4倍,DNA结合活性则从2．8和3．7倍分别降为1．1和1．5倍),而蛋白酪氨酸磷酸酶抑制剂矾酸钠则能明显地促进他们的增高(磷酸化水平从2．0倍增到3．4倍,DNA结合活性从3．1倍增为5．1倍)。这些结果提示,蛋白酪氨酸激酶和蛋白酪氨酸磷酸酶可能共同参与了缺血诱导STAT3的激活调控,STAT3的激活可能有助于海马神经元适应氧化应激。     

前列腺癌组织及PC-3细胞中STAT3及磷酸化STAT3的表达

目的　研究前列腺癌组织及前列腺癌细胞株PC- 3 中STAT3 蛋白及磷酸化STAT3 蛋白的表达。方法　常规石蜡包埋切片SABC免疫组化法检测45例前列腺癌组织、20例前列腺增生组织中STAT3 及磷酸化STAT3 表达, 细胞免疫化学法检测前列腺癌细胞株PC 3细胞STAT3及磷酸化STAT3表达。结果　STAT3在前列腺癌及前列腺增生组织表达阳性率分别为77. 8%和50. 0%, 两者间具有显著差异; 磷酸化STAT3在前列腺癌及前列腺增生组织表达阳性率分别为68. 9%和35. 0%, 两者间具显著差异(P<0 .05); PC- 3细胞中STAT3及磷酸化STAT3表达阳性。结论　STAT3蛋白在前列腺癌中高表达且持续激活, 可能与前列腺癌的发生具有密切联系。     

Nix is a selective autophagy receptor for mitochondrial clearance

Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin‐like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP‐L1 to damaged mitochondria through its amino‐terminal LC3‐interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.     

Sequential activation of inflammatory signaling pathways during graft-versus-host disease (GVHD): early role for STAT1 and STAT3

The aim of this study was to delineate the temporal and spatial sequence of STAT1 and STAT3 activation during development of GVHD following fully Major Histocompatibility Complex (MHC)-mismatched allogeneic Bone Marrow Transplantation (BMT). Activation of inflammatory signaling pathways in GVHD target organs was assessed by western blotting, phospho-flow cytometry and electromobility shift assays (EMSA). Development of GVHD was associated with significant expansion of phospho[p]-STAT1 and p-STAT3 expressing CD4⁺ T cells and CD8⁺ T cells. GVHD-specific STAT3/STAT1 activation preceded activation of Nuclear Factor-κB (NF-κB) and Mitogen Activated Protein Kinase (MAPK) and was associated with subsequent induction of STAT1 or STAT3-dependent inflammatory gene-expression programs (e.g. expression of IRF-1, SOCS1, IL-17). Our studies may help to establish a functional hierarchy of the signaling events leading to the development of GVHD and could be helpful in designing new molecularly targeted treatment approaches for GVHD.     

The role of autophagy in maintaining intestinal mucosal barrier

The intestinal mucosal barrier is the first line to defense against luminal content penetration and performs numerous biological functions. The intestinal epithelium contains a huge surface that is lined by a monolayer of intestinal epithelial cells (IECs). IECs are dominant mediators in maintaining intestinal homeostasis that drive diverse functions including nutrient absorption, physical segregation, secretion of antibacterial peptides, and modulation of immune responses. Autophagy is a cellular self-protection mechanism in response to various stresses, and accumulating studies have revealed its importance in participating physiological processes of IECs. The regulatory effects of autophagy depend on the specific IEC types. This review aims to elucidate the myriad roles of autophagy in regulating the functions of different IECs (stem cells, enterocytes, goblet cells, and Paneth cells), and present the progress of autophagy-targeting therapy in intestinal diseases. Understanding the involved mechanisms can provide new preventive and therapeutic strategies for gastrointestinal dysfunction and diseases.     

The interplay between autophagy and tumorigenesis: exploiting autophagy as a means of anticancer therapy

In wild‐type cells, autophagy represents a tumour‐suppressor mechanism, and dysfunction of the autophagy machinery increases genomic instability, DNA damage, oxidative stress and stem/progenitor expansion, which are events associated with cancer onset. Autophagy occurs at a basal level in all cells depending on cell type and cellular microenvironment. However, the role of autophagy in cancer is diverse and can promote different outcomes even in a single tumour. For example, in hypoxic tumour regions, autophagy emerges as a protective mechanism and allows cancer cell survival. By contrast, in cancer cells surrounding the tumour mass, the induction of autophagy by radio‐ or chemotherapy promotes cell death and significantly reduces the tumour mass. Importantly, inhibition of autophagy compromises tumorigenesis by mechanisms that are not entirely understood. The aim of this review is to explain the apparently contradictory role of autophagy as a mechanism that both promotes and inhibits tumorigenesis using different models. The induction/inhibition of autophagy as a mechanism for cancer treatment is also discussed.