Focal adhesion kinase-related non-kinase ameliorates liver fibrosis by inhibiting aerobic glycolysis via the FAK/Ras/c-myc/ENO1 pathway

BACKGROUND Hepatic stellate cell(HSC)hyperactivation is a central link in liver fibrosis development.HSCs perform aerobic glycolysis to provide energy for their activation.Focal adhesion kinase(FAK)promotes aerobic glycolysis in cancer cells or fibroblasts,while FAK-related non-kinase(FRNK)inhibits FAK phosphorylation and biological functions.AIM To elucidate the effect of FRNK on liver fibrosis at the level of aerobic glycolytic metabolism in HSCs.METHODS Mouse liver fibrosis models were established by administering CCl4,and the effect of FRNK on the degree of liver fibrosis in the model was evaluated.Transforming growth factor-β1 was used to activate LX-2 cells.Tyrosine phosphorylation at position 397(pY397-FAK)was detected to identify activated FAK,and the expression of the glycolysis-related proteins monocarboxylate transporter 1(MCT-1)and enolase1(ENO1)was assessed.Bioinformatics analysis was performed to predict putative binding sites for c-myc in the ENO1 promoter region,which were validated with chromatin immunoprecipitation(ChIP)and dual luciferase reporter assays.RESULTS The pY397-FAK level was increased in human fibrotic liver tissue.FRNK knockout promoted liver fibrosis in mouse models.It also increased the activation,migration,proliferation and aerobic glycolysis of primary hepatic stellate cells(pHSCs)but inhibited pHSC apoptosis.Nevertheless,opposite trends for these phenomena were observed after exogenous FRNK treatment in LX-2 cells.Mechanistically,the FAK/Ras/c-myc/ENO1 pathway promoted aerobic glycolysis,which was inhibited by exogenous FRNK.CONCLUSION FRNK inhibits aerobic glycolysis in HSCs by inhibiting the FAK/Ras/c-myc/ENO1 pathway,thereby improving liver fibrosis.FRNK might be a potential target for liver fibrosis treatment.

Developing selective PI3K degraders to modulate both kinase and non-kinase functions

For the first time,proteolysis-targeting chimeras(PROTAC)technology was utilized to achieve the isoform-selective degradation of class I phosphoinositide 3-kinases(PI3Ks)in this study.Through screening and optimization,the PROTAC molecule ZM-PI05 was identified as a selective degrader of p110αin multiple breast cancer cells.More importantly,the degrader can down-regulate p85 regulatory subunit simultaneously,thereby inhibiting the non-enzymatic functions of PI3K that are independent on p110catalytic subunits.Therefore,compared with PI3K inhibitor copanlisib,ZM-PI05 displayed the stronger anti-proliferative activity on breast cancer cells.In brief,a selective and efficient PROTAC molecule was developed to induce the degradation of p110αand concurrent reduction of p85 proteins,providing a tool compound for the biological study of PI3K-αby blocking its enzymatic and non-enzymatic functions.

FRNK抑制人乳腺癌细胞体外增殖及机制研究

目的:研究黏着斑相关非激酶(focal adhesion kinase related non-kinase,FRNK)对人乳腺癌MCF-7细胞增殖的抑制作用及相关机制。方法:通过RT-PCR方法克隆目的基因FRNK,构建pcDNA3.1-FRNK重组质粒;经脂质体分别介导重组质粒(pcDNA3.1-FRNK)、阳性对照质粒(pcDNA3.1-GFP)和空质粒(pcDNA3.1)转染MCF-7细胞,以正常MCF-7细胞作为空白对照;通过检测转染后细胞FRNK蛋白的表达,并结合转染pcDNA3.1-GFP后细胞表达荧光的多寡来评估转染效率;采用MTT法研究转染后12、24、48和72h各时间点细胞的增殖情况;转染质粒48h后,采用流式细胞术分析MCF-7细胞周期,Westernblot法检测细胞核内NF-κBp65的表达。结果:pcDNA3.1-FRNK质粒可经脂质体介导高效转染MCF-7细胞,促进细胞FRNK的表达,FRNK表达量在转染后48h达高峰;转染pcDNA3.1-FRNK后,MCF-7细胞增殖趋缓,且这种抑制增殖呈一定的时间依赖性;转染FRNK基因后,S+G2/M期的细胞比例较正常细胞显著下降(P<0.05);转染pcDNA3.1-FRNK后MCF-7细胞核内NF-κBp65蛋白表达减少。结论:FRNK可抑制MCF-7细胞增殖,其抑制作用与下调NF-κBp65核易位相关。

Proline-rich tyrosine kinase 2 and its inhibitor PRNK

Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor protein tyrosine kinase,which is also known as Ca2 +-dependent tyrosine kinase or related adhesion focal tyrosine kinase.Pyk2 activation exerts a critical regulatory mechanism for various physiological processes including cytoskeleton function,regulation of cell growth and death,modulation of ion channels and multiple signaling events.However,mechanisms underlying the functional diversity of Pyk2 are not clear.A Pyk2 isoform that encodes only part of the C-terminal domain of Pyk2,named as PRNK (Pyk2-related non-kinase),acts as a dominant-negative inhibitor of Pyk2-dependent signaling by displacing Pyk2 from focal adhesions.Research on functional PRNK probably provides new potential inhibitory tool targeting Pyk2 and makes it possible to explore more of Pyk2 pathological mechanism.PRNK is a promising candidate targeting Pyk2 modulation.This review focuses on the functional investigation of Pyk2 and its structure and localization,including recent research with inhibitory strategies targeting Pyk2 by the method of PRNK.

Discoidin domain receptor 1 as a potent therapeutic target in solid tumors

Despite significant discoveries in basic cancer research and improvements in treatment options and clinical outcomes,cancer remains a major public health concern worldwide.Today,the main focus of cancer research is the signaling pathways that are crucial for cell survival,cell proliferation,and cell migration.The aberrant expression of proteins involved in these signaling pathways often leads to abnormal cell growth,cell metastasis,and invasion of healthy tis-sues.One such protein is discoidin domain receptor 1(DDR1)which belongs to the family of receptor tyrosine kinases(RTKs)and is activated upon collagen binding,as a result,downstream signaling pathways are stimulated which are responsible for cell survival,cell growth,adhesion,extracellular matrix remodeling,and cell migration.DDR1 is found to have abnormally elevated expression in various solid tumors,implying a critical role in cancer progression.Tradi-tional cancer treatment involves the use of cytotoxic drugs,chemotherapy,radiotherapy,and surgery,which do not pro-vide long-term survival and often result in cancer recurrence.Numerous small-molecule kinase inhibitors have been synthesized against RTKs including DDR1 and have been highly efficacious in tumor reduction.More recently,targeting the DDR1 extracellular domain(ECD)has garnered much attention from researchers,as inhibiting the DDR1-collagen binding has been attributed to maximizing the likelihood of the combined cytotoxic effect of both immune cells and tar-geted drugs.This review focuses on the structure,function,activation,and signaling partners of DDR1,its role in different solid tumors,andfinally discusses about designing more DDR1 non-kinase inhibitors as promising therapeutic strategies against DDR1-driven tumors.

Growth arrest signaling of the Raf/MEK/ERK pathway in cancer

The Raf/MEK/extraceUular signal-regulated kinase （ERK） pathway has a pivotal role in facilitating cell proliferation, and its deregulated activation is a central signature of many epithelial cancers. However paradoxically, sustained activity of Raf/MEK/ERK can also result in growth arrest in many different cell types. This anti-proliferative Raf/MEK/ERK signaling also has physiological significance, as exemplified by its potential as a tumor suppressive mechanism. Therefore, significant questions include in which cell types and by what mechanisms this pathway can mediate such an opposing context of signaling. Particularly, our understating of the role of ERK1 and ERK2, the focal points of pathway signaling, in growth arrest signaling is still limited. This review discusses these aspects of Raf/MEK/ ERK-mediated growth arrest signaling.