Kinome screening for regulators of the estrogen receptor identifies LMTK3 as a new therapeutic target in breast cancer

Therapies targeting estrogen receptor α (ERα, encoded by ESR1) have transformed the treatment of breast cancer. However, large numbers of women relapse, highlighting the need for the discovery of new regulatory targets modulating ERα pathways. An siRNA screen identified kinases whose silencing alters the estrogen response including those previously implicated in regulating ERα activity (such as mitogen-activated protein kinase and AKT). Among the most potent regulators was lemur tyrosine kinase-3 (LMTK3), for which a role has not previously been assigned. In contrast to other modulators of ERα activity, LMTK3 seems to have been subject to Darwinian positive selection, a noteworthy result given the unique susceptibility of humans to ERα+ breast cancer. LMTK3 acts by decreasing the activity of protein kinase C (PKC) and the phosphorylation of AKT (Ser473), thereby increasing binding of forkhead box O3 (FOXO3) to the ESR1 promoter. LMTK3 phosphorylated ERα, protecting it from proteasomal degradation in vitro. Silencing of LMTK3 reduced tumor volume in an orthotopic mouse model and abrogated proliferation of ERα+ but not ERα- cells, indicative of its role in ERα activity. In human cancers, LMTK3 abundance and intronic polymorphisms were significantly associated with disease-free and overall survival and predicted response to endocrine therapies. These findings yield insights into the natural history of breast cancer in humans and reveal LMTK3 as a new therapeutic target.     

The multifaceted role of lemur tyrosine kinase 3 in health and disease

In the last decade, LMTK3 (lemur tyrosine kinase 3) has emerged as an important player in breast cancer, contributing to the advancement of disease and the acquisition of resistance to therapy through a strikingly complex set of mechanisms. Although the knowledge of its physiological function is largely limited to receptor trafficking in neurons, there is mounting evidence that LMTK3 promotes oncogenesis in a wide variety of cancers. Recent studies have broadened our understanding of LMTK3 and demonstrated its importance in numerous signalling pathways, culminating in the identification of a potent and selective LMTK3 inhibitor. Here, we review the roles of LMTK3 in health and disease and discuss how this research may be used to develop novel therapeutics to advance cancer treatment.     

Designing and optimization of novel human LMTK3 inhibitors against breast cancer – a computational approach

Estrogen receptor-α (ERα) is expressed more in patients with breast cancer and its level correlated with endocrine resistance. LMTK3 is reported as breast cancer target with regulation of estrogen receptor-α (ERα) through phosphorylation. In this computational study, structure-based inhibitor screening was performed on human LMTK3 using ZINC database. ATP-binding cavity with critical residues involved in the LMTK3 phosphorylation was used as target site for the screening. From the large ligand library, the best compounds were screen with three-phase virtual screening methods in Dockblaster, AutoDock Vina and AutoDock, respectively. The evaluation of ligands was carried out by binding energy and weak interactions, such as hydrogen bond interactions and hydrophobic contacts, in the target site that favors LMTK3 inhibition. Top compounds were found to be more effective in druglikeness activity by ADME prediction. The stability and binding affinity of ligand complexes were optimized by trajectory analysis such as RMSD, Rg, SASA and interhydrogen bonds from molecular dynamics simulations. The behavior of protein motion after ligand binding was illustrated by eigenvectors from principal component analysis (PCA). In addition, binding free energy of the LMTK3–ligand complexes were calculated by MM/PBSA methods and results supported the strong binding in dynamic system. Thus, the computational studies illustrated the structural insights on LMTK3 inhibition mechanism by ligands ZINC04670539, ZINC05607079 and ZINC04344028, also proposed as potent lead candidates. Our findings step towards developing novel LMTK3 inhibitors and identified lead candidates can be future breast cancer drugs with further experimental studies.     

Overexpression of MEKK3 confers resistance to apoptosis through activation of NFkappaB

Many cancers have constitutively activated NFkappaB, the elevation of which contributes to cancer cell resistance to chemotherapeutic agent-induced apoptosis. Although mitogen-activated protein kinase/extracellular-regulated kinase kinase kinase-3 (MEKK3) has been shown to participate in the activation of NFkappaB, its relations to apoptosis and cancer are unclear. In this study, we established cell model systems to examine whether stable expression of MEKK3 could lead to increased NFkappaB activity and confer resistance to apoptosis. In addition, we investigated in breast and ovarian cancers whether MEKK3 expression may be altered and correlated with aberrant NFkappaB activity. We show that stable cell lines overexpressing MEKK3 not only had elevated levels of NFkappaB binding activity but also were more responsive to cytokine stimulation. These stable cells showed 2-4-fold higher basal expression of Bcl-2 and xIAP than the parental cells. Consistent with this increased expression of cell survival genes, MEKK3 stable cells showed reduced activation of caspases 3 and 8 and poly(ADP-ribose) polymerase cleavage and dramatically increased resistance to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand, doxorubicin, daunorubicin, camptothecin, and paclitaxel. Intriguingly, analysis of human breast and ovarian cancers showed that a significant fraction of these samples have elevated MEKK3 protein levels with corresponding increases in NFkappaB binding activities. Thus, our results established that elevated expression of MEKK3 appears to be a frequent occurrence in breast and ovarian cancers and that overexpression of MEKK3 in cells leads to increased NFkappaB activity and increased expression of cell survival factors and ultimately contributes to their resistance to apoptosis. As such, MEKK3 may serve as a therapeutic target to control cancer cell resistance to cytokine- or drug-induced apoptosis.     

Imaging Sites of Inhibition of Proteolysis in Pathomimetic Human Breast Cancer Cultures by Light-Activated Ruthenium Compound

The cysteine protease cathepsin B has been causally linked to progression and metastasis of breast cancers. We demonstrate inhibition by a dipeptidyl nitrile inhibitor (compound 1) of cathepsin B activity and also of pericellular degradation of dye-quenched collagen IV by living breast cancer cells. To image, localize and quantify collagen IV degradation in real-time we used 3D pathomimetic breast cancer models designed to mimic the in vivo microenvironment of breast cancers. We further report the synthesis and characterization of a caged version of compound 1, [Ru(bpy)₂(1)₂](BF₄)₂ (compound 2), which can be photoactivated with visible light. Upon light activation, compound 2, like compound 1, inhibited cathepsin B activity and pericellular collagen IV degradation by the 3D pathomimetic models of living breast cancer cells, without causing toxicity. We suggest that caged inhibitor 2 is a prototype for cathepsin B inhibitors that can control both the site and timing of inhibition in cancer.     

BCAR3 Regulates Src/p130Cas Association, Src Kinase Activity, and Breast Cancer Adhesion Signaling

The nonreceptor protein-tyrosine kinase c-Src is frequently overexpressed and/or activated in a variety of cancers, including those of the breast. Several heterologous binding partners of c-Src have been shown to regulate its catalytic activity by relieving intramolecular autoinhibitory interactions. One such protein, p130^Cas (Cas), is expressed at high levels in both breast cancer cell lines and breast tumors, providing a potential mechanism for c-Src activation in breast cancers. The Cas-binding protein BCAR3 (breast cancer antiestrogen resistance-3) is expressed at high levels in invasive breast cancer cell lines, and this molecule has previously been shown to coordinate with Cas to increase c-Src activity in COS-1 cells. In this study, we show for the first time using gain- and loss-of-function approaches that BCAR3 regulates c-Src activity in the endogenous setting of breast cancer cells. We further show that BCAR3 regulates the interaction between Cas and c-Src, both qualitatively as well as quantitatively. Finally, we present evidence that the coordinated activity of these proteins contributes to breast cancer cell adhesion signaling and spreading. Based on these data, we propose that the c-Src/Cas/BCAR3 signaling axis is a prominent regulator of c-Src activity, which in turn controls cell behaviors that lead to aggressive and invasive breast tumor phenotypes.     

Synthesis and biological evaluation of benzimidazole derivatives as the G9a Histone Methyltransferase inhibitors that induce autophagy and apoptosis of breast cancer cells

G9a (also known as KMT1C or EHMT2) is initially identified as a H3K9 methyltransferase that specifically mono- and dimethylates 'Lys-9’ of histone H3 (H3K9me1 and H3K9me2, respectively) in euchromatin. It is overexpressed in various human cancers and employed as a promising target in cancer therapy. We discovered a benzoxazole scaffold through virtual high-throughput screening, and designed, synthesized 24 derivatives and investigated for inhibition of G9a. After several rounds of kinase and anti-proliferative activity screening, we discovered a potent G9a antagonist (GA001) with an IC₅₀ value of 1.32 μM that could induce autophagy via AMPK in MCF7 cells. In addition, we found high concentration of GA001 could induce apoptosis via p21-Bim signal cascades in MCF7 cells. Our results highlight a new approach for the development of a novel drug targeting G9a with a potential to induce autophagy and apoptosis for future breast cancer therapy.     

LMTK2基因沉默抑制人上皮性卵巢癌细胞生长和转移的作用机制

摘要 目的：探讨狐猴酪氨酸激酶2（LMTK2）基因沉默对人上皮性卵巢癌(EOC)细胞生长和转移的抑制作用及其可能的机制。方法：通过RT-qPCR和Western-blot检测了人正常卵巢上皮细胞IOSE80和人上皮性卵巢癌细胞系（SKOV3、ES2、OVCAR-3和HEY）中LMTK2的表达,使用Lipofectamine 3000转染试剂将LMTK2的短发夹RNA（shRNA）、阴性对照shRNA、LMTK2过表达重组pcDNA3.1质粒或阴性对照质粒转染到SKOV3细胞中,并分为LMTK2-shRNA组、NC-shRNA组、LMTK2-pcDNA3.1组或NC-pcDNA3.1组。另外,使用PI3K/Akt抑制剂LY294002处理SKOV3细胞1 h。通过CCK-8法测定细胞增殖,Annexin V-FITC/PI染色法测定细胞凋亡,划痕实验评价细胞迁移,Transwell实验评价细胞侵袭。对BALB/c雌性裸鼠皮下注射转染NC-shRNA或LMTK2-shRNA的SKOV3细胞建立体内移植瘤模型,并记录接种28 d内的肿瘤体积。结果：与人正常卵巢上皮细胞IOSE80相比,卵巢癌细胞系（SKOV3、ES2、OVCAR-3和HEY）中LMTK2的mRNA和蛋白表达水平均显著升高,其中SKOV3的LMTK2 mRNA和蛋白表达水平最高（P<0.05）。与NC-shRNA组相比,LMTK2-shRNA组SKOV3细胞活力、相对迁移面积、侵袭细胞数均显著降低,而细胞凋亡率显著升高（P<0.05）。此外,与NC-shRNA组相比,LMTK2-shRNA组SKOV3细胞中Bax的蛋白表达水平显著升高,而Bcl-2、MMP2、MMP9、p-Akt的蛋白表达水平显著降低（P<0.05）。LY294002处理逆转了上调LMTK2对SKOV3细胞生长和转移的影响（P<0.05）。在接种第21天和28天时,与NC-shRNA组相比,LMTK2-shRNA组裸鼠的肿瘤体积显著降低（P<0.05）。结论：LMTK2基因沉默通过抑制PI3K/Akt信号通路降低了人上皮性卵巢癌细胞的生长和转移能力。     

Overexpression of novel lncRNA NLIPMT inhibits metastasis by reducing phosphorylated glycogen synthase kinase 3β in breast cancer

Long noncoding RNAs (lncRNAs) are considered as regulators of gene expression in cancers. However, cancer profiling has little focused on noncoding genes. Here, we reported that RP11–115N4.1 (here renamed novel lncRNA inhibiting proliferation and metastasis [NLIPMT]) was downregulated in breast cancer tissues. Ectopic expression of NLIPMT inhibited mammary cell proliferation, motility in vitro. Moreover, lnc-NLIPMT reduced the growth of implanted MDA-MB-231 cells in vivo. Mechanistically, glycogen synthase kinase 3β (GSK3β) was identified as an effector protein regulated by lnc-NLIPMT. Inhibition of GSK3β activity restored NLIPMT-induced inhibition of proliferation and motility in breast cancer cells. These data reveal that lnc-NLIPMT functions as a driver of breast cancer progression and might serve as a potential target for antimetastatic therapies.     

Evaluation of active recombinant catalytic domain of human ErbB-2 tyrosine kinase, and suppression of activity by a naturally derived inhibitor, ZH-4B

Human cancers frequently express high levels of ErbB-2 tyrosine kinase, which is associated with aggressive tumor behavior and poor prognosis. ErbB-2 is thus a promising target for cancer therapy. Here we express the catalytic domain of ErbB-2 as a soluble active kinase, and investigate the correlations between its activity and kinase concentration, ATP concentration, substrate concentration and divalent cation type. A simple and effective screening model is established to identify and evaluate potential inhibitors of ErbB-2 kinase. ZH-4B, a naturally derived small molecule compound that potently inhibits ErbB-2 kinase activity with an IC50 value of 2.45+/-0.56 microM, is identified. In SK-OV-3 human ovarian cancer cells and SK-BR-3 human breast carcinoma cells, ZH-4B blocks epidermal growth factor (EGF)-induced phosphorylation of ErbB-2 in a dose-dependent manner. Our data collectively indicate that ZH-4B is a potential novel anti-cancer agent that deserves further investigation.     

Synthesis,antiproliferative and pro-apoptotic activity of 2-phenylindoles

Breast cancer is the second most common cancer worldwide after lung cancer with the vast majority of early stage breast cancers being hormone-dependent. One of the major therapeutic advances in the clinical treatment of breast cancer has been the introduction of selective estrogen receptor modulators (SERMs). We describe the design and synthesis of novel SERM type ligands based on the 2-arylindole scaffold to selectively target the estrogen receptor in hormone dependent breast cancers. Some of these novel compounds are designed as bisindole type structures, while others are conjugated to a cytotoxic agent based on combretastatin A4 (CA4) which is a potent inhibitor of tubulin polymerisation. The indole compounds synthesised within this project such as 31 and 86 demonstrate estrogen receptor (ER) binding and strong antiproliferative activity in the ER positive MCF-7 breast cancer cell line with IC₅₀ values of 2.71 μM and 1.86 μM respectively. These active compounds induce apoptotic activity in MCF-7 cells with minimal effects on normal peripheral blood cells. Their strong anti-cancer effect is likely mediated by the presence of two ER binding ligands for 31 and an ER binding ligand combined with a cytotoxic agent for 86.     

LKB1 Catalytic Activity Contributes to Estrogen Receptor α Signaling

The tumor suppressor serine-threonine kinase LKB1 is mutated in Peutz-Jeghers syndrome (PJS) and in epithelial cancers, including hormone-sensitive organs such as breast, ovaries, testes, and prostate. Clinical studies in breast cancer patients show low LKB1 expression is related to poor prognosis, whereas in PJS, the risk of breast cancer is similar to the risk from germline mutations in breast cancer (BRCA) 1/BRCA2. In this study, we investigate the role of LKB1 in estrogen receptor α (ERα) signaling. We demonstrate for the first time that LKB1 binds to ERα in the cell nucleus in which it is recruited to the promoter of ERα-responsive genes. Furthermore, LKB1 catalytic activity enhances ERα transactivation compared with LKB1 catalytically deficient mutants. The significance of our discovery is that we demonstrate for the first time a novel functional link between LKB1 and ERα. Our discovery places LKB1 in a coactivator role for ERα signaling, broadening the scientific scope of this tumor suppressor kinase and laying the groundwork for the use of LKB1 as a target for the development of new therapies against breast cancer.     

Denbinobin suppresses breast cancer metastasis through the inhibition of Src-mediated signaling pathways

Denbinobin (5-hydroxy-3,7-dimethoxy- 1,4-phenanthraquinone), a biologically active chemical isolated from Ephemerantha lonchophylla, has been demonstrated to display anti-cancer activity. Breast cancer is the leading cause of female mortality, and the high mortality is mainly attributable to metastasis. Src kinase activity is elevated in many human cancers, including breast cancer, and is often associated with aggressive disease. In the present study, we examined the anti-metastatic effects of denbinobin through decreasing Src kinase activity in human and mouse breast cancer cells. Denbinobin caused significant block of Src kinase activity in both human and mouse breast cancer cells. Moreover, phosphorylation of the signaling molecules focal adhesion kinase, Crk-associated substrate and paxillin downstream of Src was also inhibited by denbinobin. Furthermore, denbinobin inhibited the in vitro migration, invasion and in vivo metastasis of breast cancers in a mouse metastatic model. The denbinobin-treated group showed a significant reduction in tumor metastasis, orthrotopic tumor volume, and spleen enlargement compared to the control group. In addition, transfection of breast cancer cells with a plasmid coding for a constitutively active Src prevented the denbinobin-mediated phosphorylation of Src and downstream molecules and cell migration. Our findings provide evidences that denbinobin inhibits Src-mediated signaling pathways involved in controlling breast cancer migration and metastasis, suggesting that it has therapeutic potential in breast cancer treatment.