依维莫司协同拉帕替尼降低HER2+乳腺癌对拉帕替尼耐药

研究依维莫司(Everolimus)联合拉帕替尼(Lapatinib)对HER2~+乳腺癌耐药细胞的影响。CCK-8法测定乳腺癌亲本细胞(skbr3)和耐药细胞(skbr3-LR)的细胞活性、半数抑制浓度(Half inhibitory concentration,IC_(50));CompuSyn软件计算联合作用指数(combined effect index, CI)值;流式细胞术检测对照组实验组skbr3-LR的周期;Western blotting方法检测各组AKT、m TOR、S6相关蛋白的表达量。依维莫司联合拉帕替尼使skbr3-LR对拉帕替尼的IC_(50)从(27.13±0.3)下降到(6.79±0.04);20 nmol/L依维莫司联合5μmol/L、10μmol/L、20μmol/L、40μmol/L拉帕替尼CI指数分别为0.243 67、0.278 61、0.282 19、0.366 79、0.416 67;拉帕替尼联合依维莫司组G1期细胞百分数(69.84±0.93)%显著大于对照组(52.91±1.64)%(p0.001),也显著高于拉帕替尼组(59.77±0.52)%(p0.001)和依维莫司组(58.73±0.64)%(p0.001);Western blotting结果显示skbr3-LR中p-mTOR比skbr3表达上调(p0.001),20 nmol/L依维莫司或10μmol/L拉帕替尼单独处理skbr3-LR,以及联合用药组与skbr3-LR组相比p-mTOR均有显著下调(p0.001, p0.01, p0.001)。依维莫司联合拉帕替尼可协同抑制skbr3-LR增殖,降低其拉帕替尼耐药性,其作用机制部分是通过下调AKT/mTOR/S6通路实现的。     

HER2 阳性乳腺癌转移患者最佳治疗方案的研究进展

针对人表皮生长因子受体HER2 的靶向制剂曲妥珠单抗,显著改善了HER2 阳性乳腺癌转移患者疾病的发展状况,提高了 患者的总体生存期（OS）,然而耐药现象时有发生。其它靶向制剂如帕妥珠单抗,酪氨酸激酶受体抑制剂拉帕替尼和ado-transtusumab emtansine等克服了其耐药性,为治疗提供了更多选择。这些HER2 靶向制剂单用或联用已显示出良好的临床功效,但最 佳治疗次序依然未知。随着新型靶向制剂的出现,最佳治疗方案的研究成为热点。本篇综述重点阐述了目前HER2 阳性乳腺癌转 移患者最佳治疗方案的研究进展,以及新型靶向制剂的现有状况。     

拉帕替尼通过激活p38MAPK信号通路促进NB4细胞凋亡

该文探讨了拉帕替尼对急性早幼粒细胞白血病NB4细胞增殖和凋亡的影响及相关分子机制。用p38MAPK抑制剂和不同浓度的拉帕替尼处理NB4细胞24 h,CCK-8(cell counting kit-8)实验检测细胞增殖,FITC-Annexin V/PI双染色法检测细胞凋亡,光学显微镜和Hoechst 33258染色观察细胞形态,Western blot检测Bcl-2(B cell leukemia-2)、Bax(Bcl-2 associated X protein)、caspase-3、PARP(poly-ADP-ribose polymerase)、PML-RARα(promyelocytic leukemia-retinoic acid receptor alpha)、p38MAPK(p38 mitongen-activated protein kinase)和p-p38MAPK(phosphorylated p38 mitongen-activated protein kinase)等蛋白质水平。结果显示,随着拉帕替尼药物浓度的增加,细胞增殖率显著降低,细胞凋亡数量明显增加,Hoechst 33258染色可见染色质浓缩、碎裂等凋亡现象。同时,拉帕替尼能降低Bcl-2和PML-RARα蛋白质水平,增加Bax、cleaved caspase-3、cleaved PARP和p-p38MAPK等蛋白质水平。用p38MAPK抑制剂预处理后,细胞增殖率升高,凋亡率降低,p-p38MAPK、Bax、cleaved caspase-3和cleaved PARP等蛋白质水平降低。该文结果提示,拉帕替尼能够抑制NB4细胞增殖并促进细胞凋亡,并且p38MAPK信号通路可能参与这些过程。     

茶多酚联合吉非替尼抗肺腺癌血管生成的初步研究

目的:通过观察茶多酚联合吉非替尼对人肺腺癌生长的抑制作用.验证茶多酚抗肿瘤血管生成效应.方法:建立人肺腺癌A549移植瘤模型.设立对照组、茶多酚组、吉非替尼组及两者联合组.观察对肺腺癌A549移植瘤的抑制率,检测移植瘤体的微血管密度;并观察EGFR-VEGF这一肿瘤血管生成重要通路的相关因子VEGF、AKT-2、HIF-1a和STAT-3表达水平.结果:茶多酚组、吉非替尼组对移植性人肺腺癌A549瘤体、肿瘤组织微血管密度及VEGF的表达都有明显的抑制效果;两者联合使用效果明显增强;茶多酚组、吉非替尼组能明显抑制AKT-2、HIF-1a和STAT-3表达水平,但两者联合组对AKT-2、HIF-1a作用并不明显,而能明显抑制STAT3表达.结论:茶多酚、吉非替尼对移植性人肺腺癌A549具有明显的抑制效果,两者联合使用有一定增效作用,并能够影响肿瘤血管生成通路的相关因子表达.     

靶向HER2的CAR-T细胞构建与抗肿瘤活性的体外分析

CAR-T细胞疗法通过靶向识别肿瘤细胞表面抗原,特异性杀伤肿瘤细胞,近年来已经成为肿瘤免疫疗法的研究热点。通过基因工程方法构建靶向人类表皮生长因子受体2(HER2)的CAR慢病毒表达质粒,以磷酸钙沉淀辅助多质粒共转染HEK293T细胞包装,制备CAR慢病毒颗粒lenti-car,感染人外周血单核细胞获得HER2靶向的CAR-T细胞,并分析其对HER2阳性和阴性肿瘤细胞的特异性抑制效果。研究结果表明,构建的CAR-T细胞可被HER-2阳性的肿瘤细胞特异性激活,分泌大量炎症性细胞因子IFN-γ和IL-2。在同样效靶比等处理条件下,构建的HER2靶向CAR-T细胞对HER2阳性的人卵巢癌细胞株SK-OV-3的生长抑制率为(58.47±1.72)%,显著高于对照组(P0.05);而对HER2阴性的人慢性髓原白血病细胞株K562的生长抑制率为(11.74±2.37)%,与对照组无显著差异(P0.05)。进一步,在K562细胞中转染人HER2表达载体使其成为HER2阳性,则HER2靶向CAR-T细胞对其的生长抑制率上升为(30.41±7.59)%,较HER2阴性K562具有明显差异(P0.05)。研究结果表明,构建的HER2靶向的第二代CAR-T细胞可选择性地抑制高表达HER2蛋白的肿瘤细胞的生长,暗示了其对HER2阳性肿瘤进行细胞免疫治疗的临床应用前景。     

Hsp90分子抑制剂拮抗肿瘤耐药的研究进展

Hsp90作为热休克蛋白家族中的重要一员,是一种对细胞生存所必需的分子伴侣,它发挥着稳定顾客蛋白构象、维持其功能的作用。许多顾客蛋白在肿瘤中处于过度表达或持续激活状态,与肿瘤的发生发展有着密切的关系。因此,Hsp90在近年的研究中倍受关注,已经发展为抗肿瘤治疗的良好靶点,目前已经有多个Hsp90抑制剂进入临床实验。近年随着肿瘤分子生物学的研究,肿瘤分子靶向治疗已取得明显成果,针对多种癌症已获得了多个用于靶向治疗的单克隆抗体或小分子化学物质,如用于治疗某些HER2阳性乳腺癌的曲妥珠单抗、用于治疗NSCLC的吉非替尼等。然而随着这些药物的应用,肿瘤耐药性不可避免的产生。多方面研究表明Hsp90抑制剂会引起与耐药相关的多个分子的降解,提示其在拮抗耐药方面具有重要的意义。本文就Hsp90分子抑制剂在拮抗肿瘤耐药方面的研究进行综述。     

胃癌组织中Src激酶激活和HER2、Ki-67的表达及相关性研究

目的 研究胃癌组织中非受体酪氨酸激酶c-Src激活形式p-Src(Y419)和HER2、Ki-67的表达及相互关系.方法 应用免疫组化法检测123例胃癌组织及56例癌旁组织p-Src(Y419)、HER2、Ki-67的表达,分析其临床病理意义和相互关系.结果 p-Src(Y419)在胃癌及癌旁组织中表达积分中位值分别为80和30,差异显著(P＜0.05),其表达强弱与肿瘤大小、分化程度、浸润深度、pTNM分期显著相关;HER2在胃癌组织中高表达率为39％,癌旁组织HER2无表达,差异显著(P＜0.05);分化良好,肠型,单纯腺癌的胃癌组织HER2表达率高.Ki-67在胃癌及癌旁组织中标记指数范围分别为0-98％和0-20％,其中位值分别为70％和3.5％,差异显著(P＜0.05).Ki-67表达高低与组织类型、Lauren分型、脉管癌栓显著相关,与分化程度有显著差异的趋势;胃癌组织中HER2表达与p-Src(Y419)、Ki-67表达呈正相关,而癌旁组织中无类似关系;胃癌及癌旁组织中p-Src(Y419)表达与Ki-67表达均呈正相关.结论 胃癌组织Src激活、HER2及Ki-67表达在胃癌中有重要的临床意义,可能作为胃癌生物学行为的标记物.胃癌中c-Src激活和HER2、Ki-67表达有一定的相关性.     

Spautin-1通过抑制自噬可增强舒尼替尼诱导的肾癌细胞凋亡

目的:研究spautin-1(一种自噬抑制剂)是否能抑制舒尼替尼诱导的肾癌细胞的自噬以及对舒尼替尼诱导的肾癌细胞凋亡的影响。方法:以肾癌细胞系786-O细胞为模型,Western Blot检测spautin-1对舒尼替尼诱导786-O细胞自噬的影响;Cell Counting Kit-8(CCK-8)检测spautin-1和舒尼替尼对786-O细胞增殖的影响;应用流式细胞术,检测spautin-1对舒尼替尼诱导的786-O细胞凋亡的影响;Western Blot检测spautin-1的促凋亡作用与PI3K/AKT/GSK3β信号通路及抗凋亡蛋白Bcl-2和Mcl-1的关系。结果:与舒尼替尼单独处理组相比,spautin-1能通过降低Beclin-1的表达显著抑制舒尼替尼在786-O细胞中诱导的自噬;Spautin-1和舒尼替尼联合作用明显增强舒尼替尼对786-O细胞增殖的抑制作用;Spautin-1能进一步增强舒尼替尼诱导的786-O细胞的凋亡;Spautin-1和舒尼替尼联合处理786-O细胞时,可以显著降低p-AKTSer473和p-GSK3βSer9的蛋白表达水平,增强GSK3β的活性,进而下调Bcl-2、Mcl-1的表达。结论:Spautin-1能通过抑制AKT活性并活化GSK3β,进一步降低抗凋亡蛋白Bcl-2、Mcl-1的表达,增强舒尼替尼诱导的肾癌细胞凋亡。     

厄洛替尼联合多烯紫杉醇-卡铂治疗晚期非小细胞肺癌的临床研究

目的:探讨厄洛替尼联合多烯紫杉醇和卡铂对晚期非小细胞肺癌(non-small cell lung cancer,NSCLC)的临床疗效及毒副反应。方法:选择2010年1月-2012年1月我院收治的Ⅲb或Ⅳ期非鳞状非小细胞肺癌患者共92例。所有病例均给予多烯紫杉醇(100mg/m3)+卡铂(AUC 5.5,浓度-时间曲线下面积5.5)治疗2个周期,完成2个周期治疗后,将病人随机分为对照治疗组(多烯紫杉醇+卡铂治疗)和厄洛替尼治疗组(厄洛替尼联合多烯紫杉醇+卡铂治疗),每组各46例,厄洛替尼组给予口服厄洛替尼150mg/dl/天剂量治疗。两组均继续治疗2个周期,观察厄洛替尼联合多烯紫杉醇-卡铂治疗对晚期非小细胞肺癌患者的疗效、患者中位生存期及毒副反应。结果:两组患者治疗客观有效率对照治疗组为26.1%,厄洛替尼治疗组为45.6%,差异具有统计学意义(P0.05)。厄洛替尼治疗组患者中位生存期为6.9个月。与对照治疗组相比,厄洛替尼治疗组中患者3/4级中性粒细胞降低的发生率显著降低,差异具有统计学意义(P0.05)。结论:厄洛替尼能增强多烯紫杉醇+卡铂治疗方案对晚期非小细胞肺癌的疗效,减轻化疗的毒副反应。     

EZH2在消化系统肿瘤发生发展中的作用

消化系统肿瘤是一类由多因素和多基因异常引发的恶性肿瘤,相关基因的异常表观遗传调控与其发生和发展密切相关。果蝇zeste基因增强子人类同源物(enhancer of zeste homolog 2,EZH2)是多梳蛋白抑制复合体2(polycomb repressive complex2,PRC 2)的核心亚基,也是一种重要的负性表观遗传调控蛋白质,它通过催化组蛋白H3K 27位点的甲基化而使肿瘤抑制基因表达沉默。近年来研究发现,EZH2在消化系统肿瘤组织和细胞中高表达,并与肿瘤的增殖、侵袭和转移显著性正相关,可独立用作肿瘤患者不良预后指标。对EZH2与消化系统肿瘤相互关系的深入研究,将对这类肿瘤的治疗提供新的分子靶点和新的途径。     

Src and CXCR4 are involved in the invasiveness of breast cancer cells with acquired resistance to lapatinib

Lapatinib is a dual EGFR and ErbB-2 tyrosine kinase inhibitor that has significantly improved the clinical outcome of ErbB-2-overexpressing breast cancer patients. However, patients inexorably develop mechanisms of resistance that limit the efficacy of the drug. In order to identify potential targets for therapeutic intervention in lapatinib-resistant patients, we isolated, from ErbB-2-overexpressing SK-Br-3 breast cancer cells, the SK-Br-3 Lap-R-resistant subclone, which is able to routinely grow in 1 µM lapatinib. Resistant cells have a more aggressive phenotype compared with parental cells, as they show a higher ability to invade through a matrigel-coated membrane. Lapatinib-resistant cells have an increased Src kinase activity and persistent levels of activation of ERK1/2 and AKT compared with parental cells. Treatment with the Src inhibitor saracatinib in combination with lapatinib reduces AKT and ERK1/2 phosphorylation and restores the sensitivity of resistant cells to lapatinib. SK-Br-3 Lap-R cells also show levels of expression of CXCR4 that are higher compared with parental cells and are not affected by Src inhibition. Treatment with saracatinib or a specific CXCR4 antibody reduces the invasive ability of SK-Br-3 Lap-R cells, with the two drugs showing cooperative effects. Finally, blockade of Src signaling significantly increases TRAIL-induced cell death in SK-Br-3 Lap-R cells. Taken together, our results demonstrate that breast cancer cells with acquired resistance to lapatinib have a more aggressive phenotype compared with their parental counterpart, and that Src signaling and CXCR4 play an important role in this phenomenon, thus representing potential targets for therapeutic intervention in lapatinib-resistant breast cancer patients.     

Differential sensitivity of ERBB2 kinase domain mutations towards lapatinib

Background

Overexpression of the ERBB2 kinase is observed in about one-third of breast cancer patients and the dual ERBB1/ERBB2 kinase inhibitor lapatinib was recently approved for the treatment of advanced ERBB2-positive breast cancer. Mutations in the ERBB2 receptor have recently been reported in breast cancer at diagnosis and also in gastric, colorectal and lung cancer. These mutations may have an impact on the clinical responses achieved with lapatinib in breast cancer and may also have a potential impact on the use of lapatinib in other solid cancers. However, the sensitivity of lapatinib towards clinically observed ERBB2 mutations is not known.

Methodology/Principal Findings

We cloned a panel of 8 clinically observed ERBB2 mutations, established stable cell lines and characterized their sensitivity towards lapatinib and alternative ERBB2 inhibitors. Both lapatinib-sensitive and lapatinib-resistant ERBB2 mutations were observed. Interestingly, we were able to generate lapatinib resistance mutations in wt-ERBB2 cells incubated with lapatinib for prolonged periods of time. This indicates that these resistance mutations may also cause secondary resistance in lapatinib-treated patients. Lapatinib-resistant ERBB2 mutations were found to be highly resistant towards AEE788 treatment but remained sensitive towards the dual irreversible inhibitors CL-387785 and WZ-4002.

Conclusions/Significance

Patients harbouring certain ERBB2 kinase domain mutations at diagnosis may not benefit from lapatinib treatment. Moreover, secondary lapatinib resistance may develop due to kinase domain mutations. Irreversible ERBB2 inhibitors may offer alternative treatment options for breast cancer and other solid tumor patients harbouring lapatinib resistance mutations. In addition, these inhibitors may be of interest in the scenario of secondary lapatinib resistance.     

Lapatinib Antagonizes Multidrug Resistance–Associated Protein 1–Mediated Multidrug Resistance by Inhibiting Its Transport Function

Lapatinib, a tyrosine kinase inhibitor, is used in the treatment of advanced or metastatic breast cancer overexpressing human epidermal receptor 2 (HER2). Lapatinib can modulate the function of ATP-binding cassette (ABC) transporters (ABCB1 and ABCG2), which are the major mechanism responsible for multidrug resistance (MDR) in cancer. In this study, we investigated the effect of lapatinib on multidrug resistance–associated protein 1 (MRP1 [ABCC1]), MRP2 (ABCC2), MRP4 (ABCC4) and lung relative resistance protein (LRP) drug efflux pumps. We demonstrated that lapatinib could enhance the efficacy of conventional chemotherapeutic agents in MRP1-overexpressing cells in vitro and in vivo, but no effect in MRP2-, MPR4- and LRP-overexpressing cells. Furthermore, lapatinib significantly increased the accumulation of rhodamine 123 (Rho123) and doxorubicin (DOX) in MRP1-overexpressing cells. However, lapatinib did not alter the protein or mRNA expression levels of MRP1. Further studies showed that the level of phosphorylation of AKT and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) were not altered at the indicated concentrations of lapatinib. In conclusion, lapatinib enhanced the efficacy of conventional chemotherapeutic agents in MRP1-overexpressing cells by inhibiting MRP1 transport function without altering the level of AKT or ERK1/2 phosphorylation. These findings will encourage the clinical research of lapatinib combined with conventional chemotherapeutic drugs in MRP1-overexpressing cancer patients.     

Lapatinib,a dual HER1/HER2 tyrosine kinase inhibitor,augments basal cleavage of HER2 extracellular domain (ECD) to inhibit HER2‐driven cancer cell growth

The ultimate biological and clinical meaning of shed HER2 extracellular domain (ECD) has remained largely unclear until recently. Oversecretion of soluble HER2 ECD has been shown to inhibit growth of HER2‐overexpressing cancer cells by promoting HER2 ECD dimerization with HER transmembrane receptors thus impairing their cross‐tyrosine phosphorylation and decreasing their activation status. HER2‐targeted drugs capable to enhance the occurrence of basal HER2 ECD shedding but simultaneously preventing formation of truncated cell membrane‐bound HER2 intracellular fragment, which exhibits an undesirable constitutive kinase activity, might be extremely efficient at managing HER2‐positive cancer disease. The dual HER1/HER2 Tyrosine Kinase inhibitor lapatinib, which works intracellularly and directly targets the TK domain of HER2, drastically augments basal shedding of HER2 ECD to inhibit HER2‐driven cancer cell growth. Lapatinib treatment significantly augments the concentration of the inactive (unphosphorylated) form of HER2 protein at the tumor cell membrane and promotes an exacerbated HER2 ECD shedding to the extracellular milieu of HER2‐overexpressing cancer cells. Exacerbated sensitivity of trastuzumab‐resistant cancer cells, which contain nearly undetectable levels of soluble HER2 ECD when compared with trastuzumab‐sensitive parental cells to lapatinib‐induced cell growth inhibition, takes place when lapatinib treatment fully restores high levels of basal HER2 ECD shedding. The dramatic augmentation of HER2 ECD shedding that occurs upon treatment of with lapatinib is fully suppressed in lapatinib‐refractory HER2‐positive cells. These findings, altogether, may provide crucial insights concerning clinical studies aimed to accurately describe HER2 ECD as a potential predictor of response or resistance to the HER2‐targeted drugs trastuzumab and lapatinib. J. Cell. Physiol. 226: 52–57, 2010. © 2010 Wiley‐Liss, Inc.     

IL-8 secreted by tumor associated macrophages contribute to lapatinib resistance in HER2-positive locally advanced breast cancer via activation of Src/STAT3/ERK1/2-mediated EGFR signaling

Locally advanced breast cancer (LABC) is an aggressive disease characterized by late clinical presentation, large tumor size, treatment resistance and low survival rate. Expression of EGFR/HER2 and activation of intracellular tyrosine kinase domains in LABC are associated with poor prognosis. Thus, target therapies such as the anti-receptor tyrosine kinases lapatinib drug have been more developed in the past decade. The response to lapatinib involves the inhibition of RTKs and subsequently signaling molecules such as Src/STAT3/Erk1/2 known also to be activated by the cytokines in the tumor microenvironment (TME). The aim of the present study is to identify the major cytokine that might contribute to lapatinib resistance in EGFR+/HER2+ LABC patients. Indeed, tumor associated macrophages (TAMs) are the main source of cytokines in the TME. Herein, we isolated TAMs from LABC during modified radical mastectomy (MRM). Cytokine profile of TAMs revealed that IL-8 is the most prominent highly secreted cytokine by TAMs of LABC patients. Using in-vitro cell culture model we showed that recombinant IL-8 (50 and 100 ng/mL) at different time intervals interfere with lapatinib action via activation of Src/EGFR and signaling molecules known to be inhibited during treatment. We proposed that to improve LABC patients' response to lapatinib treatment it is preferred to use combined therapy that neutralize or block the action of IL-8.     

Lapatinib Plasma and Tumor Concentrations and Effects on HER Receptor Phosphorylation in Tumor

Purpose

The paradigm shift in cancer treatment from cytotoxic drugs to tumor targeted therapies poses new challenges, including optimization of dose and schedule based on a biologically effective dose, rather than the historical maximum tolerated dose. Optimal dosing is currently determined using concentrations of tyrosine kinase inhibitors in plasma as a surrogate for tumor concentrations. To examine this plasma-tumor relationship, we explored the association between lapatinib levels in tumor and plasma in mice and humans, and those effects on phosphorylation of human epidermal growth factor receptors (HER) in human tumors.

Experimental Design

Mice bearing BT474 HER2+ human breast cancer xenografts were dosed once or twice daily (BID) with lapatinib. Drug concentrations were measured in blood, tumor, liver, and kidney. In a randomized phase I clinical trial, 28 treatment-naïve female patients with early stage HER2+ breast cancer received lapatinib 1000 or 1500 mg once daily (QD) or 500 mg BID before evaluating steady-state lapatinib levels in plasma and tumor.

Results

In mice, lapatinib levels were 4-fold higher in tumor than blood with a 4-fold longer half-life. Tumor concentrations exceeded the in vitro IC₉₀ (~ 900 nM or 500 ng/mL) for inhibition of HER2 phosphorylation throughout the 12-hour dosing interval. In patients, tumor levels were 6- and 10-fold higher with QD and BID dosing, respectively, compared to plasma trough levels. The relationship between tumor and plasma concentration was complex, indicating multiple determinants. HER receptor phosphorylation varied depending upon lapatinib tumor concentrations, suggestive of changes in the repertoire of HER homo- and heterodimers.

Conclusion

Plasma lapatinib concentrations underestimated tumor drug levels, suggesting that optimal dosing should be focused on the site of action to avoid to inappropriate dose escalation. Larger clinical trials are required to determine optimal dose and schedule to achieve tumor concentrations that maximally inhibit HER receptors.

Trial Registration

Clinical Trial Registration: NCT00359190     

89Zr-mAb3481 PET for HER3 tumor status assessment during lapatinib treatment

Treatment of human epidermal growth factor receptor 2 (HER2)-driven breast cancer with tyrosine kinase inhibitor lapatinib can induce a compensatory HER3 increase, which may attenuate antitumor efficacy. Therefore, we explored in vivo HER3 tumor status assessment after lapatinib treatment with zirconium-89 (⁸⁹Zr)-labeled anti-HER3 antibody mAb3481 positron emission tomography (PET). Lapatinib effects on HER3 cell surface expression and mAb3481 internalization were evaluated in human breast (BT474, SKBR3) and gastric (N87) cancer cell lines using flow cytometry. Next, in vivo effects of daily lapatinib treatment on⁸⁹Zr-mAb3481 BT474 and N87 xenograft tumor uptake were studied. PET-scans (BT474 only) were made after daily lapatinib treatment for 9 days, starting 3 days prior to ⁸⁹Zr-mAb3481 administration. Subsequently, ex vivo ⁸⁹Zr-mAb3481 organ distribution analysis was performed and HER3 tumor levels were measured with Western blot and immunohistochemistry. In vitro, lapatinib increased membranous HER3 in BT474, SKBR3 and N87 cells, and consequently mAb3481 internalization 1.7-fold (BT474), 1.4-fold (SKBR3) and 1.4-fold (N87). ⁸⁹Zr-mAb3481 BT474 tumor uptake was remarkably high at SUV_mean 5.6±0.6 (51.8±7.7%ID/g) using a 10 μg ⁸⁹Zr-mAb3481 protein dose in vehicle-treated mice. However, compared to vehicle, lapatinib did not affect ⁸⁹Zr-mAb3481 ex vivo uptake in BT474 and N87 tumors, while HER3 tumor expression remained unchanged. In conclusion, lapatinib increased in vitro HER3 tumor cell expression, but not when these cells were xenografted. ⁸⁹Zr-mAb3481 PET accurately reflected HER3 tumor status. ⁸⁹Zr-mAb3481 PET showed high, HER3-specific tumor uptake, and such an approach might sensitively assess HER3 tumor heterogeneity and treatment response in patients.     

Effects of anti-proliferative lichen metabolite,protolichesterinic acid on fatty acid synthase,cell signalling and drug response in breast cancer cells

BackgroundThe lichen compound (+)-protolichesterinic acid (+)-PA, isolated from Iceland moss, has anti-proliferative effects on several cancer cell lines. The chemical structure of (+)-PA is similar to a known fatty acid synthase (FASN) inhibitor C75.AimsTo test whether the anti-proliferative activity of (+)-PA is associated with effects on FASN and HER2 (human epidermal growth factor receptor 2) and major signalling pathways. Synergism between (+)-PA and lapatinib, a HER2 active drug, was also evaluated.Materials and methodsPure compound was isolated by preparative high-performance liquid chromatography (HPLC) and purity of (+)-PA analyzed by analytical HPLC. Cell viability was assessed using Crystal violet staining. FASN and HER2 expression was estimated by immunofluorescence. The Meso Scale Discovery (MSD)^® assay was used to measure activation of ERK1/2 and AKT. Synergism was estimated by the CalcuSyn software.ResultsTreatment with (+)-PA increased FASN expression in SK-BR-3 cells, which overexpress FASN and HER2, implying a compensatory response to inhibition of FASN activity. HER2 expression was decreased suggesting secondary downregulation. ERK1/2 and AKT signalling pathways were inhibited, probably due to reduced levels of HER2. No effects were observed in T-47D cells. Synergism between (+)-PA and lapatinib was observed in the SK-BR-3 cells.ConclusionResults suggest that the primary effect of (+)-PA is inhibition of FASN activity. Synergistic effects with lapatinib were seen only in SK-BR-3 cells, and not T-47D cells, further supporting the notion that (+)-PA acts by inhibiting FASN with secondary effects on HER2 expression and signalling. (+)-PA could therefore be a suitable agent for further testing, alone or in combination treatment against HER2-overexpressing breast cancer.