摘要
目的：乳腺癌是严重威胁女性健康的恶性肿瘤之一，全球每年约120万人被确诊为乳腺癌，每年约有70万人死于此类疾病，在所有癌症引发的死亡中排名第三，仅次于肺癌和胃癌。乳腺癌的发病机制十分复杂，表皮生长因子受体2（Her2）在20-30%的乳腺癌中过表达，而且与患者的不良预后密切相关。因此探索Her2相互作用分子的生物学功能、阐明相关的分子机制对于深入了解Her2阳性乳腺癌的病理生理学过程及改善患者的预后具有重要意义。
Erbin是应用酵母双杂技术研究Her2相互作用蛋白时被发现的，因其可与Her2发生直接的，特异的相互作用，故命名为Her2/ErbB2相互作用蛋白（ErBb2interaction protein），简称Erbin。Erbin广泛表达于正常的上皮组织中。已有研究证实，Erbin可以负向调控Her2下游RAS-RAF-ERK信号通路的活化。但在Her2阳性乳腺癌中Erbin的表达水平及表达模式尚不清楚，Erbin在Her2阳性乳腺癌中的生物学功能及对其恶性演进过程的影响亦未见报道。本次研究中，发现在Her2阳性乳腺癌组织中，Erbin的表达显著降低，甚至出现了表达丢失的现象。实验结果表明，在Her2阳性的乳腺癌细胞中敲低Erbin表达可明显增强细胞的迁移能力，诱导乳腺癌细胞出现侵袭表型。同时还发现，敲低Erbin表达可诱导Her2阳性的乳腺癌细胞对治疗性单抗赫赛汀产生抗性。分子机制的研究表明，敲低Erbin主要通过诱导AKT信号通路的异常活化来实现上述的生物学效应。这些结果提示，Erbin通过调控AKT信号通路影响乳腺癌细胞的迁移能力及对赫赛汀的耐药，从而可能在Her2阳性乳腺癌的恶性演进过程中发挥着重要作用。
方法：
应用Erbin特异性抗体、通过免疫组织化学染色在人乳腺癌组织芯片中检测Erbin在蛋白水平的表达情况；通过Oncomine数据库检索，探索在Her2阳性的乳腺癌组织中Erbin在mRNA水平的表达情况；通过免疫印迹法及实时定量PCR检测在乳腺癌细胞系中，Erbin在转录水平及蛋白水平的表达情况；设计并构建了Erbin shRNA表达载体，通过细胞转染、病毒感染以及蛋白质免疫印迹，检测在Her2阳性乳腺癌细胞中敲低Erbin表达对Heregulin诱导的AKT信号通路活化的影响；通过构建Erbin真核表达载体、细胞转染及免疫印迹法检测了，在Her2阳性的乳腺癌细胞中过表达Erbin对Heregulin诱导的AKT活化的影响；通过CCK8（细胞增殖检测），探索敲低Erbin表达对Her2阳性乳腺癌细胞增殖的影响；通过流式细胞术，检测敲低Erbin表达对Her2阳性乳腺癌细胞的细胞周期行进的影响；通过二维培养条件下的细胞划痕实验，检测了敲低Erbin表达对Her2阳性乳腺癌细胞迁移能力的影响；通过Matrigel细胞三维培养法，检测敲低Erbin表达对Her2阳性乳腺癌细胞侵袭表型形成的影响；应用AKT及ERK信号通路的特异性抑制剂，探索敲低Erbin影响细胞迁移及侵袭表型形成的分子机制；通过细胞增殖检测实验，检测敲低Erbin表达对靶向Her2的治疗性抗体-赫赛汀细胞增殖抑制效应的影响；通过细胞海绵三维细胞培养法，检测敲低Erbin对Her2阳性乳腺癌细胞赫赛汀耐药的影响；通过细胞锚定非依赖增殖检测法，探索敲低Erbin表达对Her2阳性乳腺癌细胞的赫赛汀敏感性的影响；应用AKT及ERK特异性抑制剂，探索Erbin影响Her2阳性乳腺癌细胞赫赛汀耐药的分子机制。
结果：
1Erbin在乳腺癌及Her2阳性乳腺癌组织中表达显著降低
免疫组化的结果表明，在所检测的10例正常乳腺组织中，8例呈现Erbin高表达（++-+++）；而在20例浸润性乳腺癌组织中，Erbin的表达水平普遍偏低，甚至出现了表达丢失的现象。15例乳腺癌组织Erbin的表达水平为“+”或阴性，仅5例为“++”。在Oncomine数据库检索时发现了一组含有53例浸润性乳腺癌的样本。通过与正常乳腺组织进行比较，发现这是一组高Her2阳性率的乳腺癌样本，在53例样本中有52例为Her2阳性，仅有1例为Her2阴性。同时检索了该组样本中Erbin在mRNA水平的表达情况。结果表明，在52例样本中Erbin mRNA的水平都显著低于正常乳腺组织，仅有一例例外。这一发现与之前在乳腺癌组织芯片中检测得到的结果相符，提示Erbin表达水平在Her2阳性乳腺癌组织中显著下降。通过对该组患者的预后进行分析发现，与那些三年内未出现肿瘤复发的患者相比，三年内出现复发的患者标本中Erbin表达水平的下调更为显著。提示Erbin在Her2阳性乳腺癌的恶性演进过程中可能发挥着重要作用。此外本次实验还应用Western-blot及实时定量RT-PCR法检测了人乳腺上皮细胞MCF-10A及5种乳腺癌细胞系（MCF-7、MCF-7/Her2、MDA-453、T47D、MDA-435）中检测了Erbin在转录水平和蛋白水平的表达情况。实验结果表明，在转录水平，MCF-7、MCF-7/Her2及T47D细胞的Erbin表达明显低于MCF-10A细胞。而MDA-453细胞Erbin表达显著高于MCF-10A细胞。在蛋白水平，与MCF-10A细胞相比，MCF-7、MCF-7/Her2、MDA-453、MDA-435细胞Erbin的表达水平相对较低。
2Erbin可在Her2阳性乳腺癌细胞中负调控AKT信号通路的活化
设计并构建了Erbin shRNA的慢病毒表达载体，利用慢病毒感染的方法获得了三株稳定敲低Erbin的乳腺癌细胞（均为Her2阳性乳腺癌细胞），分别命名为MDA-453-Erbin-sh、 SKBR3-Erbin-sh和MCF-7/Her2-Erbin-sh。Western-blot检测结果表明，在这三种感染Erbin shRNA慢病毒的细胞中，Erbin的表达被显著抑制。同时，将感染表达对照shRNA慢病毒的细胞作为对照组。通过应用Her2的激活分子Heregulin分别刺激上述乳腺癌细胞，检测Her2下游的主要促生存信号通路的变化。实验结果表明，在MCF-7/Her2对照细胞中，Heregulin的刺激可以使AKT的磷酸化发生一个先升后降，如同“抛物线”样的变化;但在MCF-7/Her2-Erbin-sh细胞中，Heregulin刺激所造成的AKT的磷酸化，无论强度还是持续的时间都明显强于对照细胞。在MDA-453细胞中，Heregulin的刺激可以使对照细胞的AKT出现一个先快速磷酸化继而缓慢下降的过程，虽然沉默Erbin的表达并没有显著提高p-AKT的水平，但是造成了AKT的持续活化，即使在刺激六小时后，其活化程度依然没有明显的衰减。在SKBR3-Erbin-sh细胞中也得到了同样的结果，即在高表达Her2的乳腺癌细胞中，Erbin表达下调可增强由Heregulin诱导的AKT的磷酸化。上述实验表明，在高表达Her2的乳腺癌细胞中，Erbin表达的缺失可增强由Heregulin诱导及Her2激活所导致的AKT的磷酸化。为进一步探讨Erbin对AKT信号通路的调控作用，实验中构建了Erbin的真核表达载体，并将其瞬时转染Her2阳性乳腺癌细胞MCF-7/Her2和MDA-453，转染48小时后用无血清的培养基饥饿细胞过夜，然后用Heregulin刺激细胞。Western Blot的结果显示，Erbin过表达可显著抑制Heregulin诱导的AKT活化。这进一步证明了，在高表达Her2的乳腺癌细胞中，Erbin对于Heregulin诱导的AKT信号通路活化具有负向调控作用。
3Erbin缺失可促进AKT信号依赖的细胞迁移及侵袭
为进一步探索Erbin表达降低在Her2阳性乳腺癌恶性演进过程中的意义。首先通过体外划痕实验进行检测敲低Erbin表达对Her2阳性乳腺癌细胞迁移能力的影响。结果表明，在MCF-7/Her2细胞中敲低Erbin的表达可以显著加速创口的愈合,而且在Heregulin存在的情况下这种促进作用更为明显。实验采用以基质胶为基础的三维细胞培养法探索Erbin对Her2阳性乳腺癌细胞侵袭能力的影响。实验结果表明，对照细胞可以在三维细胞培养体系中形成边缘规则的球形结构，而大部分（80%）的Erbin低表达细胞在三维体系中形成边缘极不规则且具有侵袭样结构的球体。上述结果表明，在Her2阳性乳腺癌细胞中沉默Erbin表达，可增强细胞迁移和侵袭能力。为进一步探索此过程中的精确分子机制，在划痕实验中使用了AKT特异性的抑制剂GDC0941。Western-blot的结果表明，GDC0941可特异性抑制AKT的磷酸化，而不影响ERK的磷酸化。划痕实验的结果表明，GDC0941能够显著逆转Erbin表达缺失对细胞迁移能力的影响，证实了AKT的活化可能在此过程中发挥了重要作用。那么ERK作为Her2下游另一条重要的信号通路，它的激活是否也参与Erbin表达缺失诱导的细胞迁移能力增强呢？为了回答这一问题，本次实验选用了ERK的特异性抑制剂PD184352。结果表明，PD184352它可以在不影响AKT磷酸化的前提下，特异性抑制ERK的磷酸化。划痕实验的结果表明，PD184352并不能逆转敲低Erbin表达所诱导的细胞迁移。以上实验结果表明，在Her2阳性的乳腺癌细胞中，Erbin表达缺失以AKT信号通路依赖的方式促进Her2阳性乳腺癌细胞迁移及侵袭表型形成。
4敲低Erbin表达诱导AKT信号依赖的赫赛汀耐药
目前靶向Her2的治疗性抗体-赫赛汀是Her2阳性乳腺癌的主要治疗手段之一。然而赫赛汀耐药是制约此药物发挥疗效及造成患者不良预后的重要原因。阐明赫赛汀耐药的相关机制、发现预测及克服耐药的分子靶标具有重要意义。应用不同浓度的赫赛汀分别处理表达对照shRNA及表达Erbin shRNA的MCF-7/Her2细胞。在连续刺激5天后，采用CCK8细胞增殖实验，检测了敲低Erbin表达对赫赛汀细胞增殖抑制效应的影响。实验结果表明，在对照组细胞中，赫赛汀可显著抑制细胞的增殖，并呈现出良好的量效关系。在敲低Erbin的MCF-7/Her2细胞中，赫赛汀虽然可以在一定程度上抑制细胞增殖并具有量效关系。但是与对照细胞相比，相同浓度赫赛汀对敲低Erbin的MCF-7/Her2细胞的增殖抑制率显著降低。这提示，敲低Erbin表达可能诱导Her2阳性乳腺癌细胞对赫赛汀产生耐药。为进一步排除此现象的细胞特异性，实验中选取MDA-453细胞重复进行了上述实验。实验结果表明，敲低Erbin表达可诱导MDA-453细胞对赫赛汀产生耐药。
由于在二维培养和三维培养条件下，肿瘤细胞对药物的敏感性可能存在差异。本次实验应用细胞海绵（cellusponge）三维细胞培养法检测了敲低Erbin表达对MDA-453细胞赫赛汀敏感性的影响。实验结果显示，赫赛汀可完全抑制对照细胞在细胞海绵中的生长。而敲低Erbin表达不但可以增加MDA-453细胞在细胞海绵中的克隆体积，还可以诱导MDA-453细胞对赫赛汀产生耐药。在接下来的实验中，采用了软琼脂细胞克隆形成实验进一步检测了敲低Erbin表达对MDA-453细胞赫赛汀抗性的影响。实验结果显示，赫赛汀可显著抑制对照细胞的锚定非依赖性生长，而在敲低Erbin的细胞中，这种抑制效应显著减弱。此结果进一步支持了这一假设，即Erbin表达缺失可诱导Her2阳性乳腺癌细胞对赫赛汀产生抗性。
为进一步探索敲低Erbin表达诱导Her2阳性乳腺癌细胞赫赛汀抗性的分子机制，本次实验检测了AKT及ERK信号通路在敲低Erbin表达诱导赫赛汀耐药过程中的作用。通过细胞增殖实验发现，在对照细胞中赫赛汀可抑制AKT的诱导剂Heregulin诱导的细胞增殖。但是在敲低Erbin的细胞中，赫赛汀不能抑制Heregulin诱导的细胞增殖。AKT的抑制剂GDC0941可显著逆转由于敲低Erbin导致的赫赛汀耐药，而ERK特异性抑制剂PD184352不能逆转敲低Erbin诱导的赫赛汀耐药。这一结果提示，Erbin表达缺失主要通过诱导AKT信号的异常活化诱导Her2阳性乳腺癌细胞赫赛汀耐药的发生。
结论：
1在乳腺癌及Her2阳性乳腺癌组织中Erbin表达水平显著下调
2在Her2阳性的乳腺癌细胞中Erbin负向调控了AKT信号通路。
3Erbin表达缺失以AKT信号通路依赖的方式促进Her2阳性乳腺癌细胞迁移及侵袭表型形成。
4Erbin表达缺失以AKT信号通路依赖的方式诱导Her2阳性乳腺癌细胞赫赛汀耐药。
Objectives: Breast cancer is one of the malignancies that threatenwomen health, with around1200000new cases per year around the world.Breast cancer is the third most common leading cause of cancer related deathfollowing lung and gastric cancer, with around700000deaths each year. Themechanism of the malignant progression of breast cancer is very complicated.ErBb2/Her2is overexpressed in20–30%breast cancer cases. Overexpresionof Her2intimatly correlates with poor prognosis of the patient with breastcancer. Consequently, identification of the biological function of Her2interacting proteins and illustration the relative mechanism are very importantfor us to understand the pathogenesis of breast cancer and better the prognosisof the patients with breast cancer.
Erbin was originally identified as a new binding partner of Her2throughyeast two-hybrid system, so named because it specifically and directlyinteracts with Her2(Her2/ErbB2interacting protein). Erbin is ubiquitouslyexpressed in normal epithelial tissues and constitutively associates with Her2at the basolateral membranes in epithelial cells. The inhibitory role of Erbin inRAS-RAF-ERK signaling has been demonstrated. However, whether theexpression of Erbin is altered in Her2-overexpressing breast cancer is unclear.There is little information regarding the function of Erbin in cancerprogression. In the present study, we demonstrate that the level of Erbin issignificantly downregulated or lost in Her2-overexpressing breast cancertissues. Knockdown of Erbin remarkably promotes cell migration, inducesinvasive phenotype of breast cancer cells with Her2overexpression andantagonized the anti-proliferative effect of therapeutic antibody Herceptin inHer2positive breast cancer cells. The data reveal that Erbin exerts the effects in Her2positive breast cancer cells mainly through a AKT dependent manner.The data also suggest that Erbin may play a role in breast cancer progression.
Methods：The breast cancer tissue array was employed to evaluate theexpression of Erbin in breast cancer tissues through the specific Erbinantibody and immunohistochemistry assay. The publicly available databaseOncomine was used to explore the expression of Erbin at transcriptional levelin Her2positive breast cancer tissues. Western-blot and Real-time RT-PCRwas employed to detect the expression of Erbin at mRNA and protein levels inbreast cancer cell lines. We designed Erbin shRNA expression vector. Theeffects of Erbin knockdown on AKT activation triggered by Heregulinstimulation in Her2positive breast cancer cells was evaluated throughtransfection, transduction and western blot assay. The eukaryotic expressionvector for Erbin overexpression was constructed. Transduction andWestern-blot assay was employed to explore the effects of Erbinoverexpression on the AKT activation induced by Heregulin in the breastcancer cells with Her2overexpression. The cell proliferation assay was used totest the effects of Erbin knockdown on the proliferation of Her2positive breastcancer cells. Flow cytometry assay was employed to analysis the effects ofErbin knockdown on the cell cycle progression of Her2positive breast cancercells. Two-dimensional cell culture and wound-healing assay was employed toevaluate the migration capacity of Her2positive breast cancer in response toErbin knockdown. Matigel based three-dimension cell culture assay was usedto evaluate the invasive structure formation of Her2positive breast cancercells in response to Erbin knockdown. The specific inhibitors of AKT andERK were employed to explore the precise mechanism about the enhancementof cell migration and invasive structure formation of Her2positive breastcancer cells triggered by Erbin knockdown. The cell proliferation assay wasused to explore the effects of Erbin knockdown on Herceptin resistance of thebreast cancer cells with Her2overexpression. The cellusponge basedthree-dimension culture system was employed to detect the sensitivity of Her2positive breast cancer cells to Herceptin treatment in response to Erbin knockdown. Anchorage-independent growth of the breast cancer cells withErbin deficiency was determined by soft agar colony formation assays. Thespecific inhibitors of AKT and ERK were employed to explore the precisemechanism about the Herceptin resistance triggered by Erbin deficiency inHer2overexpressing breast cancer cells.
Results：
1The expression of Erbin is down-regulated in the breast cancer tissuesamples and the Her2-overexpressing breast cancer tissue samples
Erbin expression was examined in breast cancer and normal breast tissuesamples by immunohistochemical labeling using the breast cancer tissue array,which containing10cases of normal breast tissues and20invasive breasttumor tissue samples. The results demonstrate that the expression of Erbin inmost normal breast tissues (8/10) was high (++–+++). However, in invasivebreast cancer tissues, Erbin expression was low (+) or even lost (15/20), inaddition to5breast tissue samples with high (++) level of Erbin. We thensearched for Erbin mRNA expression in human breast cancer tissues, in whichHer2are overexpressed in most tissue samples (52/53), through a publiclyavailable database Oncomine. Compared with normal breast tissues, the ErbinmRNA expression is significantly decreased in breast cancer tissues. Thisresult demonstrated that Erbin expression was decreased in Her2positivebreast cancer tissues. Further analysis revealed that the frequency of Erbindownregulation in the patients with recurrence within three years issignificantly higher than those surviving disease-free. The expression of ErbinmRNA and protein were assessed in a panel of human breast cancer cell lines(MCF-7, MCF-7/Her2, MDA-453, T47D, MDA-435) and a human breastepithelial cell line MCF-10A by quantitative RT-PCR and Western-blot. Thedata show that relatively high levels of Erbin mRNA and protein weredetected in MCF-10A cells. Compared with MCF-10A cells, the expression ofErbin in breast cancer cell lines was relatively low.
2Erbin negatively regulates Heregulin-induced AKT phosphorylation inHer2-overexpressing breast cancer cells
The lentiviral vector expressing Erbin shRNA was designed andconstructed. The expression of Erbin was silenced by transduction with thelentiviral vectors expressing Erbin shRNA in MDA-453, SKBR3, andMCF-7/Her2cells. The three cell lines, which expressed Erbin shRNA, werenominated as MDA-453-Erbin-sh, SKBR3-Erbin-sh, MCF-7/Her2-Erbin-sh,respectively. The cells expressing control shRNA were regarded as controlcells and nominated as MDA-453-NC, SKBR3-NC and MCF-7/Her2-NC cells.We found that the phosphorylation of AKT induced by Heregulin occurredrapidly and then gradually decreased in MCF-7/Her2-NC cells. Noticeably,Heregulin stimulation resulted in a dramatic enhancement both in intensityand duration of AKT activation in MCF-7/Her2-Erbin-sh cells and persistentactivation of AKT lasted up to6h. Although the effect of Erbin silencing onHeregulin-induced AKT activation was not remarkable in MDA-453cells, itdid prolong the duration of AKT activation. The effect of Erbin knockdown onAKT signaling was also observed in SKBR3cells. Erbin silencing caused aprominent elevation in AKT phosphorylation especially in the Heregulinstimulation. To further verify the potential role of Erbin in modulatingHeregulin-induced AKT activation, Erbin was transiently overexpressed inHer2-overexpressing breast cancer cells. To further verify the potential role ofErbin in modulating Heregulin-induced AKT activation, Erbin was transientlyoverexpressed in MCF-7/Her2and MDA-453cells. Overexpression of Erbinin MCF-7/Her2cells not only significantly decreased the intensity ofHeregulin-induced AKT phosphorylation, but also shortened its duration. Thesimilar results were also observed in MDA-453cells. These data indicate thatErbin inhibits Heregulin-induced AKT phosphorylation in Her2-overexpressing breast cancer cells.
3Loss of Erbin enhances the migratory ability of Her2-overexpressingbreast cancer cells in an AKT dependent manner
To further explore the significance of Erbin deficiency in the malignantprogression of Her2positive breast cancer, in vitro scratch assays wereperformed to evaluate the effect of Erbin knockdown on the migratory activity of breast cancer cells. An accelerated scratch wound closure was observed inErbin silencing cells in the presence or absence of Heregulin stimulation. Thisresult was further verified in Matrigel-based three-dimensional cell culturesystems. We found that MCF-7/Her2-NC cells possessed uniform sphericalarchitectures, whereas most colonies (>80%) of MCF-7/Her2-Erbin-sh cellsexhibited invasive three-dimensional structures. To confirm the role of AKTsignaling in Erbin knockdown-induced cell migration, the AKT inhibitorGDC0941was employed, which specifically repressed Heregulin inducedAKT activation, but did not influenced ERK activation triggered by Heregulinstimulation. The data showed that the treatment with GDC0941remarkablyreversed the effects of Erbin knockdown on the cell migration. To furtherconfirm the role of AKT signaling in the enhanced migration induced by Erbinknockdown, we performed the same experiment by using MEK1/2inhibitorPD184352. PD184352specifically blocked ERK activation, but did not affectAKT phosphorylation triggered by Heregulin and cell migration induced byErbin knockdown. These data may indicate that increased cell migration byErbin knockdown at least partially depends on the activation of AKT signalingpathway.
4Knockdown of Erbin induces Herceptin resistance in Her2-overexpressing breast cancer cells in an AKT dependent manner
Currently, Herceptin, a therapeutic antibody targeting Her2, is one of themost important approaches for treating Her2positive breast cancer. However,Herceptin resistance is the main obstacle impairing the therapeutic effects ofHerceptin and induces poor prognosis of the patients with Her2positive breasttumor. Illustration of the mechanism of Herceptin resistance and explorationof the molecular targets for predicting and overcoming Herceptin resistanceare very important. The cells were treated with various concentrations ofHerceptin and evaluated the proliferation activities of the cells by CCK-8assays. We found that Herceptin at various concentrations efficiently inhibitedthe growth of MCF-7/Her2-NC and MDA-453/NC cells under conventionalculture or cultured in the medium containing low FBS. Loss of Erbin dramatically antagonized the anti-proliferative effect of Herceptin inMCF-7/Her2-Erbin-sh and MDA-453/Erbin-sh cells.
The sensitivities of the cancer cells to the therapeutics are different intwo-dimensional culture and three-dimensional conditions. A three-dimensional culture system was employed to further confirm whether loss ofErbin affects the inhibitory effect of Herceptin on breast cancer cell growth.Similar to the CCK-8assays, Herceptin completely suppressed the colonyformation of MDA-453/NC cells cultured in3D cellusponge. However, thesame concentration of Herceptin only slightly inhibited the colony formationin MDA-453/Erbin-sh cells. In addition, larger colonies were noticed inMDA-453/Erbin-sh cells in3D cellusponge. To further verify the associationof Erbin deficiency with Herceptin resistance, the soft agar colony formationassays was employed. The data showed that the anchorage-independentgrowth of the Her2-overexpressing breast cancer cells was enhanced by Erbinknockdown in presence of Herceptin.
To explore the mechanism underlying Herceptin resistance induced byErbin deficiency, the cells were treated with Herceptin and Heregulinsimultaneously. We found that Heregulin greatly stimulated the proliferationactivities in both control and MCF-7/Her2-Erbin-sh cells. Treatment withHerceptin significantly repressed the growth of the control cells in thepresence of Heregulin. However, the anti-proliferative effect of Herceptin wasremarkably antagonized in MCF-7/Her2-Erbin-sh cells, reflecting strong andpersistent AKT activation due to lacking Erbin expression. In addition, AKTinhibitor GDC0941strongly reversed Herceptin resistance in MCF-7/Her2-Erbin-sh cells whereas ERK inhibitor PD184352did no exert the effect,further confirming that loss of Erbin induces Herceptin resistance inHer2-overexpressing breast cancer cells, at least partially in a AKT dependentmanner.
Conclusions：
1Erbin expression at both mRNA and protein level was significantlydecreased or even disappeared in some breast cancer tissues.
2Erbin inhibits Heregulin-induced AKT phosphorylation in Her2-overexpressing breast cancer cells.
3Loss of Erbin results in accelerated cellular migration and invasivestructure formation in Her2positive breast cancer cells in a AKT dependentmanner.
4Erbin knockdown induced Herceptin resistance in Her2-overexpressingbreast cancer cells that is mainly mediated by aberrant activation of AKTtriggered by Erbin deficiency.
引文
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