The Effects Of Erbin On The Migration Capacity And Herceptin Resistance Of Her2Positive Breast Cancer Cells And Its Relative Mechanism

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 samplesErbin 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 cellsThe 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 mannerTo 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 mannerCurrently, 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.