Trastuzumab-resistant SKBR3 Cells Research Articles

Activation of STAT3/HIF-1α/Hes-1 axis promotes trastuzumab resistance in HER2-overexpressing breast cancer cells via down-regulation of PTEN

BackgroundResistance to the HER2-targeted antibody trastuzumab remains to be a major clinical challenge in the treatment of HER2-positive breast cancer. Hyper-activation of STAT3 is proposed to be a predictive biomarker of trastuzumab resistance. However, the precise mechanism(s) remains poorly defined. Evidence is emerging that HIF-1α, a central downstream element of STAT3 pathway, serves a pivotal role in the complex signaling network with subsequent diverse cellular events. Material and methodsWe have established trastuzumab resistant SKBR3 cells (SKBR3-TR). The cell viability, apoptosis as well as western blot, siRNA transfection and co-immunoprecipitation assays were performed to evaluate the involvement of STAT3/HIF-1α in modulation of trastuzumab resistance. ResultsWe found that in SKBR3-TR cells and conditioned medium-treated parental cells, constitutive phosphorylated STAT3 coincided with prominent up-regulation of HIF-1α which was accompanied with PTEN attenuation. Moreover, the inhibition of STAT3 activation by Stattic and/or genetically STAT3 knocking down decreased HIF-1α level in SKBR3-TR cells. Additionally, treatment with Stattic and/or STAT3 siRNA engendered the up-regulation of PTEN protein in STAT3-inhibited resistant cells. Restoration of PTEN was also observed following siRNA-mediated silencing of HIF-1α expression. Moreover, down-regulation of HIF-1α caused a reduction in the HES-1 content. Further study with HES-1 specific siRNA revealed the elevation of PTEN expression in HES-1 knock-down trastuzumab resistant cells. ConclusionThe impairment of STAT3-HIF-1α-HES-1 pathway restored trastuzumab sensitivity through up-regulation of PTEN protein. General significanceThese findings highlighted the signal integrator role of HIF-1α in STAT3-mediated trastuzumab resistance induction which would be valuable in designing more efficient chemosensitization strategies.

Sensitizing effect of juglone is mediated by down regulation of Notch1 signaling pathway in trastuzumab-resistant SKBR3 cells.

Trastuzumab (Herceptin) monoclonal antibody directed against HER2 receptor has been administered as a treatment for metastatic HER2 positive breast cancer. The problematic issue in treatment of HER2 positive breast cancer cells is commonly the induction of resistance to trastuzumab which might be due to modulation of some vital signaling elements such as Notch1 and Pin1. In this study, we were aimed to investigate whether the cross talk between pin1 and Notch1 has a role in this event. Our results indicated that the expression level of Pin1 in resistant SKBR3 cells increased by about twofold relative to sensitive SKBR3 cells. Besides, Pin1 inhibition via juglone reduced the extent of proliferation, colony formation and migration capacity of resistant SKBR3 cells. In addition, despite a feed forward loop between Notch1 and Pin1 in sensitive SKBR3 cells, inhibition of Notch1 cleavage in resistant SKBR3 cells did not affect pin1 level whereas pin1 inhibition by juglone reduced the level of Hes1, p-Akt and increased the cellular content of Numb. Therefore, we concluded that pin1 inhibition could be considered as a promising sensitizing strategy to weaken trastuzumab resistance.

The voltage gated Ca(2+)-channel Cav3.2 and therapeutic responses in breast cancer.

BackgroundUnderstanding the cause of therapeutic resistance and identifying new biomarkers in breast cancer to predict therapeutic responses will help optimise patient care. Calcium (Ca2+)-signalling is important in a variety of processes associated with tumour progression, including breast cancer cell migration and proliferation. Ca2+-signalling is also linked to the acquisition of multidrug resistance. This study aimed to assess the expression level of proteins involved in Ca2+-signalling in an in vitro model of trastuzumab-resistance and to assess the ability of identified targets to reverse resistance and/or act as potential biomarkers for prognosis or therapy outcome.MethodsExpression levels of a panel of Ca2+-pumps, channels and channel regulators were assessed using RT-qPCR in resistant and sensitive age-matched SKBR3 breast cancer cells, established through continuous culture in the absence or presence of trastuzumab. The role of Cav3.2 in the acquisition of trastuzumab-resistance was assessed through pharmacological inhibition and induced overexpression. Levels of Cav3.2 were assessed in a panel of non-malignant and malignant breast cell lines using RT-qPCR and in patient samples representing different molecular subtypes (PAM50 cohort). Patient survival was also assessed in samples stratified by Cav3.2 expression (METABRIC and KM-Plotter cohort).ResultsIncreased mRNA of Cav3.2 was a feature of both acquired and intrinsic trastuzumab-resistant SKBR3 cells. However, pharmacological inhibition of Cav3.2 did not restore trastuzumab-sensitivity nor did Cav3.2 overexpression induce the expression of markers associated with resistance, suggesting that Cav3.2 is not a driver of trastuzumab-resistance. Cav3.2 levels were significantly higher in luminal A, luminal B and HER2-enriched subtypes compared to the basal subtype. High levels of Cav3.2 were associated with poor outcome in patients with oestrogen receptor positive (ER+) breast cancers, whereas Cav3.2 levels were correlated positively with patient survival after chemotherapy in patients with HER2-positive breast cancers.ConclusionOur study identified elevated levels of Cav3.2 in trastuzumab-resistant SKBR3 cell lines. Although not a regulator of trastuzumab-resistance in HER2-positive breast cancer cells, Cav3.2 may be a potential differential biomarker for survival and treatment response in specific breast cancer subtypes. These studies add to the complex and diverse role of Ca2+-signalling in breast cancer progression and treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0299-0) contains supplementary material, which is available to authorized users.

Protein tyrosine phosphatase SHP-1 sensitizes EGFR/HER-2 positive breast cancer cells to trastuzumab through modulating phosphorylation of EGFR and HER-2.

BackgroundTrastuzumab resistance in HER-2 positive breast cancer cells is closely related to overexpression of both epidermal growth factor receptor (EGFR) and human epidermal receptor (HER-2). SHP-1 has been demonstrated to downregulate tyrosine kinase activity including EGFR via its phosphatase function, but its effect on HER-2 activity is still unknown. Here, we examined the hypothesis that SHP-1 enhances the anticancer efficacy of trastuzumab in EGFR/HER-2 positive breast cancer cells through combining dual inhibition of EGFR and HER-2.MethodsTrastuzumab-resistant breast cancer SKBr-3 cells were generated by long-term in vitro culture of SKBr-3cells in the presence of trastuzumab. The SHP-1 was ectopically expressed by stable transfection. The activity and expression of EGFR, HER-2, and downstream signaling pathways were tested by Western blot. Cell viability was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was examined by flow cytometry. The binding between SHP-1 and EGFR/HER-2 was evaluated by immunoprecipitation assay and bimolecular fluorescence complementation. The effects of SHP-1 on tumorigenicity and trastuzumab sensitivity were confirmed via in vivo xenograft model.ResultsTrastuzumab-resistant SKBr-3 cells showed aberrant co-expression of EGFR and HER-2. Introduction of wild-type SHP-1 inhibited cell proliferation, clone formation, and promoted the apoptosis induced by trastuzumab. Meanwhile, SHP-1 overexpression reduced phosphorylation levels of EGFR and HER-2 both in parental and trastuzumab-resistant SKBr-3 cells. In vivo study showed an increased antitumor effect of trastuzumab in SHP-1 overexpressed xenografts. At last, we discovered that SHP-1 can make complexes with both EGFR and HER-2, and both phospho-EGFR and phosphor-HER-2 levels in wild-type SHP-1 immunoprecipitates were less than those in phosphatase-inactive SHP-1 (C453S) immunoprecipitates, indicating that EGFR and HER-2 are potential substrates of SHP-1.ConclusionTaken together, we have demonstrated that the SHP-1 is a negative regulatory factor of the tyrosine kinase activity of HER-2 and EGFR through inhibiting phosphorylation. Dual targeting of EGFR and HER-2, by combining trastuzumab with SHP-1 overexpression, may improve response in HER-2 overexpressing breast cancer cells that also express high levels of EGFR.

Abstract P6-05-08: Nuclear HER3 localisation plays a role in trastuzumab resistance in HER2-positive breast cancer

Abstract Background: HER3 is known to locate in the nucleus. Unlike HER4, nuclear HER3 translocation has not been reported to be due to a proteolytic cleavage process by ADAM17 and gamma-secretase. The mechanisms of nuclear HER3 induction and its role in relation to trastuzumab treatment and resistance for HER2-positive breast cancer is unclear. Methods: Using nuclear fractionation and confocal microscopy, nuclear HER3 localisation was investigated in response to trastuzumab with or without ADAM17 inhibitor and gamma-secretase inhibitor in a panel of HER2 expressing cell lines. We also correlated nuclear HER3 expression by immunohistochemistry with treatment response in patients who underwent window trastuzumab study as well as the survival outcome in a cohort of HER2-positive breast cancer patients using Kaplan–Meier survival curves with Log-rank test. Results: HER3 ligand heregulin and trastuzumab was found to induce nuclear HER3 translocation in HER2-positive breast cancer cell lines, including SKBR3. Nuclear HER3 was also enriched in acquired trastuzumab resistant SKBR3 cells (SKBr3-TR). Trastuzumab treatment induced several HER3 fragments and HER3100kD was found to be responsible for nuclear HER3 enrichment by fractionation. This fragment was confirmed to be a specific band of HER3 as shown by HER3 knockdown. Nuclear HER3 was reduced by inhibiting either gamma-secretase or ADAM17 inhibitor. Gamma-secretase or ADAM17 inhibitor reduced HER3100kD in both SKBr3 and SKBr3-TR cells. In HER2-positive breast cancer patients who underwent window trastuzumab study, baseline nuclear HER3 status was not a predictor of response for trastuzumab monotherapy at day 21. However, nuclear HER3 was enriched after trastuzumab treatment in a poor-responder patient. Total HER3 expression level in cytoplasm positively was correlated with poor response to trastuzumab monotherapy in HER2-positive patients (r = 0.67, p = 0.05). There was no statistically significant difference in disease-free survival between positive and negative nuclear HER3 expression but the number of patients was small (n = 87). Further validation to assess nuclear HER3 expression as a prognostic and predictive biomarker in HER2-positive breast cancer patients undergoing trastuzumab treatment will be assessed in randomized tumour samples from FinHER study. Conclusion: Heregulin and trastuzumab treatment seems to induce nuclear HER3 translocation in some of the HER2 positive breast cancer cells. This may be due to proteolytic cleavage of HER3 as it is reduced by ADAM17 or gamma-secretase inhibitor. Enriched nuclear localisation of HER3 seems to be one possible mechanism of acquired resistance to trastuzumab in HER2-positive breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-08.

IQGAP1 Protein Binds Human Epidermal Growth Factor Receptor 2 (HER2) and Modulates Trastuzumab Resistance

Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20-25% of breast cancers. Increased HER2 expression is an adverse prognostic factor and correlates with decreased patient survival. HER2-positive (HER2(+)) breast cancer is treated with trastuzumab. Unfortunately, some patients are intrinsically refractory to therapy, and many who do respond initially become resistant within 1 year. Understanding the molecular mechanisms underlying HER2 signaling and trastuzumab resistance is essential to reduce breast cancer mortality. IQGAP1 is a ubiquitously expressed scaffold protein that contains multiple protein interaction domains. By regulating its binding partners IQGAP1 integrates signaling pathways, several of which contribute to breast tumorigenesis. We show here that IQGAP1 is overexpressed in HER2(+) breast cancer tissue and binds directly to HER2. Knockdown of IQGAP1 decreases HER2 expression, phosphorylation, signaling, and HER2-stimulated cell proliferation, effects that are all reversed by reconstituting cells with IQGAP1. Reducing IQGAP1 up-regulates p27, and blocking this increase attenuates the growth inhibitory effects of IQGAP1 knockdown. Importantly, IQGAP1 is overexpressed in trastuzumab-resistant breast epithelial cells, and reducing IQGAP1 both augments the inhibitory effects of trastuzumab and restores trastuzumab sensitivity to trastuzumab-resistant SkBR3 cells. These data suggest that inhibiting IQGAP1 function may represent a rational strategy for treating HER2(+) breast carcinoma.

Trastuzumab Regulates IGFBP-2 and IGFBP-3 to Mediate Growth Inhibition: Implications for the Development of Predictive Biomarkers for Trastuzumab Resistance

Activation of insulin-like growth factor-I receptor (IGF-IR) signaling is an important mechanism for trastuzumab resistance. IGF-binding proteins (IGFBP) modulate IGF-IR signaling and play important roles in the control of breast cancer progression. In this article, we report that trastuzumab treatment enhances the expression and secretion of IGFBP-3 in SKBR3 cells, a trastuzumab-sensitive breast cancer cell line, and that this upregulation of IGFBP-3 induced by trastuzumab correlates with trastuzumab-mediated growth inhibition. We describe a new role for IGFBP-3 in the regulation of IGF-I-mediated cross-talk between IGF-IR and ErbB2 signaling pathways. In particular, treatment of SKBR3 cells with recombinant IGFBP-3 blocks IGF-I-induced activation of IGF-IR and ErbB2, and stable expression of IGFBP-3 inhibits SKBR3 cell growth. We find an inverse relationship in the levels of secreted IGFBP-3 such that high levels of IGFBP-3 are associated with trastuzumab-sensitive breast cancer cells (SKBR3 and BT-474), whereas low levels of IGFBP-3 are found in trastuzumab-resistant cells (clone 3 and JIMT-1). In contrast to IGFBP-3, the secretion and expression of IGFBP-2 are upregulated in trastuzumab-resistant SKBR3 cells. Furthermore, we show that IGFBP-2 stimulates activation of ErbB2 and that trastuzumab reduces IGFBP-2-stimulated ErbB2 activation. Based on our data, we propose a novel mechanism of action whereby trastuzumab enhances the expression and secretion of IGFBP-3, which interferes with IGF-I-mediated mitogenic signaling via autocrine and paracrine mechanisms and reduces IGFBP-2-induced ErbB2 activation to mediate growth inhibition. Changes in secretion profiles of IGFBP-2 and IGFBP-3 in trastuzumab-sensitive and trastuzumab-resistant cells may promote the development of IGFBP-2 and IGFBP-3 as predictive biomarkers for trastuzumab resistance.

Rac1 contributes to trastuzumab resistance of breast cancer cells: Rac1 as a potential therapeutic target for the treatment of trastuzumab-resistant breast cancer

Although treatment with trastuzumab improves outcomes for women with ErbB2-positive breast cancer, many patients who achieve an initial response to trastuzumab subsequently acquire resistance within 1 year. Rac1, a Ras-like small GTPase, has been implicated in the control of cell growth and morphology and is believed to be associated with breast cancer progression and metastasis. Here, we show that when parental SKBR3 cells become resistant to trastuzumab, Rac1 activity is increased, leading to altered cell morphology, which is accompanied by significant cytoskeleton disorganization. Furthermore, both trastuzumab-mediated down-regulation of ErbB2 and epidermal growth factor-induced down-regulation of epidermal growth factor receptor are impaired in the trastuzumab-resistant SKBR3 cells, indicating that the endocytic down-regulation of ErbB receptors is compromised in the resistant cells. This results in an aberrant accumulation of ErbB2 on the cell surface and enhanced ErbB2 and extracellular signal-regulated kinase activity in trastuzumab-resistant SKBR3 cells. Additionally, overexpression of constitutively active Rac1G12V in parental SKBR3 cells reduces sensitivity to trastuzumab. After reduction of Rac1 activity by NSC23766, a specific Rac1 inhibitor, trastuzumab-resistant SKBR3 cells display a cellular morphology similar to parental SKBR3 cells. Moreover, we show that NSC23766 restores trastuzumab-mediated endocytic down-regulation of ErbB2 and reduces extracellular signal-regulated kinase activity in resistant SKBR3 cells. Our findings highlight an important role for Rac1 in trastuzumab resistance of human breast cancer cells and identify the impaired trastuzumab-mediated endocytic down-regulation of ErbB2 as a novel mechanism of trastuzumab resistance. The significant effects of NSC23766 on trastuzumab-resistant SKBR3 cells warrant further study of NSC23766 as a potential treatment of trastuzumab-resistant breast cancers.

Calpain regulates sensitivity to trastuzumab and survival in HER2-positive breast cancer.

Abstract Abstract #2018 Background: Overexpression of HER2 is associated with an aggressive cancer and poor prognosis for patients. HER2-positive patients are treated with trastuzumab; however resistance is common. Mechanisms that regulate activity of HER2 or downstream kinase AKT are involved in regulation of survival. Calpains are cysteine proteases involved in transmission of signals during growth, motility and transformation in cancer. Mechanisms by which calpain regulates transmission of signals in HER2-positive breast cancer are not known. Here we investigate possibility that calpain regulates survival in HER2-positive breast cancer.
 Material and Methods: Activity of calpain in parental or resistant skbr3 cells was measured alone or in presence of inhibitors under conditions of adhesion or stimulation with trastuzumab using a fluorescently labeled substrate. To investigate regulation of HER2, assays were performed by mixing purified proteins or cell lysates with calpain1. Activity of HER2 was analyzed using phosphotyrosine-specific antibodies in Western blots while cleavage using antibody specific to the C-terminus. HER2-positive cancer tissue or lysates of cell lines were analyzed for presence of C-terminal fragments. Cells were transfected with the plasmid expressing catalytically inactive mutant of calpain1, selected for resistance to G418 and used in experiments to analyze the activity of HER2 and AKT. Sensitivity to trastuzumab was determined by measuring survival in presence of trastuzumab in a 96 well plate assay.
 Results: In skbr3 cells, activity of calpain increased following adhesion or stimulation with trastuzumab and was inhibited by inhibitors. Calpain induced cleavage of C-terminal domains and reduced activity in full length HER2. C-terminal fragments were detectable in the lysates of HER2-positive breast cancer tissue, cell lines and also overproduced by overexpression of calpain1. Conversely, in cells expressing catalytically inactive calpain1 the activity of HER2 and AKT was increased correlating with increased survival on fibronectin in the presence of trastuzumab. As another model for calpain-induced regulation of HER2 in mechanisms of resistance, we analyzed regulation in trastuzumab-resistant skbr3 cells. Compared to parental in resistant cells adhesion- or trastuzumab-dependent activity of HER2 was increased whereas cleavage was reduced suggesting mechanisms that regulate generation of C-terminal fragments during transmission of signals were altered. Consistenly, adhesion-dependent activity of calpain was reduced whereas trastuzumab failed to stimulate activation.
 Conclusion: These data are consistent with possibilities 1) HER2 is a direct target of calpain, 2) calpain induces cleavage of the cytoplasmic region during transmission of signals to regulate activity and integrity of HER2, 3) calpain-dependent mechanisms are involved in coupling HER2 to survival and 4) inhibition of calpain during adhesion-dependent transmission of signal or in the presence of trastuzumab allows increased activity of HER2 contributing to resistance. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2018.