Overcoming trastuzumab resistance in HER2-positive breast cancer using combination therapy

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC) comprises around 20–30% of all BC subtypes and is correlated with poor prognosis. For many years, trastuzumab, a monoclonal antibody, has been used to inhibit the HER2 activity. Though, the main resistance to trastuzumab has challenged the use of this drug in the management of HER2-positive BC. Therefore, the determination of resistance mechanisms and the incorporation of new agents may lead to the development of a better blockade of the HER family receptor signaling. During the last few years, some therapeutic drugs have been developed for treating patients with trastuzumab-resistant HER2-positive BC that have more effective influences in the management of this condition. In this regard, the present study aimed at reviewing the mechanisms of trastuzumab resistance and the innovative therapies that have been investigated in trastuzumab-resistant HER2-positive BC subjects.     

乳腺癌靶向治疗的分子基础

近年来,乳腺癌靶向治疗的研究取得了显著进展。分子靶向药物通过作用于乳腺癌细胞相关细胞的分子信号传导途径,特异性针对异常环节进行干预,控制细胞基因的表达,从而抑制或杀死肿瘤细胞,以此达到治疗乳腺癌的目的。本文将对乳腺癌靶向治疗的分子基础研究进展做一综述。     

C-FLIP(L) contributes to TRAIL resistance in HER2-positive breast cancer

Breast cancers with HER2 amplification have a poorer prognosis than the luminal phenotypes. TRAIL activates apoptosis upon binding its receptors in some but not all breast cancer cell lines. Herein, we investigated the expression pattern of c-FLIP(L) in a cohort of 251 invasive breast cancer tissues and explored its potential role in TRAIL resistance. C-FLIP(L) was relatively high-expressed in HER2-positive breast cancer in comparison with other molecular subtypes, co-expressed with TRAIL death receptors, and inversely correlated with the apoptosis index. Downregulation of c-FLIP(L) sensitized SKBR3 cells to TRAIL-induced apoptosis in a concentration- and time-dependent manner and enhanced the activities and cleavages of caspase-8 and caspase-3, without altering the surface expression of death receptors. Together, our results indicate that c-FLIP(L) promotes TRAIL resistance and inhibits caspase-3 and caspase-8 activation in HER2-positive breast cancer.     

HER-2/neu基因高表达及靶向治疗

HER-2/neu癌基因在许多肿瘤,如乳腺癌、卵巢癌、非小细胞肺癌等肿瘤中高表达,在肿瘤的发生与发展中起重要作用,与肿瘤的转化、转移、复发、预后差、患者生存期缩短有关。HER-2/neu在乳腺癌过度表达率约为20%～30%,编码蛋白P185HER2属生长因子受体家族,抗P185HER2单克隆抗体(Herceptin)作为靶向药物已临床应用治疗HER2/neu高表达乳腺癌。     

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。     

Basal/HER2 breast carcinomas

High rates of inherent primary resistance to the humanized monoclonal antibody trastuzumab (Herceptin) are frequent among HER2 gene-amplified breast carcinomas in both metastatic and adjuvant settings. The clinical efficacy of trastuzumab is highly correlated with its ability to specifically and efficiently target HER2-driven populations of breast cancer stem cells (CSCs). Intriguingly, many of the possible mechanisms by which cancer cells escape trastuzumab involve many of the same biomarkers that have been implicated in the biology of CS-like tumor-initiating cells. In the traditional, one-way hierarchy of CSCs in which all cancer cells descend from special self-renewing CSCs, HER2-positive CSCs can occur solely by self-renewal. Therefore, by targeting CSC self-renewal and resistance, trastuzumab is expected to induce tumor shrinkage and further reduce breast cancer recurrence rates when used alongside traditional therapies. In a new, alternate model, more differentiated non-stem cancer cells can revert to trastuzumab-refractory, CS-like cells via the activation of intrinsic or microenvironmental paths-to-stemness, such as the epithelial-to-mesenchymal transition (EMT). Alternatively, stochastic transitions of trastuzumab-responsive CSCs might also give rise to non-CSC cellular states that lack major attributes of CSCs and, therefore, can remain “hidden” from trastuzumab activity. Here, we hypothesize that a better understanding of the CSC/non-CSC social structure within HER2-overexpressing breast carcinomas is critical for trastuzumab-based treatment decisions in the clinic. First, we decipher the biological significance of CSC features and the EMT on the molecular effects and efficacy of trastuzumab in HER2-positive breast cancer cells. Second, we reinterpret the genetic heterogeneity that differentiates trastuzumab-responders from non-responders in terms of CSC cellular states. Finally, we propose that novel predictive approaches aimed at better forecasting early tumor responses to trastuzumab should identify biological determinants that causally underlie the intrinsic flexibility of HER2-positive CSCs to “enter” into or “exit” from trastuzumab-sensitive states. An accurate integration of CSC cellular states and EMT-related biomarkers with the currently available breast cancer molecular taxonomy may significantly improve our ability to make a priori decisions about whether patients belonging to HER2 subtypes differentially enriched with a “mesenchymal transition signature” (e.g., luminal/HER2 vs. basal/HER2) would distinctly benefit from trastuzumab-based therapy ab initio.     

HER2/neu expression correlates with vasculogenic mimicry in invasive breast carcinoma

Vasculogenic mimicry (VM) refers to the condition in which tumour cells mimic endothelial cells to form extracellular matrix‐rich tubular channels. VM is more extensive in more aggressive tumours. The human epidermal growth factor receptor 2 (HER2) gene is amplified in 20–30% of human breast cancers and has been implicated in mediating aggressive tumour growth and metastasis. However, thus far, there have been no data on the role of HER2 in VM formation. Immunohistochemical and histochemical double‐staining methods were performed to display VM in breast cancer specimens. Transfection in MCF7 cells was performed and clones were selected by G418. The three‐dimensional Matrigel culture was used to evaluate VM formation in the breast cancer cell line. According to statistical analysis, VM was related to the presence of a positive nodal status and advanced clinical stage. The positive rate of VM increased with increased HER2 expression. In addition, cases with HER2 3+ expression showed significantly greater VM channel count than those in other cases. The exogenous HER2 overexpression in MCF‐7 cells induced vessel‐like VM structures on the Matrigel and increased the VM mediator vascular endothelial (VE) cadherin. Our data provide evidence for a clinically relevant association between HER2 and VM in human invasive breast cancer. HER2 overexpression possibly induces VM through the up‐regulation of VE cadherin. Understanding the key molecular events may provide therapeutic intervention strategies for HER2+ breast cancer.     

Reverse-phase protein microarray highlights HER2 signaling activation in immunohistochemistry/FISH/HER2-negative breast cancers

Evaluation of: Wulfkuhle JD, Berg D, Wolff C et al. Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping. Clin. Cancer Res. 18(23), 6426–6435 (2012).

Exhaustive characterization and mapping of pivotal molecules and downstream effectors deregulated in breast cancer is of fundamental clinical value to define the most effective therapy. Wulfkuhle et al. applied reverse-phase protein microarray, a highly sensitive immunoassay able to perform quantitative and multiplexed analysis of total and/or modified cellular proteins, to assess protein levels and activation/phosphorylation status of the HER family (EGFR, HER2, HER3) and downstream signaling molecules in HER2⁺ and HER2^- breast cancers. The research was performed using laser capture microdissected tumor epithelial cells from frozen samples and formalin-fixed paraffin embedded specimens, which were also analyzed by immunohistochemistry (IHC) and FISH. This study identified a subgroup of IHC/FISH/HER2^- patients with HER2 activation/phosphorylation levels comparable with those obtained from IHC/FISH/HER2⁺ tumors. HER2 signaling activation was independent from total HER2 expression and involved HER3 and EGFR activation. These findings indicate that molecular characterization by reverse-phase protein microarray of HER2 and its partners/effectors in the signaling cascade enables the identification of a subgroup of IHC/FISH/HER2^- patients showing HER2 signaling activation. These patients, currently excluded from targeted therapy administration, could potentially benefit from this and it could improve prognosis and survival.     

Trastuzumab-monomethyl auristatin E conjugate exhibits potent cytotoxic activity in vitro against HER2-positive human breast cancer

Targeted therapy using specific monoclonal antibodies (mAbs) conjugated to chemotherapeutic agents or toxins has become one of the top priorities in cancer therapy. Antibody–drug conjugates (ADCs) are emerging as a promising strategy for cancer-targeted therapy. In this study, trastuzumab, a humanized monoclonal anti-HER2 antibody, was reduced by dithiothreitol and conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) through a valine-citrulline peptide linker (trastuzumab-MC-Val-Cit-PABC-MMAE [trastuzumab-vcMMAE]). After conjugation, ADCs were characterized by using UV–vis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and flow cytometry. The antitumor activity of the ADC was evaluated in breast cancer cells in vitro. In addition, ADCs were further characterized using purification by the protein A chromatography, followed by assessment using apoptosis and MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assays. Hydrophobic interaction chromatography was used to determine drug-to-antibody ratio species of ADCs produced. Our finding showed that approximately 5.12 drug molecules were conjugated to each mAb. H2L2, H2L, HL, H2, H, and L forms of ADCs were detected in nonreducing SDS-PAGE. The binding of trastuzumab-vcMMAE to HER2-positive cells was comparable with that of the parental mAb. The MTT assay showed that our ADCs induced significant cell death in HER2-positive cells, but not in HER2-negative cells. The ADCs produced was a mixture of species, unconjugated trastuzumab (14.147%), as well as trastuzumab conjugated with two (44.868%), four (16.886%), six (13.238%), and eight (10.861%) molecules of MMAE. These results indicated that MMAE-conjugated trastuzumab significantly increases the cytotoxic activity of trastuzumab, demonstrating high affinity, specificity, and antitumor activity in vitro. Trastuzumab-vcMMAE is an effective and selective agent for the treatment of HER2-positive breast tumors.     

曲妥珠单抗辅助或新辅助治疗HER2 阳性转移性乳腺癌的临床结果

目的:比较人表皮生长因子受体2过表达(HER2+)的患者既往接受以曲妥珠单抗为基础辅助或新辅助治疗后再次接受针曲妥珠单抗治疗的临床结果。方法:共247例(I-III期170例,IV期77例)HER2+转移性乳腺癌患者,其中首次接受曲妥珠单抗治疗患者211例(I-III期134例,IV期77例),再次接受曲妥珠单抗治疗患者36例(I-III期)。使用Cox比例风险回归和logistic回归分析首次或提前接受曲妥珠单抗治疗的患者的预后的临床结果,生存评估使用Kaplan-Meier法。结果:I-III期HER2+转移性乳腺癌患者中,未预先接受曲妥珠单抗治疗组的中位总生存期为36个月和预先接受曲妥珠单抗治疗组为28个月(危害比[HR],1.45;95%CI,1.05-2.02[P=0.012]);I-IV期HER2+转移性乳腺癌患者中,未预先使用曲妥珠单抗组的中位总生存期为37个月,客观缓解率58%;临床获益率77%;预先使用曲妥珠单抗组为25个月,客观缓解率28%;临床获益率37%;调整后的比值比为客观缓解率0.37(9%CI,0.18-0.77;P=0.010)和临床获益率0.28(95%CI,0.14-0.59;P=0.014)。单因素分析没有提前接受曲妥珠单抗治疗组中位总生存率较长(P=0.012)。多因素分析发现总生存率没有显著差异(P=0.19)。结论:当曲妥珠单抗用于转移性疾病,没有提前接受曲妥珠单抗治疗的HER2+乳腺癌患者临床结果优于提前接受曲妥珠单抗治疗的患者。     

MiR-221 promotes trastuzumab-resistance and metastasis in HER2-positive breast cancers by targeting PTEN

HER2-overexpressing breast cancers are characterized by frequent distant metastasis and often develop resistance after short-term effective treatment with the monoclonal antibody drug, trastuzumab. Here, we found that the oncogenic miRNA, miR-221, inhibited apoptosis, induced trastuzumab resistance and promoted metastasis of HER2-positive breast cancers. The tumor suppressor PTEN was identified as a miR-221 target; overexpression of PTEN abrogated the aforementioned miR-221-induced malignant phenotypes of the cells. These findings indicate that miR-221 may promote trastuzumab resistance and metastasis of HER2-positive breast cancers by targeting PTEN, suggesting its role as a potential biomarker for progression and poor prognosis, and as a novel target for trastuzumab-combined treatment of breast cancers. [BMB Reports 2014; 47(5): 268-273].     

Functional cooperation between co-amplified genes promotes aggressive phenotypes of HER2-positive breast cancer

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曲妥珠单抗治疗HER2阳性乳腺癌的耐药机制及新疗法探索

研究表明大约有20％的乳腺癌患者存在HER2过表达现象。HER2的异常表达及异常信号通路与乳腺癌的侵袭转移、治疗抵抗及不良预后密切相关。在临床上,对于HER2阳性的初期乳腺癌患者常联合曲妥珠单抗及化学药物治疗,但部分患者对曲妥珠单抗产生耐药。因此,研究其耐药机制对于HER2阳性乳腺癌患者的治疗、预后及新疗法的探索具有重要的临床意义。目前引起曲妥珠单抗抵抗的主要机制有：p95-HER2累积、P13K／AKT／mTOR信号异常激活、HER家族受体和IGF-1R信号增加、非受体酪氨酸激酶c-SRC活性增加等。将对上述机制及治疗HER2阳性乳腺癌的新疗法进行综述。     

Targeting of preexisting and induced breast cancer stem cells with trastuzumab and trastuzumab emtansine (T-DM1)

The antibody trastuzumab (Herceptin) has substantially improved overall survival for patients with aggressive HER2-positive breast cancer. However, about 70% of all treated patients will experience relapse or disease progression. This may be related to an insufficient targeting of the CD44^highCD24^low breast cancer stem cell subset, which is not only highly resistant to chemotherapy and radiotherapy but also a poor target for trastuzumab due to low HER2 surface expression. Hence, we explored whether the new antibody-drug conjugate T-DM1, which consists of the potent chemotherapeutic DM1 coupled to trastuzumab, could improve the targeting of these tumor-initiating or metastasis-initiating cells. To this aim, primary HER2-overexpressing tumor cells as well as HER2-positive and HER2-negative breast cancer cell lines were treated with T-DM1, and effects on survival, colony formation, gene and protein expression as well as antibody internalization were assessed. This revealed that CD44^highCD24^lowHER2^low stem cell-like breast cancer cells show high endocytic activity and are thus particularly sensitive towards the antibody-drug conjugate T-DM1. Consequently, preexisting CD44^highCD24^low cancer stem cells were depleted by concentrations of T-DM1 that did not affect the bulk of the tumor cells. Likewise, colony formation was efficiently suppressed. Moreover, when tumor cells were cocultured with natural killer cells, antibody-dependent cell-mediated cytotoxicity was enhanced, and EMT-mediated induction of stem cell-like properties was prevented in differentiated tumor cells. Thus our study reveals an unanticipated targeting of stem cell-like breast cancer cells by T-DM1 that may contribute to the clinical efficacy of this recently approved antibody-drug conjugate.