Acquired Tamoxifen Resistance in MCF-7 Breast Cancer Cells Requires Hyperactivation of eIF4F-Mediated Translation

While selective estrogen receptor modulators, such as tamoxifen, have contributed to increased survival in patients with hormone receptor-positive breast cancer, the development of resistance to these therapies has led to the need to investigate other targetable pathways involved in oncogenic signaling. Approval of the mTOR inhibitor everolimus in the therapy of secondary endocrine resistance demonstrates the validity of this approach. Importantly, mTOR activation regulates eukaryotic messenger RNA translation. Eukaryotic translation initiation factor 4E (eIF4E), a component of the cap-dependent translation complex eIF4F, confers resistance to drug-induced apoptosis when overexpressed in multiple cell types. The eIF4F complex is downstream of multiple oncogenic pathways, including mTOR, making it an appealing drug target. Here, we show that the eIF4F translation pathway was hyperactive in tamoxifen-resistant (TamR) MCF-7L breast cancer cells. While overexpression of eIF4E was not sufficient to confer resistance to tamoxifen in MCF-7L cells, its function was necessary to maintain resistance in TamR cells. Targeting the eIF4E subunit of the eIF4F complex through its degradation using an antisense oligonucleotide (ASO) or via sequestration using a mutant 4E-BP1 inhibited the proliferation and colony formation of TamR cells and partially restored sensitivity to tamoxifen. Further, the use of these agents also resulted in cell cycle arrest and induction of apoptosis in TamR cells. Finally, the use of a pharmacologic agent which inhibited the eIF4E-eIF4G interaction also decreased the proliferation and anchorage-dependent colony formation in TamR cells. These results highlight the eIF4F complex as a promising target for patients with acquired resistance to tamoxifen and, potentially, other endocrine therapies.     

Nuclear HER4 mediates acquired resistance to trastuzumab and is associated with poor outcome in HER2 positive breast cancer

The role of HER4 in breast cancer is controversial and its role in relation to trastuzumab resistance remains unclear. We showed that trastuzumab treatment and its acquired resistance induced HER4 upregulation, cleavage and nuclear translocation. However, knockdown of HER4 by specific siRNAs increased trastuzumab sensitivity and reversed its resistance in HER2 positive breast cancer cells. Preventing HER4 cleavage by a γ-secretase inhibitor and inhibiting HER4 tyrosine kinase activity by neratinib decreased trastuzumab-induced HER4 nuclear translocation and enhanced trastuzumab response. There was also increased nuclear HER4 staining in the tumours from BT474 xenograft mice and human patients treated with trastuzumab. Furthermore, nuclear HER4 predicted poor clinical response to trastuzumab monotherapy in patients undergoing a window study and was shown to be an independent poor prognostic factor in HER2 positive breast cancer. Our data suggest that HER4 plays a key role in relation to trastuzumab resistance in HER2 positive breast cancer. Therefore, our study provides novel findings that HER4 activation, cleavage and nuclear translocation influence trastuzumab sensitivity and resistance in HER2 positive breast cancer. Nuclear HER4 could be a potential prognostic and predictive biomarker and understanding the role of HER4 may provide strategies to overcome trastuzumab resistance in HER2 positive breast cancer.     

Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer

Trastuzumab is a monoclonal antibody targeted against the human epidermal growth factor receptor (HER) 2 tyrosine kinase receptor, which is overexpressed in approximately 25% of invasive breast cancers. The majority of patients with metastatic breast cancer who initially respond to trastuzumab, however, demonstrate disease progression within 1 year of treatment initiation. Preclinical studies have indicated several molecular mechanisms that could contribute to the development of trastuzumab resistance. Increased signaling via the phosphatidylinositol 3-kinase/Akt pathway could contribute to trastuzumab resistance because of activation of multiple receptor pathways that include HER2-related receptors or non-HER receptors such as the insulin-like growth factor 1 receptor, which appears to be involved in a cross-talk with HER2 in resistant cells. Additionally, loss of function of the tumor suppressor PTEN gene, the negative regulator of Akt, results in heightened Akt signaling that leads to decreased sensitivity to trastuzumab. Decreased interaction between trastuzumab and its target receptor HER2, which is due to steric hindrance of HER2 by cell surface proteins such as mucin-4 (MUC4), may block the inhibitory actions of trastuzumab. Novel therapies targeted against these aberrant molecular pathways offer hope that the effectiveness and duration of response to trastuzumab can be greatly improved.     

HER2/neu reduces the apoptotic effects of N-(4-hydroxyphenyl)retinamide (4-HPR) in breast cancer cells by decreasing nitric oxide production

The retinoid N-(4-hydroxyphenyl)retinamide (4-HPR also known as fenretinide) is a potent inducer of apoptosis in breast cancer cells. We observed a 4.5-fold reduction in 4-HPR-mediated apoptosis in MCF-7 breast cancer cells transfected with HER2/neu (MCF-7/HER2) as compared with the parental MCF-7 (MCF-7/WT) cells. Blocking HER2/neu with trastuzumab (Herceptin) led to a six-fold increase in 4-HPR-induced apoptosis in HER2/neu-overexpressing cells. These data indicate that HER2/neu reduces the sensitivity of breast cancer cells to 4-HPR. We showed previously that nitric oxide (NO) is essential for 4-HPR to induce apoptosis in breast cancer cells. The inhibitory effects of the 4-HPR and trastuzumab combination correlated with the amount of NO produced in HER2/neu-overexpressing cells. When a NO synthase (NOS) inhibitor was used to block NO production, decreased apoptosis by the 4-HPR and trastuzumab combination was observed. Furthermore, 4-HPR-mediated NOSII expression was lower in MCF-7/HER2 than MCF-7/WT cells, but was increased by trastuzumab in HER2/neu-overexpressing cells. Here we report the novel findings that HER2/neu reduces the ability of 4-HPR to induce apoptosis in breast cancer cells, and that one mechanism by which HER2/neu increases the resistance of breast cancer cells to 4-HPR is by decreasing NOSII-mediated NO production.     

Calpain4 is required for activation of HER2 in breast cancer cells exposed to trastuzumab and its suppression decreases survival and enhances response

Overactivation of HER2 and crosstalk of other HER family members contribute to a survival pathway of breast cancer cells exposed to trastuzumab, the therapeutic inhibitor of HER2 and thus, decrease response and promote resistance. We have explored the involvement of the intracellular cysteine protease calpain4, the common partner of isoforms calpain1 and calpain2, in the regulation of cell survival and trastuzumab-response. Increase of calpain4 expression and isoform activities were detected in breast cancer cells and HER2-positive tumors. Molecular analyses of parent and resistant cells suggested that perturbation of regulations, induced by calpain4 and of activities of HER2 and HER3, was associated with trastuzumab-resistance. The suppression of calpain4 destabilized calpain1 and calpain2, however, did not prevent the activation of HER2 and HER3 or cell proliferation in the absence of trastuzumab. To understand the significance, the survival of parent and trastuzumab-resistant cells in which calpain4 was suppressed, was assessed in the presence of trastuzumab; survival in each cell type was decreased and associated with a loss of HER2 and HER3 activity. Taken together, by contributing to the activation and the crosstalk of HER2, calpain4 promotes the survival pathway of breast cancer cells, and therefore, its suppression enhances trastuzumab-response and decreases resistance.     

ADAM10 mediates trastuzumab resistance and is correlated with survival in HER2 positive breast cancer

Trastuzumab prolongs survival in HER2 positive breast cancer patients. However, resistance remains a challenge. We have previously shown that ADAM17 plays a key role in maintaining HER2 phosphorylation during trastuzumab treatment. Beside ADAM17, ADAM10 is the other well characterized ADAM protease responsible for HER ligand shedding. Therefore, we studied the role of ADAM10 in relation to trastuzumab treatment and resistance in HER2 positive breast cancer. ADAM10 expression was assessed in HER2 positive breast cancer cell lines and xenograft mice treated with trastuzumab. Trastuzumab treatment increased ADAM10 levels in HER2 positive breast cancer cells (p≤0.001 in BT474; p≤0.01 in SKBR3) and in vivo (p≤0.0001) compared to control, correlating with a decrease in PKB phosphorylation. ADAM10 inhibition or knockdown enhanced trastuzumab response in naïve and trastuzumab resistant breast cancer cells. Trastuzumab monotherapy upregulated ADAM10 (p≤0.05); and higher pre-treatment ADAM10 levels correlated with decreased clinical response (p≤0.05) at day 21 in HER2 positive breast cancer patients undergoing a trastuzumab treatment window study. Higher ADAM10 levels correlated with poorer relapse-free survival (p≤0.01) in a cohort of HER2 positive breast cancer patients. Our studies implicate a role of ADAM10 in acquired resistance to trastuzumab and establish ADAM10 as a therapeutic target and a potential biomarker for HER2 positive breast cancer patients.     

Mechanisms of resistance to anti‐human epidermal growth factor receptor 2 agents in breast cancer

Approximately 20% of breast cancers are characterized by overexpression of human epidermal growth factor receptor 2 (HER2) protein and associated gene amplification, and the receptor tyrosine kinase is believed to play a critical role in the pathogenesis of these tumors. The development and implementation of trastuzumab, a humanized monoclonal antibody against the extracellular domain of HER2 protein, has significantly improved treatment outcomes in patients with HER2‐overexpressing breast cancer. However, despite this clinical usefulness, unmet needs for better prediction of trastuzumab’s response and overcoming primary and acquired resistance remain. In this review, we discuss several potential mechanisms of resistance to trastuzumab that have been closely studied over the last decade. Briefly, these mechanisms include: impaired access of trastuzumab to HER2 by expression of extracellular domain‐truncated HER2 (p95 HER2) or overexpression of MUC4; alternative signaling from insulin‐like growth factor‐1 receptor, other epidermal growth factor receptor family members, or MET; aberrant downstream signaling caused by loss of phosphatase and tensin homologs deleted from chromosome 10 (PTEN), PIK3CA mutation, or downregulation of p27; or FCGR3A polymorphisms. In addition, we discuss potential strategies for overcoming resistance to trastuzumab. Specifically, the epidermal growth factor receptor/HER2 tyrosine kinase inhibitor lapatinib partially overcame trastuzumab resistance in a clinical setting, so its efficacy results and limited data regarding potential mechanisms of resistance to the drug are also discussed. (Cancer Sci 2011; 102: 1–8)     

Trastuzumab-resistant HER2-driven breast cancer cells are sensitive to epigallocatechin-3 gallate

Overexpression of the epidermal growth factor receptor family member HER2 is found in approximately 30% of breast cancers and is a target for immunotherapy. Trastuzumab, a humanized monoclonal antibody against HER2, is cytostatic when added alone and highly successful in clinical settings when used in combination with other chemotherapeutic agents. Unfortunately, HER2 tumors in patients develop resistance to trastuzumab or metastasize to the brain, which is inaccessible to antibody therapy. Previously, we showed that the green tea polyphenol epigallocatechin-3 gallate (EGCG) inhibits growth and transformed phenotype of Her-2/neu-driven mouse mammary tumor cells. The different modes of action of EGCG and trastuzumab led us to hypothesize that EGCG will inhibit HER2-driven breast cancer cells resistant to trastuzumab. We studied trastuzumab-resistant BT474 human breast cancer cells, isolated by chronic trastuzumab exposure, and JIMT-1 breast cancer cells, derived from a pleural effusion in a patient who displayed clinical resistance to trastuzumab therapy. EGCG treatment caused a dose-dependent decrease in growth and cellular ATP production, and apoptosis at high concentrations. Akt activity was suppressed by EGCG leading to the induction of FOXO3a and target cyclin-dependent kinase inhibitor p27Kip1 levels. Thus, EGCG in combination with trastuzumab may provide a novel strategy for treatment of HER2-overexpressing breast cancers, given that EGCG can cross the blood-brain barrier.     

Neratinib overcomes trastuzumab resistance in HER2 amplified breast cancer

Trastuzumab has been shown to improve the survival outcomes of HER2 positive breast cancer patients. However, a significant proportion of HER2-positive patients are either inherently resistant or develop resistance to trastuzumab. We assessed the effects of neratinib, an irreversible panHER inhibitor, in a panel of 36 breast cancer cell lines. We further assessed its effects with or without trastuzumab in several sensitive and resistant breast cancer cells as well as a BT474 xenograft model. We confirmed that neratinib was significantly more active in HER2-amplified than HER2 non-amplified cell lines. Neratinib decreased the activation of the 4 HER receptors and inhibited downstream pathways. However, HER3 and Akt were reactivated at 24 hours, which was prevented by the combination of trastuzumab and neratinib. Neratinib also decreased pHER2 and pHER3 in acquired trastuzumab resistant cells. Neratinib in combination with trastuzumab had a greater growth inhibitory effect than either drug alone in 4 HER2 positive cell lines. Furthermore, trastuzumab in combination with neratinib was growth inhibitory in SKBR3 and BT474 cells which had acquired resistance to trastuzumab as well as in a BT474 xenograft model. Innately trastuzumab resistant cell lines showed sensitivity to neratinib, but the combination did not enhance response compared to neratinib alone. Levels of HER2 and phospho-HER2 showed a direct correlation with sensitivity to neratinib. Our data indicate that neratinib is an effective anti-HER2 therapy and counteracted both innate and acquired trastuzumab resistance in HER2 positive breast cancer. Our results suggest that combined treatment with trastuzumab and neratinib is likely to be more effective than either treatment alone for both trastuzumab-sensitive breast cancer as well as HER2-positive tumors with acquired resistance to trastuzumab.     

Trastuzumab (herceptin), a humanized anti-Her2 receptor monoclonal antibody, inhibits basal and activated Her2 ectodomain cleavage in breast cancer cells.

HER2 is a ligand-less tyrosine kinase receptor of the ErbB family that is frequently overexpressed in breast cancer. It undergoes proteolytic cleavage that results in the release of the extracellular domain and the production of a truncated membrane-bound fragment, p95. We show that HER2 shedding is activated by 4-aminophenylmercuric acetate (APMA), a well-known matrix metalloprotease activator, in HER2-overexpressing breast cancer cells. The HER2 p95 fragment, which appears after APMA-induced cleavage, is phosphorylated. We analyzed 24 human breast cancer specimens, and a phosphorylated M(r) 95,000 HER2 band could be detected in some of them, which indicated that the truncated receptor is also present in vivo. The activation of HER2 shedding by APMA in cells was blocked with batimastat, a broad-spectrum metalloprotease inhibitor. Trastuzumab (Herceptin; Genentech, San Francisco, CA), a humanized monoclonal antibody directed at the HER2 ectodomain, which has been shown to be active in patients with HER2-overexpressing breast cancer, inhibited basal and induced HER2 cleavage and, as a consequence, the generation of phosphorylated p95. This inhibitory effect of trastuzumab was not shared by 2C4, an antibody against a different epitope of the HER2 ectodomain. The inhibition of basal and APMA-induced cleavage of HER2 by trastuzumab preceded antibody-induced receptor down-modulation, which indicated that the effect of trastuzumab on cleavage was not attributable to a decrease in cell-surface HER2 induced by trastuzumab. We propose that the inhibition of HER2 cleavage and prevention of the production of an active truncated HER2 fragment represent a novel mechanism of action of trastuzumab.     

Erythropoietin receptor expression and its relationship with trastuzumab response and resistance in HER2-positive breast cancer cells

Resistance to trastuzumab is a major issue in the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer. Several potential resistance mechanisms have been investigated, but the results are controversial and no conclusion has been reached. Erythropoietin receptor (EPOR) may function in cell growth, and expressed in various cancer cells. Because the downstream signaling pathways for EPOR and HER2 partially overlapped, we hypothesized that EPOR may play a role in the inhibition effect of trastuzumab and resistance to trastuzumab. Here, we detected the expression of EPOR mRNA and protein in HER2-positive breast cancer cell lines and tissues. EPOR expressed in SKBR3, MDA-MB-453, and UACC-812 cell lines, but not in BT474. Of the 55 HER2-positive cancer tissues, EPOR was positive in 42 samples and highly expressed (H-score????25) in 24 by immunohistochemistry. The difference between EPOR expression and Ki67 index was significant (P?=?0.033), and EPOR expression also positively correlated with higher pathological stage (Spearman correlation coefficient?=?0.359; P?=?0.007). Exogenous EPO antagonized trastuzumab-induced inhibition of cell proliferation in HER2/EPOR dual-positive breast cancer cells. We then exposed SKBR3 cells to trastuzumab for 4?months to obtain trastuzumab-resistant SKBR3 cell line, which demonstrated higher phosphorylated EPOR level, higher EPO expression and more extracellular secretion than non-resistant parental SKBR3 cells. Downregulation EPOR expression using short hairpin RNA resensitized trastuzumab-resistant cells to this drug, and SKBR3 cells with EPOR downregulation demonstrated attenuated trastuzumab resistance after the same resistance induction. EPOR downregulation plus trastuzumab produced a synergetic action in the inhibition of cell proliferation and invasion in SKBR3 and MDA-MB-453 cell lines. Therefore, EPOR expression may be involved in tumor progression and proliferation in HER2-positive breast cancer. EPO/EPOR contributes to the mechanism of trastuzumab resistance in SKBR3 cell lines, and EPOR downregulation can reverse the resistance to trastuzumab and increase the inhibition effect of this drug.     

MUC1* is a determinant of trastuzumab (Herceptin) resistance in breast cancer cells

In the United States, 211,000 women are diagnosed each year with breast cancer. Of the 42,000 breast cancer patients who overexpress the HER2 growth factor receptor, <35% are responsive to treatment with the HER2-disabling antibody, called trastuzumab (Herceptin). Despite those statistics, women diagnosed with breast cancer are now tested to determine how much of this important growth factor receptor is present in their tumor because patients whose treatment includes trastuzumab are three-times more likely to survive for at least 5 years and are two-times more likely to survive without a cancer recurrence. Unfortunately, even among the group whose cancers originally respond to trastuzumab, 25% of the metastatic breast cancer patients acquire resistance to trastuzumab within the first year of treatment. Follow-on “salvage” therapies have prolonged life for this group but have not been curative. Thus, it is critically important to understand the mechanisms of trastuzumab resistance and develop therapies that reverse or prevent it. Here, we report that molecular analysis of a cancer cell line that was induced to acquire trastuzumab resistance showed a dramatic increase in the amount of the cleaved form of the MUC1 protein, called MUC1*. We recently reported that MUC1* functions as a growth factor receptor on cancer cells and on embryonic stem cells. Here, we show that treating trastuzumab-resistant cancer cells with a combination of MUC1* antagonists and trastuzumab, reverses the drug resistance. Further, HER2-positive cancer cells that are intrinsically resistant to trastuzumab became trastuzumab-sensitive when treated with MUC1* antagonists and trastuzumab. Additionally, we found that tumor cells that had acquired Herceptin resistance had also acquired resistance to standard chemotherapy agents like Taxol, Doxorubicin, and Cyclophosphamide. Acquired resistance to these standard chemotherapy drugs was also reversed by combined treatment with the original drug plus a MUC1* inhibitor.     

Expression of CD24 is associated with HER2 expression and supports HER2‐Akt signaling in HER2‐positive breast cancer cells

Human epidermal growth factor receptor 2 (HER2)‐positive breast cancer is treated with HER2‐targeted agents, such as trastuzumab and lapatinib, that suppress signaling by phosphatidylinositol 3‐kinase (PI3K)‐Akt and MAPK pathways. However, resistance to HER2‐targeted therapy remains a major clinical problem. Overexpression of CD24 has been detected in many cancers and is associated with a poor prognosis in women with breast cancer. HER2‐positive breast tumors are predominantly positive for CD24, suggesting that the expression of the two molecules is related. To investigate the relation between HER2 and CD24, we overexpressed HER2 in breast cancer cells that were triple‐negative for the estrogen receptor, progesterone receptor and HER2. We found that expression of CD24 was increased by stable overexpression of HER2. Flow cytometry thus revealed that the percentage of CD24‐positive cells was markedly higher in the HER2‐positive fraction than in the HER2‐negative fraction. Knockdown of CD24 in breast cancer cells positive for endogenous HER2 downregulated HER2 expression, whereas knockdown of HER2 did not affect the expression of CD24. Knockdown of CD24 also suppressed the phosphorylation of Akt, which functions downstream of HER2 and PI3K to promote cell survival. Moreover, knockdown of CD24 increased the sensitivity of HER2‐positive breast cancer cells to lapatinib treatment. Our results thus indicate that CD24 supports both the expression of HER2 and the consequent activation of PI3K‐Akt signaling. Furthermore, CD24 may contribute to resistance to HER2‐targeted therapy and, therefore, is itself a potential therapeutic target in HER2‐positive breast cancer.     

曲妥珠单抗耐药后HER 2阳性转移性乳腺癌靶向治疗策略

人表皮生长因子受体2（human epidermal growth factor receptor 2,HER 2）是一种跨膜酪氨酸激酶受体,可以激活下游一系列信号通路,导致肿瘤细胞增殖和存活。HER 2阳性乳腺癌是乳腺癌的特殊类型,恶性程度高,预后差。抗HER 2单克隆抗体曲妥珠单抗能够改善该类乳腺癌治疗效果,但最终仍难逃耐药的结局。近年来,针对曲妥珠单抗耐药机制的研究、新型靶向药物的研发以及治疗策略的探索,在克服曲妥珠单抗耐药性方面取得了重大进展,本文将对其耐药后治疗策略及临床试验数据进行综述。     

Trastuzumab plus tamoxifen: anti-proliferative and molecular interactions in breast carcinoma

HER2 overexpression has been associated with anti-estrogen resistance in human breast cancer, and it has been suggested that the combined treatment of an anti-HER2 antibody plus tamoxifen has enhanced anti-cancer efficacy in breast cancer. The detailed anti-proliferative interactions between trastuzumab and tamoxifen were analyzed with the isobologram and Chou and Talalay methods, which assess the presence of synergy, addition or antagonism. We used the breast cancer cell lines that are estrogen receptor (ER)-positive and HER2-positive. We also analyzed the molecular changes on the HER2 and (ER) signaling pathways that are induced by trastuzumab plus tamoxifen. In terms of cancer cell proliferation, the simultaneous combination of trastuzumab and tamoxifen on BT-474 cells was more growth inhibitory (44%) than the treatment with trastuzumab (24%) or tamoxifen (31%) alone. Isobologram analysis of simultaneous trastuzumab plus tamoxifen exposure showed, however, that there were antagonistic interactions at an effect level of 30% (IC30). Using Chou and Talalay analysis we also observed antagonistic interactions at lower levels of cell kill, although there were additive effects at highest levels of cell kill. Trastuzumab followed by tamoxifen showed antagonism at all effects levels. Tamoxifen followed by trastuzumab showed antagonism at lower levels of cell kill, and additivity at higher levels of cell kill. Similar interactions were observed using T47D cells. The molecular effects of the combined treatment with trastuzumab plus tamoxifen on the levels of HER2 and ER signaling showed that, with respect to HER2 protein levels, trastuzumab downregulated HER2 by 27%, tamoxifen upregulated HER2 by 40%, and the combination of trastuzumab plus tamoxifen did not induce changes in HER2 respect to control. With respect to HER2 mRNA, trastuzumab upregulated HER2 mRNA to 367%, tamoxifen to 166%, and the combination to 401%. With respect to HER2 phosphorylation, trastuzumab upregulated HER2 phosphorylation to 352%, tamoxifen to 202% and the combination to 633%. Epidermal growth factor receptor levels were not changed by trastuzumab or tamoxifen alone, and were upregulated to 138% by the combination. The protein levels and activity of extracellular recptor kinase were not modified by trastuzumab, tamoxifen or the combination. Finally, estrogen receptor protein and mRNA levels were downregulated to about 50% by trastuzumab, tamoxifen or the combination. Taken together, our results show that in ER-positive breast cancer cells overexpressing HER2, trastuzumab plus tamoxifen have antagonistic interactions when used in combination, and that this antagonism may be related with an increase in HER2 signaling pathways that occurs when tamoxifen is added to trastuzumab.     

曲妥珠单抗耐药后HER 2阳性转移性乳腺癌靶向治疗策略

人表皮生长因子受体2（human epidermal growth factor receptor 2,HER 2）是一种跨膜酪氨酸激酶受体,可以激活下游一系列信号通路,导致肿瘤细胞增殖和存活。HER 2阳性乳腺癌是乳腺癌的特殊类型,恶性程度高,预后差。抗HER 2单克隆抗体曲妥珠单抗能够改善该类乳腺癌治疗效果,但最终仍难逃耐药的结局。近年来,针对曲妥珠单抗耐药机制的研究、新型靶向药物的研发以及治疗策略的探索,在克服曲妥珠单抗耐药性方面取得了重大进展,本文将对其耐药后治疗策略及临床试验数据进行综述。     

Development and characterization of a preclinical ovarian carcinoma model to investigate the mechanism of acquired resistance to trastuzumab

Trastuzumab (Herceptin?) is a humanized monoclonal antibody designed to bind and inhibit the function of the human epidermal growth factor receptor?2 (HER2)/erbB2 receptor. Trastuzumab has demonstrated clinical activity in several types of HER2-overexpressing epithelial tumors, such as breast and metastatic gastric or gastroesophageal junction cancer. Relapse and therapeutic resistance, however, still occur in a subset of patients treated with regimens including trastuzumab, despite significant improvements in response rates, survival and quality of life. To investigate the potential mechanisms of acquired therapeutic resistance to trastuzumab, we developed a preclinical model of human ovarian cancer cells, SKOV-3 Herceptin-resistant (HR), and examined the corresponding changes in gene expression profiles. SKOV-3?HR cells were developed by in?vivo serial passaging of parental trastuzumab-sensitive SKOV-3?cells. Following four rounds of serial transplantation of 'break-through' xenograft tumors under trastuzumab treatment, significant and reproducible differences in the effects of trastuzumab treatment between SKOV-3?HR and SKOV-3 cells in?vivo and in?vitro were revealed. SKOV-3?HR cells retained HER2?protein expression but were unaffected by the antiproliferative effects of trastuzumab. The trastuzumab binding affinity for SKOV-3?HR cells was diminished, despite these cells having more binding sites for trastuzumab. Microarray expression profiling (MEP) was performed to determine the genes involved in the resistance mechanism. Functional analysis revealed the differential expression of genes potentially involved in angiogenesis, metastasis, differentiation and proliferation, such as mucin1?(MUC1). Immunohistochemical staining of SKOV-3?HR cells demonstrated a marked overexpression of MUC1. Based on these data, we hypothesize that the overexpression of MUC1 may hinder trastuzumab binding to HER2?receptors, abrogating the antitumor effects of trastuzumab and thus could contribute to resistance to therapy. Moreover, the resultant MEP preclinical gene signature in this preclinical model system may provide the basis for further investigation of potential clinical mechanisms of resistance to trastuzumab.     

曲妥珠单抗的耐药机制及其逆转策略

曲妥珠单抗(trastuzumab;商品名为Herceptin,赫赛汀)是靶向人表皮生长因子受体2(human epidermal growth factor receptor2,HER2)的人源化单克隆抗体,与化疗药物联用可显著提高患者的无进展生存期.然而,曲妥珠单抗的原发性和获得性耐药严重制约了其临床疗效及应用.PI3 K/AKT/mTOR信号通路的异常活化、HER家族成员间的相互作用、药物压力下代偿性生存信号的激活、肿瘤干细胞表型形成等均可能成为曲妥珠单抗耐药的重要机制.随着曲妥珠单抗耐药机制研究的不断深入,克服耐药的治疗手段也越来越丰富.有研究表明,曲妥珠单抗与其他靶向HER2胞外域的单克隆抗体或靶向其他HER家族成员的抗体类药物联用可增强曲妥珠单抗的疗效.应用PI3 K/AKT/mTOR信号通路的小分子抑制剂或耐药相关促生存信号通路的特异性抑制剂可有效逆转耐药发生,延长患者的疾病无进展生存期.深入研究曲妥珠单抗耐药的机制,不断探索逆转耐药的治疗策略,可为乳腺癌个性化治疗方案的建立提供依据.本文将对曲妥珠单抗耐药机制及克服耐药的研究进展做一介绍.