Targeting fatty acid synthase: potential for therapeutic intervention in her-2/neu-overexpressing breast cancer

Fatty acid synthase (FAS)-catalyzed de novo fatty acid biosynthesis, an anabolic energy-storage pathway largely considered of minor importance in humans, actively contributes to the cancer phenotype by virtue of its ability to specifically regulate the expression and activity of Her-2/neu (erbB-2) oncogene. First, a positive correlation between high levels of FAS expression and/or activity and the amplification and/or overexpression of Her-2/neu oncogene exists in human breast cancer cell lines. Second, Her-2/neu overexpression stimulates the activity of FAS gene promoter and ultimately mediates increased endogenous fatty acid biosynthesis, while this Her-2/neu-induced upregulation of breast cancer-associated FAS is inhibitable by anti-Her-2/neu antibodies such as trastuzumab (Herceptin(TM)). Third, pharmacological inhibition of FAS activity negatively regulates the expression and tyrosine-kinase activity of Her-2/neu-coded p185(Her-2/neu) oncoprotein.     

乳腺癌分子分型与拓扑异构酶Ⅱα基因异常关系的研究

目的探讨乳腺癌分子分型与拓扑异构酶Ⅱα基因(TOP2A)扩增或缺失的关系。方法采用免疫组织化学的方法,检测142例乳腺癌患者雌激素受体(ER)、孕激素受体(PR)及人类表皮生长因子受体2(Her-2),同时采用显色原位杂交(CISH)检测Her-2基因情况,根据结果进行分子分型,应用荧光原位杂交(FISH)检测TOP2A基因扩增或缺失状况。结果 142例中,LuminalA型53例,LuminalB型27例,Her-2过表达型49例,三阴型13例。TOP2A基因扩增19例,未扩增123例。不同分子分型中TOP2A基因的扩增情况差异有统计学意义(χ2=11.25,P<0.05),主要发生在Her-2型及LuminalB型。结论 TOP2A基因扩增主要存在于HER2基因扩增患者中,因此常规免疫组织化学检测之后,分子分型为Her-2型及LuminalB型的乳腺癌,应常规增加TOP2A的基因检测。     

Pharmacological inhibitors of Fatty Acid Synthase (FASN)--catalyzed endogenous fatty acid biogenesis: a new family of anti-cancer agents?

The expression and activity of Fatty Acid Synthase (FASN; the sole enzyme capable of the reductive de novo synthesis of long-chain fatty acids from acetyl-CoA, malonyl-CoA, and nicotinamide adenine dinucleotide phosphate -NADPH-) is extremely low in nearly all nonmalignant adult tissues, whereas it is significantly up-regulated or activated in many cancer types, thus creating the potential for a large therapeutic index. Since the pioneering observation that inhibition of FASN activity by the mycotoxin cerulenin preferentially kills cancer cells and retards the growth of tumors in xenografts models, numerous in vitro and in vivo studies have confirmed the potential of FASN as a target for antineoplastic intervention. Other FASN inhibitors such as the cerulenin derivative C75, the beta-lactone orlistat, the green tea polyphenol epigallocatechin-3-gallate (EGCG) and other naturally occurring flavonoids (i.e., luteolin, quercetin, and kaempferol), as well as the antibiotic triclosan, have been identified and have been shown to limit cancer cell growth by inducing apoptotic cell death. Though the exact mode of action of these FASN inhibitors is under discussion, it has been revealed that depletion of end-product fatty acids, toxic intracellular accumulation of supra-physiological concentrations of the FASN substrate malonyl-CoA and/or limited membrane synthesis and/or functioning by altered production of phospholipids partitioning into detergent-resistant membrane microdomains (lipid raft-aggregates), can explain, at least in part, the cytostatic, cytotoxic as well as the apoptotic effects occurring upon pharmacological inhibition of FASN activity in cancer cells. Moreover, several cancer-associated molecular features including nonfunctioning p53, overexpression of the Her-2/neu (erbB-2) oncogene, and hyperactivation of the PI-3'K down-stream effector protein kinase B (AKT), appear to determine an exacerbated sensitivity to FASN inhibition-induced cancer cell death. Although few of these inhibitors are expected to be "exclusively" selective for FASN, the potential of FASN as a target for antineoplastic intervention has eventually been confirmed by RNA interference (RNAi)-knockdown of FASN. Certainly, future studies should definitely elucidate the ultimate biochemical link between FASN inhibition and cancer cell death. Although the combination of FASN structural complexity and until recently the lack of X-ray crystallography data of mammalian FASN created a significant challenge in the exploitation of FASN as a valuable target for drug development, it is hoped that the improvement in the selectivity and potency of forthcoming novel FASN-targeted small molecule inhibitors by taking advantage, for instance, of the recent 4.5 A resolution X-ray crystallographic map of mammalian FASN, will direct the foundation of a new family of chemotherapeutic agents in cancer history.     

New synthetic inhibitors of fatty acid synthase with anticancer activity

Fatty acid synthase (FASN) is a lipogenic enzyme that is highly expressed in different human cancers. Here we report the development of a new series of polyphenolic compounds 5-30 that have been evaluated for their cytotoxic capacity in SK-Br3 cells, a human breast cancer cell line with high FASN expression. The compounds with an IC(50) < 50 μM have been tested for their ability to inhibit FASN activity. Among them, derivative 30 blocks the 90% of FASN activity at low concentration (4 μM), is highly cytotoxic in a broad panel of tumor cells, induces apoptosis, and blocks the activation of HER2, AKT, and ERK pathways. Remarkably, 30 does not activate carnitine palmitoyltransferase-1 (CPT-1) nor induces in mice weight loss, which are the main drawbacks of other previously described FASN inhibitors. Thus, FASN inhibitor 30 may aid the validation of this enzyme as a therapeutic target for the treatment of cancer.     

A novel positive feedback loop involving FASN/p-ERK1/2/5-LOX/LTB4/FASN sustains high growth of breast cancer cells

Aim:

To investigate the endogenous signaling pathways associated with high proliferation potential of breast cancer cells.

Methods:

Breast cancer cell lines LM-MCF-7 and MCF-7 with high and low proliferation capability were used. The promoter activity of fatty acid synthase (FASN) was examined using luciferase reporter gene assay. The expression level of FASN mRNA was measured using RT-PCR and real time PCR, respectively. The level of leukotriene B4 (LTB4) was determined with ELISA. The expression levels of 5-lipoxygenase (5-LOX) was analyzed using RT-PCR and Western blot, respectively. 5-Bromo-20-deoxyuridine (BrdU) incorporation assay was used to study the proliferation of LM-MCF-7 and MCF-7 cells.

Results:

The promoter activity of FASN was significantly higher in LM-MCF-7 cells than MCF-7 cells. Treatment of LM-MCF-7 cells with ERK1/2 inhibitor PD98059 (30–50 μmol/L) or LOX inhibitor NDGA (25 μmol/L) abolished the activation of FASN. Moreover, treatment of LM-MCF-7 cells with the specific 5-LOX inhibitor MK-886 (20–40 μmol/L) or 5-LOX siRNA (50–100 nmol/L) decreased the promoter activity of FASN. The level of LTB4, the final metabolite produced by 5-LOX, was significantly higher in LM-MCF-7 cells than MCF-7 cells. Administration of exogenous LTB4 (1–10 nmol/L) was able to stimulate the promoter activity of FASN in MCF-7 cells. Treatment of LM-MCF-7 cells with the FASN inhibitor cerulenin (10 μmol/L) reduced all the levels of p-ERK1/2, 5-LOX, and LTB4. Treatment of LM-MCF-7 cells with cerulenin, PD98059, or MK-886 abolished the proliferation. Administration of exogenous LTB4 (10 nmol/L) significantly increased BrdU incorporation in MCF-7 cells.

Conclusion:

These results suggest a novel positive feedback loop involving FASN/p-ERK1/2/5-LOX/LTB4/FASN contributes to the sustaining growth of breast cancer LM-MCF-7 cells.     

Development of HER2-specific humanized antibody Herceptin (trastuzumab)

HER2 is a member of the human epidermal growth factor receptor family, possessing protein kinase activity in its cytoplasmic domain. There were evidences indicating that (1) amplification of HER2/neu gene and HER2 protein over-expression in tumor cells was observed in 25-30% of human breast cancer and (2) amplification of HER2/neu correlated with poor prognosis, including shorter disease-free and overall survival. These evidences suggested HER2 was a promising candidate for novel molecular targets of breast cancer therapy. Herceptin is a recombinant humanized monoclonal antibody generated by Genentech, Inc. for the treatment of HER2 over-expressed/HER2 gene amplified metastatic breast cancer (MBC). Preclinical studies demonstrated that the antibody had anti-tumor activity in vivo and in vitro, and additive or synergistic enhancement of anti-tumor activity of the antibody was observed in combination with various anti-tumor agents in mouse models. In clinical studies, apparent extension of overall survival was observed in HER2 overexpressing MBC patients. Herceptin is the first anticancer drug whose use as a treatment for MBC patients is decided based on the status of the HER2 gene amplification/HER2 protein over-expression. The development and standardization of HER2 test were a key strategy in clinical development of this drug, since appropriate selection of patients with HER2 over-expression was the essential point for success.     

34例乳腺癌HER-2基因扩增与蛋白表达相关性研究

目的了解乳腺癌组织中人表皮生长因子受体2（HER-2）基因扩增与Her-2／neu蛋白表达的一致性与相关性。方法采用荧光原位杂交（FISH）法及免疫组化（IHC）法分别检测349例浸润性乳腺癌患者的HER-2基因扩增与蛋白表达情况,分析二者检测结果的一致性与相关性。结果FISH与IHC符合率为55．3％,结果存在一致性（Kappa=0．189,P=0．000）,呈正相关（r=0．434,P=0．000）。结论FISH与IHC两种检测结果存在一致性,但IHC（＋～＋＋＋）患者均应以FISH检测作为评价HER-2基因是否扩增的标准方法。     

Ras mutation, irrespective of cell type and p53 status, determines a cell's destiny to undergo apoptosis by okadaic acid, an inhibitor of protein phosphatase 1 and 2A.

Okadaic acid (OA), a toxin from the black sponge Halicondria okadai, is a specific inhibitor of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A). OA is a tumor promoter but also induces apoptosis in some tumor cell lines. In this study, we determined whether ras mutation and/or p53 status are characteristics associated with the cell's sensitivity to the induction of apoptosis by OA. Several cell lines that differed in ras and p53 mutations were treated with OA (10-100 nM). At 24 to 48 h after treatment, the percentage of cells undergoing apoptosis was quantitated. The cell lines with mutations in either H-ras (human bladder carcinoma cell line T24 and mouse keratinocyte cell line 308), or K-ras (human colon carcinoma cell lines DLD-1 and HCT116; human prostate cancer cell lines LNCaP and PC-3; human lung cancer cell lines Calu-6 and SKLU-1; and human pancreatic cancer cell line MIAPaCa2) were more sensitive to OA-induced apoptosis (3- to 10-fold) than the cell lines that lacked the ras mutation (mouse epidermal cell lines C50 and JB6; murine fibroblast cell line NIH3T3; human colon cancer cell line HT29; human kidney epithelial cell line Hs715.K; and human pancreatic cancer cell line Bx-PC3). Similarly, using isogenic cell lines we found that overexpression of mutated H-ras in NIH3T3 and in SV40 immortalized human uroepithelial cells (SVHUC) enhanced their sensitivity to undergo apoptosis in response to OA treatment. The T24, DLD-1, SKLU-1, Calu-6, and MIAPaCa2 cell lines express mutated p53. The SVHUC as well as their ras-transfected counterparts have inactive p53 due to complex formation between large "T" antigen and p53. Taken together, these results imply that OA-induced apoptosis may involve a p53-independent pathway. The transfectants (NIH3T3-ras and SVHUC-ras), which express mutated H-ras, have up-regulated PP2A activity. OA treatment inhibited in vivo the levels of PP1 and PP2A activity, and induced apoptosis in SVHUC-ras and other cell lines. We conclude that OA-induced cell death pathway in ras-activated cell lines may involve a cross talk between PP1 and PP2A and ras signaling pathways. In light of the present results, the current theory that OA promotes mouse skin tumor formation by selective expansion of initiated cells that harbor ras mutations needs reevaluation.     

Predicting the clinical course of breast cancer patients undergoing trastuzumab-based therapy: an outlook

Her-2/neu overexpressing breast cancer is associated with reduced overall survival, sex steroid receptor negativity and increased resistance to antihormonal therapy, and thus represents a subgroup with poor prognosis. The anti Her-2/neu receptor antibody trastuzumab (Herceptin), however, specifically targets this protein and provides a valuable addition to classical systemic therapies. Unfortunately, not all tumors that express Her-2/neu protein are also adequate candidates for trastuzumab therapy. Therefore, most clinicians now consider Her-2/neu oncoprotein overexpression and/or her-2/neu gene amplification a prerequisite for trastuzumab-based antineoplastic therapy. Nevertheless, due to the relatively low response rates that are observed even in this preselected patient cohort, better response predictors are clearly needed. Here, we review established parameters such as Her-2/neu immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) and their ability to predict the clinical course of trastuzumab-treated breast cancer. We also evaluate promising parameters such as serum levels of the Her-2/neu extracellular domain (ECD) and the activation status of Her-2/neu oncoprotein (pHer-2/neu), and their use in the clinical setting. Finally, novel tumor-specific such as tumor M2-PK, IGF-IR and p53 are discussed and their potential to predict the efficacy of trastuzumab is assessed.     

Reversal effect of Dioscin on multidrug resistance in human hepatoma HepG2/adriamycin cells

Multidrug resistance is a serious obstacle encountered in cancer treatment. Since drug resistance in human cancer is mainly associated with overexpression of the multidrug resistance gene 1 (MDR1), the promoter of the human MDR1 gene may be a target for multidrug resistance reversion drug screening. In the present study, HEK293T cells were transfected with pGL3 reporter plasmids containing the 2 kb of MDR1 promoter, and the transfected cells were used as models to screen for candidate multidrug resistance inhibitors from over 300 purified naturally occurring compounds extracted from plants and animals. Dioscin was found to have an inhibiting effect on MDR1 promoter activity. The resistant HepG2 cell line (HepG2/adriamycin) was used to validate the activity of multidrug resistance reversal by Dioscin. Results showed that Dioscin could decrease the resistance degree of HepG2/adriamycin cells, and significantly inhibit P-glycoprotein expression, as well as increase the accumulation of adriamycin in HepG2/adriamycin cells as measured by Flow Cytometric analysis. These results suggest that Dioscin is a potent multidrug resistance reversal agent and may be a potential adjunctive agent for tumor chemotherapy.     

乳腺癌Her-2基因基础与临床研究进展

Her-2基因是一种原癌基因,位于17号染色体长臂上,编码185KD跨膜蛋白,具有酪氨酸激酶活性,属于人类表皮生长因子受体家族。Her-2基因扩增及蛋白的过度表达与乳腺癌的预后及治疗有密切联系。所以Her-2基因及蛋白的测定极为重要,但目前常用的IHC和FISH的检测学方法仍存在一些问题,需尽快确定测定方法的统一标准。赫赛汀是针对Her-2／neu的单抗,应用于Her-2过度表达的乳腺癌治疗有十余年的发展,但对赫赛汀耐药机制仍不十分明了,有待进一步研究。     

曲妥珠单抗在Her-2高表达胃癌治疗中的研究近况

目的：了解曲妥珠单抗（赫赛汀）在人表皮生长因子受体2（Her-2）高表达胃癌治疗中的研究近况。方法：对近年来曲妥珠单抗（赫赛汀）在Her-2高表达胃癌治疗中研究情况的文献资料进行分析,并对Her-2相关的检测方法进行探讨。结果：曲妥珠单抗（赫赛汀）在体内外能够抑制Her-2高表达胃癌细胞的生长,Ⅱ、Ⅲ期临床试验结果显示它能明显提高Her-2高表达胃癌患者的疗效和生活质量,显著延长生存时间。结论：曲妥珠单抗（赫赛汀）有望成为一种新型的治疗胃癌的生物靶向药物。     

Overcoming treatment resistance in HER2-positive breast cancer: potential strategies

Human epidermal growth factor receptor (HER)-2 overexpression or amplification occurs in about 20% of all breast cancers and results in a worse prognosis. Nevertheless, anti-HER2 treatments have recently been developed, resulting in dramatic improvements in the clinical outcome of patients with HER2-positive breast cancer. Trastuzumab has shown efficacy in early and advanced breast cancer treatment and lapatinib is currently approved for the treatment of advanced disease. Other anti-HER2 agents are being investigated. Mechanisms of resistance to trastuzumab treatment include crosstalk with heterologous receptors and amplification of HER2 signalling; amplification of the phosphoinositide 3-kinase (PI3K)/AKT pathway; alteration in binding of trastuzumab to HER2; and loss of HER2 expression. Proposed mechanisms of resistance to lapatinib involve derepression and/or activation of compensatory survival pathways through increased PI3K/AKT or estrogen receptor (ER) signalling. Several strategies to overcome resistance to anti-HER2 treatment are in different phases of development and include treatment with pertuzumab, T-DM1 and mammalian target of rapamycin (mTOR) inhibitors.     

HER2基因突变晚期非小细胞肺癌治疗进展

肺癌是全球范围内一种高发病率和高死亡率的恶性肿瘤,非小细胞肺癌（NSCLC）是最常见的亚型,发生率约85%。人表皮生长因子受体2（HER2）是受体酪氨酸激酶（RTK）的重要成员之一,NSCLC中HER2基因主要表现为HER2突变、HER2扩增和HER2过表达三种形式。目前,靶向HER2突变的酪氨酸激酶抑制剂、单克隆抗体和抗体偶联药物在HER2突变的NSCLC中显示出一定的临床疗效,但相关免疫治疗疗效有限。本文就HER2突变相关NSCLC的治疗进展进行综述,以期为进一步完善HER2突变晚期NSCLC临床诊疗策略提供理论依据。     

Anti-erbB-2 antibody trastuzumab in the treatment of HER2-amplified breast cancer

Human epidermal growth factor receptor-2 (HER2/erbB-2) is a member of a family of four transmembrane receptor tyrosine kinases that regulate cell growth, survival and differentiation via multiple signal transduction pathways. Amplification of the HER2 gene occurs in 20–25% of human breast cancers. This amplification event is an independent adverse prognostic factor as well as a predictive factor for increased response to doxorubicin-based combination chemotherapy, response to trastuzumab and decreased response to hormonal therapy. Methods for detecting protein overexpression or gene amplification in clinical tumor specimens include immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) techniques, with the latter considered by some to be more accurate. Trastuzumab (Herceptin) is a recombinant humanized monoclonal antibody which targets an epitope in the extracellular domain of the HER2 protein. Preclinical models demonstrated that this antibody has significant anti-tumor activity as a single agent and has synergy with certain chemotherapeutic drugs. Phase II and III clinical trials performed in women with metastatic breast cancer that overexpress HER2 have shown that trastuzumab has clinical activity when used as first-, second- or third-line monotherapy, and improves survival when used as first-line therapy in combination with chemotherapy. Newer combinations with numerous chemotherapeutic drugs have also shown significant clinical activity in phase II studies. In all of these trials, trastuzumab was generally well-tolerated, but cardiac toxicity (particularly when the antibody was combined with anthracyclines) was an unexpected adverse effect. Although trastuzumab is currently usually administered on a weekly intravenous schedule, evidence suggests that a triple dose of the drug given once every three weeks has a pharmacokinetic profile expected to be equally efficacious. Neither the optimal schedule nor the optimal duration of trastuzumab therapy has yet been clearly defined in controlled clinical trials. Current clinical investigations of trastuzumab include its use in both the adjuvant and neoadjuvant settings as well as in combination with other chemotherapy drugs or new biologic targeted agents.     

Fatty acid synthase (FASN) as a therapeutic target in breast cancer

Introduction: Ten years ago, we put forward the metabolo-oncogenic nature of fatty acid synthase (FASN) in breast cancer. Since the conception of this hypothesis, which provided a model to explain how FASN is intertwined with various signaling networks to cell-autonomously regulate breast cancer initiation and progression, FASN has received considerable attention as a therapeutic target. However, despite the ever-growing evidence demonstrating the involvement of FASN as part of the cancer-associated metabolic reprogramming, translation of the basic science-discovery aspects of FASN blockade to the clinical arena remains a challenge.

Areas covered: Ten years later, we herein review the preclinical lessons learned from the pharmaceutical liabilities of the first generation of FASN inhibitors. We provide an updated view of the current development and clinical testing of next generation FASN-targeted drugs. We also discuss new clinico-molecular approaches that should help us to convert roadblocks into roadways that will propel forward our therapeutic understanding of FASN.

Expert opinion: With the recent demonstration of target engagement and early signs of clinical activity with the first orally available, selective, potent and reversible FASN inhibitor, we can expect Big pharma to revitalize their interest in lipogenic enzymes as well-credentialed targets for oncology drug development in breast cancer.     

Strategies to target HER2/neu overexpression for cancer therapy.

Amplification or overexpression of the HER2/neu (also known as erbB-2) gene has been noted in various types of human cancers. In addition to malignant transformation, the activation of signaling pathways of HER2/neu enhances various metastasis-associated properties and may render cancer cells resistant to conventional therapies. This, at least partially, contributes to the poor prognosis and lower survival rate of patients. Many studies have demonstrated that repression of HER2/neu overexpression suppresses the malignant phenotypes of cancer cells. Therefore, various novel HER2/neu-blocking agents have been developed, several of which have been tested in clinical trials with satisfactory results, including trastuzumab, a HER2/neu monoclonal antibody that has been approved by the FDA in the treatment of HER2/neu-overexpressing breast cancer patients. In this article, we intend to discuss the biological relevance and significance of HER2/neu overexpression in tumorigenesis, metastasis, and resistance to conventional therapy. We also summarize the currently available strategies and combination therapies targeting HER2/neu-overexpressing cancer cells. Although the optimal treatment for HER2/neu-overexpressing cancer patients remains elusive, the initial success of trastuzumab indicates that HER2/neu is a good target for cancer therapy. Further elucidation of HER2/neu-mediated pathways and downstream molecules is critical to provide alternative therapies, overcome drug resistance, and improve the therapeutic outcome for HER2/neu-overexpressing cancer patients.     

Maximizing the response to Herceptin therapy through optimal use and patient selection

The aggressiveness of human epidermal growth factor receptor-2 (HER2)-positive breast cancer and the poor prognosis of women with this disease demand the availability of accurate and reliable tests for HER2 status and the optimization of HER2-targeted therapy. The distinctive clinical pattern of HER2-positive breast cancer underlines the importance of testing for HER2 status and efforts are ongoing to validate the two major methods in use-immunohistochemistry (IHC), which measures cell membrane HER2 expression, and fluorescence in situ hybridization (FISH), which measures gene copy number. Clinical trial results demonstrate that there is an association between strong HER2 overexpression (IHC 3+) and optimal response to therapy with the novel recombinant HER2 antibody Herceptin. High levels of concordance between IHC 3+ and FISH-positive status have been observed, and response to treatment with Herceptin is similar for patients whose breast cancers are IHC 3+ and those who are FISH-positive. Observations to date have led to the formulation of an algorithm for HER2 status determination and Herceptin use which recommends that: (i) the HER2 status of all women with breast cancer be determined at presentation, (ii) all IHC 3+ and FISH-positive patients with metastatic disease should receive Herceptin, (iii) Herceptin should be used early in the course of metastatic breast cancer and preferably first line, and (iv) Herceptin therapy should be continued until disease progression.