Cardio-oncology: Understanding the different mechanisms of cardiovascular toxicity

Due to the success of finding treatments for various types of cancer, overall cancer survival has increased substantially in recent decades. Ironically, the clinical success of antineoplastic therapy is attenuated by the comorbidity of cardiovascular diseases, which appear to be the main complications of intense cancer treatment and are the second main cause of long-term morbidity and mortality among cancer survivors.Cardio-oncology is the new area of cardiology that aims to treat the specific cardiologic status of cancer patients.Most antineoplastic therapies are associated with some degree of cardiovascular toxicity, ranging from asymptomatic and transient cardiac events to clinically significant and long-lasting events.Antineoplastic agents are responsible for different cardiovascular injuries, which can be reversible or permanent. All anatomical structures of the heart can, however, be affected by transthoracic radiotherapy.Cardiotoxicity mechanisms are recognized according to the presence or absence of structural anomalies and their reversibility, being classified into Type I and Type II, aiming to distinguish the drugs that induce irreversible damage (Type I) from the drugs that predominantly induce reversible left ventricular dysfunction (Type II).While anthracyclines and anti-HER2 agents form the two major groups of cardiotoxic drugs, other antineoplastic agents, such as other monoclonal antibodies, certain tyrosine kinase inhibitors and antiangiogenic drugs can also be cardiotoxic via different mechanisms.This article presents the different pathophysiological mechanisms of cardiovascular toxicity according to the treatment regimen used.     

Combined targeting of HER2 and VEGFR2 for effective treatment of HER2-amplified breast cancer brain metastases

Brain metastases are a serious obstacle in the treatment of patients with human epidermal growth factor receptor-2 (HER2)–amplified breast cancer. Although extracranial disease is controlled with HER2 inhibitors in the majority of patients, brain metastases often develop. Because these brain metastases do not respond to therapy, they are frequently the reason for treatment failure. We developed a mouse model of HER2-amplified breast cancer brain metastasis using an orthotopic xenograft of BT474 cells. As seen in patients, the HER2 inhibitors trastuzumab and lapatinib controlled tumor progression in the breast but failed to contain tumor growth in the brain. We observed that the combination of a HER2 inhibitor with an anti–VEGF receptor-2 (VEGFR2) antibody significantly slows tumor growth in the brain, resulting in a striking survival benefit. This benefit appears largely due to an enhanced antiangiogenic effect: Combination therapy reduced both the total and functional microvascular density in the brain xenografts. In addition, the combination therapy led to a marked increase in necrosis of the brain lesions. Moreover, we observed even better antitumor activity after combining both trastuzumab and lapatinib with the anti-VEGFR2 antibody. This triple-drug combination prolonged the median overall survival fivefold compared with the control-treated group and twofold compared with either two-drug regimen. These findings support the clinical development of this three-drug regimen for the treatment of HER2-amplified breast cancer brain metastases.     

Crosstalk between the estrogen receptor and the HER tyrosine kinase receptor family: molecular mechanism and clinical implications for endocrine therapy resistance

Breast cancer evolution and tumor progression are governed by the complex interactions between steroid receptor [estrogen receptor (ER) and progesterone receptor] and growth factor receptor signaling. In recent years, the field of cancer therapy has witnessed the emergence of multiple strategies targeting these specific cancer pathways and key molecules (ER and growth factor receptors) to arrest tumor growth and achieve tumor eradication; treatment success, however, has varied and both de novo (up front) and acquired resistance have proven a challenge. Recent studies of ER biology have revealed new insights into ER action in breast cancer and have highlighted the role of an intimate crosstalk between the ER and HER family signaling pathways as a fundamental contributor to the development of resistance to endocrine therapies against the ER pathway. The aim of this review article is to summarize the current knowledge on mechanisms of resistance of breast cancer cells to endocrine therapies due to the crosstalk between the ER and the HER growth factor receptor signaling pathways and to explore new available therapeutic strategies that could prolong duration of response and circumvent endocrine resistant tumor growth.     

Examination of HER3 targeting in cancer using monoclonal antibodies

The human EGF receptor (HER/EGFR) family of receptor tyrosine kinases serves as a key target for cancer therapy. Specifically, EGFR and HER2 have been repeatedly targeted because of their genetic aberrations in tumors. The therapeutic potential of targeting HER3 has long been underestimated, due to relatively low expression in tumors and impaired kinase activity. Nevertheless, in addition to serving as a dimerization partner of EGFR and HER2, HER3 acts as a key player in tumor cells’ ability to acquire resistance to cancer drugs. In this study, we generated several monoclonal antibodies to HER3. Comparisons of their ability to degrade HER3, decrease downstream signaling, and inhibit growth of cultured cells, as well as recruit immune effector cells, selected an antibody that later emerged as the most potent inhibitor of pancreatic cancer cells grown as tumors in animals. Our data predict that anti-HER3 antibodies able to intercept autocrine and stroma–tumor interactions might strongly inhibit tumor growth, in analogy to the mechanism of action of anti-EGFR antibodies routinely used now to treat colorectal cancer patients.Growth factors and their plasma-membrane–embedded receptors regulate cellular proliferation and migration during both embryogenesis and oncogenesis (1). One example entails the epidermal growth factor (EGF) family and the corresponding human EGF receptor (HER)/ERBB family of four receptor tyrosine kinases: the EGF receptor (EGFR or ERBB1), HER2 (c-Neu or ERBB2), HER3 (ERBB3), and HER4 (ERBB4) (2). The intracellular parts of ERBB/HER receptors harbor a catalytic tyrosine kinase domain (3). After ligand binding to an ectodomain, these receptors form active homodimers or heterodimers (4–7). Unlike EGFR, HER3/ERBB3 presents very low tyrosine kinase activity (8). Nevertheless, it binds neuregulins (NRGs) and exerts profound influence on signaling pathways, primarily through dimerization with EGFR and HER2. In line with roles in cancer progression, and similarly to EGFR and HER2, mutant forms of HER3 have recently been reported in colon and gastric cancer (9).Cancer therapies that use monoclonal antibodies (mAbs) to target ERBB/HER family members are becoming a mainstay in oncology. For example, trastuzumab, which targets HER2, is currently used to treat gastric and breast cancer (10, 11). However, the majority of patients with metastatic HER2-positive breast cancer will become trastuzumab-resistant after prolonged treatment, a development significantly less common in an adjuvant or neo-adjuvant setting. It has been reported that trastuzumab-resistant tumors show elevated expression of HER3 (12), and, similarly, inhibition of HER2 using a kinase inhibitor also up-regulates HER3 (13). Likewise, HER3 has been implicated in the development of resistance to treatment with other ERBB/HER-targeted therapies (14, 15), agents blocking insulin-like growth factor receptors (16), and chemotherapeutic agents (17). For these reasons, anti-HER3 antibodies (Abs) are being developed by several laboratories, and some have reached initial clinical trials.Importantly, anticancer mechanisms of therapeutic Abs are multifactorial and incompletely understood. The involvement of Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity, as well as various cellular processes such as triggering of apoptosis, blocking angiogenesis, inhibiting tumor cell proliferation, interfering with signaling cascades, and accelerating receptor internalization, have been described (18). Our own studies proposed critical involvement of the endocytic system. In particular, combinations of two mutually noncompetitive Abs (Abs against different epitopes that do not cause steric hindrance) to EGFR (19) or to HER2 (20, 21) have been shown to accelerate receptor endocytosis and inhibit tumor growth, better than either Ab alone. Furthermore, a recently completed clinical trial that combined with chemotherapy two mutually noncompetitive anti-HER2 mAbs, for the treatment of HER2-positive breast cancer (22), confirmed the clinical benefit of combining noncompetitive mAbs.Anti-HER3 agents that are being developed or are in clinical trials show promising results, yet their efficacy might be viewed, in general, as variable and rather modest (23). Hence, it is imperative to develop new agents and resolve their molecular mechanisms of action. In this study, we generated in mice a relatively broad series of anti-HER3 mAbs and examined them for the ability to both degrade HER3 and decrease downstream signaling. The new mAbs displayed a variety of biochemical and biological attributes, such as binding affinity to HER3, ability to displace NRG and block downstream signals, sort HER3 for degradation, and recruit natural killer cells. Ultimately, the Abs were tested in vitro and in tumor-bearing animals for their ability to inhibit growth of cancer cells. We focus on an especially potent tumor-inhibitory Ab that can displace NRG, robustly sort HER3 for intracellular degradation, and also recruit immune effector cells.     

Conditional mutation of the ErbB2 (HER2) receptor in cardiomyocytes leads to dilated cardiomyopathy

The ErbB2 (Her2) proto-oncogene encodes a receptor tyrosine kinase, which is frequently amplified and overexpressed in human tumors. ErbB2 provides the target for a novel and effective antibody-based therapy (Trastuzumab/Herceptin) used for the treatment of mammary carcinomas. However, cardiomyopathies develop in a proportion of patients treated with Trastuzumab, and the incidence of such complications is increased by combination with standard chemotherapy. Gene ablation studies have previously demonstrated that the ErbB2 receptor, together with its coreceptor ErbB4 and the ligand Neuregulin-1, are essential for normal development of the heart ventricle. We use here Cre-loxP technology to mutate ErbB2 specifically in ventricular cardiomyocytes. Conditional mutant mice develop a severe dilated cardiomyopathy, with signs of cardiac dysfunction generally appearing by the second postnatal month. We infer that signaling from the ErbB2 receptor, which is enriched in T-tubules in cardiomyocytes, is crucial for adult heart function. Conditional ErbB2 mutant mice provide a model of dilated cardiomyopathy. In particular, they will allow a rigorous assessment of the role of ErbB2 in the heart and provide insight into the molecular mechanisms that underlie the adverse effects of anti-ErbB2 antibodies.     

Recent advances in the treatment of hormone receptor positive HER2 negative metastatic breast cancer

Endocrine therapy is the recommended systemic therapy for hormone receptor (HR) positive metastatic breast cancer (MBC).However so far the limited number of endocrine agents and the onset of endocrine resistance have severely limited the therapeutic options for this patients.In the last years many targeted agents have been investigated to prevent or overcome endocrine resistance; only a few of them have been found effective in HR positive MBC, such as everolimus, CK4/6 inhibitors and HDAC inhibitors.Furthermore, translational medicine studies using next generation sequencing technologies have evaluated genetic variations of a broad panel of cancer-related genes and explored their correlations with targeted agents benefit. In some studies predictive biomarkers have been identified and many ongoing studies are evaluating the efficacy of targeted drugs in HR positive MBC patients selected for biomarkers or stratified by pathways amplification.     

靶向HER2基因的siRNA联合卡铂对肺腺癌A549细胞的抑制作用

目的 探讨靶向HER2基因的siRNA联合卡铂对肺腺癌A549细胞的抑制作用.方法 设计并合成3对靶向HER2基因的siRNA(2621组、1724组、1491组),分别转染肺腺癌A549细胞,同时设阴性对照组及空白组.应用实时荧光定量PCR法和免疫组化法检测细胞内HER2 mRNA及其蛋白的表达.应用流式细胞术检测卡铂与HER2 siRNA联合应用时肺癌A549细胞的凋亡率.结果 2621组和1724组HER2 mRNA及蛋白表达均低于1491组和阴性对照组.流式细胞术检查显示,各组间细胞凋亡率比较P＜0.01,HER2 siRNA+卡铂组细胞的凋亡率均高于对应浓度的卡铂组.结论 siRNA能抑制HER2基因在肺癌A549细胞中的表达,并与卡铂协同诱导癌细胞凋亡.     

Therapy of metastatic breast cancer with humanized antibodies against the HER2 receptor protein

The HER2 protein, a member of the epidermal growth factor family, is encoded by the protooncogene c-erbB-2. Its overexpression, occurring in approximately one-third of all breast carcinomas, is associated with a poor prognosis. A humanized mouse antibody against HER2 has been developed by genetic engineering. Here an unspecific human IgG was connected to the recognizing mouse IgG fragment. The allergization typical for allogeneic antibodies does not take place in this context. The effectiveness of this antibody has been confirmed by two international prospective phase III trials that tested it alone and combined with chemotherapy. Both modes of application increased the response rates and the time to progression. Side-effects were rare except for a high rate of cardiac dysfunction when the antibody was combined with anthracyclines. The effectiveness and negligible side-effects of the chimeric antibody against HER2 (Herceptin) render it a valuable tool in the treatment of breast cancer. Received: 8 February 1999 / Accepted: 6 April 1999     

人表皮生长因子受体家族在肺癌中的表达研究

目的：检测人表皮生长因子受体（HER）及其基因（erbB)家族在非癌肺组织和肺癌组织中的表达，探讨HER过度表达与肺癌临床病理的关系。方法：采用免疫组化的方法分别检测20例非癌肺组织及有随访资料的70例肺癌（43例鳞癌和27例腺癌）组织中HER的表达。结果：HER在非癌肺组织存在阳性表达（＋），肺癌中存在HER1，HER2，HER4的过度表达，该过度表达与肺癌病理类型，分化程度不相关，而与肺癌淋巴结转移，TNM分期，患者术后生存率相关，结论：erbB1,erbB2,erbB4是晚期肺癌生长的调控基因，干预期过度表达可能是治疗晚期肺癌的有效方法。     

SGLT2 inhibitors break the vicious circle between heart failure and insulin resistance: targeting energy metabolism

Heart Failure Reviews - Heart failure (HF) often coexists with insulin resistance (IR), and the incidence of HF in type 2 diabetes mellitus (T2DM) patients is significantly higher. The reciprocal...     

Epidermal growth factor receptor/HER2/insulin-like growth factor receptor signalling and oestrogen receptor activity in clinical breast cancer

Breast cancer models of acquired tamoxifen resistance, oestrogen receptor (ER)+ /ER- de novo resistance and gene transfer studies cumulatively demonstrate the increased importance of growth factor receptor signalling, notably the epidermal growth factor receptor (EGFR)/HER2, in tamoxifen resistance. Our recent in vitro studies also suggest that EGFR signalling productively cross-talks with insulin-like growth factor receptor (IGF-1R) and, where present, activates ER on key AF-1 serine residues to facilitate acquired tamoxifen-resistant growth. This paper presents our immunohistochemical evidence that EGFR/HER2 signalling (i.e. transforming growth factor (TGF)alpha, EGFR and HER2 expression; phosphorylation of EGFR, HER2 and ERK1/2 MAP kinase) is also prominent in clinical de novo resistant and modestly increased in acquired tamoxifen-resistant states, suggesting that anti-EGFR/HER2 strategies may prove valuable treatments. Primary breast cancer samples employed were obtained for (1) patients subsequently treated with tamoxifen for advanced disease where endocrine response and survival data were available and (2) ER+ elderly patients during tamoxifen response and relapse. We also present our clinical immunohistochemical findings that IGF-1R expression, its phosphorylation on tyrosine 1316, and also phosphorylation on serine 118 of ER are not only prominent in ER+ tamoxifen-responsive disease, but are also detectable in ER+ de novo and acquired tamoxifen-resistant breast cancer, where there is evidence of EGFR/ER cross-talk. Our data suggest that agents to deplete effectively ER or IGF-1R signalling may be of value in treating ER+ de novo/acquired tamoxifen resistance in addition to tamoxifen-responsive disease in vivo. IGF-1R inhibitors may also prove valuable in ER- patients, since considerable IGF-1R signalling activity was apparent within approximately 50% of such tumours.     

HER2 in gastric cancer: Comparative analysis of three different antibodies using whole-tissue sections and tissue microarrays

AIM:To compare the performance of three commercially available anti-human epidermalgrowth factor receptor 2(HER2)antibodies in whole-tissue sections and tissue microarrays(TMAs)of a series of gastric tumors.METHODS:We present a comparative analysis of three anti-HER2 antibodies(HercepTest,4B5 and SP3)using TMA and whole-tissue sections prepared from the same paraffin blocks of 199 gastric adenocarcinomas operated upon between January 2004 and December2008 at a Brazilian cancer hospital.The data on the patients’age,sex,the anatomical location of the tumor and the Lauren’s histological classification were collected from clinical and pathological records.The immunohistochemical(IHC)results were examined by two pathologists and the cases were classified as positive(3+),equivocal(2+)and negative(0 or 1+),according to the criteria of the IHC scoring system of gastric cancer.TMAs and whole-tissue sections were evaluated separately and independently.All cases yielding discordant IHC results and/or scored as 2+were subjected to dual-color in situ hybridization in order to determine the final HER2 status.Besides determining the sensitivity and predictive value for HER2-positive status,we measured the accuracy of each antibody by calculating the area under the receiver operating characteristic(ROC)curve.The agreement between the results obtained using the TMAs and those obtained using the whole-tissue sections was assessed by means of Kappa coefficient.RESULTS:Intratumoral heterogeneity of HER2 expression was observed with all antibodies.HER2-positive expression(3+)in the whole-tissue sections was observed in 23 cases(11.6%)using the 4B5 antibody,in 18 cases(9.1%)using the SP3 antibody and in 10 cases(5.1%)using the HercepTest antibody.In the TMAs,11 positive cases(5.6%)were identified using SP3 antibody,9(4.6%)using the 4B5 antibody and 6(3%)using the HercepTest antibody.The sensitivity using whole-tissue sections and TMA,respectively,was 95.2%and 42.9%with 4B5,90.5%and 66.7%with SP3 and 47.6%and42.9%with HercepTest.T     

Tissue-specific targeting of the insulin receptor gene

The techniques to study the mechanisms that underlie the pathogenesis of disease processes have been revolutionized by the development of methods that allow spatiotemporal control of gene deletion or gene expression in transgenic and knockout animals. The ability to interfere with the function of a single protein in a specific tissue allows unprecedented flexibility for exploring gene function in both health and disease. The present review will summarize some of the different knockouts and transgenics generated recently to study type 2 diabetes and critically evaluate the techniques used to examine the function of the insulin receptor in two nonclassical insulin target tissues—the pancreatic islet and the central nervous system.     

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein–protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C₁₆H₁₅NO₄ (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors.Toll-like receptors (TLRs) are type I transmembrane receptors that detect conserved “pathogen-associated molecular patterns” from microbes, as well as host-derived “danger-associated molecular patterns” (1). TLR2 heterodimerizes with TLR6 or TLR1 to recognize diacyl lipopeptides or triacyl lipopeptides, respectively (2, 3), present in gram-positive and gram-negative bacteria (4–9).Ligand engagement of TLR2/1 or TLR2/6 activates the myeloid differentiation primary response gene 88 (MyD88)-dependent pathway (i.e., nuclear translocation of NF-κB, activation of MAPKs), resulting in production of proinflammatory cytokines (10). Dysregulated TLR2 signaling has been implicated in numerous diseases (e.g., sepsis, atherosclerosis, tumor metastasis, ischemia/reperfusion injury) (11–14). Several inhibitors of TLR2 signaling have been developed (15–18), yet none is licensed for human use. A better understanding of the Toll/IL-1 receptor resistance (TIR) domain interactions involved in TLR2 signaling could lead to novel therapeutic agents.Both TLRs and adapter proteins contain a cytoplasmic TIR domain that mediates homotypic and heterotypic interactions during TLR signaling (19). Two adapter proteins implicated in TLR2 signaling are MyD88 and TIRAP (Mal). A conserved Pro [e.g., P681 in human TLR2 (hTLR2), P712 in murine TLR4 (mTLR4), P674 in hTLR10, P804 in mTLR11] within the BB loop of almost all TIR domains is critical for signaling (20–27). More importantly, the BB loop P681H mutation in hTLR2 abolished recruitment of MyD88 and signaling (20, 26). Based on this evidence, the BB loop within the TLR2 TIR domain appears to be an ideal target for attenuation of TLR2 signaling.Visual inspection of the crystal structure of the hTLR2 TIR domain (26) revealed a pocket formed by residues on the β-B strand and α-B helix that includes the highly conserved Pro and Gly residues of the BB loop. We hypothesized that targeting this pocket with a small molecule might inhibit interaction of TLR2 with MyD88, and thereby blunt TLR2 signaling. We identified C₁₆H₁₅NO₄ (C29) and its derivative, ortho-vanillin (o-vanillin), which inhibit mTLR2 and hTLR2 signaling initiated by synthetic and bacterial agonists without cytotoxicity. Interestingly, mutation of the BB loop pocket residues revealed a differential requirement for TLR2/1 vs. TLR2/6 signaling. Our data indicate that computer-aided drug design (CADD) is an effective approach for identifying small molecule inhibitors of TLR2 signaling and has the potential to identify inhibitors for other TLR signaling pathways.