The generation of both T killer and Th cell clones specific for the tumor-associated antigen HER2 using retrovirally transduced dendritic cells

Induction of antitumor immunity involves the presence of both CD8(+) CTLs and CD4(+) Th cells specific for tumor-associated Ags. Attempts to eradicate cancer by adoptive T cell transfer have been limited due to the difficulty of generating T cells with defined Ag specificity. The current study focuses on the generation of CTL and Th cells against the tumor-associated Ag HER2 using autologous dendritic cells (DC) derived from CD34(+) hematopoietic progenitor cells which have been retrovirally transduced with the human epidermal growth factor receptor 2 (HER2) gene. HER2-transduced DC elicited HER2-specific CD8(+) CTL that lyse HER2-overexpressing tumor cells in context of distinct HLA class I alleles. The induction of both HLA-A2 and -A3-restricted HER2-specific CTL was verified on a clonal level. In addition, retrovirally transduced DC induced CD4(+) Th1 cells recognizing HER2 in context with HLA class II. HLA-DR-restricted CD4(+) T cells were cloned that released IFN-gamma upon stimulation with DC pulsed with the recombinant protein of the extracellular domain of HER2. These data indicate that retrovirally transduced DC expressing the HER2 molecule present multiple peptide epitopes and subsequently elicit HER2-specific CTL and Th1 cells. The method of stimulating HER2-specific CD8(+) and CD4(+) T cells with retrovirally transduced DC was successfully implemented for generating HER2-specific CTL and Th1 clones from a patient with HER2-overexpressing breast cancer. The ability to generate and expand HER2-specific, HLA-restricted CTL and Th1 clones in vitro facilitates the development of immunotherapy regimens, in particular the adoptive transfer of both autologous HER2-specific T cell clones in patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.     

CTLs directed against HER2 specifically cross-react with HER3 and HER4

The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated Ag by T cell-based immunotherapeutical strategies such as cancer vaccines and adoptive T cell transfer. The prerequisite for a successful T cell-based therapy is the induction of T cells capable of recognizing the HER2-expressing tumor cells. In this study, we generated human cytotoxic T cell clones directed against the HER2(369-377) epitope known to be naturally presented with HLA-A*0201. Those HER2-reactive CTLs, which were also tumor lytic, exhibited a similar lysis pattern dividing the targets in lysable and nonlysable tumor cells. Several HER2-expressing tumor cells became susceptible to CTL-mediated lysis after IFN-gamma treatment and, in parallel, up-regulated molecules of the Ag-presenting machinery, indicating that the tumor itself also contributes to the success of CTL-mediated killing. Some of the HER2(369-377)-reactive T cells specifically cross-reacted with the corresponding peptides derived from the family members HER3 and/or HER4 due to a high sequence homology. The epitopes HER3(356-364) and HER4(361-369) were endogenously processed and contributed to the susceptibility of cell lysis by HER cross-reacting CTLs. The principle of "double" or "triple targeting" the HER Ags by cross-reacting T cells will impact the further development of T cell-based therapies.     

Generation of tumor-reactive CTL against the tumor-associated antigen HER2 using retrovirally transduced dendritic cells derived from CD34+ hemopoietic progenitor cells

Ag-specific CD8+ CTL are crucial for effective tumor rejection. Attempts to treat human malignancies by adoptive transfer of tumor-reactive CTL have been limited due to the difficulty of generating and expanding autologous CTL with defined Ag specificity. The current study examined whether human CTL can be generated against the tumor-associated Ag HER2 using autologous dendritic cells (DC) that had been genetically engineered to express HER2. DC progenitors were expanded by culturing CD34+ hemopoietic progenitor cells in the presence of the designer cytokine HyperIL-6. Proliferating precursor cells were infected by a retroviral vector encoding the HER2 Ag and further differentiated into CD83+ DC expressing high levels of MHC, adhesion, and costimulatory molecules. Retroviral transduction of DC resulted in the expression of the HER2 molecule with a transduction efficiency of 15%. HER2-transduced DC correctly processed and presented the Ag, because HLA-A*0201-positive DC served as targets for CTL recognizing the HLA-A*0201-binding immunodominant peptide HER2(369-377). HER2-transduced DC were used as professional APCs for stimulating autologous T lymphocytes. Following repetitive stimulation, a HER2-specific, HLA-A*0201-restricted CTL line was generated that was capable of lysing HLA-A*0201-matched tumor cells overexpressing HER2. A CD8+ T cell clone could be generated that displayed the same specificity pattern as the parenteral CTL line. The ability to generate and expand HER2-specific, MHC class I-restricted CTL clones using HER2-transduced autologous DC in vitro facilitates the development of adoptive T cell transfer for patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.     

A novel treatment strategy of HER2-targeted therapy in combination with Everolimus for HR+/HER2- advanced breast cancer patients with HER2 mutations

The incidence of HER2 somatic mutations in breast cancer is about 2–4%, mainly occurring in the HR+/HER2- subtype. Preclinical studies suggest that HER2 mutations can lead to constitutive HER2 activation, but effective treatment options for the clinical management of patients with HER2 mutations remain obscure. Our study analyzed HER2 mutation status by performing next-generation sequencing using tumor tissues and over 300 plasma samples from 72 metastatic breast cancer patients. We observed that two patients bearing HER2 mutations (Patient #1 bearing S310F and V777L mutations, Patient #2 bearing 778insGSP mutation) achieved a durable partial response to Trastuzumab combined with Everolimus. In vitro experiments showed that T47D and MCF7 cells overexpressing these HER2 mutants (S310F, V777L, 778insGSP and L755S) were sensitive to HER2-targeted therapies combined with the mTOR inhibitor Everolimus. These findings provide a treatment option for patients with HER2 mutations by combining HER2-targeted therapies with Everolimus.     

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.     

An immunotherapy approach with dendritic cells genetically modified to express the tumor-associated antigen,HER2

Dendritic cells (DC), genetically modified to express ovalbumin by the retroviral vector GCDNsap, can elicit stronger anti-tumor immunity than those loaded with the peptides. To assess the clinical feasibility of the strategy, such DC were prepared by differentiation of hematopoietic progenitor cells transduced with the human epidermal growth factor receptor 2 (HER2). When inoculated in mice, the DC primed both HER2-specific cytotoxic T lymphocytes and type 1 T helper lymphocytes, resulting in production of HER2-specific antibody. Of importance is that the antibody mediated antibody-dependent cellular cytotoxicity and opsonization. The potent anti-tumor effects were also confirmed by results of experiments using HER2-transgenic mice. Inoculation of HER2-transduced DC resulted in longer disease-free survival of treated mice that showed significant reduction of primary and metastatic tumors. Interestingly, footpad inoculation resulted in stronger anti-tumor effects compared to subcutaneous administration and induced higher levels of the HER2-specific antibody, suggesting that an important role of humoral immunity in anti-tumor effects for malignancies with membrane-type tumor-associated antigens (TAA). Taken together, vaccination of the TAA-transduced DC may represent a promising form of therapy for breast cancers expressing HER2.     

Anti HER2 ScFv-GFP融合抗体靶向检测HER2的表达

为验证真核表达的携带绿色荧光的抗HER2单链抗体应用于临床诊断HER2阳性肿瘤细胞和病理组织的可靠性,构建融合基因Anti HER2 ScFv-GFP,重组入pFAST Bac HT A载体,在昆虫细胞Sf9中表达,以Ni2+-NTA亲和层析法纯化Anti HER2 ScFv-GFP融合蛋白,测定其浓度与纯度,将同浓度的纯化蛋白分别与3种乳腺癌细胞BT474、SKBR3和MCF7各混合12 h、24 h和48 h,分析其在不同时间段结合HER2阳性肿瘤细胞的稳定性。用纯化蛋白直接检测经抗原修复的乳腺癌病理组织,与免疫组织化学法检测结果对比。结果在昆虫细胞Sf9中可观察到明显绿色荧光,纯化的融合蛋白相对分子量约60 kDa,浓度为115.5μg/mL,纯度约97%,SKBR3和BT474鉴定为HER2阳性。结合12 h、24 h、48 h后其细胞表面均有明显绿色荧光,而HER2阴性MCF7被洗脱后无荧光,该抗体滴度为1:64,48 h内该荧光抗体仍具有稳定性。携带绿色荧光的融合抗体检测病理组织与IHC法的结果完全一致。表明成功表达的携带绿色荧光的抗HER2单链抗体可特异性检测HER2阳性乳腺癌细胞BT474和SKBR3,在HER2阳性肿瘤细胞和临床病理组织检测上具有应用前景。     

HER2 regulates Brk/PTK6 stability via upregulating calpastatin,an inhibitor of calpain

Breast tumor kinase (Brk), also known as protein kinase-6 (PTK6), is a nonreceptor protein-tyrosine kinase that has a close functional relationship with the human epidermal growth factor receptor 2 (HER2). High levels of Brk were found in HER2-positive tumor specimens from patients with invasive ductal breast cancer; however, the underlying mechanism of the co-overexpression of Brk and HER2 remains elusive. In the current study, we explored the mechanism of HER2 and Brk co-overexpression in breast cancer cells by investigating the effect of overexpression and knockdown of HER2 on the level of Brk in breast cancer cells. We found that Brk was more stable in HER2-elevated cells than in control vector-transfected cells and was less stable in HER2 siRNA-treated cells than in control siRNA-treated cells, suggesting that HER2 regulates Brk protein stability. Further studies indicated that degradation of Brk involved a calpain-1-mediated proteolytic pathway and indicated an inverse relationship between the level of HER2 expression and calpain-1 activity. We found that HER2 inhibited calpain-1 activity through upregulating calpastatin, an endogenous calpain inhibitor. Silencing of HER2 downregulated calpastatin, and the downregulation could be rescued by overexpression of constitutively active MEK. Together, these data offer novel mechanistic insights into the functional relationship between Brk and HER2.     

Primary Human Ovarian Epithelial Cancer Cells Broadly Express HER2 at Immunologically-Detectable Levels

The breadth of HER2 expression by primary human ovarian cancers remains controversial, which questions its suitability as a universal antigen in this malignancy. To address these issues, we performed extensive HER2 expression analysis on a wide panel of primary tumors as well as established and short-term human ovarian cancer cell lines. Conventional immunohistochemical (IHC) analysis of multiple tumor sites in 50 cases of high-grade ovarian serous carcinomas revealed HER2 overexpression in 29% of evaluated sites. However, more sensitive detection methods including flow cytometry, western blot analysis and q-PCR revealed HER2 expression in all fresh tumor cells derived from primary ascites or solid tumors as well as all established and short-term cultured cancer cell lines. Cancer cells generally expressed HER2 at higher levels than that found in normal ovarian surface epithelial (OSE) cells. Accordingly, genetically-engineered human T cells expressing an HER2-specific chimeric antigen receptor (CAR) recognized and reacted against all established or primary ovarian cancer cells tested with minimal or no reactivity against normal OSE cells. In conclusion, all human ovarian cancers express immunologically-detectable levels of HER2, indicating that IHC measurement underestimates the true frequency of HER2-expressing ovarian cancers and may limit patient access to otherwise clinically meaningful HER2-targeted therapies.     

A caspase-6 and anti-human epidermal growth factor receptor-2 (HER2) antibody chimeric molecule suppresses the growth of HER2-overexpressing tumors

Clinical studies have suggested that human epidermal growth factor receptor-2 (HER2) provide a useful target for antitumor therapy. We previously described the generation of a chimeric HER2-targeted immunocasp-3 protein. In this study, we extend the repertoire of chimeric proapoptotic proteins with immunocasp-6, a construct that comprises a HER2-specific single-chain Ab, a single-chain Pseudomonas exotoxin A, and an active caspase-6, which can directly cleave lamin A leading to nucleus damage and inducing programmed cell death. We demonstrate that the secreted immunocasp-6 molecule selectively recognizes and induces apoptosis in HER2-overexpressing tumor cells in vitro, but not in cells with undetectable HER2. The immunocasp-6 gene was next transferred into BALB/c athymic mice bearing human breast SK-BR-3 tumors by i.m. injection of liposome-encapsulated vectors, by intratumor injection of adenoviral vectors, or by i.v. injection of PBMC modified by retroviral infection. Regardless of the method used, expression of immunocasp-6 suppressed tumor growth and prolonged animal survival significantly. Our data show that the chimeric immunocasp-6 molecule can recognize HER2-positive tumor cells, promptly attack their nucleus, and induce their apoptotic death, suggesting the potential of this strategy for the treatment of human cancers that overexpress HER2.     

Antibody-drug conjugates in breast cancer: Marching from HER2-overexpression into HER2-low

The recent decade has witnessed a vigorous prosper of antibody-drug conjugates (ADCs) in solid tumors including breast cancer. Integrating the specificity of monoclonal antibodies and potency of cytotoxic drugs, ADCs are capable of delivering cytotoxic agents directly to tumor cells and surrounding accomplices with heterogeneous antigen expression by exerting the distinctive bystander effect. Up till now, three ADCs (T-DM1, T-DXd and SG) have attained the official approval and stepped into clinical practices in breast cancer, with numerous promising products in the pipeline. As an unprecedented breast cancer subgroup identified following solidified drug benefit, the cognitive and therapeutic paradigm of HER2-low population which was previously thought lacking definite targets and efficacious regimens has been thoroughly rewritten by ADCs, and several encouraging achievements are expected in ongoing trials. Herein, we discuss the contrived knowledge, latest advancements and future perspectives of ADCs in HER2-overexpressing and HER2-low breast cancer.     

Quantum dots-based double-color imaging of HER2 positive breast cancer invasion

It has been well recognized that human epidermal growth factor receptor 2 (HER2) level in breast cancer (BC) is closely related to the malignant biologic behaviors of the tumor, including invasion and metastasis. Yet, there has been a lack of directly observable evidence to support such notion. Here we report a quantum dots (QDs)-based double-color imaging technique to simultaneously show the HER2 level on BC cells and the type IV collagen in the tumor matrix. In benign breast tumor, the type IV collagen was intact. With the increasing of HER2 expression level, there has been a progressive decrease in type IV collagen around the cancer nest. At HER2 (3+) expression level, there has virtually been a total destruction of type IV collagen. Moreover, HER2 (3+) BC cells also show direct invasion into the blood vessels. This novel imaging method provides direct observable evidence to support the theory that the HER2 expression level is directly related to BC invasion.     

Specific targeting of HER2 overexpressing breast cancer cells with doxorubicin-loaded trastuzumab-modified human serum albumin nanoparticles

Specific targeting of tumor cells to achieve higher drug levels in tumor tissue and to overcome cardiotoxic and other secondary effects is the major goal in cancer therapy. With trastuzumab as a humanized monoclonal antibody binding, the HER2 receptor specific targeting is possible. In the present study, target-oriented nanoparticles based on biodegradable human serum albumin (HSA) loaded with cytostatic drug doxorubicin were developed. The surface of the nanoparticles was modified by covalent attachment of trastuzumab. HER2 overexpressing breast cancer cells showed a good cellular binding and uptake of these nanoparticles. The specific transport of the cytostatic drug doxorubicin with this nanoparticulate formulation into the HER2 overexpressing breast cancer cells, their release, and biological activity was demonstrated. The results indicate that these cell-type specific drug-loaded nanoparticles could achieve an improvement in cancer therapy. To our knowledge, this is the first study demonstrating a specific trastuzumab-based targeting of HER2 overexpressing breast cancer cells with doxorubicin-loaded nanoparticles.     

Cbl induced ubiquitination of HER2 mediate immune escape from HER2-targeted CAR-T

Breast cancer (BC) with high HER2 expression has higher recurrence rate and worse prognosis, and its immunotherapy is promising. Based on the high expression of HER2, develop Chimeric Antigen Receptor T-cell (CAR-T) and PDL-1 immunotherapy, and study the molecular pathways of related immune cells and recurrence. HER2-CAR-T cells were constructed using retroviruses, and their specific recognition and immune effects on HER2+ BC cells were verified by in vivo and in vitro experiments. PDL-1 was used as adjuvant immunotherapy, knocking down PDL-1 in tumor cells or dendritic cells, or depleted macrophages to study immune pathways. The negative regulation of HER2 by cbl was determined by IP, ubiquitination experiments, and segmented plasmids, elucidating the molecular mechanism of HER2+ BC recurrence after immunotherapy. HER2-CAR-T specifically recognizes HER2-positive tumor cells and inhibits tumor growth in vivo and in vitro, and anti-PDL1 treatment enhances the therapeutic effect of HER2-CAR-T on tumors. HER2-CART therapy eradicated solid tumors after PDL1 knockdown in dendritic cells. Immunotherapy of relapsed tumors lost HER2 expression by upregulating cbl. HER2-CAR-T shows specific recognition of HER2+ cells and can mediate immune response therapy with the cooperation of PDL-1.     

Specific blockade of VEGF and HER2 pathways results in greater growth inhibition of breast cancer xenografts that overexpress HER2

We have previously reported that breast cancer cells which overexpress HER2 produce higher levels of VEGF than cells with low levels of HER2. This study tested the hypothesis that dual targeting of the VEGF (with VEGF-Trap) and HER2 (with trastuzumab) pathways would result in greater growth inhibition of HER2-overexpressing breast cancer xenografts than either agent alone. In this study we found that human and murine endothelial cells expressed high levels of VEGF receptors (VEGFR1, VEGFR2, & VEGFR3). VEGF-Trap decreased levels of secreted VEGF derived from both human and murine cells and effectively blocked VEGF-induced tyrosine phosphorylation of VEGFR2. VEGF-Trap as a single treatment inhibited tumor microvessel density (MVD), tumor vasculature, cell proliferation, and tumor growth of BT474 xenografts in a dose-dependent manner from 2.5 mg/kg to 25 mg/kg. VEGF-Trap decreased levels of both human VEGF and PlGF protein in vivo. Trastuzumab as a single agent effectively inhibited BT474 tumor growth in a dose-dependent manner, associated with a decrease in human VEGF, tumor MVD and tumor cell proliferation. Treatment with a combination of VEGF-Trap (2.5-10 mg/kg) and trastuzumab (1 mg/kg) produced significantly greater inhibition of BT474tumor growth than either individual agent, associated with greater inhibition of tumor MVD and tumor cell proliferation. Thus, VEGF-Trap in combination with trastuzumab produces superior growth inhibition of tumor xenografts which overexpress HER2, which may result from inhibition of both tumor angiogenesis and proliferation. Similar mechanisms may contribute to the clinical anti-tumor activity of trastuzumab in combination with inhibitors of VEGF signaling pathway in women with breast cancers which overexpress HER2.     

Effective and persistent antitumor activity of HER2-directed CAR-T cells against gastric cancer cells in vitro and xenotransplanted tumors in vivo

Human epidermal growth factor receptor 2 (HER2) proteins are overexpressed in a high proportion of gastric cancer (GC) cases and affect the maintenance of cancer stem cell (CSC) subpopulations, which are used as targets for the clinical treatment of patients with HER2-positive GC. Despite improvements in survival, numerous HER2-positive patients fail treatment with trastuzumab, highlighting the need for more effective therapies. In this study, we generated a novel type of genetically modified human T cells, expressing a chimeric antigen receptor (CAR), and targeting the GC cell antigen HER2, which harbors the CD137 andCD3ζ moieties. Our findings show that the expanded CAR-T cells, expressing an increased central memory phenotype, were activated by the specific recognition of HER2 antigens in an MHC-independent manner, and effectively killed patient-derived HER2-positive GC cells. In HER2-positive xenograft tumors, CAR-T cells exhibited considerably enhanced tumor inhibition ability, long-term survival, and homing to targets, compared with those of non-transduced T cells. The sphere-forming ability and in vivo tumorigenicity of patient-derived gastric cancer stem-like cells, expressing HER2 and the CD44 protein, were also inhibited. Our results support the future development and clinical application of this adoptive immunotherapy in patients with HER2-positive advanced GC.     

HER2/HER3 signaling regulates NK cell-mediated cytotoxicity via MHC class I chain-related molecule A and B expression in human breast cancer cell lines

Overexpression of the receptor tyrosine kinases HER2 and HER3 is associated with a poor prognosis in several types of cancer. Presently, HER2- as well as HER3-targeted therapies are in clinical practice or evaluated within clinical trials, including treatment with mAbs mediating growth inhibition and/or activation of Ab-induced innate or adaptive cellular immunity. A better understanding of how HER2/HER3 signaling in tumors influences cellular immune mechanisms is therefore warranted. In this study, we demonstrate that HER2/HER3 signaling regulates the expression of MHC class I-related chain A and B (MICA and MICB) in breast cancer cell lines. The MICA and MICB (MICA/B) molecules act as key ligands for the activating receptor NK group 2, member D (NKG2D) and promote NK cell-mediated recognition and cytolysis. Genetic silencing of HER3 but not HER2 downregulated the expression of MICA/B, and HER3 overexpression significantly enhanced MICA expression. Among the major pathways activated by HER2/HER3 signaling, the PI3K/AKT pathway was shown to predominantly regulate MICA/B expression. Treatment with the HER3-specific ligand neuregulin 1β promoted the expression in a process that was antagonized by pharmacological and genetic interference with HER3 but not by the ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related protein kinases inhibitor caffeine. These observations further emphasize that HER2/HER3 signaling directly, and not via genotoxic stress, regulates MICA/B expression. As anticipated, stimulating HER2/HER3 enhanced the NKG2D-MICA/B-dependent NK cell-mediated cytotoxicity. Taken together, we conclude that signaling via the HER2/HER3 pathway in breast carcinoma cell lines may lead to enhanced NKG2D-MICA/B recognition by NK cells and T cells.     

Apigenin induces apoptosis through proteasomal degradation of HER2/neu in HER2/neu-overexpressing breast cancer cells via the phosphatidylinositol 3-kinase/Akt-dependent pathway

Apigenin is a low toxicity and non-mutagenic phytopolyphenol and protein kinase inhibitor. It exhibits anti-proliferating effects on human breast cancer cells. Here we examined several human breast cancer cell lines having different levels of HER2/neu expression and found that apigenin exhibited potent growth-inhibitory activity in HER2/neu-overexpressing breast cancer cells but was much less effective for those cells expressing basal levels of HER2/neu. Induction of apoptosis was also observed in HER2/neu-overexpressing breast cancer cells in a dose- and time-dependent manner. However, the one or more molecular mechanisms of apigenin-induced apoptosis in HER2/neu-overexpressing breast cancer cells remained to be elucidated. A cell survival pathway involving phosphatidylinositol 3-kinase (PI3K), and Akt is known to play an important role in inhibiting apoptosis in response to HER2/neu-overexpressing breast cancer cells, which prompted us to investigate whether this pathway plays a role in apigenin-induced apoptosis in HER2/neu-overexpressing breast cancer cells. Our results showed that apigenin inhibits Akt function in tumor cells in a complex manner. First, apigenin directly inhibited the PI3K activity while indirectly inhibiting the Akt kinase activity. Second, inhibition of HER2/neu autophosphorylation and transphosphorylation resulting from depleting HER2/neu protein in vivo was also observed. In addition, apigenin inhibited Akt kinase activity by preventing the docking of PI3K to HER2/HER3 heterodimers. Therefore, we proposed that apigenin-induced cellular effects result from loss of HER2/neu and HER3 expression with subsequent inactivation of PI3K and AKT in cells that are dependent on this pathway for cell proliferation and inhibition of apoptosis. This implies that the inhibition of the HER2/HER3 heterodimer function provided an especially effective strategy for blocking the HER2/neu-mediated transformation of breast cancer cells. Our results also demonstrated that apigenin dissociated the complex of HER2/neu and GRP94 that preceded the depletion of HER2/neu. Apigenin-induced degradation of mature HER2/neu involves polyubiquitination of HER2/neu and subsequent hydrolysis by the proteasome.