MEK inhibition leads to PI3K/AKT activation by relieving a negative feedback on ERBB receptors

The phosphoinositide 3-kinase (PI3K)/AKT and RAF/MEK/ERK signaling pathways are activated in a wide range of human cancers. In many cases, concomitant inhibition of both pathways is necessary to block proliferation and induce cell death and tumor shrinkage. Several feedback systems have been described in which inhibition of one intracellular pathway leads to activation of a parallel signaling pathway, thereby decreasing the effectiveness of single-agent targeted therapies. In this study, we describe a feedback mechanism in which MEK inhibition leads to activation of PI3K/AKT signaling in EGFR and HER2-driven cancers. We found that MEK inhibitor-induced activation of PI3K/AKT resulted from hyperactivation of ERBB3 as a result of the loss of an inhibitory threonine phosphorylation in the conserved juxtamembrane domains of EGFR and HER2. Mutation of this amino acid led to increased ERBB receptor activation and upregulation of the ERBB3/PI3K/AKT signaling pathway, which was no longer responsive to MEK inhibition. Taken together, these results elucidate an important, dominant feedback network regulating central oncogenic pathways in human cancer.     

RON confers lapatinib resistance in HER2-positive breast cancer cells

Lapatinib-resistance is a major problem for HER2-positive breast cancer treatment. SK-BR-3-LR, a lapatinib-resistant cell clone, was established from HER2-positive SK-BR-3 breast cancer cells following chronic exposure to lapatinib. The PI3K/AKT signaling pathway was demonstrated to be resistant to HER2 inhibition in SK-BR-3-LR cells. However, both small-molecular Recepteur d’Origine Nantais (RON) inhibitors and RON-targeted small interfering RNA (siRNA) effectively restored lapatinib sensitivity in these cells by inhibiting PI3K/AKT activation. Our results demonstrate for the first time the important role of RON in mediating lapatinib resistance and suggest that RON-targeted therapy may become a novel, promising therapeutic strategy after the failure of lapatinib treatment in patients with HER2-positive breast cancer.     

Resistance to HER2-directed antibodies and tyrosine kinase inhibitors: mechanisms and clinical implications

The antibody trastuzumab and the tyrosine kinase inhibitor lapatinib are approved by the FDA for the treatment of HER2-overexpressing breast cancer. These anti-HER2 drugs are changing the natural history of HER2-overexpressing breast cancer. However, therapeutic resistance to trastuzumab or lapatinib, as either single-agents or in combination with chemotherapy in the metastatic setting, typically occurs within months of starting therapy. Several mechanisms of trastuzumab-resistance have been reported that include signaling from other HER receptors, signaling from receptor tyrosine kinases (RTKs) outside of the HER (ErbB) family, increased phosphatidylinositol 3-kinase signaling, and the presence of truncated forms of HER2. Mechanisms of resistance to lapatinib also point to increased phosphatidylinositol 3-kinase signaling as well as derepression/activation of compensatory survival pathways. In this review, we discuss how these models and mechanisms enhance our understanding of the clinical resistance to HER2-directed therapies.     

HER2过表达乳腺癌曲妥珠单抗耐药后靶向治疗策略

 曲妥珠单抗与化疗药物联用后提高了人类表皮生长因子受体2（HER2）过表达乳腺癌的反应率,但在开始治疗后数月易出现治疗性耐药,限制了患者的总生存期。曲妥珠单抗耐药机制主要与HER家族以外的蛋白酪氨酸激酶受体信号通路活化、PI3K-AKT通路扩增等有关。酪氨酸激酶抑制剂、磷脂酰肌醇3-激酶抑制剂等分子靶向药物作为曲妥珠单抗耐药者的替代治疗手段,单药疗效均不甚满意。多个大型临床试验证实,新型分子靶向药物联合应用能明显限制,甚或最终消除曲妥珠单抗初始治疗耐药性问题。     

HER2 signaling modulates the equilibrium between pro- and antiangiogenic factors via distinct pathways: implications for HER2-targeted antibody therapy

We determined the impact of HER2 signaling on two proangiogenic factors, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), and on an antiangiogenic factor, thrombospondin-1 (TSP-1). Re-expression of HER2 in MCF-7 and T-47D breast cancer cells that endogenously express low levels of HER2 resulted in elevated expression of VEGF and IL-8 and decreased expression of TSP-1. Inhibition of HER2 with a humanized anti-HER2 antibody (trastuzumab, or Herceptin) or a retrovirus-mediated small interfering RNA against HER2 (siHER2) decreased VEGF and IL-8 expression, but increased TSP-1 expression in BT474 breast cancer cells that express high levels of HER2. These in vitro results were further evaluated by treatment of BT474 xenografts in immunosuppressed mice with trastuzumab. Trastuzumab inhibited growth of BT474 xenografts and decreased microvascular density associated with downregulation of VEGF and IL-8 and with upregulation of TSP-1 expression. Inhibiting the PI3K-AKT pathway decreased VEGF and IL-8 expression. AKT1 overexpession increased VEGF and IL-8 expression, but did not increase TSP-1 expression. A p38 kinase inhibitor, SB203580, instead blocked TSP-1 expression and a p38 activator, MKK6, increased TSP-1 expression. Trastuzumab stimulated sustained p38 activation and SB203580 attenuated the TSP-1 upregulation induced by trastuzumab. HER2 signaling therefore influences the equilibrium between pro- and antiangiogenic factors via distinct signaling pathways. Trastuzumab inhibits angiogenesis and tumor growth, at least in part, through activation of the HER2-p38-TSP-1 pathway and inhibition of the HER2-PI3K-AKT-VEGF/IL-8 pathway.     

PI3K inhibitors in trastuzumab-resistant HER2-positive breast cancer cells with PI3K pathway alterations

The activation of the PI3K signaling pathway resulting from genetic alterations induces carcinogenesis and resistance to anticancer therapies. Breast cancer is a major malignancy that is associated with dysregulation of the PI3K signaling pathway. PIK3CA mutations and PTEN loss occur in every subtype of breast cancer. PI3K inhibitors are being evaluated in breast cancer after the success of an alpha isoform-specific PI3K inhibitor in estrogen receptor (ER)-positive/HER2-negative metastatic breast cancer. Some preclinical data indicate the potential for PI3K/mTOR targeting in combination with trastuzumab for HER2-positive breast cancer with or without expression of the estrogen receptor. However, the role of this therapy in HER2-positive breast cancer with PIK3CA mutations and/or PTEN loss remains unclear. We examined three HER2-positive, ER-negative breast cancer cell lines to determine the efficacy of a novel alpha isoform-specific PI3K inhibitor in combination with trastuzumab. The results indicated that this combination was effective in PIK3CA-mutant or PTEN-deficient breast cancer cells by inducing apoptosis and inhibiting the expression of downstream proteins. PTEN loss by siRNA modulation in parental HER2-positive cancer cells with PI3K signaling pathway alterations could not confer resistance to alpelisib or GDC-0077 plus trastuzumab. We selected the CK-MB-1 cell line without alterations in the PI3K pathway to demonstrate that PI3K inhibitors plus trastuzumab represented a biomarker-specific treatment. In vivo effects of alpelisib plus trastuzumab were tested and confirmed in a mouse model, showing the combination strategy offered the best opportunity to achieve tumor volume reduction. With known safety profiles, this cytotoxic chemotherapy-free regimen warrants further attention as a biomarker-driven strategy for treating HER2-positive breast cancer.     

Antibody targeting of HER2/HER3 signaling overcomes heregulin‐induced resistance to PI3K inhibition in prostate cancer

Dysregulated expression and/or mutations of the various components of the phosphoinositide 3‐kinase (PI3K)/Akt pathway occur with high frequency in prostate cancer and are associated with the development and progression of castration resistant tumors. However, small molecule kinase inhibitors that target this signaling pathway have limited efficacy in inhibiting tumor growth, primarily due to compensatory survival signals through receptor tyrosine kinases (RTKs). Although members of the epidermal growth factor receptor (EGFR), or HER, family of RTKs are strongly implicated in the development and progression of prostate cancer, targeting individual members of this family such as EGFR or HER2 has resulted in limited success in clinical trials. Multiple studies indicate a critical role for HER3 in the development of resistance against both HER‐targeted therapies and PI3K/Akt pathway inhibitors. In this study, we found that the growth inhibitory effect of GDC‐0941, a class I PI3K inhibitor, is markedly reduced in the presence of heregulin. Interestingly, this effect is more pronounced in cells lacking phosphatase and tensin homolog function. Heregulin‐mediated resistance to GDC‐0941 is associated with reactivation of Akt downstream of HER3 phosphorylation. Importantly, combined blockade of HER2 and HER3 signaling by an anti‐HER2/HER3 bispecific antibody or a mixture of anti‐HER2 and anti‐HER3 antibodies restores sensitivity to GDC‐0941 in heregulin‐treated androgen‐dependent and ‐independent prostate cancer cells. These studies indicate that the combination of PI3K inhibitors with HER2/HER3 targeting antibodies may constitute a promising therapeutic strategy for prostate cancer.     

PI3K pathway inhibitors for the treatment of brain metastases with a focus on HER2+ breast cancer

The incidence of breast cancer brain metastases has increased in recent years, largely due to improved control of systemic disease with human epidermal growth factor receptor 2 (HER2)-targeted agents and the inability of most of these agents to efficiently cross the blood–blood barrier (BBB) and control central nervous system disease. There is, therefore, an urgent unmet need for treatments to prevent and treat HER2+ breast cancer brain metastases (BCBMs). Aberrant activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway is frequently observed in many cancers, including primary breast tumors and BCBMs. Agents targeting key components of this pathway have demonstrated antitumor activity in diverse cancers, and may represent a new treatment strategy for BCBMs. In preclinical studies, several inhibitors of PI3K and mTOR have demonstrated an ability to penetrate the BBB and down-regulate PI3K signaling, indicating that these agents may be potential therapies for brain metastatic disease. The PI3K inhibitor buparlisib (BKM120) and the mTOR inhibitor everolimus (RAD001) are currently under evaluation in combination with trastuzumab in patients with HER2+ BCBMs.     

Molecular basis for sensitivity and acquired resistance to gefitinib in HER2-overexpressing human gastric cancer cell lines derived from liver metastasis

Gastric cancer metastasised to the liver was found to overexpress HER2 at a significantly higher incidence than primary gastric cancers. The purpose of the present study was to investigate the possibility of molecular therapy targeting HER2 overexpression in gastric cancer liver metastasis. We developed three new HER2-overexpressing gastric cancer cell lines (GLM-1, GLM-2, GLM-4) without epidermal growth factor receptor (EGFR) mutations derived from such liver metastasis, two of which had HER2 gene amplifications. All these GLM series of cell lines were highly sensitive to gefitinib in vitro, a specific inhibitor of EGFR tyrosine kinase (Iressa) rather than anti-HER2 antibody trastuzumab (Herceptin), whereas most of the HER2 low-expressing counterparts were not. In these HER2-overexpressing GLM series, protein kinase B (Akt), but not extracellular signal-regulated kinase 1/2 (ERK1/2), was constitutively phosphorylated, and gefitinib efficiently inhibited this Akt phosphorylation, induced strong apoptosis in vitro and exhibited antitumour activity in tumour xenografts in nude mice. This gefitinib-mediated antitumour effect in xenograft was significantly potentiated by trastuzumab treatment. On the other hand, gefitinib-resistant cells (GLM-1R) exhibited increased EGFR expression, followed by constitutive activation of mitogen-activated protein kinase (MAPK) pathway. These results suggest that the antitumour effect of gefitinib is due to the effective inhibition of HER2-driven constitutive activation of phosphatidylinositol-3-kinase (PI3K)/Akt pathway, and that the acquired resistance to gefitinib is due to the constitutive activation of Ras/MAPK pathway in compensation for PI3K/Akt pathway. Gastric cancer liver metastasis with HER2 overexpression would be a potential molecular target for gefitinib and trastuzumab.     

PI3K and MEK inhibitor combinations: examining the evidence in selected tumor types

The PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways are two of the most frequently dysregulated kinase cascades in human cancer. Molecular alterations in these pathways are implicated in tumorigenesis and resistance to anticancer therapies. The PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways are known to interact with each other at several nodes, and mounting evidence suggests that dual blockade of both pathways may be required to achieve anticancer effects in certain contexts. This may include tumor types with a high frequency of RAS/RAF/MEK/ERK pathway activation, or situations in which dual pathway strategies may be required to overcome resistance to current targeted therapies. Several clinical studies are currently evaluating the combination of PI3K and MEK inhibitors in a variety of different cancers with certain types of molecular alterations. This review will summarize existing knowledge of the PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways, the cross-talk between them, and the current generation of PI3K and MEK inhibitors that target them. The preclinical rationale for dual pathway inhibition will be discussed within the context of the major tumor types currently being explored in ongoing clinical trials, namely malignant melanoma with BRAF or NRAS mutations, and colorectal, ovarian, pancreatic, and basal-like breast cancers. The emerging clinical profile of PI3K and MEK inhibitor combinations, as reported in Phase I trials, will also be discussed.     

The PI3K/AKT/mTOR Signaling Pathway: Implications in the Treatment of Breast Cancer

Epidemiologic and experimental studies support a key role of the phosphatidyl inositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway in the biology of human cancers. Alterations resulting in activation of PI3K/Akt/mTOR signaling are perhaps the most frequent events observed in solid tumors, including breast cancer, and contribute to neoplastic transformation. The PI3K/mTOR pathway can be activated by overproduction of growth factors or chemokines, loss of phosphatase and tensin homolog (PTEN) expression, or by mutations in growth factor receptors Ras, PTEN, or PI3K itself. Activation of this pathway contributes to cell cycle proliferation, growth, cell cycle entry, survival, cell motility, protein synthesis, and glucose metabolism, all important aspects of tumorigenesis. The most common genetic aberrations in breast cancer are activating somatic missense mutations in the gene encoding the p110a (PIK3CA) subunit of PI3K. The PTEN gene is often hypermethylated or decreased in expression, through as yet unclear mechanisms, in breast cancer. Studies have shown that PI3K/PTEN/AKT pathway modulation is implicated in HER2/neu-tumorigenesis and in response to the HER2-targeting antibody trastuzumab. Components of the pathway are regulated by feed-back and cross-talk to other signaling cascades and appear to be implicated with drug resistance. Over the past few years, a number of components of this signaling cascade have been the subject of intense drug-discovery activities. Rapamycin analogs have already been shown to have antitumor efficacy in some tumor types. Newer-generation PI3K, AKT, and mTOR inhibitors have shown significant promise preclinically and are now in clinical trials. This article summarizes the progress made in the elucidation of the pathway, clinical implications in pathology of breast cancer, and reviews novel drugs targeting this pathway for cancer treatment, particularly inhibitors of PI3K, AKT, and mTOR, currently undergoing clinical trials. Potential combination strategies, safety concerns, and resistance mechanisms for this new generation of anticancer agents are also discussed.     

Human epidermal growth factor receptor 2 regulates angiopoietin-2 expression in breast cancer via AKT and mitogen-activated protein kinase pathways

Abnormal activation of human epidermal growth factor receptor 2 (HER2; ErbB-2) in breast tumors results in increased metastasis and angiogenesis, as well as reduced survival. Here, we show that angiopoietin-2 (Ang-2) expression correlates with HER2 activity in human breast cancer cell lines. Inhibiting HER2 activity with anti-HER2 monoclonal antibody trastuzumab (Herceptin) or HER2 short interfering RNA in tumor cells down-regulates Ang-2 expression. Consistent with the important roles of AKT and mitogen-activated protein kinase in the HER2 signaling pathway, AKT and ERK mitogen-activated protein kinase (MAPK) kinase activity is necessary for Ang-2 up-regulation by HER2. Moreover, overexpression of HER2 protein up-regulates Ang-2 expression. Heregulin-beta1-induced Ang-2 up-regulation is abrogated when AKT and ERK kinase activity are blocked. Immunohistochemical analysis of HER2 and Ang-2 proteins in human breast carcinomas shows that Ang-2 expression in breast cancer correlates with HER2 expression. These studies provide evidence that the Ang-2 gene is regulated by HER2 activity in breast cancer, and propose an additional mechanism for HER2 contributing to tumor angiogenesis and metastasis.     

PI3K activation promotes resistance to eribulin in HER2-negative breast cancer

Background Eribulin is a microtubule-targeting agent approved for the treatment of advanced or metastatic breast cancer (BC) previously treated with anthracycline- and taxane-based regimens. PIK3CA mutation is associated with worse response to chemotherapy in oestrogen receptor-positive (ER+)/human epidermal growth factor receptor 2-negative (HER2−) metastatic BC. We aimed to evaluate the role of phosphoinositide 3-kinase (PI3K)/AKT pathway mutations in eribulin resistance.Methods Resistance to eribulin was evaluated in HER2− BC cell lines and patient-derived tumour xenografts, and correlated with a mutation in the PI3K/AKT pathway.Results Eleven out of 23 HER2− BC xenografts treated with eribulin exhibited disease progression. No correlation with ER status was detected. Among the resistant models, 64% carried mutations in PIK3CA, PIK3R1 or AKT1, but only 17% among the sensitive xenografts (P = 0.036). We observed that eribulin treatment induced AKT phosphorylation in vitro and in patient tumours. In agreement, the addition of PI3K inhibitors reversed primary and acquired resistance to eribulin in xenograft models, regardless of the genetic alterations in PI3K/AKT pathway or ER status. Mechanistically, PI3K blockade reduced p21 levels likely enabling apoptosis, thus sensitising to eribulin treatment.Conclusions PI3K pathway activation induces primary resistance or early adaptation to eribulin, supporting the combination of PI3K inhibitors and eribulin for the treatment of HER2− BC patients.Subject terms: Breast cancer, Tumour biomarkers, Predictive markers, Cancer therapeutic resistance     

Roles of BIM induction and survivin downregulation in lapatinib-induced apoptosis in breast cancer cells with HER2 amplification

Lapatinib, a dual tyrosine kinase inhibitor of the epidermal growth factor receptor and human epidermal growth factor receptor 2 (HER2), is clinically active in patients with breast cancer positive for HER2 amplification. The mechanism of this anti-tumor action has remained unclear, however. We have now investigated the effects of lapatinib in HER2 amplification-positive breast cancer cells with or without an activating PIK3CA mutation. Lapatinib induced apoptosis in association with upregulation of the pro-apoptotic protein Bcl-2 interacting mediator of cell death (BIM) through inhibition of the MEK-ERK signaling pathway in breast cancer cells with HER2 amplification. RNA interference (RNAi)-mediated depletion of BIM inhibited lapatinib-induced apoptosis, implicating BIM induction in this process. The pro-apoptotic effect of lapatinib was less pronounced in cells with a PIK3CA mutation than in those without one. Lapatinib failed to inhibit AKT phosphorylation in PIK3CA mutant cells, likely because of hyperactivation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway by the mutation. Depletion of PIK3CA (a catalytic subunit of PI3K) revealed that survivin expression is regulated by the PI3K pathway in these cells, suggesting that insufficient inhibition of PI3K-survivin signaling is responsible for the limited pro-apoptotic effect of lapatinib in HER2 amplification-positive cells with a PIK3CA mutation. Consistent with this notion, depletion of survivin by RNAi or treatment with a PI3K inhibitor markedly increased the level of apoptosis in PIK3CA mutant cells treated with lapatinib. Our results thus suggest that inhibition of both PI3K-survivin and MEK-ERK-BIM pathways is required for effective induction of apoptosis in breast cancer cells with HER2 amplification.     

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.     

MEK抑制剂在EGFR-TKI靶向治疗非小细胞肺癌中的作用

非小细胞肺癌是最常见的肺癌,最常见的基因突变是EGFR突变,EGFR-TKI已被用于治疗含这类突变的患者。然而,随着治疗进展,患者逐渐出现耐药性导致治疗失败。主要原因是EGFR信号通路下游重新激活,其中RAS/RAF/MEK/ERK和PI3K/AKT/PKC途径最重要。ERK1/2信号再激活可产生对EGFR抑制剂的抗性。目前临床研究已经发现,MEK抑制剂可以抑制ERK磷酸化,从而阻止随后的MAP激酶下游磷酸化,并因此诱导肿瘤活动的退化和停滞。大量试验表明,ERK途径的持续激活有助于获得吉非替尼耐药性。MEK抑制剂还可以诱导细胞周期阻滞和凋亡。本文总结了MEK抑制剂和EGFR-TKI的作用及其在NSCLC治疗中的作用,为肺癌分子靶向治疗提供了新思路。     

Trastuzumab has preferential activity against breast cancers driven by HER2 homodimers

In breast cancer cells with HER2 gene amplification, HER2 receptors exist on the cell surface as monomers, homodimers, and heterodimers with EGFR/HER3. The therapeutic antibody trastuzumab, an approved therapy for HER2(+) breast cancer, cannot block ligand-induced HER2 heterodimers, suggesting it cannot effectively inhibit HER2 signaling. Hence, HER2 oligomeric states may predict the odds of a clinical response to trastuzumab in HER2-driven tumors. To test this hypothesis, we generated nontransformed human MCF10A mammary epithelial cells stably expressing a chimeric HER2-FKBP molecule that could be conditionally induced to homodimerize by adding the FKBP ligand AP1510, or instead induced to heterodimerize with EGFR or HER3 by adding the heterodimer ligands EGF/TGFα or heregulin. AP1510, EGF, and heregulin each induced growth of MCF10A cells expressing HER2-FKBP. Trastuzumab inhibited homodimer-mediated but not heterodimer-mediated cell growth. In contrast, the HER2 antibody pertuzumab, which blocks HER2 heterodimerization, inhibited growth induced by heregulin but not AP1510. Lastly, the HER2/EGFR tyrosine kinase inhibitor lapatinib blocked both homodimer- and heterodimer-induced growth. AP1510 triggered phosphorylation of Erk1/2 but not AKT, whereas trastuzumab inhibited AP1510-induced Erk1/2 phosphorylation and Shc-HER2 homodimer binding, but not TGFα-induced AKT phosphorylation. Consistent with these observations, high levels of HER2 homodimers correlated with longer time to progression following trastuzumab therapy in a cohort of patients with HER2-overexpressing breast cancer. Together, our findings confirm the notion that HER2 oligomeric states regulate HER2 signaling, also arguing that trastuzumab sensitivity of homodimers may reflect their inability to activate the PI3K (phosphoinositide 3-kinase)/AKT pathway. A clinical implication of our results is that high levels of HER2 homodimers may predict a positive response to trastuzumab.     

Anti-HER3 Domain 1 and 3 Antibodies Reduce Tumor Growth by Hindering HER2/HER3 Dimerization and AKT-Induced MDM2, XIAP,and FoxO1 Phosphorylation

Blockade of the human epidermal growth factor receptor 3 (HER3) and of the downstream phosphatidylinositide 3-kinase (PI3K)/AKT pathway is a prerequisite for overcoming drug resistance and to develop novel treatments for cancers that are not eligible for the currently approved targeted therapies. To this end, we generated specific antibodies (Abs) against domain 1 (D1) and domain 3 (D3) of HER3 that recognize epitopes that do not overlap with the neuregulin-binding site. The fully human H4B-121 Ab and the mouse monoclonal Abs 16D3-C1 and 9F7-F11 inhibited tumor growth in nude mice xenografted with epidermoid, pancreatic, or triple-negative breast cancer cells. The combination of one anti-HER3 Ab and trastuzumab improved tumor growth inhibition in mice xenografted with HER2^low cancer cell lines, for which trastuzumab alone shows no or moderate efficiency. Ab-induced disruption of tumor growth was associated with G₁ cell cycle arrest, proliferation inhibition, and apoptosis of cancer cells. Anti-HER3 Abs blocked HER2/HER3 heterodimerization and HER3 phosphorylation at the cell membrane, leading to inhibition of phosphorylation of the downstream AKT targets murine double minute 2, X-linked inhibitor of apoptosis, and forkhead box O1. This study demonstrates that anti-HER3 D1 and D3 Abs could represent a new option for immunotherapy of pancreatic and triple-negative breast cancers.