Trastuzumab and lapatinib modulation of HER2 tyrosine/threonine phosphorylation and cell signaling

Cell surface transmembrane signaling receptors EGFR, HER3, and HER4 are activated by ligand-binding-mediated dimerization and phosphorylation. In contrast, HER2 amplification promotes signaling by increasing homo/heterodimerization and ligand binding. Trastuzumab or lapatinib therapy of HER2 amplicon-positive breast cancer cells induces growth inhibition and intracellular growth pathway signaling modulation. The mechanism(s) by which trastuzumab, an IgG1 humanized antibody, induces modification of cell signaling upon binding to an extracellular determinant on a ligand-less ??receptor?? membrane protein remains unexplained. Using immune detection methodology comprised of antibodies detecting three distinct domains of HER and five tyrosine/threonine phosphorylation sites, the effects of trastuzumab and lapatinib were defined during steady state growth inhibition. Here, we show that lapatinib markedly reduces HER2 tyrosine phosphorylation, while in contrast, no change in tyrosine phosphate levels is detected during trastuzumab-mediated cell growth inhibition. As trastuzumab treatment does not change either the steady state HER2 protein levels or HER2 mRNA, these findings argue against an antibody-dependent alteration in internalization kinetics. We further show a sequenced relationship between lapatinib-induced blockage of phosphorylation (6?C8?h) and induction of delayed cell death (5?C6?days), while trastuzumab-treated cells showed no evidence of cell death up to 9?days. Taken together, these results demonstrate that inhibition of HER2 phosphorylation by lapatinib is sufficient to induce apoptosis while trastuzumab binding to the extracellular HER2 domain may function by sterically modulating the detection of phosphate moieties by cytoplasmic signal transducers. This investigation also detected a 20?kD protein, which is down-regulated by lapatinib, further demonstrating the complexity of this signal transduction system.     

S-adenosylhomocysteine hydrolase inhibition by 3-deazaneplanocin A analogues induces anti-cancer effects in breast cancer cell lines and synergy with both histone deacetylase and HER2 inhibition

Epigenetic abnormalities including abnormal histone methyltransferase activity contribute to breast cancer pathogenesis. An example is over expression of the polycomb repressive complex (PRC) 2 member enhancer of zeste homolog 2 (EZH2) which is linked to epigenetic silencing and poor prognosis. Recent evidence shows that S-adenosylhomocysteine (AdoHcy) hydrolase inhibitors (AHI) such as 3-deazaneplanocin A (DZNep) modulate chromatin through indirect inhibition of histone methyltransferases including EZH2. We investigated the biological effects of AdoHcy hydrolase inhibition using DZNep and its structural analogues 3-deazaadenosine (DZA) and neplanocin A (Nep A) in breast cancer cells. EZH2 protein expression was decreased and dose dependent growth inhibition occurred with variable potencies in MCF7, MDA-MB-231 and SKBr3 breast cancer cells. Cellular proliferation was inhibited through G₂/M cell cycle arrest and apoptosis. In addition breast cancer cells accumulated cytoplasmic lipid droplets in response to AdoHcy hydrolase inhibition consistent with a differentiating effect. Each analogue induced a similar pattern of biological activity against breast cancer cells but with differences in potency (DZA > DZNep > Nep A). Co-administration with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) induced synergistic inhibition of breast cancer cell proliferation. Furthermore, the relatively AHI resistant human epidermal growth factor receptor 2 (HER2) positive cell line SKBr3 underwent synergistic growth inhibition in response to co-treatment with the HER2 directed therapeutic antibody trastuzumab. In conclusion, AHI induce growth inhibition, cell cycle arrest, apoptosis and differentiation in breast cancer cells and synergise with HDAC and HER2 inhibition. Targeting histone methyltransferase activity might be of therapeutic value in breast cancer.     

Taxanes enhance trastuzumab-mediated ADCC on tumor cells through NKG2D-mediated NK cell recognition

Recent clinical data indicate a synergistic therapeutic effect between trastuzumab and taxanes in neoadjuvantly treated HER2-positive breast cancer (BC) patients. In HER2+ BC experimental models and patients, we investigated whether this synergy depends on the ability of drug-induced stress to improve NK cell effectiveness and thus trastuzumab-mediated ADCC. HER2+ BC cell lines BT474 and MDAMB361 treated with docetaxel showed up-modulation of NK activator ligands both in vitro and in vivo, accompanied by a 15–40% increase in in vitro trastuzumab-mediated ADCC; antibodies blocking the NKG2D receptor significantly reduced this enhancement. NKG2D receptor expression was increased by docetaxel treatment in circulating and splenic NK cells from mice xenografted with tumor cells, an increase related to expansion of the CD11b⁺Ly6G⁺ cell population. Accordingly, NK cells derived from HER2+ BC patients after treatment with taxane-containing therapy expressed higher levels of NKG2D receptor than before treatment. Moreover, plasma obtained from these patients recapitulated the modulation of NKG2D on healthy donors'' NK cells, improving their trastuzumab-mediated activity in vitro. This enhancement occurred mainly using plasma from patients with low NKG2D basal expression. Our results indicate that taxanes increase tumor susceptibility to ADCC by acting on tumor and NK cells, and suggest that taxanes concomitantly administered with trastuzumab could maximize the antibody effect, especially in patients with low basal immune effector cytotoxic activity.     

Synergistic antitumor efficacy against the EGFRvIII+HER2+ breast cancers by combining trastuzumab with anti-EGFRvIII antibody CH12

Although Trastuzumab, an anti-HER2 antibody, benefits certain patients with HER2-overexpressing breast cancer, de novo or acquired trastuzumab resistance remains a haunting issue. EGFRvIII, co-expressing with HER2 in some breast tumors, indicates a poor clinical prognosis. However, the role of EGFRvIII in the function of trastuzumab is not clear. Here, we demonstrated that EGFRvIII overexpression contributed to de novo trastuzumab resistance and the feedback activation of STAT3 caused by trastuzumab also resulted in acquired resistance in EGFRvIII⁺HER2⁺ breast cancers. CH12, a highly effective anti-EGFRvIII monoclonal antibody that preferentially binds to EGFRvIII, significantly suppressed the growth of EGFRvIII⁺HER2⁺ breast cancer cells in vitro and in vivo. Importantly, CH12 in combination with trastuzumab had a synergistic inhibitory effect on EGFRvIII⁺HER2⁺ breast cancers in vitro and in vivo via attenuating the phosphorylation of EGFR and HER2 and their downstream signal pathways more effectively and reversing STAT3 feedback activation. Moreover, the combination therapy suppressed angiogenesis and induced cell apoptosis significantly. Together, these results suggested a synergistic efficacy of the combination of trastuzumab with CH12 against EGFRvIII⁺HER2⁺ breast cancers, which might be a potential clinical application in the future.     

Trastuzumab in combination with PEGylated interferon-α1b exerts synergistic antitumor activity through enhanced inhibition of HER2 downstream signaling and antibody-dependent cellular cytotoxicity

The anti-HER2 monoclonal antibody trastuzumab is the mainstay of treatment for HER2-positive breast and gastric cancer, and its combination with multiple chemotherapeutic agents has represented an effective and rational strategy in the clinic. In this study, we report that trastuzumab in combination with PEGylated interferon-α1b (IFN-α1b), a polyethylene glycol (PEG)-conjugated form of a subtype of interferon alpha (IFN-α), synergistically inhibited the proliferation of HER2-positive cells, including BT-474 and SK-BR-3 breast cancer cells and NCI-N87 gastric cancer cells, and also induced their apoptosis, but had no effect on HER2-negative MDA-MB-231 breast cancer cells. Trastuzumab inhibited phosphorylation of HER2, AKT and ERK, an effect that was enhanced by PEGylated IFN-α1b, likely owing to PEGylated IFN-α1b-mediated downregulation of HER2 through the lysosomal degradation pathway. Moreover, PEGylated IFN-α1b significantly enhanced trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) in HER2-positive cells. Importantly, trastuzumab combined with PEGylated IFN-α1b exhibited significant synergistic antitumor activity in HER2-positive BT-474 xenografts, an effect that was associated with enhanced inhibition of HER2 expression and AKT and ERK phosphorylation. Strikingly, depletion of natural killer cells with anti-Asialo GM1 antibody abrogated the synergistic antitumor activity, indicating that augmented ADCC is essential for this synergy. Taken together, our findings indicate that both enhanced inhibition of HER2 downstream signaling and augmented ADCC contribute to the synergistic antitumor activity of trastuzumab with PEGylated IFN-α1b, and imply that combining trastuzumab with PEGylated IFN-α1b could be a promising strategy for HER2-positive cancers.     

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

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

Lapatinib acts on gastric cancer through both antiproliferative function and augmentation of trastuzumab-mediated antibody-dependent cellular cytotoxicity

Background

Trastuzumab has been recently approved for clinical use to treat HER2-expressing advanced gastric cancer, and anti-HER2-targeting therapy has become a promising option for gastric cancer. Lapatinib is a dual tyrosine kinase inhibitor targeting EGFR and HER2. The aim of the present study was to explore the utility of lapatinib for gastric cancer, with a particular focus on trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC).

Methods

Nine gastric cancer cell lines were evaluated for the effects of lapatinib on the cell-surface accumulation of HER2 and analyzed for their additional effects on trastuzumab-mediated ADCC. Also, HER2 signaling with Western blot, proliferative function with the MTT assay, and apoptosis-inducing activity with 7ADD/Annexin-V were investigated when a panel of gastric cancer cell lines was treated with lapatinib.

Results

Lapatinib inhibited HER2 signaling and cell proliferation in the panel of gastric cancer cell lines. Lapatinib also induced the accumulation of HER2 on the cell surface, resulting in the enhancement of trastuzumab-mediated ADCC of gastric cancer.

Conclusions

Lapatinib exhibits inhibitory activity in gastric cancer cells, and the combination of lapatinib with trastuzumab may be a promising treatment strategy for gastric cancer patients.     

Tunicamycin enhances the antitumor activity of trastuzumab on breast cancer in vitro and in vivo

Trastuzumab, a humanized monoclonal antibody targeting HER2, has demonstrated clinical benefits for women with HER2-positive breast cancer; however, trastuzumab resistance remains the biggest clinical challenge. In this study, results showed that tunicamycin, an inhibitor of N-glycosylation, synergistically enhanced the antitumor activity of trastuzumab against HER2-overexpressing breast cancer cells through induction of cell cycle arrest and apoptosis. Combined treatment of tunicamycin with trastuzumab dramatically decreased the expression of EGFR family and its down signaling pathway in SKBR3 and MCF-7/HER2 cells. Tunicamycin dose-dependently inhibited tumor growth in both of SKBR3 xenografts and MCF-7/HER2 xenografts. Optimal tunicamycin without inducing ER stress in liver tissue significantly increased the antitumor effect of trastuzumab in MCF-7/HER2 xenografts. Combinations of trastuzumab with N-glycosylation inhibitors tunicamycin may be a promising approach for improving clinical efficacy of trastuzumab.     

Exosomal pMHC-I complex targets T cell-based vaccine to directly stimulate CTL responses leading to antitumor immunity in transgenic FVBneuN and HLA-A2/HER2 mice and eradicating trastuzumab-resistant tumor in athymic nude mice

One of the major obstacles in human epidermal growth factor receptor 2 (HER2)-specific trastuzumab antibody immunotherapy of HER2-positive breast cancer is the development of trastuzumab resistance, warranting the search for other therapeutic strategies. Using mouse models, we previously demonstrated that ovalbumin (OVA)-specific dendritic cell (DC)-released exosome (EXO_OVA)-targeted CD4⁺ T cell-based (OVA-T_EXO) vaccine stimulates efficient cytotoxic T lymphocyte (CTL) responses via exosomal peptide/major histocompatibility complex (pMHC)-I, exosomal CD80 and endogenous IL-2 signaling; and long-term CTL memory by means of via endogenous CD40L signaling. In this study, using two-photon microscopy, we provide the first visual evidence on targeting OVA-T_EXO to cognate CD8⁺ T cells in vivo via exosomal pMHC-I complex. We prepared HER2/neu-specific Neu-T_EXO and HER2-T_EXO vaccines using adenoviral vector (AdVneu and AdV_HER2)-transfected DC (DCneu and DC_HER2)-released EXOs (EXOneu and EXO_HER2), and assessed their stimulatory effects on HER2/neu-specific CTL responses and antitumor immunity. We demonstrate that Neu-T_EXO vaccine is capable of stimulating efficient neu-specific CTL responses, leading to protective immunity against neu-expressing Tg1-1 breast cancer in all 6/6 transgenic (Tg) FVBneuN mice with neu-specific self-immune tolerance. We also demonstrate that HER2-T_EXO vaccine is capable of inducing HER2-specific CTL responses and protective immunity against transgene HLA-A2⁺HER2⁺ BL6-10_A2/HER2 B16 melanoma in 2/8 double Tg HLA-A2/HER2 mice with HER2-specific self-immune tolerance. The remaining 6/8 mice had significantly prolonged survival. Furthermore, we demonstrate that HER2-T_EXO vaccine stimulates responses of CD8⁺ T cells capable of not only inducing killing activity to HLA-A2⁺HER2⁺ BL6-10_A2/HER2 melanoma and trastuzumab-resistant BT474_A2 breast cancer cells in vitro but also eradicating 6-day palpable HER2⁺ BT474_A2 breast cancer (3–4 mm in diameter) in athymic nude mice. Therefore, the novel T cell-based HER2-T_EXO vaccine may provide a new therapeutic alternative for women with HER2⁺ breast cancer, especially for trastuzumab-resistant HER2⁺ breast cancer patients.     

The Global Need for a Trastuzumab Biosimilar for Patients With HER2-Positive Breast Cancer

Trastuzumab improves survival outcomes for patients with HER2-positive (HER2⁺) breast cancer, yet not all such women receive this important therapy. Trastuzumab was approved by the US Food and Drug Administration in 1998 and the European Medicines Agency in 2000 as treatment for HER2⁺ metastatic breast cancer (MBC). Observational studies between 2000 and 2015 in patients with HER2⁺ MBC suggest that nearly 12% in the United States, 27% to 54% in Europe, and 27.1% to 49.2% in China did not receive trastuzumab or any other HER2-targeted agent as first- and/or later-line for treatment of metastatic disease. In 2006, both agencies approved trastuzumab as adjuvant therapy for patients with HER2⁺ early breast cancer (EBC). Observational studies on real-world treatment patterns for HER2⁺ EBC between 2005 and 2015 suggest that 19.1% to 59.5% of patients across regions of North America, Europe, Australia, New Zealand, and China did not receive (neo)adjuvant trastuzumab. Data suggest that some patient subgroups, including older patients, those with HER2⁺/hormone receptor-positive disease, and women with small and/or node-negative HER2⁺ tumors, were less likely to receive anti-HER2 therapy. Barriers to accessing trastuzumab are multifactorial and include issues related to drug funding and high treatment costs for patients that have been reported worldwide. Herein, we review available literature on the use of, and barriers to, treatment with trastuzumab in patients with HER2⁺ breast cancer. We also discuss how the availability of safe and effective biosimilars might increase access to trastuzumab and allow greater use of anti-HER2 therapy, potentially improving patient outcomes.     

HER2/neu increases the expression of Wilms' Tumor 1 (WT1) protein to stimulate S-phase proliferation and inhibit apoptosis in breast cancer cells

High levels of the Wilms' Tumor 1 (WT1) protein and mRNA had been associated with aggressive phenotypes of breast tumors. Here we report that the HER2/neu oncogene increases WT1 expression. Approximately threefold higher levels of WT1 protein were observed in MCF-7 breast cancer cells transfected with the HER2/neu oncogene than in parental MCF-7 cells. Conversely, inhibition of HER2/neu with the anti-HER2/neu trastuzumab (Herceptintrade mark) antibody decreased WT1 protein levels in HER2/neu-overexpressing BT-474 and SKBr3 cells. We also found that HER2/neu engages Akt to regulate WT1 levels since inhibition of Akt reduced WT1 levels. Decreased expression of WT1 protein led to cell cycle arrest at the G1 phase and increased apoptosis in HER2/neu-overexpressing cells, which is correlated with decreased cyclin D1 and Bcl-2 levels. Our data indicate that HER2/neu engages Akt to increase WT1 expression, and that WT1 protein plays a vital role in regulating cell cycle progression and apoptosis in HER2/neu-overexpressing breast cancer cells.     

Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression

Although HER2-targeting antibody trastuzumab confers a substantial benefit for patients with HER2-overexpressing breast and gastric cancer, overcoming trastuzumab resistance remains a large unmet need. In this study, we revealed a STAT3-centered positive feedback loop that mediates the resistance of trastuzumab. Mechanistically, chronic exposure of trastuzumab causes the upregulation of fibronection (FN), EGF and IL-6 in parental trastuzumab-sensitive breast and gastric cells and convergently leads to STAT3 hyperactivation. Activated STAT3 enhances the expression of FN, EGF and IL-6, thus constituting a positive feedback loop which amplifies and maintains the STAT3 signal; furthermore, hyperactivated STAT3 signal promotes the expression of MUC1 and MUC4, consequently mediating trastuzumab resistance via maintenance of persistent HER2 activation and masking of trastuzumab binding to HER2 respectively. Genetic or pharmacological inhibition of STAT3 disrupted STAT3-dependent positive feedback loop and recovered the trastuzumab sensitivity partially due to increased apoptosis induction. Combined trastuzumab with STAT3 inhibition synergistically suppressed the growth of the trastuzumab-resistant tumor xenografts in vivo. Taken together, our results suggest that feedback activation of STAT3 constitutes a key node mediating trastuzumab resistance. Combinatorial targeting on both HER2 and STAT3 may enhance the efficacy of trastuzumab or other HER2-targeting agents in HER2-positive breast and gastric cancer.     

HDAC inhibitor SNDX-275 enhances efficacy of trastuzumab in erbB2-overexpressing breast cancer cells and exhibits potential to overcome trastuzumab resistance

Trastuzumab (or Herceptin), as the first erbB2-targeted therapy, has been successfully used to treat breast cancer patients with erbB2-overexpressing tumors. However, resistances to trastuzumab frequently occur, and novel strategies/agents are urgently needed to abrogate the resistant phenotype. Our current study explores the potential of SNDX-275, a class I HDAC inhibitor, to overcome trastuzumab resistance and investigates the combinational effects of SNDX-275 and trastuzumab on both sensitive and resistant breast cancer cells. Cell proliferation assays showed that SNDX-275 significantly enhanced trastuzumab-induced growth inhibition in trastuzumab-sensitive, erbB2-overexpressing breast cancer cells. Importantly, SNDX-275 at its therapeutic range re-sensitized trastuzumab-resistant cells to trastuzumab-mediated growth inhibition. SNDX-275 in combination with trastuzumab resulted in a dramatic reduction of erbB3 and its phosphorylation (P-erbB3), and inhibition of Akt signaling. Apoptotic-ELISA and western blot analyses confirmed that the combinations of SNDX-275 and trastuzumab as compared to SNDX-275 alone significantly enhanced DNA fragmentation and induced more PARP cleavage and caspase-3 activation in both trastuzumab-sensitive and -resistant breast cancer cells. Furthermore, co-immunoprecipitation assays revealed that SNDX-275 mainly attenuated the interactions of erbB2 and erbB3 receptors, but had no significant effect on erbB2/IGF-1R or erbB3/IGF-1R associations in the trastuzumab-resistant breast cancer cells. These data indicated that SNDX-275 enhanced trastuzumab efficacy against erbB2-overexpressing breast cancer cells, and exhibited potential to overcome trastuzumab resistance via disrupting erbB2/erbB3 interactions and inactivating PI-3K/Akt signaling. SNDX-275 may be included in erbB2-targeted regimen as a novel strategy to treat breast cancer patients whose tumors overexpress erbB2.     

Differential signaling by an anti-p185(HER2) antibody and heregulin

To understand the molecular mechanisms by which anti-p185HER2 antibody and the ligand heregulin inhibit tumor growth, we have investigated several signaling proteins and pathways. We report here that anti-p185HER2 monoclonal antibody ID5 induced tyrosine phosphorylation of HER2 in SKBr3 breast cancer cells that overexpress p185HER2. Heregulin beta1 induced phosphorylation of both HER3 and HER2. ID5 produced a greater association of phospholipase C (PLC)-gamma1 with HER2 than did heregulin. Concordantly, ID5, but not heregulin, increased PLC-gamma1 activity. However, the G1 cell cycle arrest and induction of p27Kip1 produced by ID5 were not affected by the inhibition of PLC-gamma. ID5 preferentially induced binding of the Mr 46,000 isoform of SHC to HER2, whereas heregulin preferentially induced binding of the Mr 52,00 isoform of SHC to HER3. Heregulin, but not ID5, induced the p85 subunit of phosphatidylinositol 3'-kinase (PI3-K) to interact with HER3. Heregulin induced sustained activation of P13-K signaling, whereas ID5 had only a transient effect. Heregulin, but not ID5, activated the c-Jun-NH2-terminal kinase cascade. Pretreatment of SKBr3 cells with ID5 decreased heregulin-induced association of HER2 with HER3 as well as the activation of c-Jun-NH2-terminal kinase and PI3-K activities. Inhibition of the mitogen-activated protein kinase pathway in SKBr3 cells did not affect heregulin-induced G2-M-phase arrest, apoptosis, and differentiation. Heregulin-induced apoptosis could be blocked by inhibition of p70s6k, but not by inhibition of PI3-K. Heregulin-induced differentiation could be eliminated by inhibition of PI3-K. We conclude that ID5 and heregulin signal via different pathways, although both agents can inhibit the clonogenic growth of cells that overexpress HER2.     

Selective death of human breast cancer cells by lytic immunoliposomes: Correlation with their HER2 expression level

Trastuzumab (Herceptin™) targets the human epidermal growth factor receptor 2 (HER2), which is overexpressed in 20–30% of breast and ovarian cancers carrying a bad prognosis. Our purpose was to target HER2-overexpressing human breast cancer cells with pegylated immunoliposomes bearing trastuzumab and containing melittin, which has recently shown anticancer properties. Using a panel of human breast cancer cells with different HER2 expression levels, these immunoliposomes decreased cancer cells viability in a dose–response manner and in correlation to their level of HER2 expression. Specific binding of the immunoliposomes to SKBr3 breast cancer cells was shown by ImageStream-based analysis. The morphological changes observed in the treated cells suggested a cytolytic process. This preclinical approach may suppose an effective strategy for the treatment of HER2-overexpressing tumors, and can support the development of an early phases I–II clinical trial. Trastuzumab resistant breast cancer cells (JIMT-1), can also be targeted using this approach.     

Met receptor contributes to trastuzumab resistance of Her2-overexpressing breast cancer cells

Her2 is overexpressed in 20% to 30% of breast tumors and correlates with reduced disease-free and overall patient survival. Trastuzumab, a humanized monoclonal antibody directed against Her2, represents the first Her2-targeted therapy, which decreases the risk of relapse and prolongs patient survival. Resistance to trastuzumab, both inherent and treatment-acquired, represents a significant barrier to the effective treatment of Her2 (+) breast cancer. The Met receptor tyrosine kinase is aberrantly expressed in breast cancer and predicts poor patient prognosis. In this study, we find that Met is frequently expressed in Her2-overexpressing breast cancer cells, as well as Her2 (+) breast cancer. Importantly, Met contributes to trastuzumab resistance, as inhibition of Met sensitizes cells to trastuzumab-mediated growth inhibition, whereas Met activation protects cells against trastuzumab by abrogating p27 induction. Remarkably, Her2-overexpressing breast cancer cells rapidly up-regulate Met expression after trastuzumab treatment, promoting their own resistance. Our study suggests that a subset of Her2 (+) patients may benefit from combined inhibition of Her2 and Met.     

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.     

Formation of the eIF4F translation-initiation complex determines sensitivity to anticancer drugs targeting the EGFR and HER2 receptors

Elucidating how cancer cells respond to antagonists of HER receptor family members is critical to understanding mechanisms of therapeutic resistance that arise in patients. In large part, resistance to such agents appears to arise from deregulation of the phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR pathway. mTOR-dependent phosphorylation of the translation repressor 4E-BP1 leads to its dissociation from eIF4E, thereby causing an increase in the formation of the eIF4F complex, which also comprises eIF4G and eIF4A. In this study, we show that trastuzumab, cetuximab, and erlotinib all decrease the formation of the eIF4F complex in breast, colon, and head and neck cancer cells, respectively. Ectopic expression of eIF4E restores the trastuzumab-dependent defect in eIF4F formation, renders cells resistant to the trastuzumab-mediated decrease in cell proliferation, and rescues breast cancer xenografts from inhibition by trastuzumab. In breast tumor specimens, the level of eIF4E expression is associated with the therapeutic response to a trastuzumab-based regimen. Together, our findings suggest that formation of the eIF4F complex may be a critical determinant of the response to anticancer drugs that target HER2 and epidermal growth factor receptor.