Inhibition of heregulin mediated MCF-7 breast cancer cell growth by the ErbB3 binding protein EBP1

The ErbB2/3 heterodimer plays a critical role in breast cancer genesis and progression. EBP1, an ErbB3 binding protein, inhibits breast cancer growth but its effects on ErbB3 ligand mediated signal transduction or ErbB receptors is not known. We report here that ectopic expression of EBP1 in MCF-7 and AU565 breast cancer cell lines inhibited HRG-induced proliferation. ErbB2 protein levels were substantially decreased in EBP1 transfectants, while ErbB3 levels were unchanged. HRG-induced AKT activation was attenuated in EBP1 stable transfectants and transfection of a constitutively activated AKT partially restored the growth response to HRG. Down-regulation of EBP1 expression in MCF-7 cells by shRNA resulted in increased cell growth in response to HRG and increased cyclin D1 and ErbB2 expression. These results suggest that EBP1, by down-regulating ErbB signal transduction, attentuates HRG-mediated growth of breast cancer cells.     

Endocrine resistance associated with activated ErbB system in breast cancer cells is reversed by inhibiting MAPK or PI3K/Akt signaling pathways

Endocrine therapy resistance is one of the main challenges in the treatment of estrogen receptor positive (ER+) breast cancer patients. This study showed that two ER+ human breast carcinoma cell lines derived from MCF‐7 (MVLN cells) that have acquired under OH‐Tamoxifen selection two distinct phenotypes of endocrine resistance both displayed constitutive activation of the PI3K/Akt and MAPK pathways. Aberrant expression and activation of the ErbB system (phospho‐EGFR, phospho‐ErbB2, phospho‐ErbB3, over‐expression of ErbB4 and over‐expression of several ErbB ligands) were also observed in the two resistant cell lines, suggesting the existence of an autocrine loop leading to constitutive activation of MAPK and PI3K/Akt survival pathways. The recent clinical use of specific signal transduction inhibitors is one of the most promising therapeutic approaches in breast cancers. The MEK inhibitor PD98059 and the PI3K inhibitor LY294002 were both able to enhance the cytostatic effect of OH‐Tamoxifen or fulvestrant on MVLN sensitive cells. In the two resistant cell lines, inhibition of the MAPK or the PI3K/Akt pathways associated with endocrine therapy was sufficient to reverse OH‐Tamoxifen or fulvestrant resistance. Investigating the effect of a combination of both inhibitors on the reversion of OH‐Tamoxifen and fulvestrant resistance in the two resistant cell lines suggested that, in clinical practice, a strategy combining the two inhibitors would be the best approach to target the different endocrine resistance phenotypes possibly present in a tumor. In conclusion, the combination of MAPK and PI3K inhibitors represents a promising strategy to overcome endocrine therapy resistance in ER+ breast cancer patients.     

Novel therapeutic strategies targeting the epidermal growth factor receptor (EGFR) family and its downstream effectors in breast cancer.

From the early experience with tamoxifen to the current use of Herceptin, targeted therapy has been proven to be an important part of breast cancer (BC) treatment. In the last decade, advances in molecular biology have allowed scientists to design highly individualized, 'smart' pharmaceuticals, capable of manipulating the growth factor pathways and the genes that are involved in the development and maintenance of the malignant phenotype. The epidermal growth factor receptor (EGFR) family, as one of the best studied growth factor pathways in cancer, resembles a 'treasure island' by providing a wide range of biologically relevant targets involved in breast carcinogenesis. While a large number of new agents targeting this pathway are continuingly being tested in preclinical experiments, clinicians are witnessing the migration of some of these agents to daily practice. The aim of this review is to provide clinicians with an updated synopsis of the most advanced anti-erbB therapeutic strategies with activity against BC.     

Breast cancer cells with acquired resistance to the EGFR tyrosine kinase inhibitor gefitinib show persistent activation of MAPK signaling

Although the epidermal growth factor receptor (EGFR) is frequently expressed in human primary breast carcinoma, the majority of breast cancer patients do not respond to treatment with EGFR tyrosine-kinase inhibitors such as gefitinib. We isolated through a stepwise dose escalation of the drug two gefitinib-resistant SK-Br-3 clones, ZD6 and ZD10 (ZD) cells, which showed, respectively, a three- to five-fold increase in the IC₅₀ for gefitinib as compared with parental cells. The levels of expression of EGFR were increased in ZD cells as compared with wild-type SK-Br-3 cells. The phosphorylation of EGFR, ErbB-2, ErbB-3 and Akt was significantly reduced in gefitinib-resistant cells. In contrast, ZD cells showed levels of MAPK phosphorylation similar to untreated wild-type cells when cultured in presence of gefitinib. Persistent activation of MAPK was also observed in gefitinib-resistant clones isolated from MDA-MB-175 and MDA-MB-361 breast cancer cell lines. ZD cells showed an increased sensitivity to the MEK inhibitor PD98059 as compared with SK-Br-3 cells, and a synergistic anti-tumor effect was observed when ZD cells were treated with a combination of gefitinib and PD98059. Overexpression of a constitutively activated form of p42-MAPK in SK-Br-3 cells resulted in an approximately 50% increase in the IC₅₀ to gefitinib. Finally, culture of ZD10 resistant cells in absence of gefitinib led to reversion of the resistant phenotype. These observations suggest that MAPK signaling might play a role in the resistance that develops in breast cancer cells after long-term exposure to gefitinib. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Trastuzumab-induced recruitment of Csk-homologous kinase (CHK) to ErbB2 receptor is associated with ErbB2-Y1248 phosphorylation and ErbB2 degradation to mediate cell growth inhibition

The inhibitory effect of trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of ErbB2, is associated with its ability to induce ErbB2-Y1248 phosphorylation, and the status of phosphorylated ErbB2-Y1248 (ErbB2-pY1248) may correlate with the sensitivity of breast cancers to trastuzumab. The mechanisms of which remain unclear. Here, we show that binding of trastuzumab to ErbB2 activates ErbB2 kinase activity and enhances ErbB2-Y1248 phosphorylation in trastuzumab-sensitive breast cancer cells. This in turn increases the interaction between ErbB2 and non-receptor Csk-homologous kinase (CHK), leading to growth inhibition of breast cancer cells. Overexpression of CHK mimics trastuzumab treatment to mediate ErbB2-Y1248 phosphorylation, Akt downregulation, and growth inhibition of trastuzumab-sensitive breast cancer cells. CHK overexpression combined with trastuzumab exerts an additive effect on cell growth inhibition. We further demonstrate that positive ErbB2-pY1248 staining in ErbB2-positive breast cancer biopsies correlates with the increased trastuzumab response in trastuzumab neoadjuvant settings. Collectively, this study highlights an important role for ErbB2-pY1248 in mediating trastuzumab-induced growth inhibition and trastuzumab-induced interactions between CHK and ErbB2-pY1248 is identified as a novel mechanism of action that mediates the growth inhibition of breast cancer cells. The novel mechanistic insights into trastuzumab action revealed by this study may impact the design of next generation of therapeutic monoclonal antibodies targeting receptor tyrosine kinases, as well as open new avenues to identify novel targets for the treatment of ErbB2-positive cancers.     

MiR-21 overexpression is associated with acquired resistance of EGFR-TKI in non-small cell lung cancer

Background and purpose

With the increasing use of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) in patients with advanced non-small cell lung cancer (NSCLC), its acquired resistance has become a major clinical problem. Recent studies revealed that miR-21 was involved into the resistance of cytotoxic agents. The aim of this study was to investigate its role in the acquired resistance of NSCLC to EGFR-TKI.

Methods

EGFR-TKI-sensitive human lung adenocarcinoma cell line PC9 and the acquired resistant cell line, PC9R, were used. Lentiviral vectors were used to infect PC9 or PC9R to regulate the miR-21 expression. The expression of targeted proteins PTEN and PDCD4 was controlled by RNA interference. MicroRNA array, RT-PCR and TaqMan MicroRNA Assays were used to detect miR-21 expression. The MTT and Annexin V assays were used to determine proliferation and apoptosis. Western Blot and immunohistochemistry were used to analyze target protein expression (PTEN, PDCD4, Akt, p-Akt). We also constructed PC9R xenograft tumor model to observe the relationship between miR-21 and EGFR-TKI resistance in vivo and validated it in the clinical serum specimens of NSCLC patients treated with EGFR-TKI.

Result

MiR-21 was overexpressed in the EGFR-TKI resistant cell line PC9R relative to PC9. The level of miR-21 was reversely correlated with the expression of PTEN and PDCD4 and positive correlated with PI3K/Akt pathway. Inhibiting miR-21 with lentivirus vector induces apoptosis in PC9R cell line and inhibiting miR-21with ASO suppressed tumor growth in nude mice treated with EGFR-TKI. Furthermore, serum miR-21 expression in NSCLC patients treated with EGFR-TKI was significantly higher at the time of acquiring resistance than at baseline (p < 0.01).

Conclusion

miR-21 is involved in acquired resistance of EGFR-TKI in NSCLC, which is mediated by down-regulating PTEN and PDCD4 and activating PI3K/Akt pathway.     

Heregulin-HER3-HER2 signaling promotes matrix metalloproteinase-dependent blood-brain-barrier transendothelial migration of human breast cancer cell lines

HER2-positive breast tumors are associated with a high risk of brain relapse. HER3 is thought to be an indispensible signaling substrate for HER2 (encoded by ERBB2) and is induced in breast cancer-brain metastases, though the molecular mechanisms by which this oncogenic dimer promotes the development of brain metastases are still elusive. We studied the effects of the HER3-HER2 ligand, heregulin (neuregulin-1, broadly expressed in the brain), on luminal breast cancer cell lines in vitro. Treatment of SKBr3 (ERBB2-amplified), MDA-MB-361 (ERBB2-amplified, metastatic brain tumor-derived) and MCF7 (HER2-positive, not ERBB2-amplified) cells with exogenous heregulin increased proliferation and adhesive potential, concomitant with induction of cyclin D1 and ICAM-1, and suppression of p27. All three cell lines invaded through matrigel toward a heregulin chemotactic signal in transwell experiments, associated with activation of extracellular cathepsin B and matrix metalloproteinase-9 (MMP-9). Moreover, heregulin induced breast cancer cell transmigration across a tight barrier of primary human brain microvascular endothelia. This was dependent on the activity of HER2, HER3 and MMPs, and was completely abrogated by combination HER2-HER3 blockade using Herceptin^® and the humanized HER3 monoclonal antibody, EV20. Collectively these data suggest mechanisms by which the HER3-HER2 dimer promotes development of metastatic tumors in the heregulin-rich brain microenvironment.     

Pentagalloylglucose inhibits estrogen receptor alpha by lysosome-dependent depletion and modulates ErbB/PI3K/Akt pathway in human breast cancer MCF-7 cells

Estrogens and estrogen receptors (ER) play important roles in estrogen-dependent and ER-positive breast cancer development. Inhibitors against estrogen biosynthesis or anti-estrogens have been used in breast cancer treatment for many years. The aim of this study was to determine whether pentagalloylglucose (5GG) has inhibitory effects on ER function. In the present study, we found that 5GG significantly reduced the growth of estrogen-responsive human breast cancer MCF-7 cells, and suppressed the phosphorylation and protein level of estrogen receptor alpha (ERalpha). Interestingly, 5GG decreased ERalpha protein levels by promoting the degradation of ERalpha protein in the lysosome. The ERalpha can be activated through a ligand-dependent and/or a ligand-independent pathway. The activated Akt kinase was shown to directly phosphorylate ERalpha at its serine residues and cause ligand independent activation. Our results showed that 5GG might inhibit the phosphatidylinositol 3-kinase (PI3K)/Akt pathway either through directly inhibiting Akt kinase activity or through inhibiting phosphorylation of the upstream receptor tyrosine kinases. The depletion of ErbB family receptors, including epidermal growth factor receptor (EGFR), ErbB2, and ErbB3, was also observed. 5GG treatment also led to a dose-dependent decrease in the expression of the estrogen-activated cyclin D1 expression. These findings suggested that 5GG might be a useful chemopreventive or therapeutic agent for hormone-dependent breast cancer through suppressing the functions of ERalpha by lysosome-dependent depletion and modulating the ErbB/PI3K/Akt pathway.     

Activation of PI3K/Akt signaling and hormone resistance in breast cancer

Akt is a serine/threonine kinase that has been demonstrated to play an important role in survival when cells are exposed to different apoptotic stimuli. Recent studies show that aberrant activation of Akt in breast carcinoma is associated with a poor prognosis and resistance to endocrine therapy and chemotherapy. The Akt signaling pathway is currently attracting considerable attention as a new target for effective therapeutic strategies. We investigated the incidence of Akt activation in 252 primary breast carcinomas and relationships among the activation of Akt, HER2 overexpression, hormone receptor expression, and alteration of the PTEN gene. Eighty-four cases (33.3%) were positive for pAkt expression. pAkt was significantly associated with HER2 overexpression (p<0.0001) and LOH at the PTEN gene locus (p<0.01). There was an inverse correlation between pAkt and PR (p<0.05). We also retrospectively examined the relationship between Akt activation and the efficacy of endocrine therapy for metastatic breast cancer. Of these 36 metastatic breast cancer cases, 12 cases (33.4%) were considered to show positive pAkt expression. In the pAkt-positive patients, endocrine therapy demonstrated worse efficacy than in pAkt-negative patients (p<0.01). In addition, the clinical benefit was the smallest in the patients positive both for HER2 and pAkt (p<0.01). The clinical benefit rate of estrogen deprivation therapy with AI or LH-RH agonist was significantly lower in the pAkt-positive patients than that in the pAkt-negative ones (p<0.05), and there was a tendency for the clinical benefit of SERM to be smaller in the pAkt-positive patients (p=0.09). These findings therefore suggest that Akt activation induces endocrine resistance in metastatic breast cancer, irrespective of the kind of endocrine agents that were administered. Our findings indicate that the activation of Akt in the downstream pathway of HER2 plays an important role in resistance to endocrine therapy for breast cancer. Our findings suggest that pAkt may be a useful predictor of resistance to endocrine therapy for breast cancer, while also suggesting that the inhibition of Akt may increase the efficacy of endocrine therapy.     

Nuclear HER4 mediates acquired resistance to trastuzumab and is associated with poor outcome in HER2 positive breast cancer

The role of HER4 in breast cancer is controversial and its role in relation to trastuzumab resistance remains unclear. We showed that trastuzumab treatment and its acquired resistance induced HER4 upregulation, cleavage and nuclear translocation. However, knockdown of HER4 by specific siRNAs increased trastuzumab sensitivity and reversed its resistance in HER2 positive breast cancer cells. Preventing HER4 cleavage by a γ-secretase inhibitor and inhibiting HER4 tyrosine kinase activity by neratinib decreased trastuzumab-induced HER4 nuclear translocation and enhanced trastuzumab response. There was also increased nuclear HER4 staining in the tumours from BT474 xenograft mice and human patients treated with trastuzumab. Furthermore, nuclear HER4 predicted poor clinical response to trastuzumab monotherapy in patients undergoing a window study and was shown to be an independent poor prognostic factor in HER2 positive breast cancer. Our data suggest that HER4 plays a key role in relation to trastuzumab resistance in HER2 positive breast cancer. Therefore, our study provides novel findings that HER4 activation, cleavage and nuclear translocation influence trastuzumab sensitivity and resistance in HER2 positive breast cancer. Nuclear HER4 could be a potential prognostic and predictive biomarker and understanding the role of HER4 may provide strategies to overcome trastuzumab resistance in HER2 positive breast cancer.     

Increased signalling of EGFR and IGF1R, and deregulation of PTEN/PI3K/Akt pathway are related with trastuzumab resistance in HER2 breast carcinomas

Background:

Trastuzumab resistance hampers its well-known efficacy to control HER2-positive breast cancer. The involvement of PI3K/Akt pathway in this mechanism is still not definitively confirmed.

Methods:

We selected 155 patients treated with trastuzumab after development of metastasis or as adjuvant/neoadjuvant therapy. We performed immunohistochemistry for HER2, ER/PR, epidermal growth factor 1-receptor (EGFR), α-insulin-like growth factor 1-receptor (IGF1R), phosphatase and tensin homologue (PTEN), p110α, pAkt, pBad, pmTOR, pMAPK, MUC1, Ki67, p53 and p27; mutational analysis of PIK3CA and PTEN, and PTEN promoter hypermethylation.

Results:

We found 46% ER/PR-positive tumours, overexpression of EGFR (15%), α-IGF1R (25%), p110α (19%), pAkt (28%), pBad (22%), pmTOR (23%), pMAPK (24%), MUC1 (80%), PTEN loss (20%), and PTEN promoter hypermethylation (20%). PIK3CA and PTEN mutations were detected in 17% and 26% tumours, respectively. Patients receiving adjuvant trastuzumab with α-IGF1R or pBad overexpressing tumours presented shorter progression-free survival (PFS) (all P⩽0.043). Also, p110α and mTOR overexpression, liver and brain relapses implied poor overall survival (OS) (all P⩽0.041). In patients with metastatic disease, decreased PFS correlated with p110α expression (P=0.024), whereas for OS were the presence of vascular invasion and EGFR expression (P⩽0.019; Cox analysis).

Conclusion:

Our results support that trastuzumab resistance mechanisms are related with deregulation of PTEN/PI3K/Akt/mTOR pathway, and/or EGFR and IGF1R overexpression in a subset of HER2-positive breast carcinomas.     

Chemoresistant tumor cell lines display altered epidermal growth factor receptor and HER3 signaling and enhanced sensitivity to gefitinib

Deregulated signaling through the epidermal growth factor receptor (EGFR) is involved in chemoresistance. To identify the molecular determinants of sensitivity to the EGFR inhibitor gefitinib (Iressa, ZD1839) in chemoresistance, we compared the response of matched chemosensitive and chemoresistant glioma and ovarian cancer cell lines. We found that chemoresistant cell lines were 2- to 3-fold more sensitive to gefitinib growth-inhibitory effects, because of decreased proliferation rather than survival. Sensitivity to gefitinib correlated with overexpression and constitutive phosphorylation of HER2 and HER3, but not EGFR, altered HER ligand expression, and enhanced activation of EGF-triggered EGFR pathway. No activating mutations were found in EGFR. Gefitinib fully inhibited EGF-induced and constitutive Akt activation only in chemoresistant cells. In parallel, gefitinib downregulated constitutively phosphorylated HER2 and HER3, and activated GSK3beta with a concomitant degradation of cyclin D1. Ectopically overexpressed HER2 on its own was insufficient to sensitize chemonaive cells to gefitinib. pHER3 coimmunoprecipitated with p85-PI3K in chemoresistant cells and gefitinib dissociated these complexes. siRNA-mediated inhibition of HER3 decreased constitutive activation of Akt and sensitivity to gefitinib in chemoresistant cells. Our study indicates that in chemoresistant cells gefitinib inhibits both an enhanced EGF-triggered pathway and a constitutive HER3-mediated Akt activation, indicating that inhibition of HER3 together with that of EGFR could be relevant in chemorefractory tumors. Furthermore, in combination experiments gefitinib enhanced the effects of coadministered drugs more in chemoresistant than chemosensitive ovarian cancer cells. Combined treatment might be therapeutically beneficial in chemoresistant tumors from ovary and likely from other tissues.     

Acquired resistance to anti-EGFR mAb ICR62 in cancer cells is accompanied by an increased EGFR expression,HER-2/HER-3 signalling and sensitivity to pan HER blockers

Background:

The human epidermal growth factor receptor (EGFR) is an important target for cancer treatment. Currently, only the EGFR antibodies cetuximab and panitumumab are approved for the treatment of patients with colorectal cancer. However, a major clinical challenge is a short-term response owing to development of acquired resistance during the course of the treatment.

Methods:

In this study, we investigated the molecular mechanisms underlying development of acquired resistance in DiFi colorectal cancer cells to the anti-EGFR mAb ICR62 (termed DiFi62) and to the small molecule tyrosine kinase inhibitor (TKI) gefitinib (termed DiFiG) using a range of techniques.

Results:

Compared with the findings from parental DiFi and DiFiG cells, development of acquired resistance to anti-EGFR mAb ICR62 in DiFi62 cells was accompanied by an increase in cell surface EGFR and increased phosphorylation of HER-2 and HER-3. Interestingly, DiFi62 cells also acquired resistance to treatment with anti-EGFR mAbs cetuximab and ICR61, which bind to other distinct epitopes on the extracellular domain of EGFR, but these cells remained equally sensitive as the parental cells to treatment with pan-HER inhibitors such as afatinib.

Conclusions:

Our results provide a novel mechanistic insight into the development of acquired resistance to EGFR antibody-based therapy in colorectal cancer cells and justify further investigations on the therapeutic benefits of pan-HER family inhibitors in the treatment of colorectal cancer patients once acquired resistance to EGFR antibody-based therapy is developed.