Rita Nahta

The HER-2-Targeting Antibodies Trastuzumab and Pertuzumab Synergistically Inhibit the Survival of Breast Cancer Cells

Trastuzumab (herceptin) and pertuzumab (Omnitarg, 2C4) are recombinant humanized monoclonal antibodies that target different extracellular regions of the HER-2 tyrosine kinase receptor. We explored combination effects of these agents in the HER-2-overexpressing BT474 breast cancer cell line. Trastuzumab and 2C4 synergistically inhibited the survival of BT474 cells, in part, because of increased apoptosis. Trastuzumab increased 2C4-mediated disruption of HER-2 dimerization with the epidermal growth factor receptor and HER-3. Combination drug treatment reduced levels of total and phosphorylated HER-2 protein and blocked receptor signaling through Akt but did not affect mitogen-activated protein kinase. These results suggest that combining HER-2-targeting agents may be a more effective therapeutic strategy in breast cancer rather than treating with a single HER-2 monoclonal antibody.

HER2 therapy: Molecular mechanisms of trastuzumab resistance

Trastuzumab is a monoclonal antibody targeted against the HER2 tyrosine kinase receptor. The majority of patients with metastatic breast cancer who initially respond to trastuzumab develop resistance within one year of treatment initiation, and in the adjuvant setting 15% of patients still relapse despite trastuzumab-based therapy. In this review, we discuss potential mechanisms of antitumor activity by trastuzumab, and how these mechanisms become altered to promote therapeutic resistance. We also discuss novel therapies that may improve the efficacy of trastuzumab, and that offer hope that the survival of breast cancer patients with HER2-overexpressing tumors can be vastly improved.

P27kip1 Down-Regulation Is Associated with Trastuzumab Resistance in Breast Cancer Cells

Trastuzumab (Herceptin) is a recombinant humanized monoclonal antibody directed against HER-2. The objective response rate to trastuzumab monotherapy is 12–34% for a median duration of 9 months, by which point most patients become resistant to treatment. We created two trastuzumab-resistant (TR) pools from the SKBR3 HER-2-overexpressing breast cancer cell line to study the mechanisms by which breast cancer cells escape trastuzumab-mediated growth inhibition. Both pools maintained her-2 gene amplification and protein overexpression. Resistant cells demonstrated a higher S-phase fraction by flow cytometry and a faster doubling time of 24–36 h compared with 72 h for parental cells. The cyclin-dependent kinase inhibitor p27kip1 was decreased in TR cells, and cyclin-dependent kinase 2 activity was increased. Importantly, exogenous addition of p27kip1 increased trastuzumab sensitivity. Additionally, resistant cells displayed heightened sensitivity to the proteasome inhibitor MG132, which induced p27kip1 expression. Thus, we propose that trastuzumab resistance may be associated with decreased p27kip1 levels and may be susceptible to treatments that induce p27kip1 expression.

Lapatinib induces apoptosis in trastuzumab-resistant breast cancer cells: effects on insulin-like growth factor I signaling

The majority of breast cancer patients who achieve an initial therapeutic response to the HER2-targeted antibody trastuzumab will show disease progression within 1 year. Thus, the identification of novel agents that effectively inhibit survival of cancer cells that have progressed on trastuzumab is critical. In the current study, we show that the dual epidermal growth factor receptor (EGFR)/human EGFR-2 (HER2) kinase inhibitor lapatinib induces apoptosis in trastuzumab-resistant cells derived from the HER2-overexpressing SKBR3 breast cancer line. Lapatinib inhibited EGFR and HER2 signaling in resistant cells, blocking activation of downstream Akt, mitogen-activated protein kinase, and S6 kinases and inducing expression of p27kip1. Importantly, lapatinib also inhibited insulin-like growth factor I (IGF-I) signaling and growth-promoting effects in parental and resistant cells, and the cytotoxic effects of lapatinib were further enhanced by the IGF-I receptor–blocking antibody αIR3. As increased IGF-I receptor signaling has been implicated in trastuzumab resistance, our data strongly support further study of lapatinib as a potential therapeutic in breast cancers that have progressed on trastuzumab. [Mol Cancer Ther 2007;6(2):667–74]

Prognostic significance of phosphorylated P38 mitogen‐activated protein kinase and HER‐2 expression in lymph node‐positive breast carcinoma

Chemotherapy‐induced p38 mitogen‐activated protein kinase (MAPK) phosphorylation reportedly leads to increased apoptosis in breast carcinoma cells. The goals of the current study were to assess the incidence of activated phosphorylated p38 MAPK (P‐p38) expression in invasive breast carcinoma, correlate expression of P‐p38 MAPK with HER‐2, and estimate the prognostic value of this marker in patients with lymph node‐positive breast carcinoma treated with adjuvant chemotherapy.

B cell translocation gene 1 contributes to antisense Bcl-2-mediated apoptosis in breast cancer cells

The antiapoptotic protein Bcl-2 is overexpressed in a majority of breast cancers, and is associated with a diminished apoptotic response and resistance to various antitumor agents. Bcl-2 inhibition is currently being explored as a possible strategy for sensitizing breast cancer cells to standard chemotherapeutic agents. Antisense Bcl-2 oligonucleotides represent one method for blocking the antiapoptotic effects of Bcl-2. In this study, we show that antisense Bcl-2 efficiently blocks Bcl-2 expression, resulting in the apoptosis of breast cancer cells. Antisense Bcl-2-mediated cytotoxicity was associated with the induction of the B cell translocation gene 1 (BTG1). Importantly, knockdown of BTG1 reduced antisense Bcl-2-mediated cytotoxicity in breast cancer cells. Furthermore, BTG1 expression seems to be negatively regulated by Bcl-2, and exogenous expression of BTG1 induced apoptosis. These results suggest that BTG1 is a Bcl-2-regulated mediator of apoptosis in breast cancer cells, and that its induction contributes to antisense Bcl-2-mediated cytotoxic effects. [Mol Cancer Ther 2006;5(6):1593–601]

Adenovirus type 5 E1A gene therapy for ovarian clear cell carcinoma: a potential treatment strategy

Resistance of ovarian clear cell carcinoma (CCC) to platinum-based chemotherapy is associated with poor prognosis, and an effective treatment for advanced disease is urgently needed. HER2/neu is up-regulated more often in CCC than in other histologic types of epithelial ovarian cancer. The purpose of this study was to assess possible treatment for ovarian CCC with the anti-HER2 antibody trastuzumab or human adenovirus type 5 E1A. We treated 10 CCC cell lines with trastuzumab or E1A and assessed cell viability, proliferation, and colony formation and the expression of HER2 and wild-type p53 proteins and molecules downstream of those signaling pathways. HER2 protein was detected at various levels in all 10 cell lines by Western blotting and in 5 CCC cell lines by immunohistochemical staining; HER2 gene amplification was detected (by fluorescence in situ hybridization) in only one cell line (RMG-I). Trastuzumab did not inhibit proliferation in any of the four CCC cell lines tested (RMG-I, SKOV-2, OVTOKO, and OVSAYO). However, transfection with E1A (as compared with control vectors) reduced colony formation in all 10 CCC cell lines regardless of HER2 expression level. Infection of RMG-I and SMOV-2 cells with an adenoviral vector encoding E1A led to significant (P < 0.05) suppression of proliferation and enhancement of cell death; this effect required stabilization of p53 (but not p73) protein and was associated with the up-regulation of Bax and the cleavage of caspase-9. Other mechanisms, such as p53-independent apoptosis, may also be involved in E1A-mediated cell death in CCC. Finally, treatment with E1A prolonged survival in a CCC xenograft model (P < 0.001). E1A gene therapy, because of its ability to stabilize wild-type p53, is worth exploring as a treatment modality for women with ovarian CCC, which typically expresses wild-type p53. [Mol Cancer Ther 2007;6(1):227–35]

Signal Transduction Inhibitors in the Treatment of Breast Cancer

Signal transduction pathways are frequently altered in human breast cancer and are the targets of several novel therapies currently in clinical trials. Therapeutic strategies include extracellular blockade of tyrosine kinase receptors with the monoclonal antibodies C225 and trastuzumab. Competitive inhibitors of adenosine triphosphate binding sites on tyrosine and serine/threonine kinases are also being evaluated in phase I/II trials; these include ZD1839, OSI-774 and CI-1033. Flavopiridol and UCN-01 are nonspecific cell cycle kinase antagonists with preliminary evidence of breast cancer cell growth inhibition. Several inhibitors of mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling are also in various stages of preclinical or clinical development. Additionally, inhibitors of farnesyl transferase have demonstrated activity in breast cancer cells irrespective of ras status. Current evidence suggests that targeting of signaling molecules is a promising new approach to treatment of breast cancer.

Novel pharmacological approaches in the treatment of breast cancer

Breast cancer is the most common malignancy among women. Novel pharmacological agents, including hormonal, cytotoxic and biological therapies, are currently being developed and tested in clinical trials and may offer patients more treatment options and an improved chance of long-term survival. Signal transduction inhibitors that block endocrine or growth factor pathways have demonstrated exciting antitumour effects in clinical trials. In addition to new chemotherapeutic drugs, numerous biological agents including growth factor receptor-directed monoclonal antibodies and tyrosine kinase inhibitors that target specific molecular lesions are being examined as potential breast cancer treatments.