Linda X.H. Yuan

The majority of breast cancer patients who achieve an initial therapeutic response to the human epidermal growth factor receptor 2 (HER-2)-targeted antibody trastuzumab will show disease progression within 1 year. We previously reported the characterization of SKBR3-derived trastuzumab-resistant pools. In the current study, we show that HER-2 interacts with insulin-like growth factor-I receptor (IGF-IR) uniquely in these resistant cells and not in the parental trastuzumab-sensitive cells. The occurrence of cross talk between IGF-IR and HER-2 exclusively in resistant cells is evidenced by the IGF-I stimulation resulting in increased phosphorylation of HER-2 in resistant cells, but not in parental cells, and by the inhibition of IGF-IR tyrosine kinase activity leading to decreased HER-2 phosphorylation only in resistant cells. In addition, inhibition of IGF-IR tyrosine kinase activity by I-OMe-AG538 increased sensitivity of resistant cells to trastuzumab. HER-2/IGF-IR interaction was disrupted on exposure of resistant cells to the anti-IGF-IR antibody alpha-IR3 and, to a lesser extent, when exposed to the anti-HER-2 antibody pertuzumab. Heterodimer disruption by alpha-IR3 dramatically restored sensitivity to trastuzumab and resistant cells showed a slightly increased sensitivity to pertuzumab versus parental cells. Neither alpha-IR3 nor pertuzumab decreased HER-2 phosphorylation, suggesting that additional sources of phosphorylation other than IGF-IR exist when HER-2 and IGF-IR are not physically bound. Our data support a unique interaction between HER-2 and IGF-IR in trastuzumab-resistant cells such that cross talk occurs between IGF-IR and HER-2. These data suggest that the IGF-IR/HER-2 heterodimer contributes to trastuzumab resistance and justify the need for further studies examining this complex as a potential therapeutic target in breast cancers that have progressed while on trastuzumab.

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 [corrected] 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 alphaIR3. 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.

INTRODUCTION: The human epidermal growth factor receptor 2 (HER2) is a validated therapeutic target in breast cancer. Heterodimerization of HER2 with other HER family members results in enhanced tyrosine phosphorylation and activation of signal transduction pathways. HER2 overexpression increases the translation of fatty acid synthase (FASN), and FASN overexpression markedly increases HER2 signaling, which results in enhanced cell growth. However, the molecular mechanism and regulation of HER2 and FASN interaction are not well defined. Lapatinib is a small-molecule tyrosine kinase inhibitor that blocks phosphorylation of the epidermal growth factor receptor and HER2 in breast cancer cells, resulting in apoptosis. We hypothesized that FASN is directly phosphorylated by HER2, resulting in enhanced signaling and tumor progression in breast cancer cells. METHODS: Using mass spectrometry, we identified FASN as one of the proteins that is dephosphorylated by lapatinib in SKBR3 breast cancer cells. Immunofluorescence, immunoprecipitation, Western blotting, a kinase assay, a FASN enzymatic activity assay, an invasion assay, a cell viability assay and zymography were used to determine the role of FASN phosphorylation in invasion of SKBR3 and BT474 cells. The FASN inhibitor C75 and small interfering RNA were used to downregulate FASN expression and/or activity. RESULTS: Our data demonstrated that FASN is phosphorylated when it is in complex with HER2. FASN phosphorylation was induced by heregulin in HER2-overexpressing SKBR3 and BT474 breast cancer cells. Heregulin-induced FASN phosphorylation resulted in increased FASN enzymatic activity, which was inhibited by lapatinib. The FASN inhibitor C75 suppressed FASN activity by directly inhibiting HER2 and FASN phosphorylation. Blocking FASN phosphorylation and activity by lapatinib or C75 suppressed the activity of matrix metallopeptidase 9 and inhibited invasion of SKBR3 and BT474 cells. CONCLUSIONS: FASN phosphorylation by HER2 plays an important role in breast cancer progression and may be a novel therapeutic target in HER2-overexpressing breast cancer cells.

PURPOSE: The purpose is to evaluate expression levels of Jun activation domain-binding protein 1 (JAB1) in breast cancer tissue and adjacent normal tissue, to determine whether JAB1 expression is associated with p27(Kip1) expression in invasive breast carcinomas, and to evaluate the prognostic significance of JAB1 and p27(Kip1) in node-negative breast cancer. EXPERIMENTAL DESIGN: JAB1 levels were measured in 10 matched pairs of invasive breast tumor tissue and adjacent normal tissue using Western blot analysis. We also investigated the immunoreactivity of JAB1 and p27(Kip1) levels in paraffin-embedded tissue specimens from 220 patients with node-negative breast cancer who had not received adjuvant systemic therapy. The median follow-up was 15 years. RESULTS: JAB1 was expressed at higher levels in invasive tumors than in adjacent normal tissue (P = 0.01). JAB1 overexpression was observed in 57% of invasive breast cancers. Low levels of p27(Kip1) were noted in 70% of the tumor specimens. We found an inverse correlation between JAB1 and p27(Kip1) expression levels (P = 0.01). JAB1 overexpression was associated with patient age of at least 50 years (P = 0.03) and tumor size of </=2 cm (P = 0.01). Elevated levels of p27(Kip1) were associated with low nuclear grade (P = 0.01). At 5 years of follow-up, neither JAB1 nor p27(Kip1) expression was related to disease-free survival. CONCLUSIONS: These data indicate that JAB1 is commonly overexpressed in invasive breast carcinomas. JAB1 overexpression is associated with low levels of p27(Kip1) in node-negative breast cancer. In this study, JAB1 and p27(Kip1) were not independent prognostic factors.

PURPOSE: To determine the role of PEA-15 in breast cancer. EXPERIMENTAL DESIGN: A reverse-phase protein array was used to measure PEA-15 expression levels in 320 human breast cancers; these levels were correlated with clinical and tumor characteristics. PEA-15 was overexpressed by an adenovirus vector or by stably expressing PEA-15 in different breast cancer cell lines. The effects on breast cancer cell survival and on the downstream apoptotic signaling pathway were measured in terms of cell proliferation (trypan blue for cell viability, bromodeoxyuridine incorporation for DNA synthesis), anchorage-independent growth (soft agar colony formation), and apoptosis (fluorescence-activated cell sorter analysis). The preclinical efficacy of Ad.PEA-15 given intratumorally was evaluated in nude mice bearing tumors from s.c. implanted human MDA-MB-468 triple-negative breast cancer cells. RESULTS: In human breast cancers, low levels of PEA-15 expression correlated with high nuclear grade (P < 0.0001) and with negative hormone receptor status (P = 0.0004). Overexpression of PEA-15 in breast cancer cells resulted in growth inhibition, reduction in DNA synthesis, and onset of caspase-8-dependent apoptosis. In athymic nude mice bearing MDA-MB-468 xenografts, tumor volumes were significantly smaller in mice treated intratumorally with Ad.PEA-15 than in control mice (P < 0.0001). Tumors from mice treated with Ad.PEA-15 had increased levels of activated (phosphorylated) extracellular signal-regulated kinase and reduced levels of Ki-67 compared with tumors from nontreated or control-adenovirus-treated mice. CONCLUSION: PEA-15 has therapeutic potential in breast cancer. Further preclinical and clinical exploration of PEA-15 as a druggable target is warranted.