Sailaja Pidaparthi

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.

A novel proximity assay for the detection of proteins and protein complexes: quantitation of HER1 and HER2 total protein expression and homodimerization in formalin-fixed, paraffin-embedded cell lines and breast cancer tissue.

The availability of drugs targeting the EGFR/HER/erbB signaling pathway has created a need for diagnostics that accurately predict treatment responses. We have developed and characterized a novel assay to provide sensitive and quantitative measures of HER proteins and homodimers in formalin-fixed, paraffin-embedded (FFPE) cell lines and breast tumor tissues, to test these variables. In the VeraTag assay, HER proteins and homodimers are detected through the release of fluorescent tags conjugated to specific HER antibodies, requiring proximity to a second HER antibody. HER2 protein quantification was normalized to tumor area, and compared to receptor numbers in 12 human tumor cell lines determined by fluorescence-activated cell sorting (FACS), and with HER immunohistochemistry (IHC) test categories and histoscores in cell lines and 170 breast tumors. HER1 and HER2 expression levels determined by the VeraTag assay are proportional to receptor number over more than a 2 log10 range, and HER homodimer levels are consistent with crosslinking and immunoprecipitation results. VeraTag HER2 measurements of breast tumor tissue and cell lines correlate with standard IHC test categories (P<0.001). VeraTag HER2 levels also agree with IHC histoscores at lower HER2 protein levels, but are continuous and overlapping between IHC test categories, extending the dynamic range 5-fold to 10-fold at higher HER2 levels. The VeraTag assay specifically and reproducibly measures HER1 and HER2 protein and homodimers in FFPE tissues. The continuous measure of HER2 protein levels over a broad dynamic range, and the novel HER2 homodimer measure, are presently being assessed as predictive markers for responses to targeted HER2 therapy.

Proximity-based assays for the detection of activated HER3, HER2/HER3 heterodimers and HER3/PI3K complexes in formalin-fixed, paraffin-embedded cell line controls and tumors.

Abstract #4040 Although kinase-defective, the role of the HER3 receptor in driving tumor growth through dimerization with other HER family members is well recognized. There is increasing interest in developing therapeutic strategies to interfere with signaling through this receptor, either directly or indirectly. At the same time, there is recognition that the activation of HER3 could be an important biomarker of drug response and resistance. In particular the HER2/HER3 heterodimer has been extensively studied and, among all the possible homo and heterodimers formed by HER receptors, is considered the most mitogenic because of the ability of HER3 to effectively couple to the pro-survival PI3K/Akt pathway.
 Using the VeraTag™ technology, we have developed a suite of assays designed to detect quantitatively the activation status of the HER3 receptor by measuring HER3-specific phosphorylation and formation of HER3 signaling complexes in formalin-fixed, paraffin-embedded (FFPE) cell line controls and tissue samples.These assays are dual antibody-based and measure complexes of proteins in close association since signal is generated only when the antibodies are bound to target-specific epitope sites proximal to one another. A capillary electrophoresis instrument equipped with laser-induced fluorescence provides a highly sensitive and quantitative read-out of the assay. We report data on assays which detect a ligand-induced, 2-5-fold change in HER2/HER3 heterodimer formation, a 10-20 fold change in phosphorylated HER3 signal and as high as a 10 fold change in HER3/PI3K complex formation in a series of cell line controls, spanning a wide range of HER2 and HER3 receptor levels. FFPE data were verified with results from co-immunoprecipitation using antibodies identical to those used in the VeraTag assay. The influence of the HER2 and HER3 levels on the formation of HER2/HER3 heterodimers and correlation with HER3 phosphorylation and formation of the PI3K adaptor complex will be discussed.
 In addition, we have used these assays to landscape HER3 activation in a number of tissues, including breast and ovarian tumors, focusing particularly on HER2 positive breast cancer, where the HER2/HER3 heterodimer is thought to play a particularly significant role. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4040.