Abstract
Background: Within all invasive breast cancers, 20-30% harbor HER2 gene amplification. HER2 activates signaling pathways promoting survival and cell proliferation resulting in an aggressive phenotype. Even with the development of trastuzumab (anti-HER2 therapy) this subtype has one of the worst out-comes of all breast cancers. Primary and acquired resistance to trastuzumab is increasingly recognized as a major obstacle in the clinical management of HER2 disease. Reported mechanisms of resistance involve a truncated HER2 receptor, mutations in the PI3K/AKT pathway, and up-regulation of alternative tyrosine kinase receptors.
In our prospective IRB-approved study of breast cancers, we observed an over-representation of TIMP-4 positive cancers among HER2-positive patients with disease progression while receiving, or shortly after completing, treatment. Secreted TIMP-4 acts through the tetraspanin CD63 on tumor cells to initiate the same signaling pathways as HER2. Based on this observation we hypothesized that TIMP-4 provides an alternative mechanism to promote continued growth and spread of HER2 positive tumors in the presence of anti-HER2 therapy and might be used as a predictive biomarker for treatment resistance.
Methods: Cell culture experiments using human breast cancer cell lines with HER2 amplification, with or without mutations in PIK3CA and PTEN, were used to assess the effects of anti-HER2 treatment on cell growth and downstream signaling pathways. Cells were grown in the presence or absence of human recombinant TIMP-4 and used to determine the effects on growth, AKT/MAPK activation, apoptosis and response to neutralizing antibodies against HER2 alone or in combination with anti-TIMP4 antibody.
Results: Under normal growth conditions, SK-BR-3 cells (HER2 amplified, wild type PI3K and PTEN) demonstrated a significant growth reduction in the presence of anti-HER2, while anti-TIMP4 had no effect. However, in TIMP-4 elevated conditions cells were resistant to anti-HER2 treatment while anti-TIMP4 had a modest effect. Combining the two antibodies reduced the growth significantly. Cells with mutations in either PI3K (MDA-MB-453) or PTEN (ZR-75-1) were insensitive to anti-HER2 treatment under normal growth conditions. In the presence of TIMP-4, cells with PI3K mutation were still insensitive, while cells harboring PTEN mutation responded to anti-HER2 treatment. Cells with either mutation pattern had a modest response to anti-TIMP4 and the combination treatment. Both antibody treatments were associated with reduced activation of growth and survival promoting enzymes as determined by Western blotting.
Conclusions: Elevated TIMP-4 levels could contribute to what clinically appears as resistance to trastuzumab. While regular anti-HER2 treatment caused diminished growth among cells with wild type PI3K and PTEN, the presence of high TIMP-4 levels necessitated treatment with both antibodies for the same growth reduction. Tumor cells insensitive to anti-HER2 treatment due to mutations in growth and survival promoting pathways acquired a modest response when treated with both anti-HER2 and anti-TIMP4 antibodies. We are currently evaluating the possible mechanism for the observed conversion in sensitivity.
Citation Format: Brady AL, Isaac KM, Adams P, Zemba-Palko V, Wallon UM. TIMP-4 as a biomarker and alternative activator of growth and survival in trastuzumab-resistant cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-07.
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