Abstract B070: TIMP-4 – prognostic marker and treatment target for triple-negative breast cancers
Authors: Kunkel Emily K. , DuHadaway James , Sutanto-Ward Erika , Birnhak Lauren N. , Ringqvist Jenny R. , Ali Zonera A. , Gilman Paul B. , Prendergast George C. , Wallon U. Margaretha 
Published: May 2015

Abstract
Background: Tissue inhibitor of metalloproteinase-4 (TIMP-4) is a secreted multi-functional protein associated with poor survival prognosis among early-stage triple-negative breast cancers (TNBC)a. Triple-negative breast cancers (TNBC) represent a highly aggressive form of this disease with few treatment options available. Current standard chemotherapy (CTX) includes taxane or adriamycin based regiments.
Extracellular TIMP-4 binds to the membrane bound tetraspanin CD63 and induces the activation of PI3K/AKT/mTOR survival pathway. Here we report that TIMP-4 induced aggressive tumor growth and metastasis can be adverted by targeting TIMP-4 either directly by sequestering extracellular pools of TIMP-4 or indirectly by blocking the activation of downstream survival pathways.
Methods: Prospectively collected patient samples, in accordance with the IRB approved protocol, were tested for circulating levels of TIMP-4 using a commercially available ELISA assay in samples from time of surgery and at each treatment cycle. The medical oncology staff recommended therapy without knowledge of TIMP-4 status.
The role of elevated TIMP-4 in TNBC cell behavior was tested in cell culture and animal experiments using the human breast cancer line MDA-MB-468. Cells with or without TIMP-4 added to the medium were used to determine the effects on growth, clonogenic survival and response to chemotherapeutic agents such as adriamycin, Taxol, the new anti-TIMP-4 antibodyb and the PI3K/AKT/mTOR inhibitor GDC-0941. The same cell-line was used to induce tumor growth in nude mice with or without TIMP-4 containing slow-release pellets implanted into the mammary fatpad (mfp). Tumor growth and response to therapy was followed over a six-week period.
Results: Results from patient samples demonstrated that circulating TIMP-4 levels in breast cancer patients remain unaffected after surgical removal of the primary tumor. Adriamycin containing regiments was the only CTX to suppress the TIMP-4 levels independent of primary tumor size and nodal status.
Adding TIMP-4 to cell culture medium or the mfp of mice resulted in an 1.5-fold increased tumor growth rate. Elevated TIMP-4 in mice also resulted in liver metastasis in 25% of animals (N=8).
In cell cultures, the TIMP-4 induced effects were completely adverted by addition of GDC-0941. Adding the TIMP-4 antibody, to cell culture medium or i.p. injections to mice, resulted in a decelerated growth rate and no metastasis.
Conclusions: On the basis of these clinical and experimental data we suggest that TIMP-4 may represent a simple prognostic and predictive marker for TNBC patients at highest risk. The presence of TIMP-4 identifies a patient population likely to recur quickly based on the continuous activation of the PI3K/AKT/mTOR pathway. Though adriamycin therapy can reduce the TIMP-4 levels, the toxicity of this agent suggests that targeted therapy of the PI3K/AKT pathway and/or a biological therapeutic approach directed against TIMP-4 may be of benefit in this subset of pts and should be further explored.
a Liss, M et.al. Am. J. Pathol. 2009
b Donover, P et.al. J. Cell. Biochem. 2010
Citation Format: Emily K. Kunkel, James DuHadaway, Erika Sutanto-Ward, Lauren N. Birnhak, Jenny R. Ringqvist, Zonera A. Ali, Paul B. Gilman, George C. Prendergast, U. Margaretha Wallon. TIMP-4 – prognostic marker and treatment target for triple-negative breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B070.