Abstract P6-08-54: TIMP-4 is a prognostic and predictive marker in triple-negative breast cancers
Authors: Wallon U  Margaretha , Sabol Jennifer L , Zemba-Palko Vlasta , DuHadaway James S , Sutanto-Ward Erica , Ali Zonera A , Gilman Paul B , Ciocca Robin M , Carp Ned Z , Prendergast George C 
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). TNBC represent a highly aggressive form of this disease with an unmet need for effective predictive markers and targeted therapy.
Extracellular TIMP-4 binds to the membrane bound tetraspanin CD63 and induces the activation of the tumor promoting PI3K/AKT/mTOR pathway.
Here we report that TIMP-4 induced aggressive tumor growth and metastasis can be adverted by directly targeting TIMP-4 using a newly developed monoclonal antibody (mAb) to sequester TIMP-4 and the varied responses to common chemotherapy (CTX) regimen.
METHODS – 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, and the new TIMP-4 mAb. 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. Lungs, liver, spleen and mfp were collected and analyzed for presence of human cells using a specific anti-human MHC I mAb.
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 collected prior to chemotherapy and at each treatment cycle. The medical oncology staff recommended therapy without knowledge of TIMP-4 status.
RESULTS – Augmentation of TIMP-4 levels in cell culture medium or the mfp of mice resulted in similar tumor phenotype as in the clinic; fast growing tumors with accelerated disease progression.
Elevated TIMP-4 levels in the tumor environment resulted in a 1.5-fold increased growth rate with liver and/or lung metastasis in 25% of animals (N=16). No metastases were found in animals with normal TIMP-4 levels. Treating cell cultures or tumor-bearing mice (i.p. injections) with our TIMP-4 mAb resulted in decelerated growth rate and no detectable metastatic disease in the animals.
Results from patient samples demonstrated that circulating TIMP-4 levels in breast cancer patients remain elevated after definitive surgery, indicating that TIMP-4 might continuously stimulate any remaining disseminated tumor cells. Adriamycin containing regiments was the only CTX to suppress the TIMP-4 levels independent of primary tumor size and nodal status.
CONCLUSIONS – Based on these clinical and experimental data we suggest that TIMP-4 may represent a prognostic and predictive marker, and a therapeutic target for TNBC patients at highest risk. The presence of TIMP-4 identifies a patient population likely to recur quickly due to 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.
Citation Format: U Margaretha Wallon, Jennifer L Sabol, Vlasta Zemba-Palko, James S DuHadaway, Erica Sutanto-Ward, Zonera A Ali, Paul B Gilman, Robin M Ciocca, Ned Z Carp, George C Prendergast. TIMP-4 is a prognostic and predictive marker in triple-negative breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-08-54.