Abstract P4-03-10: Prognostic and predictive marker in the tumor microenvironment of breast cancers
Authors: Wallon UM , Brady AL , Isaac KM , Ali ZA , Gilman PB , Shevade A 
Published: March 2017

Abstract
Background: Though genetic alterations initiates' tumor formation, extracellular signals from the tumor microenvironment can modify the growth and spread of cancer cells. We are studying a small modifier protein, tissue inhibitor of metalloproteinase-4 (TIMP-4) that can accelerate tumor growth and metastatic potential as seen in both cell culture, animal experiments and prospective studies of clinical subjects. TIMP-4 is synthesized by stromal cells and secreted into the tumor microenviroment. It binds to the membrane-bound tetraspanin CD63 on tumor epithelial cells and induces the activation of the tumor promoting PI3K/AKT pathway. This modifier has its strongest effect on the so-called triple-negative breast cancers (TNBC) and breast cancers with HER2 gene amplification.
Methods: Cell culture experiments using the human MDA-MB-468 (TNBC) and SK-BR-3 (HER2 amplified) cell-lines were used to assess the effects of elevated levels of TIMP-4 on downstream targets. Cells with or without human recombinant TIMP-4 added to the growth medium were used to determine the effects on growth, AKT activation, apoptosis and response to anti-TIMP-4 treatment. Tumor growth in nude mice with or without TIMP-4 containing slow-release pellets implanted into the mammary fatpad was followed for six-week period to assess response to anti-TIMP-4 therapy. Lungs, liver, spleen and mammary fatpads were collected and analyzed. Prospectively collected patient samples, in accordance with the IRB approved protocol, were tested for circulating levels of TIMP-4 and the cancer marker CA15-3, using commercially available ELISA kits, in samples collected prior to chemotherapy and at each treatment cycle.
Results: Augmentation of TIMP-4 levels in the cell culture medium of TNBC and HER2-amplified cells resulted in accelerated growth and increased activation of AKT as determined by Western blotting for pAKTSer 473 and pAKTThr 308. Adding an anti-TIMP4 therapeutic antibody to TNBC cells grown in the presence of TIMP-4 resulted in reduced growth. HER2-positive cells grown under identical condition and treated with either an anti-TIMP-4 or a neutralizing ErbB2 antibody demonstrated no effects on growth. Only dual blockade of HER2 and TIMP-4 resulted in reduced growth. Using the anti-TIMP-4 therapy in animals resulted in stable disease with no signs of metastatic growths. Patients undergoing treatment for TNBC or HER2-positive breast cancer that had continuous elevated TIMP-4 levels while on treatment had a higher recurrence rate than patients with diminishing TIMP-4 levels in response to therapy. The TIMP-4 levels preceded and correlated better with disease progression than the traditional CA15-3 marker.
Conclusion: Together these clinical and laboratory data suggests that TIMP-4 could be a prognostic as well as a predictive marker. Our preliminary clinical data indicates that TIMP-4 might be a more reliable marker for recurrence/progression than CA15-3. Also, our cell and animal studies indicate that TIMP-4 could be therapeutic target for TNBC and HER2-positive breast cancer patients that test positive for this marker. Thus providing a targeted therapy for TNBC patients and HER2-positive patients that do not have full clinical benefit of HER2-targeted therapies alone.
Citation Format: Wallon UM, Brady AL, Isaac KM, Ali ZA, Gilman PB, Shevade A. Prognostic and predictive marker in the tumor microenvironment of breast cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-03-10.