Elevated circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) levels are associated with neuroendocrine differentiation in castration resistant prostate cancer

BACKGROUND. Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a 28.5kDa secreted glycoprotein that inhibits matrix metalloproteinase (MMP) activity. Our group has previously shown that elevated plasma TIMP-1 levels predict poor survival in metastatic castrationresistant prostate cancer (CRPC) patients; however, the underlying source and impact of elevated circulating TIMP-1 protein is unknown. METHODS. In this study, we used qRT-PCR, ELISA and immunohistochemistry to evaluate TIMP-1 expression in androgen-sensitive and resistant prostate cancer (PC) cell lines, tumor tissues and patient sera, and to correlate TIMP-1 levels to expression of chromogranin A (CGA), an established marker of neuroendocrine differentiation (NED). We also explored the relationship between TIMP-1 overexpression and induction of NED by overexpressing TIMP-1 in androgen-sensitive LNCaP cells, as well as by inducing NED of LNCaP cells with IL-6. RESULTS. Patients with CRPC have significantly higher serum TIMP-1 levels compared to patients with hormone-sensitive disease. Although circulating TIMP-1 levels were increased, peripheral blood cells were not the source of elevation. Instead, elevated TIMP-1 expression was associated with higher expression of CGA in both blood and metastatic tumor tissue. We further show that androgen receptor (AR) and PSA non-expressing prostate cancer cell lines known to display NED phenotypes such as PC-3, PC-3M, and DU145 cells, expressed high levels of TIMP-1, in contrast to AR (þ) and PSA (þ) adenocarcinoma cell lines such as LNCaP, VCaP, and LAPC-4, which had barely detectable levels of TIMP-1. In addition, ectopic overexpression of TIMP-1 in LNCaP cells did not induce NED. However, TIMP-1 mRNA expression was elevated >10-fold during IL-6-induced NED of LNCaP cells, suggesting that TIMP-1 overexpression accompanies, but is not the driving force for NED. Finally, we show that conditioned media from androgen-resistant PC-3, PC-3M, and DU145 cells induced TIMP-1 mRNA expression in primary prostate stromal fibroblasts in an ERK and NF-kB dependent manner. CONCLUSIONS. We provide in vitro and clinical evidence to support the association between NED and elevated circulating TIMP-1 expression in CRPC. Our observation