Interleukin-6 is a potent inducer of S100P, which is up-regulated in androgen-refractory and metastatic prostate cancer

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Abstract

Elevated circulating interleukin-6 (IL6) and up-regulated S100P in prostate cancer (PCa) specimens correlate independently with progression to androgen-independent and metastatic PCa. The cause of up-regulated S100P levels in advanced PCa remains to be determined. We investigated the possibility that IL6 is an inducer of S100P. Determination of mRNA and protein levels by real-time PCR and Western blotting revealed that IL6 is a more potent inducer of S100P than the synthetic androgen, R1881, in the LNCaP/C4-2B model of PCa progression. IL6 did not require androgen to induce S100P in these cells, which express a functional androgen receptor (AR). Like R1881, IL6 was unable to induce S100P in PC3 cells that lack a functional AR. IL6 did not strongly induce the AR-dependent genes PSA and KLK2 and, contrary to R1881, down-regulated Cyr61/CCN1, a potential marker that is down-regulated in PCa. Epidermal growth factor (EGF), which like IL6 is a non-androgen activator of the AR, did not induce S100P. The data identifies a unique gene-induction profile for IL6 and suggests that IL6 may require a functional AR for S100P induction. A link between elevated IL6 and up-regulated S100P in androgen-refractory and metastatic PCa is postulated.

Introduction

Prostate cancer (PCa) is a leading cause of cancer deaths in men in the Western world. The progression of PCa from organ-confined growth to metastatic disease is initially androgen-dependent. The transition to hormone refractory (androgen-independent) PCa, which is currently incurable and is not fully understood, may be expedited by androgen-independent activation of the androgen receptor (AR) by cytokines and growth factors, including interleukin-6 (IL6) and epidermal growth factor (EGF) (Grossmann, Huang, & Tindall, 2001). The specific gene induction profiles of these non-ligand AR activators may hold a key to new prognostic and diagnostic tools and therapeutic targets for improved management of PCa.

Recently, a striking association between PCa progression and up-regulated S100P was identified. Tissue microarray analysis with 544 clinical specimens from different stages of disease progression showed that S100P mRNA and protein levels were significantly increased in hormone refractory and metastatic PCa (Mousses et al., 2002). S100P, which is a member of the S100 family involved in calcium signaling (Becker, Gerke, Kube, &Weber, 1992) has been linked to resistance to chemotherapeutic drugs and loss of senescence in cancer cell lines (Bertram, Palfner, Hiddemann, & Kneba, 1998; Guerreiro Da Silva et al., 2000). The expression of S100P is known to be regulated by androgen (Averboukh, Liang, Kantoff, & Pardee, 1996; Amler et al., 2000), but the factors leading to its dysregulation in hormone refractory PCa are unknown. Similar to the association of S100P with PCa progression, serum levels of IL6 are significantly elevated in patients with metastatic and hormone-refractory PCa (Adler et al., 1999; Drachenberg, Elgamal, Rowbotham, & Murphy, 1999). High serum IL6 correlates with PCa morbidity and cachexia and is a candidate prognostic factor for mortality (Twillie et al., 1995, Nakashima et al., 2000, Pfitzenmaier et al., 2003). IL6 is a member of the gp130 family of cytokines that have overlapping biological activities in the normal immune, hemopoietic and neural systems (Simpson, Hammacher, Smith, Matthews, & Ward, 1997; Taga & Kishimoto, 1997). The specific IL6 receptor (IL6R) is expressed in benign prostatic tissue and a majority of tumor cells obtained from radical prostatectomy of PCa patients (Hobisch et al., 2000). Activation of the IL6 signaling receptor, gp130, like S100P, has been linked to resistance to chemotherapeutic drugs (Bertram et al., 1998, Borsellino et al., 1999).

We have used the LNCaP/C4-2B model of PCa progression to show that IL6 is a potent inducer of S100P. In contrast to the androgen-responsive LNCaP cells, the LNCaP-derived C4-2B cells are androgen-independent and metastasize preferentially to bone (Thalmann et al., 1994). We identify a unique gene-induction profile for IL6 in prostate cancer cells that have a functional AR and postulate a link between up-regulated S100P and increased serum IL6 in androgen-refractory and metastatic PCa.

Section snippets

Tissue culture and media

The cells were maintained in T-medium containing 5% FBS (C4-2B cells) or 10% heat-inactivated FBS (LNCaP cells) in a 37 °C, humidified atmosphere at 5% CO2. For assays in steroid-free conditions, the cells were cultured in phenol red-free Minimal Essential Medium Alpha Medium with charcoal-stripped FBS from Gibco BRL (Invitrogen, Melbourne, VIC, Australia).

RNA extraction and cDNA preparation

The cells were seeded at 0.4–1 × 106 cells per 75 cm2 flask (Interpath Services, West Heidelberg, VIC) and allowed to adhere prior to 24 h

S100P mRNA is up-regulated by IL6 and R1881

Initial experiments showed that IL6 up-regulates S100P mRNA in a time-dependent fashion. Typically, significant S100P mRNA increases were detectable from around 24 h to at least 3 days’ stimulation of the cells with 10 ng/ml IL6, with the 48 h levels in between 24 and 72 h (Fig. 1A and B and data not shown). The up-regulation of S100P in C4-2B and LNCaP cells by IL6 was also dose-dependent (Fig. 1C and data not shown).

The synthetic androgen, R1881, induced dose-dependent increases of S100P mRNA in

Acknowledgements

This work was supported by project grant 143709 from the National Health and Medical Research Council of Australia (to AH). We thank Michelle Alejandrino, Christine Henderson and Tony Blick for technical assistance, Joe Pereira for the Cyr61/CCN1 oligonucleotides, Dr. Richard Simpson for the IL6, Mr. Anthony Penington for statistical advice and Dr. John Price for critically reading the manuscript.

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