Cancer Letters

Cancer Letters

Volume 307, Issue 2, 28 August 2011, Pages 200-210
Cancer Letters

Insulin-like growth factor-binding protein-3 suppresses tumor growth via activation of caspase-dependent apoptosis and cross-talk with NF-κB signaling

https://doi.org/10.1016/j.canlet.2011.04.004Get rights and content

Abstract

Nuclear factor-kappaB (NF-κB) is constitutively activated in a variety of human cancers including prostate cancer and involved in tumorigenesis, tumor progression and chemo-resistance. Insulin-like growth factor-binding protein-3 (IGFBP-3) is a potent tumor suppressor and is significantly suppressed in a variety of cancers. Diverse biological effects of IGFBP-3 have been reported to be both dependent and independent of the IGF/IGF-I receptor (IGF-IR) axis. The precise underlying mechanisms of IGF/IGF-IR-independent, antiproliferative actions of IGFBP-3 are yet to be elucidated. We found an inverse correlation between NF-κB activity and IGFBP-3 expression during prostate cancer progression using an in vitro prostate cancer progression model. Restoration of IGFBP-3 resulted in significant inhibition of constitutively elevated NF-κB activity in prostate cancer cells. IGFBP-3 further inhibited the expression of NF-κB-regulated angiogenic factors such as VEGF and IL-8, and cell adhesion molecules, ICAM-1 and VCAM-1. This inhibitory action of IGFBP-3 was IGF/IGF-IR-independent since IGFBP-3 mutant devoid of IGF binding affinity had a similar inhibitory effect. We identified that IGFBP-3 degrades the key NF-κB regulatory molecules–IκBα and p65-NF-κB proteins through activation of caspase-8 and -3/-7, thereby inhibiting elevated NF-κB activity in prostate cancer. Finally intratumoral administration of IGFBP-3 resulted in significant tumor suppression as well as sensitization of antitumor effect of doxorubicin. Our findings indicate that IGFBP-3 exerts antitumor effects via IGF-independent mechanisms which involve activation of caspase-dependent apoptosis and cross-talk with NF-κB signaling. The use of IGFBP-3 as a cancer therapeutic with this distinctive suppression mechanism may offer alternate means to treat chemotherapy resistant tumors.

Introduction

NF-κB is an inducible and ubiquitously expressed transcription factor for genes involved in cell survival, cell adhesion, inflammation, growth, and angiogenesis [1]. NF-κB is activated in response to a wide variety of stimuli. Its activation is through IKK-dependent phosphorylation and degradation of IκB proteins, thus unmasking the nuclear localization sequence of NF-κB allowing NF-κB to enter the nucleus and bind κB-regulatory elements [2]. It has been shown that NF-κB is constitutively activated in a variety of human cancers including prostate cancer in vitro and in vivo [3]. In comparison to normal prostate tissue, a high proportion of cancerous prostate tissues are characterized by strong nuclear staining indicative of constitutive NF-κB activity [4], [5], [6] and this nuclear staining has been associated with clinical stage and patient outcomes [7]. Blocking NF-κB activity resulted in inhibition of angiogenesis, invasion, and metastasis of prostate cancer cells where concomitant inhibition of VEGF and IL-8 expression was also observed [8], [9]. Other studies have demonstrated that NF-κB suppression sensitized prostate cancer cells to chemotherapeutic agents and to ionizing radiation [10], [11]. Furthermore, prostate specific antigen (PSA) expression was up-regulated by NF-κB in androgen-independent prostate cancer cells, implying pathophysiological significance of NF-κB signaling in prostate cancer [12]. Taken together, these studies strongly support that NF-κB plays an important role in prostate tumor progression.

The insulin-like growth factor binding proteins (IGFBPs) are components of the IGF system that includes ligands, IGF-I, IGF-II, and transmembrane receptors, IGF-I and IGF-II receptors (IGF-IR and IGF-IIR) [13]. IGFBPs modulate IGF binding to its receptor and subsequent biological effects. Recent evidence indicates that some IGFBPs may themselves have specific biological effects independent of the IGF/IGF-IR axis in a variety of cancer cell systems, including human prostate cancer cells. In particular, IGFBP-3, the most abundant IGFBP in circulation, has been shown to function as a major growth-suppressing factor through IGF/IGF-IR-independent mechanism. The intrinsic biological effect of IGFBP-3 was demonstrated through the studies of IGF-IR-null murine fibroblast [14] and IGFBP-3 mutant devoid of IGF binding affinity [15]. The first evidence for cell surface binding of IGFBP-3 was demonstrated using chick embryo fibroblast where cell growth inhibition occurred regardless of IGF [3]. Furthermore, up-regulation of IGFBP-3 expression was correlated with TGF-β-induced cell growth inhibition in breast [16] and prostate cancer cells [17], followed by the study proposing IGFBP-3 as a functional ligand for the type V TGF-β receptor to induce cell growth inhibition [13]. IGFBP-3 has also been shown to translocate to the nucleus to interact with nuclear receptors such as retinoid X receptor-α, retinoic acid (RA) receptor, and Nur77 to induce apoptosis [18]. Recent report indicates that IGFBP-3 also interacts with BAX at mitochondria to induce apoptosis [19]. These studies demonstrate that IGFBP-3 induces apoptosis in an IGF/IGF-IR-independent manner in a variety of cancer cells.

Recent studies have provided some evidence for potential interaction between IGFBPs and NF-κB signaling cascade. IGFBP-5, but not IGFBP-3, sensitized MDA-MB-231 breast cancer cells to the inhibitory effects of TNF-α through blockage of TNF-α-induced nuclear translocation of Rel A [20]. Another study, however, reported that IGFBP-3 inhibits TNF-α-induced NF-κB activity in human colonic carcinoma cells [20]. IGFBP-3 has also been shown to significantly enhance TRAIL-induced cell death in colonic carcinoma-derived cell lines by inhibiting NF-κB activation in response to the induction of apoptosis [21]. However, the underlying mechanisms of action by which IGFBP-3 achieves inhibition of NF-κB signaling cascades are yet to be elucidated. Nevertheless, since a persistent constitutive nuclear activation of NF-κB has shown to induce resistance to various chemotherapeutic agents and radiotherapy [22], [23], IGFBP-3 may be used to treat tumors which have developed therapeutic-resistance associated with activated NF-κB.

In the present study, we described the cross-talk between IGFBP-3 and NF-κB signaling pathway in vitro and in vivo, identified a novel mechanism for IGFBP-3-mediated inhibition of NF-κB activity, and demonstrated the functional effect of IGFBP-3 on NF-κB-mediated cell survival and proliferation.

Section snippets

Cell culture and treatment

BPH-1, CAFTD-02 and CAFTD-04 were cultured in RPMI 1640 medium containing 10% FBS, whereas P69SV40T (P69), M2182, and M12 were cultured in RPMI 1640 medium supplemented with 10 ng/ml epidermal growth factor, 0.02 mM dexamethasone, 5 μg/ml insulin, 5 μg/ml transferrin, 5 μg/ml selenium, and 10 μg/ml gentamicin at 37 °C under 5% CO2. Infection was conducted in appropriate wells with the adenovirus (Ad:IGFBP-3, mutant-Ad:IGFBP-3GGG or Ad:EV) at indicated m.o.i. After 24 h, IKK 2 inhibitor IV (1 or 5 μM)

IGFBP-3 is suppressed while NF-κB is activated during cancer progression

Elevated NF-κB activity has been implicated in cell proliferation, angiogenesis, invasion, metastasis, and chemo-resistance of prostate cancer. To investigate the potential role of IGFBP-3 on NF-κB activity, we utilized two distinct well-characterized experimental prostate cancer progression systems: (1) lineages of human SV40Tag-immortalized nonmalignant prostate epithelium cell lines; the P69SVT40 (P69) lineage, its tumorigenic derivative M2182 and its tumorigenic and metastatic derivative

Discussion

Recent epidemiologic studies indicate that higher IGF-1 and lower IGFBP-3 levels are independently associated with a greater risk of common cancers including prostate cancer [29], [30], [31], [32]. It is reported that there was a significant reduction in expression of IGFBP-3 in prostate tumors with a high Gleason score as compared to those with low scores and benign prostate tissues [33]. In addition, plasma IGFBP-3 levels proved to be independent predictors of disease progression in

Conflict of interest

The authors have nothing to declare.

Acknowledgment

This work was supported by US Department of Defense Grants PC040971 and PC061151 (to Y.O).

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