Satoko Kojima md phd, Department of Urology, Teikyo University Chiba Medical Center, 3426-3 Anesaki, Ichihara-city, Chiba 299-0111, Japan. Email: email@example.com
In the last decade, abundant evidence has suggested that the insulin-like growth factor (IGF) family comprises a multi-component network of molecules involved in the regulation of both physiological and pathological growth processes in the prostate. The IGF axis plays an important role in the tumorigenesis and neoplastic growth of prostate cancer. Epidemiological observations indicate that circulating IGF-I levels are positively associated with increased risk of prostate cancer. Activation of IGF-I receptor (IGF-IR) by IGF-I has mitogenic and anti-apoptotic effects on normal and malignant prostate cells. Therapeutic alternatives in men with progressive prostate cancer after androgen ablation are very limited and more effective therapies are needed for such patients. Inactivation of the IGF-I axis represents a potential target to treat androgen-independent prostate cancer. This review addresses epidemiological studies of IGF-I and therapeutic strategies including reduction of IGF-I levels, inhibition of IGF-IR and the signaling mechanisms involved.
Prostate cancer (PCa) is the most frequently diagnosed cancer and second leading cause of cancer deaths among men in developed countries.1 Although more than 90% of PCa patients initially respond to androgen deprivation therapy, most tumors became refractory and progress to androgen-independent (AI) PCa. The mechanisms of growth in AI PCa have been investigated for decades, indicating that non-androgenic growth factors are involved.2 Insulin-like growth factor (IGF)-I plays an important role in cell proliferation, inhibiting cell death in many malignant cells, including prostate,3,4 breast,5 colon6 and lung cancer.7 Growing evidence accumulated over the last decade suggests that the IGF axis is involved in both mitogenic and anti-apoptotic events in malignant cells.8,9 This axis also appears to represent a critical determinant of response to numerous cancer therapies.10 The potential roles of IGF-I receptor (IGF-IR) in prostate carcinogenesis11,12 and associations with resistance to androgen deprivation have been described, but is not well established.13 This review describes the role of the IGF axis in PCa epidemiology, androgen-independent progression and methods of blocking IGF signal transduction as a potential cancer therapy for PCa.
The IGF system
Insulin-like growth factors exert multiple effects on glucose, fat, and protein metabolism. IGFs also play important roles in regulating cell proliferation, differentiation, apoptosis and transformation.10,14 The IGF family consists of two ligands (IGF-I and IGF-II), IGF-IR and IGF-II receptor (IGF-IIR), six high-affinity IGF-binding proteins (IGFBP1-6), and other low-affinity IGFBP-related proteins (IGFBPrP).1 IGFBP3 is the most abundant of the six IGFBPs and binds approximately 75–90% of circulating IGF-I, together with the acid-labile subunit (ALS). IGFBP3 thus controls the amount of IGF-I available to enter target tissues and reach IGF-IRs.15 IGF-I and IGF-II display approximately equal affinities for IGF-IR in most systems, and IGF-IR is thought to transduce the effects of both ligands. IGF-IIR only binds IGF-II. IGFBPs provide another level of control of IGF actions by modulating the bioavailability and physiological activities of the IGF ligand.15
Signal transduction by IGF-IR
The effects of IGF-I are mediated by the receptors, IGF-IR and IGF-IIR. IGF-IR exhibits a hierarchy of binding affinities favoring IGF-I over IGF-II, and IGF-II over insulin. IGF-IR is a type 2 tyrosine kinase receptor that is involved in the proliferation and differentiation of normal cells during development, as well as progression of malignant cells and establishment of the transformed phenotype for a variety of cancer cells. IGF-IR requires ligand binding to trigger the appropriate downstream pathways. IGF-IR overexpression alone is insufficient to cause receptor activation.16 IGF-IIR has no tyrosine kinase activity and its role in IGF signaling remains unclear.
Ligand binding induces a conformational change in receptor subunits, resulting in activation of the intrinsic tyrosine kinase of the cytoplasmic domain of IGF-IR. The kinase phosphorylates the receptor, resulting in the phosphorylation of IGF-IR substrates, insulin receptor substrate (IRS)-1 or -2, Src- and collagen-homology (SHC), and growth factor receptor-binding protein 2 (Grb2). Coupled with Grb-2/Sos interaction, phosphorylated IRS and SHC activate the Ras/Raf/mitogen-activated protein kinase (MAPK) cascade, in turn stimulating cell growth and proliferation (Fig. 1).17,18 IRS-I also phosphorylates phosphatidylinositol 3′-kinase (PI3K) and Akt,19 which blocks survival-mediating targets including Bad and caspase 9. Activated AKT also activates nuclear factor-κB (NF-κB) transcriptional activity and mammalian target of rapamycin (mTOR).20 Activation of the MAPK pathway is considered critical for cell proliferation, while the PI3K pathway is important for mediating the metabolic and anti-apoptotic signals of IGF-I. Angiotensin II (Ang-II) has been reported to be a growth factor to inducing proliferation of PCa cells by activation of MAPK, and Ang-II receptor blocker could inhibit the proliferation of hormone-refractory PCa.21 Phosphatase and the Tensin homolog gene deleted on chromosome 10 (PTEN) is a lipid phosphatase that opposes IGF-IR signaling by dephosphorylating phosphatidylinositol-3,4,5-triphosphate (PIP3) and phosphatidylinositol-3,4-biphosphate (PIP2), thereby decreasing the activation of Akt. This tumor suppressor gene is often deleted in PCa.22
The IGF family and epidemiology of PCa
The number of PCa patients and the effect of PCa on public health are increasing, but the etiology of the disease remains poorly understood with the only recognized risk factors being age, heredity, and ethnicity.23,24 An association between serum testosterone levels and the PCa risk has been controversial and inconsistent.25 The presence of IGF-I in PCa cells and potential roles in the growth and development of cancer suggest that IGF-I may serve as a predictor of PCa or a potential target for PCa therapy. Approximately a dozen studies have examined the relationship between serum levels of IGF-I and PCa risk.26,27 Some studies have shown a positive relationship between IGF-I and PCa development, whereas others have shown an inverse relationship or no relationship at all (Table 1). No correlation has been identified between IGF-I and benign prostatic hyperplasia (BPH), but some studies support a significant association between elevated IGF-I levels and the risk of developing PCa.28–30 IGFBP3 is upregulated in PCa and breast cancer, and is known to be induced by p53, transforming growth factor (TGF)-β, all-trans retinoic acid, vitamin D analogs and tumor necrosis factor (TNF)-α.39 IGFBP3 has a growth-inhibitory regulatory role, not only by regulating amounts of circulating IGF-I, but also via IGF-independent mechanisms.40 Data on circulating IGFBP3 levels in association with PCa risk are controversial. Some reports have shown that elevated IGFBP3 levels are associated with increased risk of PCa,41,42 whereas other studies have reported null29 or non-significant reductions in risk with IGFBP3.31,35,36 A large nested case-control study by the Cancer Research UK Epidemiology Unit in 2007, which included 630 incident cases of PCa and 630 matched control subjects, concluded that serum IGF-I concentration is not strongly associated with PCa risk, with a small increase in risk for advanced-stage disease, and no association for IGFBP3.33
Table 1. Studies on the relationship between insulin-like growth factor-I (IGF-I) and prostate cancer (PCa) development
Insulin-like growth factor receptor is expressed in normal prostate, BPH, and most PCa cell lines.43 IGFs exert mitogenic and anti-apoptotic effects on both normal and transformed prostate epithelial cells in vitro and in vivo.44 PC3 cells showed significantly higher proliferation in IGF-I-expressing hosts than in IGF-I-deficient hosts.41 IGF-I promotes not only growth, but also metastatic (migration and invasion) behaviors of PCa cells.45 IGF-I cannot stimulate the growth of LNCaP cells in steroid- and growth hormone-free medium, supporting the fact that IGF-I requires interactions with other serum factors to promote cell growth.46
Insulin-like growth factor-binding protein 3 is the most abundant IGFBP, and plays a growth inhibitory role via IGF-dependent and -independent pathways. IGFBP3 expression is increased in response to androgen deprivation in AI C4-2 cells,47 and the addition of IGFBP3 to LNCaP cells induces inhibition of cell growth.48 Significant pathways mediating IGF-independent anti-proliferative actions of IGFBP3 remain unclear. IGFBP2 is the second most abundant IGFBP in the circulation, and has also been shown to have a growth inhibitory effect on normal prostate epithelial cells, while exerting potent stimulatory effects on PCa cells. Patients with high IGFBP2 levels following 3 months of neoadjuvant hormonal therapy (NHT) showed much fewer biochemical recurrences than those with low IGFBP2 levels.49
Role of the IGF family in AI progression
Approximately 80% of PCa patients respond to androgen-deprivation therapy; however, most progress to AI cancer.50 Multiple mechanisms contributing to AI progression have been described or hypothesized, including: (i) mutation or amplification of the androgen receptor (AR) to increase sensitivity to androgens; (ii) mutations of AR to allow AR activation by other steroids and anti-androgens; (iii) coactivators to increase the sensitivity of the AR; (iv) enhanced AR signaling through activation of AR by peptide growth factors and cytokines; and (v) bypass of AR signaling.51,52 Androgens and growth factors like IGF and epidermal growth factor (EGF) represent important mitogens for PCa cells, but function through different mechanisms. IGF and EGF bind to a tyrosine kinase receptor at the cell surface and activate downstream signal cascades (PI3K/AKT cascade) to increase PCa cell growth and proliferation. Growth factors may activate AR transcriptional activity, but ligand-independent activation of AR remains unclear. Previous studies have indicated the importance of the PI3K/AKT pathway in PCa development and progression to AI cancer.53–55 AKT phosphorylation is increased in recurrent or AI PCa. Furthermore, numerous studies have shown that AR transcriptional activity, stability and AR expression are controlled through the PI3K/AKT pathway.56–58 Several different models of interaction between AKT and AR have been suggested, including: (i) direct phosphorylation of AR by AKT; (ii) regulation of AR by AKT via the wint/GSK-3β/β-catenin pathway; (iii) cross-talk between AKT and AR with NF-κB; and (iv) interaction of AR with the forkhead box-O (FOXO) family of transcription factors.59 These studies have suggested that inhibition of IGF in combination with androgen deprivation may offer an effective therapeutic approach for PCa.
Several important interactions between AR and IGF-I have been reported recently (Table 2). Wu et al. showed that IGF-I can enhance AR nuclear translocation in the absence of androgens and that this effect is inhibited by A12, an IGF-IR inhibitory antibody.60 They showed that IGF-I decreases phosphorylation of AR in AR-transfected M12 cells. These data suggest that in castrated patients, the increase in AR expression coupled with intact IGF-IR signaling can lead to AR-mediated AI PCa progression. They also reported that PCa human xenograft models with inhibition of IGF-IR using A12 show a decreased rate of tumor growth from androgen dependent (AD) to AI tumors.44 These data mark IGF-IR as a potential therapeutic target in post-castration PCa.
Table 2. Summary of interactions between insulin-like growth factor-I receptor (IGF-IR) and androgen receptor (AR) in prostate cancer (PCa)56
Androgens increase IGF-IR levels in prostate epithelial cells
IGF-IR signaling alters AR phosphorylation, AR transcription profile
IGF-IR signaling effects translocation of AR to the nucleus
Inhibition of IGF-IR in conjunction with castration
The IGF family and PCa bone metastases
Prostate cancers metastasize most commonly to well-vascularized areas of skeleton, such as the vertebral column, ribs, skull, and proximal ends of the long bones. IGF-I and -II are the most abundant growth factors stored in the skeleton. These hormones are produced and secreted by bone cells and bone cells express both IGF-IR and IGF-IIR.61 The biggest effect of skeletal IGF is acceleration of bone formation and resorption. IGF-I upregulates expression of both receptor activator of NF-κB (RANK) ligand (RANKL) and osteoblasts that bind to RANK. The ligand-receptor interaction activates NF-κB, which stimulates differentiation of osteoclast precursors to osteoclasts and represses osteoprotegerin (OPG) in co-cultures of osteoblasts and PCa cells.62 IGF-I may act as a coupling factor in bone modeling by activating both bone formation and resorption; with the latter effect mediated through the OPG/RANKL system in bone.63 IGF-I also appears to strongly affect OPG/RANKL equilibrium in a manner that predisposes toward osteoclast recruitment.63–65 Investigation of KM1468, a monoclonal antibody against human IGF-I and -II, showed inhibited development of new bone formation and progression of established bone tumors induced by MDA PCa2b cells in a mouse-human adult bone model.66 These investigations strongly suggest that IGF-I plays important roles in PCa cell metastasis to bone.
Targeting of IGF-I signaling
Various strategies can be used to disrupt IGF-I signaling in PCa, including administration of growth hormone (GH)-releasing hormone (GHRH) antagonist, GH antagonist, anti-IGF-I antibodies, antisense oligonucleotide (ASO) and small interfering RNA (siRNA) against IGF-IR (Fig. 2). Since increased levels of IGF-I have been associated with risk of PCa and mice with low levels of IGF-I show decreased tumor incidence and growth,67,68 lowering circulating serum IGF-I levels could affect established cancers as well as treatment of PCa.
Growth hormone-releasing hormone is secreted by the hypothalamus to stimulate the release of GH from the anterior pituitary. GH binds to GH receptor, then regulates the hepatic synthesis of IGF-I. GHRH antagonists inhibit the release of GH, and function to block IGF-I signaling. Early GHRH antagonists MZ-4-71 and MZ-5-156, and newer antagonists MZ-J-7-118 and MZ-J-7-138, inhibit the growth of human AI PC3 and DU145 PCa xenografts and MZ-J-7-118 suppressed intraosseous growth of PC3 cells.69–72 MZ-J-7-118b showed no effects on LNCaP or MDA-PCa-2b PCa cells, but exerted inhibitory effects under androgen deprivation.73,74 These results show that GHRH antagonists can greatly potentiate the tumor growth inhibition induced by androgen deprivation. GHRH antagonist is a considerable agent for the management of both AD and AI PCa. GHRH antagonist is used for the treatment of acromegaly,75 and toxicity is considered favorable, including hot flushes and erythema and no changes in hematological and biochemical blood tests.76 Blockage of GH offers another method for reducing serum IGF-I levels. Pegvisomant is a polyethylene glycol derivative of GH that acts as a GH antagonist, and is used for the treatment of acromegaly,77 but no reports have described the use of pegvisomant for PCa. The oncological activity of pegvisomant is very limited in any case.78
Neutralization of IGF-I
As IGF-IR is activated by both IGF-I and -II, neutralization of ligands can inhibit activation of receptors. The binding affinity of IGFBPs is higher for IGF-I than for IGF-IR, and the use of IGFBPs to neutralize IGF-I has been investigated.79 IGFBP1 has been reported to inhibit IGF-I effects in an IGF-I-dependent manner to induce apoptosis in LNCaP cells.46
The other way to neutralize the ligand is to use antibodies against IGF-I and -II. KM1468 antibody against human IGF-I and -II has been reported to inhibit IGF-stimulated growth of MDA PCa 2b cells both in vitro and in vivo using new bone tumor models.66,79
Reduction of expression of IGF-IR
Recent research has suggested that inhibiting IGF-IR might have significant anti-neoplastic activity through enhancement of apoptosis, rendering this receptor an attractive target for anticancer therapy.80,81 Several approaches have been used to decrease IGF-IR expression in order to inhibit PCa proliferation and growth, including ASO,82 siRNA83 and inhibition of IGF-IR activation by small molecule inhibition.68,79,81
Antisense oligonucleotides are short segments of DNA synthesized to be complementary to a target mRNA transcript, thus hybridizing in anti-parallel orientation to inactivate the mRNA. The mRNAs are then digested by RNAse H, resulting in inhibition of protein synthesis.82 Transfection of ASO against IGF-IR into PCa cells reduced endogenous expression of IGF-IR with concomitant inhibition of IGF-I-stimulated cellular proliferation. Transfection of DU145 cells with ASO to IGF-IR suppressed IGF-IR protein levels to 30–50% of those in control cells and constitutive expression of IGF-IR antisense clone-transfected DU145 tumor xenograft showed no tumorigenicity. Furthermore, reduction of IGF-IR expression was associated with enhanced sensitivity of these cells to cisplatin, mitoxantrone and paclitaxel.84,85 In addition, siRNAs against IGF-IR have recently been described to decrease IGF-IR levels and IGF-IR signaling in cancer cells.86 The use of siRNA on the DU145 and PC3 PCa cell lines has been reported to increase chemosensitivity to mitoxantrone, etoposide and nitrogen mustard.1,86 Although the specificity of gene silencing is a significant advantage of siRNA gene targeting, progress is still required in the delivery and duration of the effect.1
Inhibition of IGF-IR activation
Several antagonistic anti-IGF-IR monoclonal antibodies have been reported, including IR3,87 IH7,88 MAB 391, scFv-Fc,89 EM164,81 Fab IgG1 m61090 and A12.91 IR3 has been found to inhibit IGF-IR in several cancer cell lines, although treatment with IR3 antibody did not inhibit the growth of MCF7 tumor xenograft in athymic mice.87 In another study, IH7 inhibited growth of cancer cells, but these antibodies have limited clinical utility because of the weak inhibition of cancer cell proliferation.88 As a single-chain antibody against IGF-IR, scFv-Fc effectively inhibits IGF-IR function in MCF-7 breast and PC3 PCa cells.89 EM164 is a most potent IGF-IR antagonist when compared with previously reported antibodies.81 EM164 has been shown to decrease expression of the IGF-IR β chain and reduce both phosphorylation of AKT and downstream signaling of IGF-IR. Inhibition of proliferation of human cancer cells has been reported, including in the PC3 PCa cell line.81 Fab IgG1 m610 is a novel monoclonal antibody to IGF-II that potently inhibits IGF-IR signal transduction, and is reported to inhibit growth of both DU 145 and MCF-7 PCa cells.90 More recently, A12, a fully human antibody to IGF-IR, has been reported to effectively inhibit human xenograft tumor growth, including AD and AI PCa.60,92 A12 blocks ligand signaling by two processes: (i) antagonism of ligand binding; and (ii) rapid induction of receptor internalization and degradation. Thus, A12 may greatly enhance suppression of IGF-IR signaling in tumor cells.44 A12 also reportedly enhances the therapeutic effects of docetaxel in androgen-independent PCa models in vivo.60
Small molecule tyrosine kinase inhibitors, which are low molecular mass inhibitors of IGF-IR kinase activity, reportedly inhibit IGF-IR activation in several cancers including lung, colon, breast, pancreas and PCa. INSM-18, a small-molecule tyrosine kinase inhibitor, is currently in a phase I clinical study for patients with relapsed PCa.1
Insulin-like growth factor-I receptor expression is detectable by immunofluorescence on circulating tumor cells (CTC) in PCa patients.93 CTCs and IGF-IR positive CTCs were most frequently detected in hormone-refractory prostate cancer (HRPC) patients.94 A previous study reported CTC counts as the most significant variable predictive of survival of HRPC patients. CP-751871 is a human monoclonal antibody currently undergoing phase I clinical trials.93 Detectable IGF-IR expression on CTCs before treatment with CP-751871 and docetaxel was associated with prostate-specific antigen (PSA) decline by >50%.93 Only a monoclonal antibody against IGF-IR (CP-751871)93 and IGF-IR tyrosine kinase inhibitor (Insm-18)1 are currently entered in phase I studies, but several preclinical studies are underway.
The IGF family plays an important role in PCa cell proliferation, survival, bone metastasis, and interactions with AR and AI progression, and is thus likely to become a therapeutic target for antisense oligonucleotides, siRNA and monoclonal antibodies to IGF-IR either alone, or in combination with other chemotherapeutic agents. AI PCa is regulated by several mitogenic pathways and IGF pathways may crosstalk with a variety of growth regulatory systems. The development of anti-IGF strategies seems likely to become important in the treatment of AI PCa.