Since 1957, IGF-1 and IGF-2 (historically referred to as somatomedins A and C, respectively) were identified as second messengers of growth hormone (GH) capable of promoting insulin-like anabolic effects upon normal somatic tissues such as skeletal muscle and bone [1]. Since then, they and their cognate receptors have been demonstrated to affect a diverse range of cancers by facilitating malignant transformation, altering cell differentiation, and promoting cancer growth, metastasis, and chemotherapy resistance. However, only within the last decade have physicians had at their disposal both a host of clinically relevant anti-IGF-1R targeted therapies (small molecules and antibody-based inhibitors against IGF-1R or IGF-1) and a number of diverse high throughput technology platforms capable of readily teasing apart the multifaceted pharmacodynamic effects exerted by such IGF-1R antagonism.

Whilst a number of excellent reviews have thoroughly discussed the impact of IGF-1R stimulation upon normal and malignant cells [2-7], or highlighted the myriad therapeutic options under preclinical and clinical investigation, few have concisely narrowed the focus to the complex interplay that exists between IGF-1R and a host of redundant signaling pathways (e.g., integrins, her2/neu, EGFR) potentially responsible for both de novo and acquired resistance to IGF-1R and its downstream targets. Following a necessarily brief summary of the IGF-1R family and its cancer promoting effects, the crux of this review has been dedicated to mechanisms of IGF-1R resistance and dual-targeted strategies aimed at circumventing them. We conclude by drawing upon the success of other targeted therapies (such as trastuzumab or imatinib) and suggest a rational path forward in IGF-1R centric trial design that integrates pharmacodynamic biomarkers to improve patient selection for likely responders and enhance our monitoring of the molecular changes induced by IGF-1R targeting.

Given the capacity of IGF-1R to initiate both normal and pathological signaling cascades, IGF-1R and its downstream mediators have, naturally, been widely implicated in contributing to various malignancies and been investigated as potential therapeutic targets now for more than two decades. High IGF-1 levels have been found in several sarcoma subtypes [32,33] and IGF-1R overexpression in breast, lung, prostate, or colon cancer has been shown to accelerate cancer progression [34-37] and enable anchorage-independent growth [17]. Conversely, congenital syndromes resulting in IGF-1 or growth hormone deficiency likely confer protective effects [38,39].

The inner workings of the IGF-1R molecular machinery share considerable similarities between carcinomas and sarcomas, both from a myopic view at the level of biologically conserved protein-protein interactions that can be understood with reasonable specificity, and from the higher vantage point of a ‘signaling network’ that can be anticipated to progress, if not deterministically, at least under stochastic rules gleaned from years of scientific scrutiny. Yet, considerable biologically complexity exists not only from one cancer type to the next but also, as is often the case, among asynchronously responding tumors within the same patient.

In addition to the intrinsic differences present within the IGF-1 pathway itself (via altered expression of IGF-1R, its ligand, the IGFBPs, or downstream effectors, for example), such changes only partially explain the varied sensitivity to IGF-1 oriented therapies. Extrinsic differences independent of the IGF-1R pathway, such as her2/neu or EGFR activation for breast or lung cancer, respectively, likely play a larger role since those pathways often serve as the primary regulator of their malignant phenotype. Under that scenario, the IGF-1 pathway would theoretically be redundant, and largely quiescent, until the primary pathway becomes impaired, either spontaneously or secondary to therapeutic intervention. Ultimately, then, the importance of IGF-1 signaling can vary tremendously across cancer types and even temporally within a patient's tumor if selective pressure is applied through therapeutic targeting.

Notwithstanding a clear benefit observed in a small subset of patients treated with single-agent IGF-1R antagonists, enthusiasm for single-agent IGF-1R targeting has waned and most active or developing clinical trials have evolved to use IGF-1R-targeted therapies together with others that surmount anticipated mechanisms of resistance. Of course the resistance mechanisms themselves have only partially been enumerated and, as discussed previously, they likely vary from one cancer type to another, subject to the predominant oncogenic driver (Table 1).

Putative mechanisms of resistance may be conceptually grouped by two broad categories:

Primary independence from IGF-1R activation, presumably through myriad pathways that bypass IGF-1R (i.e., upstream plasma membrane bound receptors including alternative RTKs and hybrid receptor combinations that also activate Grb2, Sos, or IRS-1) or downstream molecules capable of intrinsic self-activation of MAPK and Akt/mTOR.

With respect to the first category or resistance, cross talk via alternative RTK or non-receptor transmembrane signalers (such as integrins) could potentially bypass the need for IGF-1R signaling. In addition to EGFR, PDGF-β [93], NGF-R [10], and HER2 expression [94], some sarcomas have been shown to express c-kit [93,94]. Imatinib-induced shutdown of c-kit receptor phosphorylation leads to a 20–30% reduction in EWS cell proliferation and suppressed tumor growth in xenograft models, albeit at doses 20-fold higher than that used for treatment of gastrointestinal stromal tumors (18–22 μM) [93,95]. Used alone, less than 5% of EWS patients achieve a partial response to single-agent imatinib (440 mg/m²/day) [96]. Dasatinib, a multi-targeted tyrosine kinase inhibitor (TKI) of c-kit and PDGF-β has also shown activity, again at high concentrations [97]. Given the partial overlap IGF-1R antagonists and of the c-kit or PDGF-β TKIs (which predominately suppress MAPK), one may hypothesize that c-kit or PDGF-β up-regulation is a potential mechanism of IGF-1R resistance. The synergy observed in vitro between small molecule antagonists of the IGF-1R (such as NVP-ADW742 or NVP-AEW541) and imatinib, through apoptotic mechanisms, supports this hypothesis although, to our knowledge, secondary up-regulation of those receptors in IGF-1R-resistant cells has yet to be shown [98].

Other receptors, including the epidermal growth factor receptor (EGFR), the vascular endothelial growth factor receptor-2 (VEGFR-2), and rearranged in transformation (RET) kinase receptor have been evaluated and another, macrophage-stimulating 1 receptor tyrosine kinase (MST1R) has just recently been identified as potential means to induce IGF-1R-independent stimulation [99,100]. Though gefitinib (an EGFR kinase inhibitor) and vandetanib (an inhibitor of VEGFR-2, VEGFR-3, and RET kinase) inhibited EWS growth at high concentrations (greater than 5 μM), nonspecific effects were suspected since the phosphorylation state of MAPK and Akt were unchanged. Scotlandi et al. has reported HER2 expression in 16% of EWS specimens, however gene amplification was absent and little antiproliferative response to trastuzumab (Herceptin) was observed [94]. In summary, of the experience of non-IGF-1R tyrosine kinase inhibitors for EWS treatment, none has significant single-agent activity in the setting of functional IGF-1R. This does not, of course, rule out their role in IGF-1R-resistant tumors; the additive and/or synergistic effects reported in combination with either of the Novartis's pyrrolo[2,3-d]pyrimidine derivatives or Bristol Myers Squibb's pyrrolecarboxaldehydes (BMS-554417 or BMS-536924), in fact, suggests compensatory signaling could occur under IGF-1R-null conditions, as has been recently reported by Helman [101]. Adding a layer of complexity, since insulin and IGF-1 half-receptors have been reported to form heterodimers with members of the EGFR family in lung cancer, this adds another layer of complexity in assessing TKI-mediated resistance [72].

Regarding the second category of IGF-1R resistance, complex counterregulatory loops in the IGF-1R autocrine circuit, including the receptors, ligands, and binding-proteins, may be involved. One such example is the autoregulatory loop between Mdm2 and p53. Froment et al. have reported that Mdm2, a protein antagonist of p53, can physically bind IGF-1R and target it for ubiquitination-induced degradation independent of p53 [102,103]. Interestingly, whereas wild type-p53 down-regulates transcription of IGF-1R at the promoter level, mutant p53 induced the opposite effect in osteosarcoma and rhabdomyosarcoma cells. Since p53 mutations are observed in less than 5% of EWS primary tumors [104], it remains to be determined whether mutant-p53-induced up-regulation of IGF-1R exists as a resistance mechanism for IGF-1R targeted therapy.

The level or activation status of members of the IGF-1R family may affect resistance. Since neither mutation nor amplification is common, this is not the most likely contributor to antibody resistance. Though not yet confirmed to be prognostic in EWS, high levels of IGF-1R appear to confer sensitivity in rhabdomyosarcoma, and may serve as a valid prognostic biomarker for that cancer [105]. Low levels of IGF-1R may, conversely, confer resistance in at least two ways: (1) IGF-1R-low-expressing cells would theoretically be less reliant or ‘addicted’ upon IGF-1R for growth and; (2) targeted therapies generally requires a paired target for effectiveness [106]. Paradoxically, high IGF-1R levels, when stabilized by Heat Shock Protein-90 (HSP90; a chaperone protein that helps maintain stability, renature unfolded proteins, or targeted their degradation), may also confer at least short-term resistance as hypothesized by Martins et al. [95]. In evaluating why HSP-90 was transiently elevated in ADW742-resistant A673 EWS cells, it was suggested that client-protein stabilization of activated IGF-1R or and Akt by HSP90, maintained downstream signaling of the Akt/mTOR pathway.

In the most recent and comprehensive report of IGF-1R resistance mechanisms to use genetic and proteomic profiling, Helman compared BMS-536924-resistant sarcoma and neuroblastoma cells to sensitive ones, thereby identifying gene and protein subsets that significantly correlated with de novo drug sensitivity. Although members of the IGF-1R family did not reach statistical significance for a priori inclusion within those subgroups, high IGF-1R, IGF-1, or IGF-2 levels portended sensitivity whereas elevated IGFBPs 3 and 6 were higher in resistant cell lines. Unexpectedly, the combination of IGF-1 and IGF-2 into a single model was better than either used alone in predicting response, suggesting an active role for both ligands in IGF-1R signaling. While an IGF-2-mediated effect may not be intuitive, since IGF-2 has twenty-fold less affinity for IGF-1R, it has recently been reported that malignancies can shift their reliance from the paradigmatic IGF-1-stimulated IGF-1R pathway instead to an IGF-2-stimulated one that acts upon the IGF-1R/IR-α hybrid receptor already mentioned [107].

As suggested earlier, given the capacity for tumor-associated stoma to secrete IGF-1 or IGF-2, paracrine loops may also affect the efficacy of IGF-1R targeted therapy. Gorlick, Houghton, and others have reported relative insensitivity to IGF-1R- or mTOR-targeted therapies in vitro compared to xenografts models of similar tumor types, supporting our hypothesis that extracellular mechanisms of resistance are important [108]. Since tumor regrowth (after initial response) is a near universal occurrence in xenograft models (reportedly with continued IGF-1R downregulation and maintained p-Akt) [108] and patients treated with single-agent IGF-1R targeted therapies to date, a fresh approach must seek to obviate not only IGF-1R signaling but also the cancer type-specific resistance mechanism(s) as well.

Although many of the IGF-1R resistance mechanisms described above pertain to sarcomas, major mechanisms of resistance can be found across the spectrum of diverse cancer types. At other times, the mechanism(s) of resistance are unique and specific to the individual features that distinguish one cancer from another, as identified for the most common cancer types within Table 1.

Even before the precise mechanisms of single-agent IGF-1R success, and in some cases failure, are thoroughly scrutinized, a limited number of preclinical studies and mostly early phase clinical trials have begun to assess the safety and efficacy of dual targeting of IGF-1R and putative secondary targets suspected of enabling acquired IGF-1R resistance (Table 2). Paralleling the defined mechanisms of resistance highlighted above, multi-targeted therapy can target key components intrinsic to the IGF-1R receptor family (the receptors, ligands, or IGFBPs in various combinations) or extrinsic ones.

An example of intrinsic targeting includes MEDI-573, a dual IGF-1/2 targeted neutralizing antibody that can theoretically prevent activation of both IGF-1R and IGF-1R/IR-A hybrid receptors. Similar in effect, small molecule inhibitors of IR and IGF-1R, such as OSI-906, have generated significant enthusiasm, given anecdotal reports of clinical response [109-111]. Though not in clinical trials, yet another approach combines two antibodies that together target divergent epitopes within the ligand binding sites of IGF-1R [112]. Each of those therapeutic strategies offer to inhibit IGF-1R function while countering the compensatory IR-mediated crosstalk inherent in IR-A and its pairing with IGF-1R [88]. Whereas both IGF-1 and IGF-2 ligands bind and activate IGF-1R and IGF-1R/IR-A hybrid receptors, the additional suppression of IGF-2 may limit unopposed IR-A signaling [113]. Given the increased expression IR-A within neoplastic tissues and preferential affect upon IGF-induced mitogenic signaling, as opposed to IR-B that exerts greater influence upon glucose hemostasis within normal liver, muscle and fat, one could conceivably target just IGF-1R and the oncogenic IR-A splice variant while minimizing hyperglycemia and untoward side effects associated with down-regulated IR-B. In practice, however, this hypothesis remains to be proven and, to our knowledge, there are no selective IR-A inhibitors. Because elevated levels of plasma IGF-1 and insulin occur as respective feedback mechanisms induced through selective IGF-1R targeting and off-target effects upon IR-B, it may be necessary to target both IGF ligands, insulin, IGF-1R, both hybrid receptor types, and IR-A in unison to have the greatest clinical impact while avoiding the unintended pharmacodynamic consequences. Finally, to the extent insulin can promote IR-A mediated oncogenic effects, one could hypothesize its use for the treatment of iatrogenic hyperglycemia should be avoided in patients harboring IGF-1R driven malignancies when other pharmacological options exist.

Of course a number of preclinical and clinical studies utilize a dual-targeting approach aimed at IGF-1R and extrinsic cascade-initiating RTKs or downstream mediators. Co-targeting c-kit and IGF-1R appears to be synergistic in EWS and small cell lung cancer (SCLC) cells [114]. A novel small molecule inhibitor of the IGF-1R/IR/ALK triad, GSK1838705A, has shown antitumor activity in human tumor models and should help elucidate the relationship of IGF-1R pathway activation in ALK-positive tumors noted within subtypes of NSCLC, lymphoma, and sarcoma [115]. And several phase I/II trials investigating mTOR/IGF-1R co-targeting have just been completed; everolimus/figitumumab [116] and cixutumumab/temsirolimus (Naing, personal communication), and ganitumab/rapamycin is on the horizon [117].

Certainly with respect to cancers of the lung, prostate and colon, which rely in part upon EGFR signaling for tumor growth and survival, dual targeting of IGF-1R and EGFR has garnered much interest, given the fact that reciprocal inhibition of one RTK in epithelial cancers often enhances expression of the other. Bispecific antibodies capable of binding both IGF-1R and EGFR are undergoing investigation [118] and numerous studies have combined IGF-1R targeted therapies with others against EGFR [118-121]. Such RTK crosstalk has also been observed for the human EGF receptor 2 (HER2), the target of trastuzumab in breast cancer, and preclinical studies indicate synergy with dual IGF-1R/HER2 targeting [122,123]. Finally, significant crosstalk between IGF-1R and the androgen receptor in prostate cancers [50,124] or estrogen receptor in breast cancers [125-129] has been observed, though this combination remains to be validated clinically. Of course, many preclinical studies, and some clinical ones, have assessed the role of IGF-1R antagonists in combination with traditional cytotoxic chemotherapy. However, this topic is beyond the scope of this review.

After a flourish of clinical trials designed to investigate the role of single-agent IGF-1R targeted therapy, much of the initial optimism has been tempered by the realization that only limited subsets of patients respond and, when they occur, such responses too often are unsustained beyond a few months. Not surprisingly, without significant response rates observed for common cancers (i.e., breast, colon, lung, or prostate cancer), many pharmaceutical companies have ceased, or at a minimum delayed, clinical development of their respective IGF-1R inhibitors.

Though this deceleration in clinical trial implementation will assuredly limit patient access to IGF-1R targeted therapies in the short term, over the longer term it may actually serve a benefit by allowing the necessary preclinical science to be mature before committing a substantial number of patients to empiric, lengthy, and potentially suboptimal treatment. Drawing upon the lesions learned from other biologically targeted therapies such as trastuzumab, a number of questions must be answered if we are to make significant strides forward. Among just a few are as follows: (a) what predictive biomarkers allow for effective patient enrichment for those most likely to benefit; (b) what pharmacodynamic effects are associated with tumor control, and finally; (c) how should combinatorial therapies be advanced to avoid acquired resistance and maximize response duration. As the scientific community races to find answers, one anticipates in the not too distant future that IGF-1R antagonists will prove an essential weapon in the oncologist's arsenal to be wielded in unison with other biologically targeted agents.