Insulin-Like Growth Factors and Breast Cancer Therapy

Despite improvements in breast cancer therapy in recent years, additional therapies need to be developed. New therapies may have activity by themselves or may have utility in combination with other agents. Population, preclinical, and basic data suggest the insulin-like growth factor (IGF) system functions to maintain the malignant phenotype in breast cancer. Since the IGFs act via transmembrane tyrosine kinase receptors, targeting of the key receptors could provide a new pathway in breast cancer. In addition, IGF action enhances cell survival, so combination of anti-IGF therapy with conventional cytotoxic drugs could lead to synergistic effects. In this review, we will discuss the rationale for targeting the IGF system, potential methods to disrupt IGF signaling, and identify potential interactions between IGF inhibitors and other anti-tumor strategies. We will also identify important issues to consider when designing clinical trials.

Introduction

Breast cancer is the most common cancer in women responsible for over 40.000 deaths in the United States.¹ Since cancer death is almost always caused by growth of breast cancer in metastatic sites, systemic cytotoxic and endocrine therapies are commonly given. In operable breast cancer, systemic adjuvant therapy is employed to reduce the risk of recurrence and prolong overall survival. In women with metastatic disease, systemic therapy is given to control growth of breast cancer in distant organs. Both treatments essentially target metastatic disease; in the adjuvant setting, the goal is to eradicate any microscopic disease that is not clinically detectable while treatment of metastatic disease targets clinically obvious sites.

Though systemic treatment is clearly effective in both adjuvant and advanced settings, therapy is far from completely effective. In the adjuvant setting, administration of chemotherapy reduces the relative risk of recurrence by approximately 30%.² Identifying methods to enhance the cytotoxicity of chemotherapy are clearly needed. Since normal and cancer cells receive survival and proliferative signals from their extracellular environment, targeting of these signals could enhance the clinical benefit of chemotherapy. Indeed, trastuzumab, an antibody directed against the human epidermal growth factor receptor-2 (HER2), is commonly used in combination with chemotherapy for women with advanced cancer.³ While the exact mechanism for the synergy between trastuzumab and chemotherapy is not completely understood, it is likely that trastuzumab renders cells more sensitive to the apoptotic effects of chemotherapy by attenuating cells survival pathways.

Identifying additional survival pathways could also be used to enhance the benefits of chemotherapy. Insulin-like growth factors (IGFs) and the IGF signaling pathways play a role in development of the normal mammary gland. Numerous studies have now demonstrated that the IGF system regulates all of the key metastatic phenotypes in breast cancer cells: survival, proliferation, and metastasis.

Extensive data are available on the importance of IGF system in growth regulation of breast cancer cell lines.⁴ The type I insulin-like growth factor receptor (IGF-IR) is significantly overexpressed⁵ or hyperphosphorylated in tumor cells relative to normal breast epithelium and benign tumors.^5,6 In addition, several clinical studies also support a role for IGF-I in breast cancer risk.^7,8 In cell line model systems, IGF-IR conferred resistance to trastuzumab-induced growth inhibition⁹ and a kinase inhibitor to IGF-IR increased radiosensitivity in some breast cancer cells lines.¹⁰ Thus, data from population and laboratory studies provide a rationale for targeting IGF-IR in breast cancer. Moreover, disruption of IGF-IR may render cells more sensitive to apoptotic stimuli. Several reviews have already addressed the important role of the IGF system in breast cancer.^11-13 Here we will summarize the potential role of IGF action on breast cancer chemotherapy.

The IGF System

The IGF system involves the coordination of growth factors (IGF-I and IGF-II), cell surface receptors (IGF-IR, IGF-IIR, and the insulin receptor IR), six high affinity binding proteins (IGFBP-1 to 6), IGFBP proteases, and the downstream proteins involved in intracellular signaling distal to IGF-IR.

IGF-I and IGF-II are single-chain 7.5 kDa polypeptide growth factors with a high degree of homology to insulin. The main function of IGF-I is to act as an effector molecule of growth hormone (GH), which is fundamental to linear growth and development.¹⁴ During puberty, pulsatile GH release from the pituitary stimulates expression of IGF-I in the liver. In addition to its endocrine role, it has been suggested that IGF-I may also have an important role in prenatal growth. Mice with a homozygous deletion of the IGF-I gene have a birth weights less than 60% of their wildtype littermates, these mice have a high post-natal mortality rate.¹⁵ Thus, besides its endocrine role, IGF-I plays an important paracrine and autocrine role during normal development and growth of the organism. IGF-II expression is not regulated by GH. However, IGF-II has proliferative and antiapoptotic actions similar to IGF-I. In addition, IGF-II plays a fundamental role in embryonic and fetal growth, this was proven by IGF-II gene knockout mice, which survive but remain smaller than their wildtype littermates.¹⁶ Interestingly, size at birth and height at age 14 have been linked to increase breast cancer risk suggesting an etiologic role for the IGFs and breast cancer development.¹⁷

The actions of IGFs can be modulated by interaction with a family of six insulin-like growth factor-binding proteins, IGFBP-1 to IGFBP-6, which share 40-60% amino acid identity. IGFBP3 is the largest and most abundant IGFBP, more than 75% IGF is confined to the vascular compartment as a ternary complex with IGFBP3 and the acid labile subunit. By binding IGF-I and IGF-II, IGFBPs regulated the bioavailability of IGFs in the circulation. IGFBP-3 has also been shown to competitively inhibit IGF action at the cellular level in the absence of IGF binding and exert IGF-independent proapoptotic and antiproliferative effects through the activation of caspases involved in a death receptor-mediated pathway.¹⁸

Although at least two receptors for IGFs exist, the primary signaling receptor through which both IGF-I and IGF-II exert their biological actions is the IGF-IR. Hybrid IGF-IR/Insulin receptors also exist and could mediate IGF and insulin action in breast cancer cells.^19,20 Presence of the hybrid receptor adds an additional layer of complexity to IGF signaling.

Binding of ligand to the receptor induces autophosphorylation and activation of multiple downstream cell survival and proliferation signaling pathway via recruitment and tyrosine phosphorylation of specific adaptor/effector molecules.²¹ While individual pathways have been linked to specific cancer phenotypes, it is clear that the intracellular signals initiated after IGF-IR activation constitute a network of interacting molecular events. It is likely oversimplistic to ascribe a specific behavior to a specific pathway. Nonetheless, activation of PI3 kinase downstream of the insulin receptor substrate-1 (IRS-1) adaptor protein has been linked to cell survival and regulation of several proteins involved in apoptosis.²² Induction of cell growth and proliferation has been linked to both the PI3 kinase pathway and the MAP kinase pathway downstream of IGF-IR and its phosphorylated substrates, IRS-1 and Src-collagen homology (Shc) protein. The type II insulin-like growth factor receptor (IGF-IIR) lacks tyrosine kinase activity and appears to exert antiproliferative and proapoptotic activities by sequestration of IGF-II, reducing its availability for interaction with the IGF-IR.²³

IGFs and Normal Mammary Tissue

IGFs play a key role in proliferation and survival in the mammary gland, particularly during puberty and pregnancy.^24,25 IGF-I is a potent mitogen for mammary epithelial cells and in combination with mammotrophic hormones, such as estrogen receptor (ER), it induces ductal growth in mammary gland explant cultures.²⁶ IGF-IR null mice have deficient mammary development with reductions in the number of terminal end buds, ducts and the per cent of the fat pad occupied by glandular elements. This phenotype is partially restored by administration of des(1-3) IGF-I.²⁷ In addition, IGF-I also plays a role in the maintenance of the adult mammary gland during lactation, lactating transgenic mice overexpressing the Igf1 gene undergo ductal hypertrophy and fail to show normal mammary gland involution following weaning.²⁸ The same group also demonstrated that IGF-I slows the apoptotic loss of mammary epithelial cells during the declining phase of lactation.²⁹

It is known that IGFs is one of the developmental/essential survival factors for the mammary gland, although other factors such as epidermal growth factor (EGF) and its homologues also deliver intracellular signals that suppress apoptosis. Direct evidence for IGFs as survival factors comes from culture studies.^24,30,31 IGF-I or IGF-II can suppress the apoptosis of mammary epithelial cells induced by serum withdrawal. It has been recently established that this is achieved through PI3K and MAPK signals that ultimately inhibit activity of a proapoptotic protein, BAD and enhance expression of another antiapoptotic protein Bcl-xL.^24,30 Indirect evidence came from the transgenic mice overexpressing IGFBP-5 in the mammary gland, these mice had reduced numbers of alveolar end buds, with decreased ductal branching and increased expression of the pro-apoptotic molecule caspase-3,and decreased expression of pro-survival molecules of the Bcl-2 family.³²

IGF and Breast Cancer

The IGFs and IGF-IR function to promote proliferation, inhibit death and stimulate transformation in breast cancer cells.¹¹ High levels of serum IGF-I are associated with an increased risk of breast cancer in premenopausal women.³³ There is also substantial evidence that IGF expression occurs locally in breast cancer tissues. Although IGF-I is rarely expressed in primary breast cancer, IGF-II message is more frequently detectable in breast cancer cells compared to normal cells.³⁴ Moreover, the stroma is a rich source of both IGFs.

Studies in transgenic mice have revealed an important role of IGF-I in mammary tumorigenesis. Transgenic mice expressing human des(1-3) IGF-I (under the control of the rat whey acid protein) display an increased incidence of mammary tumors, with 53% of the mice developing mammary adenocarcinomas by 23 months of age.^35,36 Furthermore, data in a transgenic mouse system suggest that mice deficient in liver-expressed IGF-I have a reduced ability to develop mammary tumors.³⁷ In human studies, circulating IGF-I levels are higher in breast cancer patients than in controls. In addition, cohort studies have shown that higher levels of circulating IGF-I are associated with an increased risk of breast cancer in premenopausal women.³⁸

The IGF-IR, the primary mediator of the biological actions of IGF-I, has been detected in a majority of primary breast tumor samples with overexpression in 30% to 40% of breast cancers.³⁹ Furthermore, IGF-IR autophosphorylation has been found to be elevated in human breast cancer suggesting that this is an active pathway in primary breast cancer.^6,40 Interestingly, a high level of IGF-IR in patients with breast cancer is associated with a greater recurrence risk of recurrence after local radiation therapy.⁴¹

Insulin receptor substrate-1 (IRS-1), the primary signaling molecule activated in response to IGF in MCF-7 human breast cancer cells, is reported to be overexpressed in some primary breast tumors and a high IRS-1 are associated with a decreased disease-free survival in a subset of patients with all tumors.^42,43 Activation of specific IRS species are associated with distinct biological effects.⁴⁴ Activation of IRS-1 signaling was associated with cell growth, whereas insulin receptor substrate-2 (IRS-2) signaling was associated with cell motility.^44,45 Nagle et al showed that mammary tumor cells obtained from IRS-2 knock-out mice were less invasive and more apoptotic in response to growth factor deprivation than their WT counterparts. In contrast, IRS-1(-/-) tumor cells, which express only IRS-2, were highly invasive and were resistant to apoptotic stimuli.⁴⁶ These data suggest that signaling pathways downstream of IGF-IR may ultimately be responsible for the malignant phenotype mediated by this growth factor signaling system.

Breast cancer cells also produce several IGFBPs that could modulate IGF action. In addition to the indirect modulation of IGF's mitogenic and antiapoptotic signals by ligand sequestration, IGFBPs also exert IGF-independent effects on cell survival. For example, IGFBP-4 and IGFBP-5 can rescue cells from ceramide or integrin-mediated apoptosis, which may account for the poor prognosis in breast cancers with high IGFBP-4 expression^13,47 whereas IGFBP-3 has been found to directly inhibit breast cancer cell growth without interacting with IGFs.^13,48

Conventional Chemotherapy for Breast Cancer

It is well established that most chemotherapeutic agents eliminate cells by triggering apoptosis. Most currently approved agents target specific molecules required for a cell to traverse the cell cycle. Targets range from DNA itself, to enzymes (topoisomerases), or structural proteins (tubulin) required for cell division. There are three kinds of cells in a solid tumor: dividing cell that are continuously cycling, resting cell which may potentially enter the cell cycle, and those cells no longer able to divide. In breast cancer, Clarke et al have suggested that only a minority of cells contained within a tumor have the capacity to contribute to all these subpopulations of tumor cells leading to the idea that cancer stem cells exist.⁴⁹ Essentially only dividing cells are susceptible to the currently available chemotherapy drugs. It is the existence of resting or stem cells that makes it difficult to completely eradicate advanced tumors by chemotherapy; even after a substantial clinical response, a population of cells may still exist that have full capacity to enter the cell cycle.

The primary therapy of localized-early stage I and II- breast cancer is either breast-conserving surgery and radiation therapy or mastectomy with or without reconstruction.⁵⁰ Systemic adjuvant therapies designed to eradicate clinically undetectable microscopic deposits of cancer cells that may have spread from the primary tumor result in decreased recurrences and improved survival.^2,51 Adjuvant therapies include chemotherapy and hormonal therapy. In the adjuvant setting, chemotherapy is usually given in combination for 4-6 months. A wide variety of agents have been effective in breast cancer including DNA alkylators (cyclophosphamide), topoisomerase inhibitors/DNA intercalators (doxorubicin), anti-metabolites (5-fluorouracil, methotrexate), and tubulin interacting agents (paclitaxel).² Adjuvant chemotherapy effectively reduces the odds of recurrence and death by approximately 20% to 60% of patients. However, since this reduction of risk is not complete, substantial research effort is directed toward improving the benefits of chemotherapy. New target discovery and combination of new agents with “conventional” agents represent an active area of investigation.

IGF Signaling Confers Resistance to Chemotherapy

Besides inducing cell cycle progression, IGF-I also protects breast cancer cells from drug-induced apoptosis.^52-54 In fibroblasts, protection from apoptosis requires the tyrosine kinase activity of IGF-IR, as kinase defective receptors do not protect fibroblasts from apoptosis.⁵⁵ In addition to drug-induced apoptosis, IGF-IR activation appears to block other stimuli as well. For example, BNIP3 (Bcl-2/E1B 19 kDa interacting protein) is a proapoptotic member of the Bcl-2 family expressed in hypoxic regions of tumors. Treatment of the breast cancer cell line MCF-7 cells with IGF effectively protected these cells from BNIP3-induced cell death⁵⁶ likely via activation of PI3K and the Akt/PKB pathways.^55,57 Akt/PKB phosphorylates BAD, a member of the Bcl-2 family of proapoptotic proteins, phosphorylated BAD cannot heterodimerize with Bcl-2 or Bcl-Xl, remains in the cytosol and cell death is inhibited.⁵⁸ It has also been suggested that IGF can inhibit apoptosis by increasing the expression of Bcl-X_L at both the mRNA and protein level.⁵⁹ Thus, signaling from IGF-IR to multiple proteins involved in the intrinsic apoptotic pathways suggest a mechanism for protection from cell death signals.

IGFs provide resistance of breast cancer cells to chemotherapeutic agents.⁵³ IGF-I alters drug sensitivity of HBL100 human breast cancer cell by inhibition of apoptosis induced by diverse anticancer drugs, it increased cell survival of HBL100 cells treated with 5-FU, methotrexate, tamoxifen or camptothecin, but no changes were observed in Bcl-2 protein or Bax mRNA levels.⁶⁰ IGF-I signaling is also associated with resistance to the growth-inhibitory actions of trastuzumab by upregulation of ubiquitin-related p27^kip1 degradation and activation of the PI3K signaling pathway.⁶¹ In breast cancer cells, IGF-I can activate JNK which is generally associated with a pro-apoptotic response. However, activation of Akt seems to override pro-apoptotic effects of JNK activation.⁶² Thus targeting IGF-IR could enhance a pro-apoptotic response initiated by many different agents.

IGF-IR and DNA Repair

Several reports indicate that IGF-IR activation is also important in regulating DNA repair. Fibroblasts can be rescued from DNA damage by IGF-I via activation of the p38 MAP kinase signaling pathway.^63,64 Increasing IGF-IR expression increased radioresistance in breast tumor cells,⁶⁵ and delayed UVB-induced apoptosis by enhancing repair of DNA cyclobutane thymidine dimers in keratinocytes.^66,67 IGF-I stimulation supports homologous recombination-directed DNA repair (HRR) via an interaction between IRS-1 and Rad51, a key enzyme of HRR.⁶⁸ In contrast, IGF-I may actually inhibit the ability of A549 cells to repair potentially lethal DNA damage induced by radiation.⁶⁹ Though these observations are somewhat conflicting as they suggest IGF-IR may both enhance and inhibit DNA repair, it is possible that these differences relate to the varied experimental model systems and cell types studied. However, these experiments support a link between DNA repair and IGF-I action and the exact IGF effects may be context dependent.

Effects of Breast Cancer Therapy on the IGF System

On the other hand, breast cancer chemotherapy also affects IGFs. It has been reported that serum IGFBP-3 falls significantly following initiation of chemotherapy in breast cancer patients, those individuals with a decrease in IGFBP-3 greater than the median had significantly poorer survival (median survival 5.5 months vs 18 months).⁷⁰ Another clinical trial showed that plasma IGF-I concentration significantly decreased after the first cycle of cyclophosphamide, methotrexate and 5-fluorouracil adjuvant chemotherapy in breast cancer patients.⁷¹ Retinoids such as fenretinide (4-HPR) inhibit breast cell growth while decreasing IGF-I and increase IGFBP-3.^72,73

Proline analogues of melphalan can be effectively transported into the MDA-MB 231 cells, evoking higher cytotoxicity, with reduction in IGF-I receptor and MAP kinase expression.⁷⁴ Tamoxifen also affects the IGF system. IGFBP-3,4,6 levels are lower in breast cancer patients compared to normal controls and levels increased after tamoxifen treatment.^75,76 Raloxifene, a selective estrogen receptor modulator being tested in cancer prevention trials, significantly decreased IGF-I and IGF-I/IGFBP-3 ratios when compared to placebo.⁷⁷ Urokinase plasminogen activator(uPA) inhibitor -17 AAG inhibit MDA-MB-231 cell growth by inhibiting the IGF-IR and ultimately uPA, while expression of the IGF-IR and uPA in breast cancer is associated with poor survival.⁷⁸

Thus, it is clear that anti-proliferative agents affect IGF system signaling. These associations do not prove a cause and effect relationship, however, given the role of IGF signaling in cell survival, the downregulation of this signaling pathway is consistent with the effects of many anti-cancer drugs.

Anti-IGF Strategies in Breast Cancer

Given the role for IGF signaling in many aspects of the malignant phenotype, it would be valuable to have reagents to disrupt IGF action. Several strategies to interrupt IGF signaling are currently under investigation, including endocrine maneuvers to suppress IGF production; antisense oligonucleotides to reduce functional IGF-IR levels; monoclonal antibodies, dominant negatives, and tyrosine kinase inhibitors to inhibit IGF-IR activation; and neutralization of IGF action using IGFBPs.^79,80

Combination of Anti-IGF Strategy with Chemotherapy

In theory, inhibition of survival pathways by blocking IGF-IR signaling while enhancing apoptotic stimuli has appeal. Combination of anti-IGFIR antibody αIR3 with doxorubicin resulted in increased cytotoxicity in IGF-I stimulated cells than with chemotherapy alone.¹⁰² Similar enhancement of chemotherapy effects have been shown in Ewing's sarcoma cells.¹⁰³ Tyrphostin AG1024 (a tyrosine kinase inhibitor of IGF-IR) demonstrated a marked enhancement in radiosensitivity and amplification of radiation-induced apoptosis which was associated with increased expression of Bax, p53 and p21, and a decreased expression of Bcl-2.¹⁰ Another study demonstrated that cotargeting IGF-IR and c-kit synergistic inhibit proliferation and induction of apoptosis in H209 small cell lung cancer cells.¹⁰⁴ There is also evidence that somatostatin analogues may enhance the effect of tamoxifen in animal models by suppressing plasma IGF-I and –II levels.⁹

In addition to conventional agents, it is also possible that anti-growth factor receptor strategies can be combined. Recent evidence shows that increased levels of IGF-IR signaling appears to interfere with the action of trastuzumab in breast cancer cell models that overexpress HER2/neu.⁹ Thus, strategies that target IGF-IR signaling may prevent or delay development of resistance to trastuzumab.

Conclusion

The IGF-IR is a promising target in breast cancer therapy because it signals to multiple pathways required for maintenance of the malignant phenotype. Given the role for IGF-IR in cell survival, it is logical to combine anti-IGF therapies with conventional agents. Indeed, the preclinical data suggest that blockade of IGF-IR induces apoptosis and lowering a “survival threshold” with disruption of this signaling system should enhance chemotherapy efficacy.

However, there are several challenges that will need to be addressed before the idea that combination anti-IGF therapy and chemotherapy display synergy. First, there are many ways that IGF signaling could be targeted. As recently noted by Professor Baserga,¹⁰⁵ the potential anti-IGF strategies have gone from “rags to riches” in the course of a few short years. Clinical trials to test the most effective strategy will need to be completed before combination trials can begin. Second, the phenotypes regulated by IGF-IR are not restricted to survival alone. Since proliferation is also affected by IGF-IR, it will be important to consider scheduling and choice of chemotherapeutic agent when designing appropriate combinations. For example, it is possible that anti-metabolites would be less efficacious when combined with anti-IGF because of the requirement for cells in S-phase for anti-metabolites to function. Indeed, interference between hormonal therapy and chemotherapy has been noted in breast cancer¹⁰⁶ and it is possible that such interference could exist between anti-IGF therapy and certain drugs. On the other hand, agents that have a different mechanism of action, such as DNA alkylators or therapies that induce DNA strand breaks, may be enhanced by blocking IGF-IR due to the receptor's role in DNA repair. Careful preclinical studies will need to be performed before clinical trials should proceed with testing anti-IGF therapy with conventional cytotoxics. Lastly, the idea that multiple molecules are involved in growth factor signaling leads to the potential for “combination targeted therapy” trials. As mentioned, blockade of both HER2 and IGF-IR may have benefit in preclinical systems. It is also highly likely that blockade of IGF-IR and downstream signaling events (MAPK, Akt, etc), could be synergistic. Given the complexity of the cross-talk and feedback between these systems, preclinical studies should also be able to guide us with designing the optimal therapies.

However, it is clear that anti-IGF therapies will soon find their way into clinical trials. Hopefully, the vast experience with preclinical model systems will guide us in the optimal development of these agents.

Acknowledgements

This work was supported by Public Health Service grants CA74285, CA89652, and Cancer Center Support Grant P30 CA77398.

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