Alexandra Eyzaguirre

Rapamycin synergizes with the epidermal growth factor receptor inhibitor erlotinib in non–small-cell lung, pancreatic, colon, and breast tumors

The receptor for epidermal growth factor (EGFR) is overexpressed in many cancers. One important signaling pathway regulated by EGFR is the phosphatidylinositol 3′-kinase (PI3K)-phosphoinositide-dependent kinase 1-Akt pathway. Activation of Akt leads to the stimulation of antiapoptotic pathways, promoting cell survival. Akt also regulates the mammalian target of rapamycin (mTOR)-S6K-S6 pathway to control cell growth in response to growth factors and nutrients. Recent reports have shown that the sensitivity of non–small-cell lung cancer cell lines to EGFR inhibitors such as erlotinib (Tarceva, OSI Pharmaceuticals) is dependent on inhibition of the phosphatidylinositol 3′-kinase-phosphoinositide-dependent kinase 1-Akt-mTOR pathway. There can be multiple inputs to this pathway as activity can be regulated by other receptors or upstream mutations. Therefore, inhibiting EGFR alone may not be sufficient for substantial inhibition of all tumor cells, highlighting the need for multipoint intervention. Herein, we sought to determine if rapamycin, an inhibitor of mTOR, could enhance erlotinib sensitivity for cell lines derived from a variety of tissue types (non–small-cell lung, pancreatic, colon, and breast). Erlotinib could inhibit extracellular signal-regulated kinase, Akt, and S6 only in cell lines that were the most sensitive. Rapamycin could fully inhibit S6 in all cell lines, but this was accompanied by activation of Akt phosphorylation. However, combination with erlotinib could down-modulate rapamycin-stimulated Akt activity. Therefore, in select cell lines, inhibition of both S6 and Akt was achieved only with the combination of erlotinib and rapamycin. This produced a synergistic effect on cell growth inhibition, observations that extended in vivo using xenograft models. These results suggest that combining rapamycin with erlotinib might be clinically useful to enhance response to erlotinib. [Mol Cancer Ther 2006;5(11):2676–84]

Feedback Mechanisms Promote Cooperativity for Small Molecule Inhibitors of Epidermal and Insulin-Like Growth Factor Receptors

Epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR) can cooperate to regulate tumor growth and survival, and synergistic growth inhibition has been reported for combined blockade of EGFR and IGF-IR. However, in preclinical models, only a subset of tumors exhibit high sensitivity to this combination, highlighting the potential need for patient selection to optimize clinical efficacy. Herein, we have characterized the molecular basis for cooperative growth inhibition upon dual EGFR and IGF-IR blockade and provide biomarkers that seem to differentiate response. We find for epithelial, but not for mesenchymal-like, tumor cells that Akt is controlled cooperatively by EGFR and IGF-IR. This correlates with synergistic apoptosis and growth inhibition in vitro and growth regression in vivo upon combined blockade of both receptors. We identified two molecular aspects contributing to synergy: (a) inhibition of EGFR or IGF-IR individually promotes activation of the reciprocal receptor; (b) inhibition of EGFR-directed mitogen-activated protein kinase (MAPK) shifts regulation of Akt from EGFR toward IGF-IR. Targeting the MAPK pathway through downstream MAPK/extracellular signal-regulated kinase kinase (MEK) antagonism similarly promoted IGF-driven pAkt and synergism with IGF-IR inhibition. Mechanistically, we find that inhibition of the MAPK pathway circumvents a negative feedback loop imposed on the IGF-IR- insulin receptor substrate 1 (IRS-1) signaling complex, a molecular scenario that parallels the negative feedback loop between mTOR-p70S6K and IRS-1 that mediates rapamycin-directed IGF-IR signaling. Collectively, these data show that resistance to inhibition of MEK, mTOR, and EGFR is associated with enhanced IGF-IR-directed Akt signaling, where all affect feedback loops converging at the level of IRS-1.

Loss of homotypic cell adhesion by epithelial-mesenchymal transition or mutation limits sensitivity to epidermal growth factor receptor inhibition.

Overexpression and enhanced activation of the epidermal growth factor receptor (EGFR) is frequently observed in human carcinomas. Inhibitors of EGFR signaling have shown clinical utility; however, understanding response at the molecular level is important to define patient subsets most likely to benefit, as well as to support the rational design of drug combinations. Pancreatic and colorectal tumor cell lines insensitive to EGFR inhibition were those that had lost or mutated the epithelial junction constituents E-cadherin and γ-catenin, had lost homotypic adhesion, and often gained proteins associated with an epithelial to mesenchymal–like transition, such as vimentin, zeb1, or snail. In matched pairs of colorectal tumor cells, the epithelial lines showed an average 7-fold greater sensitivity than mesenchymal-like lines. In human pancreatic and colorectal tumor tissues, gain of mesenchymal characteristics and loss of epithelial characteristics correlated with advancing tumor stage. These data indicate an especially sensitive patient subset as well as a rationale for the combination of EGFR antagonists with agents that affect the epithelial to mesenchymal–like transition process as a mechanism to enhance sensitivity for more advanced mesenchymal-like tumors. [Mol Cancer Ther 2007;6(2):532–41]

Bypassing cellular EGF receptor dependence through epithelial-to-mesenchymal-like transitions

Over 90% of all cancers are carcinomas, malignancies derived from cells of epithelial origin. As carcinomas progress, these tumors may lose epithelial morphology and acquire mesenchymal characteristics which contribute to metastatic potential. An epithelial-to-mesenchymal transition (EMT) similar to the process critical for embryonic development is thought to be an important mechanism for promoting cancer invasion and metastasis. Epithelial-to-mesenchymal transitions have been induced in vitro by transient or unregulated activation of receptor tyrosine kinase signaling pathways, oncogene signaling and disruption of homotypic cell adhesion. These cellular models attempt to mimic the complexity of human carcinomas which respond to autocrine and paracrine signals from both the tumor and its microenvironment. Activation of the epidermal growth factor receptor (EGFR) has been implicated in the neoplastic transformation of solid tumors and overexpression of EGFR has been shown to correlate with poor survival. Notably, epithelial tumor cells have been shown to be significantly more sensitive to EGFR inhibitors than tumor cells which have undergone an EMT-like transition and acquired mesenchymal characteristics, including non-small cell lung (NSCLC), head and neck (HN), bladder, colorectal, pancreas and breast carcinomas. EGFR blockade has also been shown to inhibit cellular migration, suggesting a role for EGFR inhibitors in the control of metastasis. The interaction between EGFR and the multiple signaling nodes which regulate EMT suggest that the combination of an EGFR inhibitor and other molecular targeted agents may offer a novel approach to controlling metastasis.

Compensatory insulin receptor (IR) activation on inhibition of insulin-like growth factor-1 receptor (IGF-1R): rationale for cotargeting IGF-1R and IR in cancer.

Insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) and critical activator of the phosphatidylinositol 3-kinase–AKT pathway. IGF-1R is required for oncogenic transformation and tumorigenesis. These observations have spurred anticancer drug discovery and development efforts for both biological and small-molecule IGF-1R inhibitors. The ability for one RTK to compensate for another to maintain tumor cell viability is emerging as a common resistance mechanism to antitumor agents targeting individual RTKs. As IGF-1R is structurally and functionally related to the insulin receptor (IR), we asked whether IR is tumorigenic and whether IR-AKT signaling contributes to resistance to IGF-1R inhibition. Both IGF-1R and IR(A) are tumorigenic in a mouse mammary tumor model. In human tumor cells coexpressing IGF-1R and IR, bidirectional cross talk was observed following either knockdown of IR expression or treatment with a selective anti–IGF-1R antibody, MAB391. MAB391 treatment resulted in a compensatory increase in phospho-IR, which was associated with resistance to inhibition of IRS1 and AKT. In contrast, treatment with OSI-906, a small-molecule dual inhibitor of IGF-1R/IR, resulted in enhanced reduction in phospho-IRS1/phospho-AKT relative to MAB391. Insulin or IGF-2 activated the IR-AKT pathway and decreased sensitivity to MAB391 but not to OSI-906. In tumor cells with an autocrine IGF-2 loop, both OSI-906 and an anti–IGF-2 antibody reduced phospho-IR/phospho-AKT, whereas MAB391 was ineffective. Finally, OSI-906 showed superior efficacy compared with MAB391 in human tumor xenograft models in which both IGF-1R and IR were phosphorylated. Collectively, these data indicate that cotargeting IGF-1R and IR may provide superior antitumor efficacy compared with targeting IGF-1R alone. Mol Cancer Ther; 9(10); 2652–64. ©2010 AACR.

A novel, potent, and selective insulin-like growth factor-I receptor kinase inhibitor blocks insulin-like growth factor-I receptor signaling in vitro and inhibits insulin-like growth factor-I receptor–dependent tumor growth in vivo

Insulin-like growth factor-I receptor (IGF-IR) and its ligands, IGF-I and IGF-II, are up-regulated in a variety of human cancers. In tumors, such as colorectal, non–small cell lung, ovarian, and pediatric cancers, which may drive their own growth and survival through autocrine IGF-II expression, the role of IGF-IR is especially critical. Here, we present a novel small-molecule IGF-IR kinase inhibitor, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which displayed a cellular IC50 of 19 nmol/L for inhibition of ligand-dependent autophosphorylation of human IGF-IR with 14-fold cellular selectivity relative to the human insulin receptor. PQIP showed minimal activity against a panel of 32 other protein kinases. It also abolished the ligand-induced activation of downstream phosphorylated AKT and phosphorylated extracellular signal-regulated kinase 1/2 in both IGF-IR transfectant cells and a GEO human colorectal cancer cell line. Analysis of GEO cells revealed a significant level of both phosphorylated IGF-IR and IGF-II expression. Furthermore, inactivation of IGF-II in conditioned GEO culture medium by a neutralizing antibody diminished IGF-IR activation, indicating the presence of a functional IGF-II/IGF-IR autocrine loop in GEO cells. Once daily oral dosing of PQIP induced robust antitumor efficacy in GEO xenografts. The antitumor efficacy correlated with the degree and duration of inhibition of tumor IGF-IR phosphorylation in vivo by this compound. Moreover, when mice were treated for 3 days with a dose of PQIP that maximally inhibited tumor growth, only minor changes in blood glucose were observed. Thus, PQIP represents a potent and selective IGF-IR kinase inhibitor that is especially efficacious in an IGF-II–driven human tumor model. [Mol Cancer Ther 2007;6(8):2158–67]

Inactivation of Akt by the epidermal growth factor receptor inhibitor erlotinib is mediated by HER-3 in pancreatic and colorectal tumor cell lines and contributes to erlotinib sensitivity

Signaling through the receptor for epidermal growth factor receptor (EGFR) is frequently deregulated in solid tumors. Erlotinib (Tarceva, OSI-774, OSI Pharmaceuticals, Inc., Melville, NY) is a low molecular weight, orally bioavailable inhibitor of the EGFR that has been approved for both non–small cell lung cancer and pancreatic cancers. Previous studies have indicated that sensitivity to EGFR antagonists correlated with HER-3 signaling for non–small cell lung cancer. Herein, we have sought to understand the signaling pathways that mediate erlotinib sensitivity for pancreatic and colorectal cancers. In a panel of 12 pancreatic tumor cell lines, we find that EGFR is coexpressed with HER-3 in all cell lines sensitive to erlotinib but not in insensitive cell lines. Erlotinib can block HER-3 phosphorylation in these sensitive cell lines, suggesting that HER-3 is transactivated by EGFR. Knockdown of HER-3 in BxPC3, an erlotinib-sensitive pancreatic tumor cell line, results in inhibition of the phosphorylation for both Akt and S6 and is associated with a decrease in cell proliferation and reduced sensitivity to erlotinib. Therefore, EGFR transactivation of HER-3 mediates Akt signaling and can contribute to erlotinib sensitivity for pancreatic tumors. We extended our analysis to a panel of 13 colorectal tumor cell lines and find that, like pancreatic, HER-3 is coexpressed with EGFR in the most erlotinib-sensitive cell lines but not in erlotinib-insensitive cell lines. These studies suggest that HER-3 could be used as a biomarker to select patients who are most likely to respond to erlotinib therapy. [Mol Cancer Ther 2006;5(8):2051–9]

Mechanisms of resistance to EGFR tyrosine kinase inhibitors: implications for patient selection and drug combination strategies

The receptor for epidermal growth factor (EGFR, ErbB1, HER1) supports the growth and maintenance of a broad range of human tumor types, and EGFR-targeting drugs are approved for the treatment of several advanced stage cancers, including non-small cell lung cancer (NSCLC), pancreatic cancer, squamous cell cancer of the head and neck (SCCHN), and colorectal cancer. Recent years have witnessed significant advances in our understanding of dysregulated signal transduction in cancer cells resulting from changes in the expression and/or mutational status of key signaling molecules that modulate sensitivity to drugs targeting EGFR. Based on this knowledge, we have an exciting opportunity to maximize the benefit provided to cancer patients by EGFR inhibitors. In this review article, we describe molecular determinants of sensitivity or resistance to EGFR-targeted agents, with specific emphasis on EGFR tyrosine kinase inhibitors (TKIs). The impact of these findings on our ability to evaluate candidate predictive biomarkers and to design robust mechanism-based combination strategies is also discussed.

Abstract 1654: Compensatory insulin receptor (IR) activation upon inhibition of insulin-like growth factor receptor (IGF-1R): Rationale for co-targeting IGF-1R and IR in cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase (RTK) and a critical mediator of signaling through the PI3K-AKT pathway. IGF-1R is required for oncogenic transformation and tumorigenesis, and inhibiton of IGF-1R results in reduced proliferation and survival of tumor cells. These observations have spurred intense drug discovery and development efforts for both biologic and small molecule IGF-1R inhibitors for the treatment of cancer. The ability for one RTK to compensate for another to maintain growth and survival signaling in tumor cells is emerging as a common mechanism of resistance to anti-tumor agents that selectively target individual RTKs. As IGF-1R is structurally and functionally related to the insulin receptor (IR), and IR can also activate tumor cell AKT signaling and cellular transformation, we asked whether IR signaling can contribute to resistance to IGF-1R inhibition in tumor cells. In a panel of human tumor cell lines, IGF-1R/IR crosstalk was observed after treatment with a selective anti-IGF-1R monoclonal antibody, MAB391. Tumor cells treated with MAB391 responded with a compensatory increase in phospho-IR, which was also associated with an inability to fully inhibit phospho-IRS1 and phospho-AKT. In contrast, treatment with OSI-906, a small molecule dual kinase inhibitor of IGF-1R and IR, resulted in enhanced inhibition of the IRS1-AKT signaling pathway. OSI-906 showed superior efficacy compared to MAB391 in human tumor xenograft models where both phospho-IR and phospho-IGF-1R were detectable, and presumably both receptors were required by tumor cells for growth and/or survival. Both insulin and IGF-2 can activate the IR-AKT pathway and we show that treatment with either growth factor resulted in decreased sensitivity of tumor cells to MAB391, but not OSI-906. In tumor cells with an autocrine IGF-2 signaling loop, both OSI-906 and an anti-IGF-2 neutralizing antibody reduced phospho-IR and phospho-AKT levels, whereas MAB391 was ineffective. Collectively, these data indicate that OSI-906, which co-targets IGF-1R and IR, may provide superior efficacy as compared to agents that selectively target only IGF-1R. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1654.

Utilizing combinations of molecular targeted agents to sensitize tumor cells to EGFR inhibitors

EGFR inhibitors have achieved clinical antitumor activity as single agents. The specific inhibition of EGFR and associated pathways provides a mechanism for efficacious inhibition of tumor cell growth while minimizing toxicities often associated with chemotherapeutic drugs. The challenge of using targeted cancer drugs as single agents is the potential for de novo or acquired resistance due to established or adapted alternate signal transduction pathways. In this chapter, we will describe how cancer drug combinations with EGFR inhibitors can be rationally identified by utilizing our understanding of the molecular mechanism and related biomarkers of EGFR inhibitor sensitivity. Such combinations may ultimately provide better efficacy and reduced toxicity for patients.