Genomic and Molecular Characterization of EGFR and IGF1R as Key Potential Therapeutic Targets in Malignant Peripheral Nerve Sheath Tumors |
An ESUN Article
Introduction
Malignant peripheral nerve sheath tumors (MPNSTs) are a type of soft tissue sarcoma of ectomesenchymal origin. They are highly malignant and account for approximately 5%–10% of the soft tissue sarcomas. They arise from major or minor peripheral nerve branches or sheaths of peripheral nerve fibers and are derived from Schwann cells or pluripotent cells originating in the neural crest. The Schwann cell is thought to be the major contributor to the formation of benign, as well as malignant neoplasm of the nerve sheath. The World Health Organization coined the term MPNST in 2002 to replace previous heterogeneous and often confusing terminology, such as "malignant schwannoma," "malignant neurilemmoma," "neurogenic sarcoma," and "neurofibrosarcoma."
MPNSTs pose significant clinical challenges because there is great variation in terms of tumor location and clinical presentation. Histologically, MPNSTs resemble fibrosarcoma in their basic organization: spindle cell tumors with long processes disposed in whorls or storiform patterns. Immunohistochemical analysis of markers, particularly S100, plays an important role in diagnosis. At the molecular level, loss of the NF1 gene and high levels of Ras activity are known hallmarks of MPNST. Despite multidisciplinary therapy, the 5-year survival rate of patients with MPNSTs still ranges from 30% to 50%. Several studies have examined the epidemiological, etiological, clinical, pathological, and molecular characteristics of MPNSTs to find new therapeutic methods. However, no significant progress has been made.
 |
EGFR and IGFR1 as therapeutic targets for MPNSTs
Recent array comparative genomic hybridization (aCGH) studies showed that the epidermal growth factor receptor (EGFR) gene is amplified in MPNSTs. Furthermore, EGFR expression has been reported in several investigations, and the data show that EGFR expression is very important in NF-1, neurofibroma, and MPNSTs because of its significant correlation with the tumor formation and development. EGFR is a transmembrane tyrosine kinase receptor that has been implicated in a multiplicity of cancer-related signal transduction pathways, such as those regulating cellular proliferation, adhesion, and migration; neoangiogenesis; and apoptosis inhibition, which are all important features of tumorigenesis and tumor progression. EGFR induced tyrosine kinase activity plays a central role in mediating these processes and has been intensely studied to exploit it as a therapeutic target. Some small-molecule tyrosine kinase inhibitors of EGFR such as gefitinib (Iressa) and erlotinib have shown anticancer effects in patients with non-small cell lung cancer.
In our previous study, we characterized genetic alterations in MPNST tissues using genome-wide aCGH. Bioinformatic analysis revealed that a large part of the MPNST cases had amplifications of IGF1R and EGFR. Furthermore, in our recent validation studies using immunohistochemical analysis, we found overexpression of IGF1R and EGFR in most of the MPNSTs that we analyzed. Most importantly, IGF1R and EGFR expression significantly correlated with tumor metastasis and disease recurrence. We found that the patients with IGF1R and/or EGFR expression had significantly worse disease-free survival and a significantly higher risk of tumor development than those without expression of these receptors. These are important, clinically relevant findings, as anti-EGFR– and anti-IGF1R–targeted therapies, such as gefitinib, erlotinib, and MK-0646, are being tested in clinical trials in several different types of cancer, including lung cancer and gastrointestinal stromal tumors (GISTs).
IGF1R is a multifunctional tyrosine kinase receptor involved in several biological processes, including cell proliferation and differentiation, DNA repair, and cell survival. It is also implicated in the development and maintenance of malignant phenotypes, and interruption of IGF1R signaling inhibits cancer cell growth and motility in both in vitro and in vivo models in several types of carcinoma. In comparison, there have been few investigations of IGF1R-targeted therapy in sarcomas. In GISTs, two investigations have shown that IGF1R is a potential therapeutic target, and the inhibition of IGF1R activity in vitro with NVP-AEW541 or IGF1R-specific siRNA led to cytotoxicity and apoptosis in GIST cell lines through AKT and MAPK signaling. However, there are limited data regarding IGF1R expression, its prognostic significance in MPNST, and the effect of IGF1R inhibition.
EGFR and IGF1R may cooperate to regulate tumor growth and survival. Inhibition of either EGFR or IGF1R may promote activation of the other receptor. The complicated crosstalk between the EGFR and IGF1R pathways could significantly affect the efficacy of EGFR-targeted therapy. On the basis of the crosstalk between the IGF1R and EGFR pathways, studies of combination therapies that target both pathways should be performed to determine whether this combination enhances tumor-growth inhibition. These investigations would not only provide a strategy for testing combinations of EGFR inhibitors with IGF1R-targeted therapies to achieve improved patient outcomes, but also satisfy the urgent need to explore the mechanisms of intrinsic and acquired drug resistance to enhance the antitumor efficacy of these therapies.
In this proposed translational research project, we plan to further functionally characterize IGF1R and EGFR as key targetable molecules in MPNST. First, we will use MK-0646, an IGF1R monoclonal antibody, and IGF1R-specific siRNA to treat MPNST cell lines (T265p21, ST88-14, and STS26T) to determine whether knockdown and/or inhibition of IGF1R leads to apoptosis and/or reduced cell proliferation and migration/invasion. We will examine the changes in the IGF1R pathway and its associated PI3K/AKT and MAPK pathways. For EGFR, similar approaches will be used, including experiments with siRNA interference and targeted inhibition with gefitinib. If these in vitro experiments are effective, we will test whether knockdown or inhibition of both IGF1R and EGFR can suppress the growth of MPNST xenograft tumors in nude mice. These studies will provide key evidence of whether IGF1R and EGFR activity is important for the growth of MPNSTs and whether targeting IGF1R and/or EGFR would constitute an effective targeted therapy.
Purpose of the specific investigation
On the basis of our preliminary data and reported studies in the literature, we hypothesize that IGF1R and EGFR are potential therapeutic targets for the treatment of MPNSTs. We will test this hypothesis with the following three specific aims that first examine the receptors individually and then in combination.
Specific Aim 1
We will use siRNA specific for IGF1R and the IGF1R monoclonal antibody MK-0646 (made by Merck) to treat MPNST cell lines T265p21, ST88-14, and STS26T to determine whether knockdown and/or inhibition of IGF1R leads to increased cell death and reduced cell proliferation and migration/invasion. We will also evaluate the effect on molecules in the IGF1R signaling pathway, including IRS-1, PI3K, AKT, Erk, Ras, and Bad.
Specific Aim 2
In this aim, we will use an approach similar to the one outlined in specific aim 1 to evaluate the effect of inhibiting EGFR on MPNSTs. We will use siRNA specific for EGFR and the anti-EGFR antibody gefitinib to treat MPNST cell lines to determine whether knockdown and/or inhibition of EGFR leads to apoptosis, reduced cell proliferation, and attenuated cell migration and invasion. The functional assays used for this aim will be similar to those described in specific aim 1.
Specific Aim 3
Accumulating evidence suggests that often more than one key pathway is involved in the proliferation of cancer cells. Thus, inhibition of one pathway is often ineffective. Our preliminary studies showed that EGFR and IGF1R are often co-amplified in MPNSTs. Therefore, simultaneously blocking both pathways is likely a more effective therapeutic strategy. In this aim, we will treat cells with either siRNAs specific for both genes or antibodies against both targets and evaluate the effect on MPNST cells by analyzing the same functional endpoints as described in specific aim 1.
Significance of this translational research project
MPNSTs are highly malignant tumors with a high rate of local recurrence and a significant tendency to metastasize. The survival rate of patients with MPNST remains low. The dismal outcome highlights the importance of identifying clinicopathological and molecular factors that impact MPNST prognosis and points to the urgent need to establish better therapeutic strategies for patients with MPNSTs. Our studies will provide key evidence for whether IGF1R and EGFR represent key signaling pathways for MPNSTs and whether cotargeting IGF1R and EGFR would constitute an effective therapeutic strategy. This will provide critical information for the design of a new clinical trial using anti-IGF1R and anti-EGFR therapies to treate MPNSTs.
Editor's Note: This study is funded by a $50,000 grant from the Liddy Shriver Sarcoma Initiative and the Reid R. Sacco Memorial Foundation.
References
1. Buck E, Eyzaguirre A, Rosenfeld-Franklin M, Thomson S, et al. Feedback mechanisms promote cooperativity for small molecule inhibitors of epidermal and insulin-like growth factor receptors. Cancer Res. 2008; 68(20):8322-32.
2. Hewish M, Chau I, Cunningham D.Insulin-like growth factor 1 receptor targeted therapeutics: novel compounds and novel treatment strategies for cancer medicine. Recent Pat Anticancer Drug Discov. 2009;4(1):54-72.
3. Hu YP, Patil SB, Panasiewicz M, Li W, Hauser J, Humphrey LE, Brattain MG. Heterogeneity of receptor function in colon carcinoma cells determined by cross-talk between type I insulin-like growth factor receptor and epidermal growth factor receptor. Cancer Res. 2008;68(19):8004-13.
4. Huang F, Greer A, Hurlburt W, et al. The mechanisms of differential sensitivity to an insulin-like growth factor-1 receptor inhibitor (BMS-536924) and rationale for combining with EGFR/HER2 inhibitors. Cancer Res. 2009; 69(1):161-70.
5. Jiang Y, Wang L, Gong W, et al. A high expression level of insulin-like growth factor I receptor is associated with increased expression of transcription factor Sp1 and regional lymph node metastasis of human gastric cancer. Clin Exp Metastasis. 2004;21(8):755-64.
6. Keizman D, Issakov J, Meller I, Meimon N, Ish-Shalom M, Sher O, Merimsky O. Expression and significance of EGFR in malignant peripheral nerve sheath tumor. J Neurooncol. 2009;94(3):383-8.
7. Klinakis A, Szabolcs M, Chen G, Xuan S, Hibshoosh H, Efstratiadis A. Igf1r as a therapeutic target in a mouse model of basal-like breast cancer. Proc Natl Acad Sci U S A. 2009; 106(7):2359-64.
8. Kwon J, Stephan S, Mukhopadhyay A, Muders MH, Dutta SK, Lau JS, Mukhopadhyay D. Insulin receptor substrate-2 mediated insulin-like growth factor-I receptor overexpression in pancreatic adenocarcinoma through protein kinase Cdelta. Cancer Res. 2009;69(4):1350-7.
9. Liao Y, Abel U, Grobholz R, Hermani A, Trojan L, Angel P, Mayer D. Up-regulation of insulin-like growth factor axis components in human primary prostate cancer correlates with tumor grade. Hum Pathol. 2005;36(11):1186-96.
10. Lin CT, Huang TW, Nieh S, Lee SC.Treatment of a Malignant Peripheral Nerve Sheath Tumor. Onkologie. 2009;32(8-9):503-505.
11. Ling BC, Wu J, Miller SJ, Monk KR, Shamekh R, Rizvi TA, Decourten-Myers G, Vogel KS, DeClue JE, Ratner N. Role for the epidermal growth factor receptor in neurofibromatosis-related peripheral nerve tumorigenesis. Cancer Cell. 2005;7(1):65-75.
12. Ludovini V, Bellezza G, Pistola L, et al. High coexpression of both insulin-like growth factor receptor-1 (IGFR-1) and epidermal growth factor receptor (EGFR) is associated with shorter disease-free survival in resected non-small-cell lung cancer patients. Ann Oncol. 2009;20(5):842-9.
13. Pantaleo MA, Astolfi A, Di Battista M, Heinrich MC, Paterini P, Scotlandi K, Santini D, Catena F, Manara MC, Nannini M, Maleddu A, Saponara M, Lolli C, Formica S, Biasco G. Insulin-like growth factor 1 receptor expression in wild-type GISTs: A potential novel therapeutic target. Int J Cancer. 2009 Dec 15;125(12):2991-4
14. Pappano WN, Jung PM, Meulbroek JA, Wang YC, Hubbard RD, Zhang Q, Grudzien MM, Soni NB, Johnson EF, Sheppard GS, Donawho C, Buchanan FG, Davidsen SK, Bell RL, Wang J. Reversal of oncogene transformation and suppression of tumor growth by the novel IGF1R kinase inhibitor A-928605. BMC Cancer. 2009;9(1):314
15. Sachdev D, Yee D. Disrupting insulin-like growth factor signaling as a potential cancer therapy. Mol Cancer Ther 2007;6:1–12.
16. Tawbi H, Thomas D, Lucas DR, Biermann JS, Schuetze SM, Hart AL, Chugh R, Baker LH. Epidermal growth factor receptor expression and mutational analysis in synovial sarcomas and malignant peripheral nerve sheath tumors. Oncologist. 2008 Apr;13(4):459-66.
17. Williams JP, Wu J, Johansson G, Rizvi TA, Miller SC, Geiger H, Malik P, Li W, Mukouyama YS, Cancelas JA, Ratner N. Nf1 mutation expands an EGFR-dependent peripheral nerve progenitor that confers neurofibroma tumorigenic potential. Cell Stem Cell. 2008 Dec 4;3(6):658-69.
18. Zhou J, Huang W, Tao R, Ibaragi S, Lan F, Ido Y, Wu X, Alekseyev YO, Lenburg ME, Hu GF, Luo Z. Inactivation of AMPK alters gene expression and promotes growth of prostate cancer cells. Oncogene. 2009;28(18):1993-2002.
19. Zou C, Smith KD, Liu J, Lahat G, Myers S, Wang WL, Zhang W, McCutcheon IE, Slopis JM, Lazar AJ, Pollock RE, Lev D. Clinical, pathological, and molecular variables predictive of malignant peripheral nerve sheath tumor outcome. Ann Surg. 2009, 249(6):1014-22.
V7N3 ESUN Copyright © 2010 Liddy Shriver Sarcoma Initiative.