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Cancer Therapy: Preclinical |
Authors' Affiliations: 1 Drug Discovery, Incyte Corporation, Wilmington, Delaware and 2 Department of Otolaryngology, University of Pittsburgh Medical Center and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania
Requests for reprints: Jordan S. Fridman, Drug Discovery, Experimental Station, Incyte Corporation, E400, Route 141 and Henry Clay Road, Wilmington, DE 19880. Phone: 302-498-6930; Fax: 302-425-2760; E-mail: jfridman{at}incyte.com.
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Abstract |
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Experimental Design: A series of biochemical and cell-based assays were established to identify selective inhibitors of ErbB ligand shedding. The therapeutic potential of these compounds was assessed in multiple in vivo models of cancer and matrix metalloprotease–related toxicity.
Results: INCB3619 was identified as a representative selective, potent, orally bioavailable small-molecule inhibitor of a subset of ADAM proteases that block shedding of ErbB ligands. Administration of INCB3619 to tumor-bearing mice reduced ErbB ligand shedding in vivo and inhibited ErbB pathway signaling (e.g., phosphorylation of Akt), tumor cell proliferation, and survival. Further, INCB3619 synergized with clinically relevant cancer therapeutics and showed no overt or compounding toxicities, including fibroplasia, the dose-limiting toxicity associated with broad-spectrum matrix metalloprotease inhibitors.
Conclusions: Inhibition of ErbB ligand shedding offers a potentially novel and well-tolerated therapeutic strategy for the treatment of human cancers and is currently being evaluated in the clinic.
Despite the fact that certain tumor traits have been identified that may be associated with clinical responses to EGFR TKIs (e.g., EGFR gene amplification or a subset of somatic mutations in exons 18 to 21, or mesenchymal characteristics), they can account for only a fraction of those patients achieving clinical benefit from treatment. Substantial preclinical and clinical evidence exist, suggesting that the different ErbB pathways may compensate for one another, thus providing a mechanism of resistance against an agent targeting an individual pathway. For instance, increased levels of the EGFR ligand transforming growth factor-
(TGF-) have been observed in trastuzumab-treated breast cancer patients with progressive disease (6). Similar results were obtained in a preclinical model of trastuzumab resistance that showed a concordant increased sensitivity to an EGFR-selective TKI (7). Moreover, a nonbiased analysis of patients receiving gefitinib described significantly elevated expression of the ErbB ligands amphiregulin and TGF- in nonresponders, agreeing with in vitro data suggesting that high ErbB ligand concentrations can circumvent the effectiveness of an EGFR-targeted agent (8, 9)3 or the less selective ErbB kinase inhibitor GW572016 (10). These data are consistent with the preclinical observation that targeting the EGFR pathway at multiple points (ligand binding and ATP pocket) may be more efficacious than either method in isolation (11, 12)—a hypothesis currently being evaluated clinically. Finally, even in patients who do respond to EGFR-selective TKIs, the emergence of secondary mutations in the EGFR kinase domain affords resistance to the ATP-competitive TKIs (13–16). Although this is an evolving field, these and other data support the following concepts: (a) although ErbB-targeted agents have shown clinical promise, there is significant room for improvement; (b) a multifaceted approach to inhibiting multiple ErbB pathways would be expected to improve therapeutic response; and (c) inhibiting the activation of multiple ErbB ligands represents a logical and perhaps clinically relevant point of intervention.
Although certain broad spectrum matrix metalloprotease (MMP) inhibitors (e.g., marimastat) can inhibit EGFR ligand shedding in vitro (among other activities), this has not been investigated in vivo nor has the therapeutic hypothesis been tested clinically due to the lesser potency of these drugs against key ADAM protease—relative to MMPs (see below)—and their inability to be tolerated at higher doses (17). Here, we describe the discovery and preclinical development of selective inhibitors of ErbB ligand shedding and show that these compounds inhibit ErbB ligand shedding and tumor growth in vivo without inducing joint fibroplasias, the dose-limiting toxicity of many MMP inhibitors. These compounds possess the pharmacologic properties making them ideal candidates to test the therapeutic potential of sheddase inhibition for the treatment of cancer in a clinical setting.
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Materials and Methods |
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Cell culture and generation of stable cell lines. Cell lines were obtained from American Type Culture Collection (Manassas, VA) and were cultured as recommended by the supplier. Retroviral transduction was used to generate derivatives of the NMuMG and Rat-1 cells as described previously (20). The coding region of the human cDNA for TGF- was cloned into pMSCVpuro (BD Biosciences, Mountain View, CA) following amplification from a pool of human cDNA (BD QUICK-Clone). Human umbilical vascular endothelial cells were obtained from Cambrex Corp (East Rutherford, NJ) and treated following the manufacturer's protocols.
Analysis of ErbB ligand shedding and its effect. The TGF- cleavage assay used SCC9 cells stimulated with 10 µmol/L phorbol 12-myristate 13-acetate (Sigma-Aldrich, St. Louis, MO) for 30 min in serum-free medium following a 30-min pretreatment with DMSO or metalloprotease inhibitor. Supernatants were collected and assayed for soluble TGF- by a commercially available ELISA kit (R&D Systems). Alternatively, NMuMG or Rat-1 cells were grown for 24 h in the presence of DMSO or sheddase inhibitor, and supernatants were analyzed in a similar manner. Amphiregulin release was measured in supernatants of HCT116 stimulated with phorbol 12-myristate 13-acetate (1 µmol/L, 30 min) using an ELISA kit (R&D Systems). The heparin-binding EGF-like growth factor (HB-EGF) cleavage assay used Chinese hamster ovary cells stably transfected with human placental alkaline phosphatase–tagged HB-EGF (In Vitro Biology Department of Incyte Corporation, Wilmington, DE). The transfected Chinese hamster ovary cells were grown in 96-well plates and treated with DMSO or compound for 10 min followed by phorbol 12-myristate 13-acetate stimulation (2 µmol/L, 45 min). Alkaline phosphatase activity was assessed using a luminescent substrate (Roche Molecular Biochemicals, Indianapolis, IN). Heregulin cleavage was assessed using Chinese hamster ovary-K1 cells transiently transfected with the human heregulin-ß1 cDNA in pCDNA3.2-DESTTM (In Vitro Biology Department of Incyte Corporation) using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Following transfection, the cells were grown in serum-free medium for 24 h. Cells were pretreated with DMSO or compound for 15 min and stimulated with phorbol 12-myristate 13-acetate (1 µmol/L, 60 min). Heregulin-ß1 was quantified in the supernatants using a commercially available human NRG1-ß1/HRG1-ß1 ELISA kit (R&D Systems) according to the manufacturer's instructions.
Conditioned medium experiments used NMuMG-T cells grown in the presence of DMSO or INCB3619 for 24 h. Medium was collected and concentrated (Amicon Centriplus, Millipore Corp., Billerica, MA) and applied to serum-deprived (6 h) A431 cells for 30 min. Protein extracts were then subjected to immunoblotting using antibodies against total EGFR and phosphorylated EGFR (Tyr1173) antibodies (Cell Signaling, Beverly, MA).
Primary head and neck cancer cells were obtained from a consenting patient with squamous cell carcinoma of the buccal mucosa (approved by the University of Pittsburgh Institutional Review Board). The patient was not previously treated with standard therapies. Cells were maintained in DMEM, 10% fetal bovine serum, and 2 mmol/L glutamine, and passed 1:10 weekly. Epithelial cells were separated from fibroblasts using selective trypsinization. For proliferation assays, cells were seeded at 103 per well in the in a 96-well plate and allowed to attach overnight. The medium was then replaced with 100 µL of low serum medium (2% fetal bovine serum) containing compound or DMSO and maintained for 5 days. Viability was assessed following manufacturer's protocols (Promega Cell-Titer Glo, Madison, WI) and percentage inhibition of growth was determined relative to the DMSO control. Amphiregulin shedding was determined from 4 x 105 cells per well in 12-well plates, which were allowed to attach overnight. The medium was replaced with low serum medium (2% fetal bovine serum)–containing compound or DMSO and incubated overnight. Centrifuged medium was collected and analyzed following manufacturer's protocols by ELISA (R&D Systems-Human Amphiregulin Duoset). Percentage inhibition was calculated relative to a DMSO control.
Animal studies. Animal studies were done under Animal Welfare Regulation Guidelines in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. BALB/c or CD-1 nu/nu mice (Charles River, Wilmington, MA) were injected s.c. with logarithmically growing cells suspended in either serum-free culture medium, or a 1:1 mixture medium and Matrigel (BD Biosciences). The MCF-7 tumor model required implantation of estradiol pellets (17-ß estradiol, 0.72 mg/pellet, 60-day release; Innovative Research of America, Sarasota, FL). NMuMG-T tumors were implanted as s.c. tumor fragments (2 x 2 mm). Tumor size was followed using a caliper to measure the length (l) and width (w) of tumors to calculate an approximate volume [(l x w2) / 2]. In efficacy studies, treatment was usually initiated when mean tumor volumes were 
150 mm3, whereas in some pharmacodynamic marker studies, tumors were twice to thrice larger at the time of treatment. Tumor growth delay represents the difference in time for the treated group (T) and a control group (C) to reach a predetermined mean tumor size and was calculated for long-term studies (TGD = T – C). Alternatively, tumor growth inhibition (TGI) was calculated throughout the experiment [TGI = (1 – T / C) x 100]. S.c. implantation of osmotic pumps (Alzet, Cupertino, CA) was done under isofluorane anesthesia following manufacturer's protocol. These pumps were used to administer INCB3619 or marimastat in all animal studies.
Determination of circulating TGF- levels in mice was done in immunocompromised mice bearing s.c. well-established Rat-1-T tumors (>200 mm3). Briefly, tumor-bearing animals were randomized into two groups with similar mean tumor volumes. Plasma was then collected via retro-orbital bleeding 2 days after implantation of s.c. Alzet pumps delivering vehicle or INCB3619 (120 mg/kg/d). TGF- levels were determined from plasma samples in similar fashion as described above.
The murine fibroplasia model was adapted from Renkiewicz et al. (21). Synovial fibroplasias was scored on a scale of 0 (no change) to 3 [severe collagen deposition and fibroblast proliferation resulting in an increase (>10-fold) in the ratio of synovium to subsynovial tissue mass].
Hematology and clinical chemistry analyses were done by DuPont Haskell Laboratories (Newark, DE).
Evaluation of compound levels in plasma was done using standard liquid chromatography-tandem mass spectrometry techniques.
Ex vivo analysis of tumor material. Tissues were fixed in 10% neutral buffered formalin for at least 24 h at room temperature before standard processing and treatment with 3% H2O2 for 15 min to quench any endogenous peroxidase activity. Preheated citrate buffer (Antigen Unmasking Solution, Vector Laboratories, Burlingame, CA) was used for antigen retrieval and background staining was reduced using the Avidin/Biotin Blocking kit (Vector Laboratories) and CytoQ Background Buster (Innovex, Richmond, VA) following the manufacturer's protocols. Primary antibodies, rabbit anti-Ki67 (Novocastra Laboratories, Newcastle upon Tyne, United Kingdom) or rabbit anti–phospho-AKT:Ser473 (Cell Signaling), were incubated overnight at 4°C in a humidified chamber. A biotinylated secondary antibody (goat anti-rabbit, Vector Laboratories) was visualized using R.T.U. Vectastain Elite ABC Reagent and 3,3'-diaminobenzidine (Vector Laboratories). Alternatively, sections were examined for the presence of apoptotic cells by terminal deoxyribonucleotide transferase–mediated nick-end labeling (TUNEL) staining (Apoptag, Chemicon International, Temecula, CA). Following staining, sections were washed with H2O, counterstained with hematoxylin, dehydrated, and mounted. Observations were made on a Nikon Eclipse E800 system and images captured with ACT-1 software (Nikon Corporation, Melville, NY).
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Results |
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), (b) those that bind both EGFR and ErbB4 (e.g., HB-EGF), and (c) those that bind ErbB3 and/or ErbB4 (e.g., heregulin; reviewed in refs. 5, 22). Importantly, the ultimate goal was to discover molecules with drug-like properties, including oral bioavailability and good pharmacokinetics. To this end, we synthesized a number of novel hydroxamic acid-derived metalloprotease inhibitors (18),3 culminating in the identification of a series of molecules exemplified by INCB3619 (Fig. 1A
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converting enzyme) and ADAM10 (22–29). We thus established biochemical assays using recombinant proteins and fluorogenic peptide substrates and screened proprietary hydroxamic-based compounds (19) for inhibition of ADAM10/ADAM17 enzyme activity. Hydroxamic acids have previously been shown to affect HB-EGF shedding through inhibition of an unknown enzyme (30). Compounds from our collection that showed >50% inhibition of ADAM10 and/or ADAM17 at a concentration of 200 nmol/L were further characterized against a larger panel of metalloproteases and ADAM proteins to assess selectivity. A structure-activity relationship was established and provided direction for the synthesis or more potent and/or selective compounds. Using this strategy, INCB3619 was identified as a potent inhibitor of ADAM17 and ADAM10 activity with IC50 values of 14 and 22 nmol/L, respectively. Moreover, INCB3619 shows excellent selectivity both within the ADAM family (e.g., IC50 for ADAM9 is >5 µmol/L) and against a panel of MMPs (Table 1
). Functional cell-based assays confirmed the ability of INCB3619 to inhibit the shedding of TGF-, HB-EGF, amphiregulin, and heregulin with nanomolar potencies (Fig. 1B). Compounds that possess MMP activity but lack ADAM10/17 activity did not block ErbB ligand shedding in these assays (see below).
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In vitro and in vivo sequelae of selective ADAM inhibition. To begin to assess the biological effect of inhibiting ErbB ligand shedding, we first engineered a cellular model reliant on TGF- expression for its transformed phenotype (Fig. 2A
). NMuMG cells (immortalized mammary epithelial cells) were transduced with either an empty expression vector (NMuMG-V) or a vector encoding TGF- (NMuMG-T). After confirming the ability of transfected cells to produce TGF-, we tested the ability of INCB3619 to inhibit the shedding of TGF- from the cell surface by analyzing tissue culture supernatants from cells grown in the presence or absence of compound (Fig. 2B). In contrast to INCB3619, which inhibits ADAM10 and ADAM17, neither INCB8765 (ref. 9; an ADAM10 selective inhibitor; Fig. 2B, 
) nor an inhibitor of MMPs that is both ADAM10 and ADAM17 sparing (Fig. 2B, 
; ref. 9) inhibited TGF- shedding. This agrees with the knockout data implicating ADAM17 as the enzyme responsible for TGF- shedding (24, 31). The ability to potently inhibit ErbB ligand cleavage was not specific to INCB3619 as additional compounds having similar enzymatic activities as INCB3619 were also able to do so (data not shown). Moreover, this pharmacology was not limited to cells of epithelial origin as nearly identical results were obtained using immortalized fibroblasts (Fig. 2C). To extend these findings beyond contrived cell lines, we investigated the effect of sheddase inhibition on EGFR ligand release and cell proliferation in primary tumor cells surgically isolated from a consenting patient with squamous cell carcinoma of the buccal mucosa. This tumor type was chosen for investigation based on the clinical efficacy observed with antibodies that inhibit EGFR ligand binding. Using these cells, we show that INCB3619 can inhibit the release of amphiregulin (Fig. 2D) in primary tumor cells in addition to ErbB ligands in established cell lines. Treatment with INCB3619 resulted in a significant decrease in proliferation of these primary tumor cells (Fig. 2E). The EC50 for proliferation was estimated to be near the EC90 for amphiregulin shedding, suggesting that the ADAM sheddases may require marked inhibition for efficacy in this model system. Although the results in these primary cells are consistent with our cell line data, we cannot conclude that similar effects will be observed in all cancer cells. In contrast, in normal cells (human umbilical vascular endothelial cells or primary fibroblasts), there was no effect on proliferation at concentrations >10 times those effective on the tumor cells (data not shown).
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can reduce EGFR pathway activation, we treated the EGF-responsive A431 cells with conditioned medium harvested from INCB3619-treated NMuMG-T cells. Treatment of NMuMG-T cells with INCB3619 markedly inhibited activation of EGFR in the A431 cells, confirming that NMuMG-T cells release a soluble factor capable of activating EGFR and that this is prevented with a selective sheddase inhibitor (Fig. 2F). Similar experiments in which A431 cells were first incubated with an EGFR-neutralizing antibody showed no EGFR activation after addition of conditioned medium from NMuMG-T cells grown in the presence or absence of INCB3619 (data not shown), consistent with the conclusion that selective sheddase inhibition prevents activation of an autocrine TGF- loop. Likewise, we have previously shown in multiple autonomous cell-based systems that pharmacologic or genetic inhibition of ADAM10/17 can inhibit ErbB receptor phosphorylation and downstream signaling (9). However, as none of the cell lines studied here overexpress HER2, we did not detect significant changes to the amount of HER2 receptor phosphorylation (data not shown).
The ability of sheddase inhibition to reduce TGF- shedding in vivo (Fig. 3A
) as well as EGFR-driven tumor growth was investigated in both tumors of fibroblast (Fig. 3B; 42% TGI) and epithelial origin (Fig. 3C; 38% TGI). In agreement with data that has been generated using less selective compounds, the direct effect of sheddase inhibition addition on ex vivo cellular proliferation of established cell lines was not dramatic (refs. 32–35; data not shown)—similar to what was observed by others using the C225 anti-EGFR antibody. This was not unexpected as any of a number of signaling pathways may be sufficient to drive proliferation in in vitro established ell culture systems and/or to activate the ErbB receptors via crosstalk among signaling pathways. However, three-dimensional tumor growth in vivo is different than growth in culture and ErbB signaling may play a more crucial role. Supporting this hypothesis, it has been shown that xenografts arising from cultured cells significantly up-regulate key players in ErbB signaling (36). Therefore, evaluating the effect of sheddase inhibition in the more clinically relevant in vivo setting was imperative.
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Because particular cancer types have been linked to elevated ErbB ligand expression, and hence increased EGFR activity (reviewed in ref. 38), we evaluated the activity of INCB3619 in a xenograft model of one such tumor type, head and neck squamous cell carcinoma. Subcutaneous tumors were established using the SCC4 cell line. As platinum-based therapeutic regimens are the standard of care for inoperable tumors of this type, we compared the efficacy of INCB3619 to the cytotoxic agent cisplatin, alone or in combination with INCB3619. INCB3619, as a single agent, nearly completely inhibited tumor growth during the treatment period (30% TGI) and subsequently resulted in a 13-day TGD (Fig. 5A ). In contrast, cisplatin was mostly ineffective (0% TGI and a 2-day TGD) although it was administered near its maximally tolerated dose (Fig. 5A and data not shown). Although cisplatin was lackluster as a single agent, the addition of INCB3619 was synergistic (66% TGI and a 24-day TGD), producing sporadic tumor regressions (in three of seven mice). Importantly, INCB3619 did not exacerbate the toxic effects of cisplatin, as assessed by animal weights and clinical observations (data not shown). To understand the mechanism behind the observed synergy, we analyzed similarly treated tumor samples ex vivo. Immunohistochemical analyses indicated that INCB3619 alone was sufficient to modestly decrease tumor cell proliferation nearly by nearly 10% (Fig. 5B, top) and activation of the Akt/PKB survival signaling pathway (Fig. 5B, bottom). This latter effect likely accounts for the increased responsiveness to the cytotoxic insult induced by treatment with cisplatin (Fig. 5A and C). However, even as a single agent, INCB3619 was sufficient to induce tumor cell apoptosis (Fig. 5C, top right). Similar synergism was observed in a breast cancer xenograft model (MDA-MB-435) when INCB3619 was used in combination with paclitaxel, thus indicating that these effects were not particular to a specific tumor type or chemotherapeutic combination (paclitaxel, 10-day TGD; INCB3619, 20-day TGD; combination, 50-day TGD; Fig. 5D and E). INCB3619 also improved the therapeutic responses to other cytotoxic agents in models of pancreatic, non–small cell lung, and colon cancer (data not shown).
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Discussion |
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There is strong evidence that ligand shedding is critical for the transforming potential of EGFR (reviewed in ref. 44) and increased ErbB ligand expression is a common event in human cancers (38). This latter observation my be explained, in part, by observations demonstrating metalloprotease-dependent ErbB ligand shedding is increased upon activation of a number of diverse signaling pathways linked to human cancers (e.g., WNT, prostaglandin E2, and LPA; refs. 3, 5, 22, 23). Further, correlative clinical and preclinical data suggest that ErbB pathways may compensate for one another, thus providing a potential mechanism of "resistance" to selective ErbB-targeted therapies. Although this observation has yet to be conclusively proven, it suggests that sheddase inhibitors should synergize with TKIs (or other targeted therapies) because they inhibit unique aspects of a common pathway. Our studies demonstrating that INCB3619 enhances gefitinib sensitivity (Fig. 4C; ref. 9) support this concept and may represent a well-tolerated alternative to combining EGFR TKIs with antibodies specific solely for the EGFR and its ligands. Clearly, the understanding of intrinsic sensitivity to ErbB inhibition in the clinic remains an evolving field. Although EGFR amplification and/or mutation may affect response rates, multiple mechanisms that affect sensitivity may exist either in parallel or in series.
Selective sheddase inhibition seems to be well tolerated and can be equally efficacious as a TKI administered at a maximally tolerated dose. The mechanism of this apparent differential tolerability is an active area of study; however, clearly, inhibiting ligand-stimulated receptor activation is different than inhibiting basal kinase activity through direct kinase inhibition. For example, ligand-independent activation of ErbB receptors, as has been observed upon integrin-mediated binding to extracellular matrix, is unlikely to be perturbed by sheddase inhibition (45). Alternatively, a number of reports have shown that expression of tissue inhibitor of metalloproteinase-3 (TIMP-3), a gene encoding for an endogenous sheddase inhibitor, is often silenced in solid tumors, via promoter methylation (46–48). TIMP-3, as opposed to TIMP-1, TIMP-2, and TIMP-4, is the only endogenous inhibitor known to inhibit both ADAM10 and ADAM17 (ref. 49 and references therein) and may, therefore, regulate basal levels of ErbB ligand production. Perhaps, selective sheddase inhibitors have been so well tolerated because they merely complement or compensate for a loss of TIMP-3 activity in a pathologic setting.
Although INCB3619 and related molecules are zinc-dependent metalloprotease inhibitors, they are highly selective against a broad panel of MMPs and thus do not share their musculoskeletal side effects. In addition, unlike broad-spectrum MMP inhibitors, the sheddase-selective compounds do not show efficacy in a xenograft model, which has a mutated K-ras, and do not show efficacy in in vitro angiogenesis systems (i.e., inhibition of human umbilical vascular endothelial cell proliferation, migration, invasion, and tube formation; data not shown), further distinguishing their mechanism of tumor growth inhibition from that of MMP inhibitors. It remains unclear why this latter class of agents failed in the clinic but it is intriguing to find that some MMP activities have been linked to the generation of antiangiogenic molecules (e.g., endostatin and tumstatin; ref. 50). It is therefore tempting to speculate that the effect of these antiangiogenic products of MMP activity may be greater in human cancer than in the mouse or that in the preclinical mouse models, the concentration of MMP inhibitors was high enough to affect the sheddases, thus partly explaining their efficacy against some tumor xenografts.
In this report, we have shown that selective sheddase inhibitors can prevent the release of ErbB ligands from cell lines and primary patient tumor cells and that this can result in inhibition of ErbB signaling pathways in vitro and in vivo. Although we cannot conclude that inhibition of sheddases in addition to ADAM10 and ADAM17 can be important for this activity (e.g., ADAM12 or ADAM15), we have clearly shown that MMP sparing protease inhibition is sufficient for efficacy. Moreover, our previous in vitro studies have shown that selective genetic (RNA interference) inhibition of ADAM17 and not ADAM9 or ADAM15 is sufficient to affect ErbB signaling pathways (9). These molecules thus have antitumor activity in tumor models reported to be sensitive to perturbations in ErbB pathways, but not elsewhere (e.g., MDA-MB-231 tumors). Although sheddase inhibitors do possess significant single-agent antitumor activity, their ability to inhibit survival pathways such as Akt and our preclinical findings demonstrating additive or synergistic effects support the use of combination therapy in the clinic. This is further substantiated by their excellent tolerability and their lack of exacerbating the toxicity associated with standard chemotherapeutics or targeted agents. Further, combining sheddase inhibitors with other ErbB inhibitors may thwart multiple mechanisms of resistance, such as kinase (gatekeeper) mutations or increased ligand expression. As such, selective sheddase inhibitors may complement the existing arsenal of anticancer agents; hence, clinical investigation of sheddase inhibition is undoubtedly warranted.
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Acknowledgments |
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Footnotes |
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Conflict of interest statement. The authors (excluding S.M. Thomas and J.R. Grandis) declare they all currently are, or have been, employed by Incyte Corporation.
3 C. Arteaga, personal communication. 
4 Yao W, Zhu J, Burns DM, et al. Discovery of a potent, selective, and orally active human epidermal growth factor receptor-2 sheddase inhibitor for the treatment of cancer. J Med Chem. In press 2007.
5 J.S. Fridman and M. Pan, unpublished data.
6 W. Yao, unpublished observations.
7 J.S. Fridman and M. Hansbury, unpublished observations.
Received 8/24/06; revised 11/17/06; accepted 11/30/06.
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expression impairs trastuzumab-induced HER2 downregulation. Oncogene 2005;24:3002–10.[CrossRef][Medline]. Nature 1997;385:733–6.[CrossRef][Medline] converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability. J Biol Chem 2002;277:12838–45.This article has been cited by other articles:
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  K. Horiuchi, T. Kimura, T. Miyamoto, K. Miyamoto, H. Akiyama, H. Takaishi, H. Morioka, T. Nakamura, Y. Okada, C. P. Blobel, et al. Conditional Inactivation of TACE by a Sox9 Promoter Leads to Osteoporosis and Increased Granulopoiesis via Dysregulation of IL-17 and G-CSF J. Immunol., February 15, 2009; 182(4): 2093 - 2101. [Abstract] [Full Text] [PDF]
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 M. J. Duffy, E. McKiernan, N. O'Donovan, and P. M. McGowan Role of ADAMs in Cancer Formation and Progression Clin. Cancer Res., February 15, 2009; 15(4): 1140 - 1144. [Abstract] [Full Text] [PDF]
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Y. Zhang and I. Pastan High Shed Antigen Levels within Tumors: An Additional Barrier to Immunoconjugate Therapy Clin. Cancer Res., December 15, 2008; 14(24): 7981 - 7986. [Abstract] [Full Text] [PDF]
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 P. M. McGowan, E. McKiernan, F. Bolster, B. M. Ryan, A. D. K. Hill, E. W. McDermott, D. Evoy, N. O'Higgins, J. Crown, and M. J. Duffy ADAM-17 predicts adverse outcome in patients with breast cancer Ann. Onc., June 1, 2008; 19(6): 1075 - 1081. [Abstract] [Full Text] [PDF]
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C. Badoual, G. Bouchaud, N. E. H. Agueznay, E. Mortier, S. Hans, A. Gey, F. Fernani, S. Peyrard, P. L. -Puig, P. Bruneval, et al. The Soluble {alpha} Chain of Interleukin-15 Receptor: A Proinflammatory Molecule Associated with Tumor Progression in Head and Neck Cancer Cancer Res., May 15, 2008; 68(10): 3907 - 3914. [Abstract] [Full Text] [PDF]
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) or one expressing TGF-
). Pooled populations of transduced cells were injected s.c. into immunocompromised mice, and tumor growth was followed over time. B, ErbB ligand shedding was evaluated in NMuMG cells expressing TGF-
), the ADAM10 selective compound INCB8765 (
). Tumors were measured twice weekly using calipers. Points, mean tumor volumes (n = 7/group); bars, SE.
