ErbB2-overexpressing Cell Lines Research Articles

Abstract 4708: Potency and selectivity of ERBB2-targeting antibody drug conjugates in vitro

Abstract Previously we have reported on the relevance of the joint ProLiFiler and Cancer DataMiner platforms when studying the antitumor efficacy of novel antibody-drug conjugates (ADCs), such as Sacituzumab govitecan. In this study, we use our platforms to investigate and compare the selectivity of two FDA-approved Trastuzumab-based ADCs targeting ERBB2: Kadcyla, the first-in-class ADC employing a microtubule inhibitor bound via a non-cleavable linker, and Enhertu, which combines a topoisomerase inhibitor with a protease-cleavable linker. We performed a cell proliferation and survival assay using the ProLiFilerTM (Reaction Biology) to characterize the in vitro antitumor effects of Enhertu, Kadcyla, and their individual cytotoxins, deruxtecan & mertansine on 160 human cancer cell lines (CLs). The resulting data were uploaded to 4HF´s Cancer DataMiner platform for data analysis. First, we confirmed high potency and limited selectivity of mertansine, as it displayed an IC50 < 250 nM (mean: 27.3 nM) in 95% of cell lines (CLs). A COMPARE analysis confirmed that mertansine data correlated best with those of other microtubule inhibitors in our drug databases (e.g., MMAE, Spearman rho = 0.79, p=9.6E-27). Conversely, a paired analysis showed that CLs were less sensitive to Kadcyla than to mertansine except for CLs overexpressing ERBB2. The 160-CL panel included a total of nine CLs (six mammary, two gastric, one ovarian) with strong ERBB2 overexpression (Affymetrix data >10), mostly associated with ERBB2 gene amplification; seven of them were the most sensitive CLs to Kadcyla (IC50 < 6 nM). Focusing solely on breast cancer cell lines (breast cancer is the approved indication for Kadcyla), we observed a strong correlation between sensitivity to Kadcyla and ERBB2 expression (Spearman r = -0.78, p = 0.003). Interestingly, in line with published data, the ERBB2-overexpressing cell lines SNU-216 (stomach cancer) and JIM-T1 (breast cancer) were less sensitive to Kadcyla. In non-ERBB2-amplified CLs, Kadcyla sensitivity did not correlate with ERBB2 expression levels. Focusing on ERBB2-negative CLs such as hematological CLs, we unexpectedly observed consistent cell inhibition (mean IC50 =33 nM) with Kadcyla, suggesting that the ADC was cleaved, and the payload released into the medium. Overall, this study has demonstrated the relevance of our platforms to study and compare newly developed ADCs. Cytotoxic activities recorded for conjugates and free payloads, along with molecular data for the test cell lines, provides insight in the mode of action of conjugates and their target cell populations and hence can support the development of ADCs. Investigations are underway with Enhertu and its payload deruxtecan, and results will be compared with those obtained for Kadcyla and mertansine. Citation Format: Anne-Lise Peille, Kaja Holtorf, Pablo Anton Garcia, Nadine Obier, Daniel Feger, Thomas Metz, Sebastian Dempe, Heinz-Herbert Fiebig, Jan Ehlert, Vincent Vuaroqueaux. Potency and selectivity of ERBB2-targeting antibody drug conjugates in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4708.

Abstract 347: Developing a novel immunotoxin that targets cells overexpressing ErbB2

Abstract Immunotoxins are chimeric proteins comprising a specific cellular targeting domain linked to a cytotoxic factor. Here we describe the design and use of a novel, peptide-based immunotoxin that can initiate selective cytotoxicity on ErbB2-positive cells. ErbB2 is a receptor tyrosine kinase that is overexpressed in the tumor cells of approximately 30% of breast cancer patients. Immunotoxin candidates were designed to incorporate a targeting ligand with affinity for ErbB2 along with a membrane lysin-based toxin domain. The use of a membrane lysin as the toxin domain distinguishes our immunotoxins from numerous others in that internalization of the toxin is not required to induce cell death. One particular peptide candidate, NL1.1-PSA, demonstrated selective cytotoxicity towards ErbB2-overexpressing cell lines. We utilized a bioengineering strategy to show that recombinant NL1.1-PSA immunotoxin expression by Escherichia coli also conferred selective cytotoxicity towards ErbB2-overexpressing cells. Our findings hold significant promise for the use of effective immunotoxins in cancer therapeutics. While Herceptin treatment has been effective in patients with ErbB2 positive breast cancer, our novel immunotoxin may ultimately prove to be a novel and efficacious addition to the arsenal of approaches used to eliminate Herceptin-resistant breast cancer cells. Citation Format: Luqun Shen, Kelsey Weigel, Clayton Thomas, Lauren Drapalik, Zachary T. Schafer, Shaun Lee. Developing a novel immunotoxin that targets cells overexpressing ErbB2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 347.

Em08red, a dual functional antiproliferative emodin analogue, is a downregulator of ErbB2 expression and inducer of intracellular oxidative stress.

Expression of ErbB2 protein is inversely correlated with the prognosis in cancer patients. Consequently, strategies targeting ErbB2 remain an attractive option in treating several types of malignancies, including oral cancer. In addition, many studies have shown that emodin and emodin derivatives are able to inhibit growth of ErbB2-overexpressing tumor cells. In this study, a series of computer modeling-generated emodin analogues were synthesized and tested for their antiproliferative activity against oral cancer cell lines overexpressing ErbB2. Among these analogues, em08red (1,8-dihydroxy-9(10H)-anthracenone) demonstrated potent antiproliferative activity against all three tested ErbB2-overexpressing cell lines, ie, FaDu, HSC3, and OECM1. Treatment with em08red significantly downregulated activation of ErbB2 as well as the ErbB2 protein expression level in the tested cell lines and induced G2 arrest. Antiapoptosis protein (Bcl-xl and Bcl-2) expression levels were also downregulated, and active caspase-3 and caspase-9 was detected in cells after treatment with em08red. Moreover, treatment with em08red stimulated production of cytotoxic reactive oxygen species in treated cells, and this could be partially reversed by pretreatment with N-acetylcysteine. Overall, we demonstrated inhibition of ErbB2 function and induction of reactive oxygen species in tumor cells by em08red, which prevented proliferation of tumor cells and induced apoptotic cell death.

Design and evaluation of a peptide-based immunotoxin for breast cancer therapeutics

Immunotoxins are chimeric proteins comprising a specific cellular targeting domain linked to a cytotoxic factor. Here we describe the design and use of a novel, peptide-based immunotoxin that can initiate selective cytotoxicity on ErbB2-positive cells. ErbB2 is a receptor tyrosine kinase that is overexpressed in the tumor cells of approximately 30% of breast cancer patients. Immunotoxin candidates were designed to incorporate a targeting ligand with affinity for ErbB2 along with a membrane lysin-based toxin domain. One particular peptide candidate, NL1.1-PSA, demonstrated selective cytotoxicity towards ErbB2-overexpressing cell lines. We utilized a bioengineering strategy to show that recombinant NL1.1-PSA immunotoxin expression by Escherichia coli also conferred selective cytotoxicity towards ErbB2-overexpressing cells. Our findings hold significant promise for the use of effective immunotoxins in cancer therapeutics.

The ErbB2-targeting antibody trastuzumab and the small-molecule SRC inhibitor saracatinib synergistically inhibit ErbB2-overexpressing gastric cancer

The anti-ErbB2 antibody trastuzumab has shown significant clinical benefits in ErbB2-overexpressing breast and gastric cancer, but resistance to the drug is common. Here, we investigated the antitumor activity of the combination of trastuzumab and the SRC inhibitor saracatinib in ErbB2-overexpressing trastuzumab-resistant gastric cancer. The ErbB2-overexpressing human gastric cancer cell line NCI-N87 was treated with trastuzumab to obtain the trastuzumab-resistant cell line NCI-N87R. The NCI-N87R cell line showed a marked increase in SRC activity and ErbB signaling compared with the NCI-N87 cell line. Our data demonstrated that trastuzumab plus saracatinib was much more potent than either agent alone in reducing the phosphorylation of ErbB3 and AKT in both NCI-N87 and NCI-N87R gastric cancer cell lines. Trastuzumab and saracatinib synergistically inhibited the in vitro growth of NCI-N87 and NCI-N87R cell lines. Further data showed that combination therapy of trastuzumab with saracatinib resulted in a significant benefit over either agent alone in both NCI-N87 and NCI-N87R xenograft models, suggesting its potential use for treating ErbB2-overexpressing gastric cancer.

ErbB2 Dephosphorylation and Anti-Proliferative Effects of Neuregulin-1 in ErbB2-Overexpressing Cells; Re-evaluation of Their Low-Affinity Interaction

Neuregulin-1 binds to ErbB3 and ErbB4 and regulates cancer proliferation and differentiation. Neuregulin-1 had been suggested to also react with ErbB2, but this argument becomes controversial. Here, we re-evaluated the cellular responses and ErbB2 interaction of neuregulin-1 in ErbB2 overexpressing cell lines. In a competitive ligand-binding assay, we detected significant replacement of [35S]-labeled neuregulin-1 with nano molar ranges of cold neuregulin-1 in L929 cells expressing ErbB2 alone and SKOV3 cells carrying sulf-1 cDNA but not in these parental cells. The concentration of neuregulin-1 significantly decreased thymidine incorporation and phosphorylation of ErbB2 (Tyr877, Tyr1396, and Tyr1121) in ErbB2-overexpressing cancer cells as well as in L929 cells expressing ErbB2. A crosslinking assay ascertained the presence of neuregulin-1 immunoreactivity in the ErbB2 immune complexes of L929 expressing ErbB2 alone. These results suggest that the higher concentrations of neuregulin-1 exert an anti-oncogenic activity to attenuate ErbB2 auto-phosphorylation potentially through its low-affinity interaction with ErbB2.

SKLB1206, a Novel Orally Available Multikinase Inhibitor Targeting EGFR Activating and T790M Mutants, ErbB2, ErbB4, and VEGFR2, Displays Potent Antitumor Activity Both In Vitro and In Vivo

Anti-epidermal growth factor receptor (EGFR) treatment has been successfully applied in clinical cancer therapy. However, the clinical efficacy of first-generation reversible EGFR inhibitors, such as gefitinib and erlotinib, is limited by the development of drug-resistant mutations, including the gatekeeper T790M mutation and upregulation of alternative signaling pathways. Second-generation irreversible EGFR inhibitors that were designed to overcome the drug resistance due to the T790M mutation have thus far had limited success. Here, we report a novel reversible EGFR inhibitor, SKLB1206, which has potent activity against EGFR with gefitinib-sensitive and -resistant (T790M) mutations. In addition, SKLB1206 has also considerable inhibition potency against some other related oncokinases, including ErbB2, ErbB4, and VEGF receptor 2 (VEGFR2). SKLB1206 exhibited highly antiproliferative activity against a range of EGFR-mutant cell lines, including gefitinib-sensitive and -resistant cell lines, and EGFR or ErbB2-overexpressing cell lines. SKLB1206 also showed a potent antiangiogenesis effect in vitro, in a zebrafish embryonic angiogenesis assay, and in an alginate-encapsulate tumor cell assay. In vivo, oral administration of SKLB1206 showed complete tumor regression in gefitinib-sensitive HCC827 and PC-9 xenograft models and showed a considerable antitumor effect on the gefitinib-resistant H1975 model as well as other EGFR/ErbB2-overexpressing or -dependent tumor models including A431, LoVo, and N87 established in athymic mice. SKLB1206 also showed a very good oral bioavailability (50.1%). Collectively, these preclinical evaluations may support clinical development of SKLB1206 for cancers with EGFR-activating/resistance mutations or EGFR/ErbB2 overexpressed.

Studies on the Secondary Metabolites of a Pseudoalteromonas sp. Isolated from Sediments Collected at the Northeastern Coast of Brazil

Continuing search for anticancer compounds from the marine environment, we have studied microorganisms that inhabit intertidal sediments of the northeastern Brazilian coast. Of the 32 strains isolated, 13 were selected for biological evaluation of their crude extracts. The acetate extract obtained from a Gram-negative bacterium was strongly active against cancer cell lines with IC(50) values that ranged from 0.04 (HL60 leukemia cells) to 0.26 μg/ml (MDA MB-435 melanoma cells). The bacterium was identified as a Pseudoalteromonas sp. based on 16S rRNA gene sequencing. A bioassay-guided fractionation of the active extract led to the isolation of prodigiosin, a well-known tripyrrole red pigment with immunosuppressive and anticancer activities. Further experiments with ErbB-2 overexpressing cell lines, including HB4a-C3.6 (moderate overexpression), HB4a-C5.2 (high overexpression), and the parental HB4a cell line, were performed. Prodigiosin was moderately active toward HB4a cells with an IC(50) of 4.6 μg/ml, while it was 115 and 18 times more active toward HB4a-C3.6 cells (IC(50) of 0.04 μg/ml) and HB4a-C5.2 (IC(50) of 0.26 μg/ml) cells, respectively. These data suggest that, in spite of its previously described apoptosis-inducing properties, prodigiosin can selectively recognize cells overexpressing ErbB-2, which could be highly appealing in human breast cancer therapy.

AST1306, A Novel Irreversible Inhibitor of the Epidermal Growth Factor Receptor 1 and 2, Exhibits Antitumor Activity Both In Vitro and In Vivo

Despite the initial response to the reversible, ATP-competitive quinazoline inhibitors that target ErbB-family, such a subset of cancer patients almost invariably develop resistance. Recent studies have provided compelling evidence that irreversible ErbB inhibitors have the potential to override this resistance. Here, we found that AST1306, a novel anilino-quinazoline compound, inhibited the enzymatic activities of wild-type epidermal growth factor receptor (EGFR) and ErbB2 as well as EGFR resistant mutant in both cell-free and cell-based systems. Importantly, AST1306 functions as an irreversible inhibitor, most likely through covalent interaction with Cys797 and Cys805 in the catalytic domains of EGFR and ErbB2, respectively. Further studies showed that AST1306 inactivated pathways downstream of these receptors and thereby inhibited the proliferation of a panel of cancer cell lines. Although the activities of EGFR and ErbB2 were similarly sensitive to AST1306, ErbB2-overexpressing cell lines consistently exhibited more sensitivity to AST1306 antiproliferative effects. Consistent with this, knockdown of ErbB2, but not EGFR, decreased the sensitivity of SK-OV-3 cells to AST1306. In vivo, AST1306 potently suppressed tumor growth in ErbB2-overexpressing adenocarcinoma xenograft and FVB-2/Nneu transgenic breast cancer mouse models, but weakly inhibited the growth of EGFR-overexpressing tumor xenografts. Tumor growth inhibition induced by a single dose of AST1306 in the SK-OV-3 xenograft model was accompanied by a rapid (within 2 h) and sustained (≥24 h) inhibition of both EGFR and ErbB2, consistent with an irreversible inhibition mechanism. Taken together, these results establish AST1306 as a selective, irreversible ErbB2 and EGFR inhibitor whose growth-inhibitory effects are more potent in ErbB2-overexpressing cells.

Abstract 654: MM-111, an ErbB2/ErbB3 bispecific antibody, effectively combines with lapatinib to inhibit growth of ErbB2-overexpressing tumor cells

Abstract The oncogenic receptor ErbB2 (HER2) is frequently overexpressed in a variety of cancer indications, including breast, gastric, bladder, and lung. ErbB3 (HER3), the preferred dimerization partner of ErbB2, is critical for the survival and growth of ErbB2-overexpressing tumors, but is poorly inhibited by current ErbB2-targeted therapies. MM-111 is a novel bispecific antibody designed to specifically inhibit function of the ErbB2/ErbB3 heterodimer in ErbB2-overexpressing tumors. MM-111 binds simultaneously to ErbB2 and ErbB3 and blocks the interaction of ErbB3 with its ligand, heregulin. Inhibiting the ErbB2/ErbB3 heterodimer prevents ligand-induced activation of the phosphotidylinositol 3-kinase (PI3K) pro-survival pathway, altering cell cycle progression and inhibiting tumor growth. Upregulation of both heregulin and ErbB3 has been observed in tumors that have acquired resistance to the ErbB2-targeted therapies trastuzumab and lapatinib. In addition, ErbB2 and ErbB3 are frequently upregulated in estrogen receptor positive breast cancers that have developed resistance to anti-estrogen therapies. These data indicate that the ErbB2/ErbB3 receptor heterodimer potently activates growth of tumors characterized by ErbB2 overexpression and implicate ErbB3 as a mechanism of resistance to current therapies. Because of its ability to inhibit ErbB3 activity, MM-111 could be effective in combination with ErbB2-targeted therapies by overcoming ErbB3-mediated resistance. Previous data demonstrating that MM-111 acts in concert with trastuzumab to inhibit growth of ErbB2-overexpressing human tumor models support this hypothesis. We now show that MM-111 effectively combines with lapatinib to inhibit growth of tumors driven by the ErbB2/ErbB3 heterodimer. MM-111, as a single agent, is more effective than lapatinib at inhibiting heregulin-driven activation of ErbB3 and AKT in ErbB2-overexpressing cancer cell lines. In these same cell lines, the combination of MM-111 and lapatinib results in greater AKT inhibition than either treatment alone. In addition, we have demonstrated in ErbB2-overexpressing 3D spheroid cancer models that treatment with the MM-111/ lapatinib combination increases the number of cells in early phase apoptosis as compared to single-drug treatments. Finally, in an ErbB2-overexpressing cancer xenograft model, the combination of MM-111 and lapatinib results in greater inhibition of tumor growth than either therapy alone. In conclusion, we show that MM-111 and lapatinib positively combine to inhibit the growth of ErbB2-overexpressing tumors. Our results suggest that concurrent treatment with MM-111 and lapatinib may provide a potent therapeutic regimen for cancer patients whose tumors overexpress ErbB2 by deterring ErbB3-mediated resistance to lapatinib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 654. doi:10.1158/1538-7445.AM2011-654

Abstract 418: Cellular mechanisms underlying the evasion of detachment-induced cell death in inflammatory breast cancer cells

Abstract Inflammatory Breast Cancer (IBC) is a rare and aggressive form of breast cancer that results from cancerous epithelial cells that invade and block the lymphatic vessels. Because the cancerous cells have already migrated from the epithelium into the lymphatic system, these cells are inherently metastatic and have the ability to survive in the absence of attachment to the extracellular matrix (ECM). Non-cancerous epithelial cells require ECM attachment for normal cell signaling and survival, however IBC cells are able to persist without ECM attachment in the lymphatic vessels. Here we demonstrate that both the SUM149 and the ErbB2-overexpressing SUM190 cell lines derived from primary IBC tumors have acquired mechanisms to aid in their survival in detached environments. Using an in vitro model of ECM detachment, we reveal that detached IBC cells exhibit a dramatic evasion of anoikis (detachment-induced apoptosis). In the SUM149 cells, this evasion of anoikis seems to be mediated by the MAPK pathway as inhibition of this pathway with UO126 abrogates the inhibition of detachment-induced caspase activation. However, the evasion of anoikis in SUM190 cells is unaffected by U0126 treatment and is instead mediated by the PI(3)K pathway. Furthermore, when compared to the SUM149 cells, the SUM190 cells are much more effective at inhibiting anoikis. Interestingly, SUM190 cells exhibit a complete inhibition of the ECM-detachment-induced increase in pro-caspase-3, a mechanism that may explain the more efficient anoikis inhibition in these cells. Additionally, both the SUM149 cells and the SUM190 cells are able to overcome detachment-induced metabolic stress. This is demonstrated through the ability of both the SUM149 and SUM190 cells to maintain ATP levels upon detachment. However, while western blotting clearly demonstrates detachment-induced PI(3)K activation in both cell lines, we have discovered that this upregulation is necessary for metabolic rescue only in the SUM149 cell line. Neither the MAPK nor PI(3)K pathway is involved in the maintenance of ATP following ECM detachment, suggesting that these cells may utilize an alternative signaling pathway to maintain ATP levels. These results show that IBC cells have the capacity to alter both apoptotic and metabolic pathways to survive in detached environments. Further elucidation of these mechanisms may reveal new chemotherapeutic targets that permit the specific elimination of detached IBC cells within the lymphatic system. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 418. doi:10.1158/1538-7445.AM2011-418

X-Linked Inhibitor of Apoptosis Protein Inhibits Apoptosis in Inflammatory Breast Cancer Cells with Acquired Resistance to an ErbB1/2 Tyrosine Kinase Inhibitor

Inflammatory breast cancer (IBC) is a highly aggressive subtype of breast cancer that is often characterized by ErbB2 overexpression. ErbB2 targeting is clinically relevant using trastuzumab (anti-ErbB2 antibody) and lapatinib (small-molecule ErbB1/2 inhibitor). However, acquired resistance is a common outcome even in IBC patients who show an initial clinical response, which limits the efficacy of these agents. In the present study, using a clonal population of GW583340 (lapatinib analogue, ErbB1/2 inhibitor)-resistant IBC cells, we identified the overexpression of an antiapoptotic protein, X-linked inhibitor of apoptosis protein (XIAP), in acquired resistance to GW583340 in both ErbB2-overexpressing SUM190 and ErbB1-activated SUM149 cell lines derived from primary IBC tumors. A marked decrease in p-ErbB2, p-ErbB1, and downstream signaling was evident in the GW583340-resistant cells (rSUM190 and rSUM149) similar to parental counterparts treated with the drug, suggesting that the primary mechanism of action of GW583340 was not compromised in resistant cells. However, rSUM190 and rSUM149 cells growing in GW583340 had significant XIAP overexpression and resistance to GW583340-mediated apoptosis. Additionally, stable XIAP overexpression using a lentiviral system reversed sensitivity to GW583340 in parental cells. The observed overexpression was identified to be caused by IRES-mediated XIAP translation. XIAP downregulation in rSUM190 and rSUM149 cells using a small-molecule inhibitor (embelin), which abrogates the XIAP/procaspase-9 interaction, resulted in decreased viability, showing that XIAP is required for survival of cells with acquired resistance to GW583340. These studies establish the feasibility of development of an XIAP inhibitor that potentiates apoptosis for use in IBC patients with resistance to ErbB2-targeting agents.

Abstract 4961: Analysis of genomic alterations in breast cancer using an automated aCGH workstation

Abstract The current sample preparation process for aCGH tests is tedious leading to errors that confound clinical results and increase re-test rates and costs. To overcome this problem, we have developed an aCGH workstation that automates the bench work from DNA labeling to array scanning. A robotic liquid handling and incubation system (ArrayPrep® Target Preparation System) automates the labeling and magnetic bead purification of 8-96 gDNA samples then loads them onto microarrays for hybridization using a rotating incubation system (Mai Tai® Hybridization System). Arrays are then post-processed using a robotic instrument (Little Dipper® Processor) for washing and drying. Previous studies have shown that the aCGH workstation generates high quality labeled gDNA and highly reproducible array data from batches of 8-96 samples. It requires less than 1hr hands-on technician time and can substantially enhance test reproducibility and lower cost (1). In this study, we examined the ability of the aCGH workstation to reproducibly detect genomic alterations in four well characterized breast cancer cell lines, MCF-7, SK-BR-3, BT-474 and MDA-MB-231 and in breast cancer tissue samples with known Her2/neu status. For each sample, replicate gDNA samples were processed using the aCGH workstation and Agilent 44K feature microarrays designed for genome-wide DNA copy number variation (CNV) profiling. The resulting array data were then analyzed for previously reported copy number alterations (CNAs) including loci within chromosomes 1q, 8q, 11q13, 17q and 20q13 (2, 3) and components of the epidermal growth factor receptor pathway affected by copy number changes (3). The results confirm that samples processed on the aCGH workstation reproducibly detect previously reported complex alterations involving multiple levels of change on chromosome arms 1p, 8q, 9p, 11q, 15q, 17q and 20q. Our data also confirmed that copy number alteration of multiple genetic loci involved in the EGF family of pathways is common in all four cell lines. The ERBB2 locus is highly amplified in the two known ERBB2-overexpressing cell lines (BT-474 and SK-BR-3). These two cell lines share amplifications at five additional gene loci, MAP2K6, CHN2, PRKCA, LIMK1 and cMYC, as previously reported (3). In summary, this initial study has shown the successful automation of the aCGH laboratory workflow for detection of genetic abnormalities at high resolution in breast cancer samples. This automated platform holds the potential to significantly improve test reliability and lower the cost of routine genetic analysis of clinical samples.

Phage-Displayed Combinatorial Peptide Libraries in Fusion to β-Lactamase as Reporter for an Accelerated Clone Screening: Potential Uses of Selected Enzyme-Linked Affinity Reagents in Downstream Applications

Phage-display selection of combinatorial libraries is a powerful technique for identifying binding ligands against desired targets. Evaluation of target binding capacity of multiple clones recovered from phage display selection to a specific target is laborious, time-consuming, and a rate-limiting step. We constructed phage-display combinatorial peptide libraries in fusion with a beta-lactamase enzyme, which acts as a reporter. Linear dodecapeptide and cysteine-constrained decapeptide libraries were created at the amino-terminus of the Enterobacter cloacae P99 cephalosporinase molecule (P99 beta-lactamase). The overall and positional diversity of amino acids in both libraries was similar to other phage-display systems. The libraries were selected against the extracellular domain of ErbB2 receptor (ErbB2(ECD)). The target-selected clones were already conjugated to an enzyme reporter, therefore, did not require subcloning or any other post-panning modifications. We used beta-lactamase enzyme activity-based assays for sample normalizations and clone binding evaluation. Clones were identified that bound to purified ErbB2(ECD) and ErbB2-overexpressing cell-lines. The peptide sequences of the selected binding clones shared significant motifs with several rationally designed peptide mimetics and phage-display derived peptides that have been reported to bind ErbB2(ECD). beta-Lactamase fusion to peptides saved time and resources otherwise required by the phage-ELISA of a typical phage display screening protocol. The beta-lactamase enzyme assay protocols is a one-step process that does not require secondary proteins, several steps of lengthy incubations, or washings and can be finished in a few minutes instead of hours. The clone screening protocol can be adopted for a high throughput platform. Target-specific beta-lactamase-linked affinity reagents selected by this procedure can be produced in bulk, purified, and used, without any modification, for a variety of downstream applications, including targeted prodrug therapy.

Tumor necrosis factor transactivates ErbB2 in breast cancer cells.

Abstract Abstract #4056 We have previously shown that TNFα induces proliferation of the murine mammary adenocarcinoma C4HD through activation of the PI-3K/Akt signaling pathway that converges on the transcriptional activation of NF-κB. Since C4HD tumor overexpresses ErbB2 and given that this tyrosine kinase plays a critical role in C4HD cell proliferation, we wondered whether interactions between TNFα and ErbB2 could be taking place. Our findings revealed that treatment of C4HD cells with the ErbB2 inhibitor AG825 blocked TNFα-induced proliferation. Similar results were obtained using the human ErbB2-overexpressing cell line SK-BR-3. Then, we studied the effect of TNFα on ErbB2 phosphorylation in C4HD and SK-BR-3 cells. We found that TNFα increased total ErbB2 tyrosine phosphorylation in C4HD and SK-BR-3 cells, as well as on the specific residues tyrosines 927 and 1172 in murine cells and on its human homologues 877 and 1222. These effects where not caused by the release of ErbBs ligands from the cell membrane by TNFα, since treatment with the metalloprotease inhibitor GM6001 did not affect TNFα-induced ErbB2 phosphorylation. We then studied if c-Src was involved in the above effect given that it is known to directly phosphorylate ErbB2 in the Tyr 877 residue. We found that addition of PP2, a Src family inhibitor, completely inhibited phosphorylation of Tyr 927/877 ErbB2, and that to a lesser degree it inhibited Tyr 1172/1222 ErbB2 in both cell types. We also performed an in vitro cold phosphorylation assay where we observed that c-Src immunoprecipitated from C4HD cells treated with TNFα was able to phosphorylate ErbB2 on Tyr 927 residue. Taken together, these results indicate that TNFα induces phosphorylation of ErbB2 at Tyr877/927 residue and that c-Src is the tyrosine kinase involved in this effect. In addition, we observed that upon TNFα stimulation, ErbB2 associated with ErbB3 leading to PI-3K/Akt pathway activation. Treatment of cells with AG825 inhibited Akt and NF-κB activation by TNFα, as evidenced by western blots of phospho proteins and reporter gene studies, respectively. The above data for the first time identify TNFα as a cytokine able to transactivate ErbB2, disclosing c-Src involvement in such effect. Also we demonstrated that TNFα ability to activate Akt and NF-κB transcriptional activation is dependent on ErbB2 phosphorylation in breast cancer cells that overexpress ErbB2. Interestingly, TNFα appears as a new player in ErbB2-overexpressing breast tumors, and its eventual worth as a prognostic factor in anti ErbB2 therapy is yet to be determined. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4056.

Dual-Agent Molecular Targeting of the ErbB2 Receptor: Killing One Bird With Two Stones

The phase I study by Storniolo et al in this issue of Journal of Clinical Oncology, which evaluates the combination of lapatinib (a selective small-molecule ErbB1 and ErbB2 tyrosine kinase inhibitor) and trastuzumab (an antibody blocking the extracellular domain of ErbB2) in patients with advanced ErbB2-positive breast cancer, is the first published study to our knowledge examining dual-agent molecular targeting of the ErbB2 receptor. The study demonstrated that there was clinical activity in a group of heavily pretreated patients and—using a toxicity end point—determined an optimally tolerated regimen for this combination of agents. The epidermal growth factor family of receptors plays a key role in regulating growth, survival, and resistance to chemotherapy in many epithelial malignancies. Approximately 25% of breast cancers overexpress ErbB2 or demonstrate ErbB2 gene amplification; ErbB2 is a key mediator of tumor cell growth and survival, and a predictor of poor outcome. Potential strategies to block peptide growth factor signaling pathways include (1) blocking or neutralizing the growth factor or its production, (2) blocking the receptor-binding site or prevention of receptor dimerization, (3) blocking the receptor tyrosine kinase domain, and (4) blocking downstream signaling pathways. The mechanism of action of trastuzumab is multifactorial and includes (1) antibody-dependent cellular cytotoxity, (2) modulation of ErbB2 signaling, (3) internalization of ErbB2 cell-surface protein, (4) reduction in proteolytic cleavage of the ErbB2 extracellular domain, and (5) interference with DNA damage repair. Small-molecule inhibitors such as lapatinib have an advantage over antibodies because they inhibit multiple signaling pathways at plasma concentrations achieved in the clinic. This is relevant because the survival of epithelial tumors is often dependent on the presence of redundant signaling pathways. Combining anti-ErbB2 antibodies with small-molecule tyrosine kinase inhibitors is an appealing strategy because both therapies exert their antitumor effects via distinct mechanisms of action and target ErbB2 at different sites. Combining distinct classes of inhibitors may not only potentiate cellular cytotoxicity, but may also assist in overcoming inherent or acquired resistance to one class of inhibitors. Mechanisms of resistance identified to date include activating mutations in the domain of the adenosine triphosphate–binding pocket for small-molecule tyrosine kinase inhibitors and the presence of truncated receptors for monoclonal antibodies. Dual-agent molecular targeting has been examined in preclinical models using the ErbB1 pathway as well as the ErbB2 pathway. With respect to the ErbB1 pathway, Huang et al and Matar et al studied the effect of cetuximab (an antibody that binds to the extracellular domain of ErbB1) in combination with either gefitinib or erlotinib (small-molecule inhibitors of the tyrosine kinase domain or ErbB1) against a panel of human cancer cell lines in culture and in tumor xenografts. The combination of cetuximab plus gefitinib or erlotinib enhances growth inhibition, in culture and in vivo, over that observed with either agent alone. Phosphorylation inhibition of downstream effector molecules (mitogen-activated protein kinase and AKT), and apoptosis was enhanced with the combination. In lung and head and neck tumor cells highly resistant to cetuximab, gefitinib, and erlotinib, but not cetuximab, retained the capacity to inhibit these tumor cells and were also able to further inhibit the activation of downstream effectors of ErbB1 signaling. When tested in vivo, single-agent therapy resulted in transient tumor regression only at the highest doses, whereas suboptimal doses of gefitinib and cetuximab in combination resulted in complete and permanent regression of large tumors. Contradictory results were obtained by Fishel et al who determined that gefitinib and cetuximab, when used in combination, were antagonistic when tested against two ErbB1expressing epidermoid cell lines. The combination caused less apoptosis than gefitinib alone, and this appeared to be the result of overexpression of functional ErbB1 associated with the combination. It is likely that these discordant results were a result of the particular cell lines studied. Most relevant to the study being reviewed are the preclinical models examining dual targeting of the ErbB2 pathway. Xia et al treated ErbB2-overexpressing breast cancer cell lines with a combination of lapatinib and either trastuzumab or a polyclonal anti-ErbB2 antibody. The combination treatments led to enhanced tumor cell apoptosis, whereas treatment with the same doses of lapatinib, trastuzumab, or polyclonal anti-ErbB2 antibody alone led to minimal or no increase in apoptosis. They demonstrated that the enhanced tumor cell apoptosis after combination ErbB2-targeted therapies was most closely associated with downregulation of survivin protein. Konecny et al studied the combination of lapatinib and trastuzumab in a panel of human breast cancer cell lines. Synergistic activity was seen when the combination was tested in ErbB2-overexpressing cell lines. Of JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 26 NUMBER 20 JULY 1

EGFR and ErbB2 are functionally coupled to CD44 and regulate shedding, internalization and motogenic effect of CD44

Activation of the ErbB family of receptor tyrosine kinases is involved in a range of human cancers. Transmembrane signaling mediated by ErbB proteins is stimulated by peptide growth factors and is blocked by monoclonal antibodies such as trastuzumab and pertuzumab. ErbB receptors exert their function in conjunction with non-ErbB proteins, e.g. CD44. Here we show that epidermal growth factor (EGF) and heregulin induce CD44 shedding in JIMT-1, an ErbB2-overexpressing cell line resistant to trastuzumab, accompanied by internalization and intramembrane proteolysis of CD44 and enhanced cellular motility. These effects of EGF and heregulin are blocked by pertuzumab. Trastuzumab inhibits the heregulin- and hyaluronan oligosaccharide-induced shedding and internalization of CD44 and their motogenic effect. Trastuzumab also blocks CD44 shedding from JIMT-1 xenograft tumors in vivo. At the same time the internalization rate of trastuzumab is increased by hyaluronan oligosaccharide treatment in vitro. Our experiments point to an unexpected, but potentially important mechanism of action of ErbB receptor-targeted monoclonal antibodies used in the treatment of cancer.

A novel activating role of SRC and STAT3 on HGF transcription in human breast cancer cells

We have previously determined that the HGF promoter can be transactivated by a combination of activated Src and wild-type Stat3 in the mouse breast cell lines HC11 and SP1. To determine if this pathway is of relevance for the human disease, a series of human breast and other human cells lines were examined, and the status of key proteins in these cells determined. All of the human breast cell lines exhibited strong transactivation by a combination of activated Src and Stat3. This activation was dependent on a Stat3 recognition element present at nt-95. The exception was the ErbB2 over-expressing cell line SK-BR-3 where Stat3 alone could transactivate HGF though Src augmented this effect. Increased phosphorylation of Stat3 tyrosine 705 was also observed in this line. Analysis of three ovarian cell lines revealed that Src/Stat3 expression was not able to activate the HGF promoter in two of these lines (SKOV3 and IOSE-80PC). Src/Stat3 expression did activate HGF transcription in OVCAR3 cells, but this effect was not mediated by the Stat3 site at nt-95. Stat3 phosphorylation at tyrosine 705 was observed in IOSE-80PC cells, but was insufficient to allow for activation of the HGF promoter. Human kidney (HEK293) and cervical carcinoma (HeLa) cells were also not Src/Stat3 permissive, despite high levels of Stat3 phospho-Y705. These results suggest that human breast cells are a uniquely permissive environment for HGF transactivation by Src/Stat3 which may allow for the inappropriate activation of HGF transcription during the early stages of breast transformation. This could lead to paracrine or autocrine activation of the Met receptor in breast carcinoma cells.

AEE788

Aberrant epidermal growth factor receptor (EGFR) and ErbB2 expression are associated with advanced disease and poor patient prognosis in many tumor types (breast, lung, ovarian, prostate, glioma, gastric, and squamous carcinoma of head and neck). In addition, a constitutively active EGFR type III deletion mutant has been identified in non-small cell lung cancer, glioblastomas, and breast tumors. Hence, members of the EGFR family are viewed as promising therapeutic targets in the fight against cancer. In a similar vein, vascular endothelial growth factor (VEGF) receptor kinases are also promising targets in terms of an antiangiogenic treatment strategy. AEE788, obtained by optimization of the 7H-pyrrolo[2,3-d]pyrimidine lead scaffold, is a potent combined inhibitor of both epidermal growth factor (EGF) and VEGF receptor tyrosine kinase family members on the isolated enzyme level and in cellular systems. At the enzyme level, AEE788 inhibited EGFR and VEGF receptor tyrosine kinases in the nm range (IC(50)s: EGFR 2 nm, ErbB2 6 nm, KDR 77 nm, and Flt-1 59 nm). In cells, growth factor-induced EGFR and ErbB2 phosphorylation was also efficiently inhibited (IC(50)s: 11 and 220 nm, respectively). AEE788 demonstrated antiproliferative activity against a range of EGFR and ErbB2-overexpressing cell lines (including EGFRvIII-dependent lines) and inhibited the proliferation of epidermal growth factor- and VEGF-stimulated human umbilical vein endothelial cells. These properties, combined with a favorable pharmacokinetic profile, were associated with a potent antitumor activity in a number of animal models of cancer, including tumors that overexpress EGFR and or ErbB2. Oral administration of AEE788 to tumor-bearing mice resulted in high and persistent compound levels in tumor tissue. Moreover, AEE788 efficiently inhibited growth factor-induced EGFR and ErbB2 phosphorylation in tumors for >72 h, a phenomenon correlating with the antitumor efficacy of intermittent treatment schedules. Strikingly, AEE788 also inhibited VEGF-induced angiogenesis in a murine implant model. Antiangiogenic activity was also apparent by measurement of tumor vascular permeability and interstitial leakage space using dynamic contrast enhanced magnetic resonance imaging methodology. Taken together, these data indicate that AEE788 has potential as an anticancer agent targeting deregulated tumor cell proliferation as well as angiogenic parameters. Consequently, AEE788 is currently in Phase I clinical trials in oncology.

Achieving selectivity between highly homologous tyrosine kinases: a novel selective erbB2 inhibitor

The discovery of small molecule kinase inhibitors for use as drugs is a promising approach for the treatment of cancer and other diseases, but the discovery of highly specific agents is challenging because over 850 kinases are expressed in mammalian cells. Systematic modification of the 4-anilino functionality of a selective quinazoline inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase can invert selectivity to favor inhibition of the highly homologous erbB2 tyrosine kinase. The selectivity pattern was demonstrated in assays of recombinant kinases and recapitulated in measures of kinase activity in intact cells. The most potent and selective erbB2 inhibitor of the analog series has anti-proliferative activity against an erbB2-overexpressing cell line that was lacking in the original EGFR-selective compound. Subtle changes to the molecular structure of ATP-competitive small molecule inhibitors of tyrosine kinases can yield dramatic changes in potency and selectivity. These results suggest that the discovery of highly selective small molecule inhibitors of very homologous kinases is achievable.