Binding and functional profiling of antibody mutants guides selection of optimal candidates as antibody drug conjugates.

An increasingly appreciated conundrum in the discovery of antibody drug conjugates (ADCs) is that an antibody that was selected primarily for strong binding to its cancer target may not serve as an optimal ADC. In this study, we performed mechanistic cell-based experiments to determine the correlation between antibody affinity, avidity, internalization and ADC efficacy. We used structure-guided design to assemble a panel of antibody mutants with predicted Her2 affinities ranging from higher to lower relative to the parent antibody, Herceptin. These antibodies were ranked for binding via SPR and via flow-cytometry on high-Her2 SKOV3 cells and low-Her2 MCF7 cells, the latter acting as a surrogate for low-Her2 normal cells. A subpanel of variants, representative of different Her2-binding affinities (2 strong, 2 moderate and 3 weak), were further screened via high-content imaging for internalization efficacies in high versus low-Her2 cells. Finally, these antibodies were evaluated in ADC cytotoxicity screening assays (using DM1 and MMAE secondary antibodies) and as antibody-drug conjugates (DM1 and PNU159682). Our results identified specific but weak Her2-binding variants as optimal candidates for developing DM1 and PNU ADCs since they exhibited high potencies (low to sub-nM) in high-Her2 SKOV3 cells and low toxicities in low-Her2 cells. The 2 strong-affinity variants were highly potent in SKOV3 cells but also showed significant toxicities in low-Her2 cells and therefore are predicted to be toxic in normal tissues. Our findings show that pharmacological profiling of an antibody library in multiple binding and functional assays allows for selection of optimal ADCs.

Correction: A novel highly potent trivalent TGF-ß receptor trap inhibits early-stage tumorigenesis and tumor cell invasion in murine Pten-deficient prostate glands.

[This corrects the article DOI: 10.18632/oncotarget.13343.].

Assisted Design of Antibody and Protein Therapeutics (ADAPT).

Effective biologic therapeutics require binding affinities that are fine-tuned to their disease-related molecular target. The ADAPT (Assisted Design of Antibody and Protein Therapeutics) platform aids in the selection of mutants that improve/modulate the affinity of antibodies and other biologics. It uses a consensus z-score from three scoring functions and interleaves computational predictions with experimental validation, significantly enhancing the robustness of the design and selection of mutants. The platform was tested on three antibody Fab-antigen systems that spanned a wide range of initial binding affinities: bH1-VEGF-A (44 nM), bH1-HER2 (3.6 nM) and Herceptin-HER2 (0.058 nM). Novel triple mutants were obtained that exhibited 104-, 46- and 32-fold improvements in binding affinity for each system, respectively. Moreover, for all three antibody-antigen systems over 90% of all the intermediate single and double mutants that were designed and tested showed higher affinities than the parent sequence. The contributions of the individual mutants to the change in binding affinity appear to be roughly additive when combined to form double and triple mutants. The new interactions introduced by the affinity-enhancing mutants included long-range electrostatics as well as short-range nonpolar interactions. This diversity in the types of new interactions formed by the mutants was reflected in SPR kinetics that showed that the enhancements in affinities arose from increasing on-rates, decreasing off-rates or a combination of the two effects, depending on the mutation. ADAPT is a very focused search of sequence space and required only 20-30 mutants for each system to be made and tested to achieve the affinity enhancements mentioned above.

Binding Properties of the Transforming Growth Factor-ß Coreceptor Betaglycan: Proposed Mechanism for Potentiation of Receptor Complex Assembly and Signaling.

Transforming growth factor (TGF) ß1, ß2, and ß3 (TGF-ß1-TGF-ß3, respectively) are small secreted signaling proteins that each signal through the TGF-ß type I and type II receptors (TßRI and TßRII, respectively). However, TGF-ß2, which is well-known to bind TßRII several hundred-fold more weakly than TGF-ß1 and TGF-ß3, has an additional requirement for betaglycan, a membrane-anchored nonsignaling receptor. Betaglycan has two domains that bind TGF-ß2 at independent sites, but how it binds TGF-ß2 to potentiate TßRII binding and how the complex with TGF-ß, TßRII, and betaglycan undergoes the transition to the signaling complex with TGF-ß, TßRII, and TßRI are not understood. To investigate the mechanism, the binding of the TGF-ßs to the betaglycan extracellular domain, as well as its two independent binding domains, either directly or in combination with the TßRI and TßRII ectodomains, was studied using surface plasmon resonance, isothermal titration calorimetry, and size-exclusion chromatography. These studies show that betaglycan binds TGF-ß homodimers with a 1:1 stoichiometry in a manner that allows one molecule of TßRII to bind. These studies further show that betaglycan modestly potentiates the binding of TßRII and must be displaced to allow TßRI to bind. These findings suggest that betaglycan functions to bind and concentrate TGF-ß2 on the cell surface and thus promote the binding of TßRII by both membrane-localization effects and allostery. These studies further suggest that the transition to the signaling complex is mediated by the recruitment of TßRI, which simultaneously displaces betaglycan and stabilizes the bound TßRII by direct receptor-receptor contact.

A novel highly potent trivalent TGF-ß receptor trap inhibits early-stage tumorigenesis and tumor cell invasion in murine Pten-deficient prostate glands.

The effects of transforming growth factor beta (TGF-ß) signaling on prostate tumorigenesis has been shown to be strongly dependent on the stage of development, with TGF-ß functioning as a tumor suppressor in early stages of disease and as a promoter in later stages. To study in further detail the paradoxical tumor-suppressive and tumor-promoting roles of the TGF-ß pathway, we investigated the effect of systemic treatment with a TGF-ß inhibitor on early stages of prostate tumorigenesis. To ensure effective inhibition, we developed and employed a novel trivalent TGF-ß receptor trap, RER, comprised of domains derived from the TGF-ß type II and type III receptors. This trap was shown to completely block TßRII binding, to antagonize TGF-ß1 and TGF-ß3 signaling in cultured epithelial cells at low picomolar concentrations, and it showed equal or better anti-TGF-ß activities than a pan TGF-ß neutralizing antibody and a TGF-ß receptor I kinase inhibitor in various prostate cancer cell lines. Systemic administration of RER inhibited prostate tumor cell proliferation as indicated by reduced Ki67 positive cells and invasion potential of tumor cells in high grade prostatic intraepithelial neoplasia (PIN) lesions in the prostate glands of Pten conditional null mice. These results provide evidence that TGF-ß acts as a promoter rather than a suppressor in the relatively early stages of this spontaneous prostate tumorigenesis model. Thus, inhibition of TGF-ß signaling in early stages of prostate cancer may be a novel therapeutic strategy to inhibit the progression as well as the metastatic potential in patients with prostate cancer.

Analysis of acquired resistance to metronomic oral topotecan chemotherapy plus pazopanib after prolonged preclinical potent responsiveness in advanced ovarian cancer.

An alternative or follow-up adjunct to conventional maximum tolerated dose (MTD) chemotherapy now in advanced phase III clinical trial assessment is metronomic chemotherapy--the close regular administration of low doses of drug with no prolonged breaks. A number of preclinical studies have shown metronomic chemotherapy can cause long term survival of mice with advanced cancer, including metastatic disease, in the absence of overt toxicity, especially when combined with targeted antiangiogenic drugs. However, similar to MTD chemotherapy acquired resistance eventually develops, the basis of which is unknown. Using a preclinical model of advanced human ovarian (SKOV-3-13) cancer in SCID mice, we show that acquired resistance can develop after terminating prolonged (over 3 months) successful therapy utilizing daily oral metronomic topotecan plus pazopanib, an oral antiangiogenic tyrosine kinase inhibitor (TKI). Two resistant sublines were isolated from a single mouse, one from a solid tumor (called KH092-7SD, referred to as 7SD) and another from ascites tumor cells (called KH092-7AS, referred to as 7AS). Using these sublines we show acquired resistance to the combination treatment is due to tumor cell alterations that confer relative refractoriness to topotecan. The resistant phenotype is heritable, associated with reduced cellular uptake of topotecan and could not be reversed by switching to MTD topotecan or to another topoisomerase-1 inhibitor, CPT-11, given either in a metronomic or MTD manner nor switching to another antiangiogenic drug, e.g. the anti-VEGFR-2 antibody, DC101, or another TKI, sunitinib. Thus, in this case cross resistance seems to exist between MTD and metronomic topotecan, the basis of which is unknown. However, gene expression profiling revealed several potential genes that are stably upregulated in the resistant lines, that previously have been implicated in resistance to various chemotherapy drugs, and which, therefore, may contribute to the drug resistant phenotype.

IGFBP-4 anti-angiogenic and anti-tumorigenic effects are associated with anti-cathepsin B activity.

Insulin-like growth factor-binding protein 4 (IGFBP-4/IBP-4) has potent IGF-independent anti-angiogenic and antitumorigenic effects. In this study, we demonstrated that these activities are located in the IGFBP-4 C-terminal protein fragment (CIBP-4), a region containing a thyroglobulin type 1 (Tg1) domain. Proteins bearing Tg1 domains have been shown to inhibit cathepsins, lysosomal enzymes involved in basement membrane degradation and implicated in tumor invasion and angiogenesis. In our studies, CIBP-4 was shown to internalize and co-localize with lysosomal-like structures in both endothelial cells (ECs) and glioblastoma U87MG cells. CIBP-4 also inhibited both growth factor-induced EC tubulogenesis in Matrigel and the concomitant increases in intracellular cathepsin B (CatB) activity. In vitro assays confirmed CIBP-4 capacity to block recombinant CatB activity. Biodistribution analysis of intravenously injected CIBP-4-Cy5.5 in a glioblastoma tumor xenograft model indicated targeted accumulation of CIBP-4 in tumors. Most importantly, CIBP-4 reduced tumor growth in this animal model by 60%. Pleiotropic anti-angiogenic and anti-tumorigenic activities of CIBP-4 most likely underlie its observed therapeutic potential against glioblastoma.

Roles for endothelin receptor B and BCL2A1 in spontaneous CNS metastasis of melanoma.

Metastatic spread of melanoma to the central nervous system (CNS) is a common and devastating manifestation of disease progression, which, despite its clinical importance, remains poorly understood with respect to underlying molecular mechanisms. Using a recently developed preclinical model of spontaneous melanoma CNS metastasis, we have identified alterations in expression of endothelin receptor B (EDNRB) as a potential factor that influences brain metastatic potential. Induced overexpression of this gene mediated enhanced overall metastatic disease, and resulted in an increased incidence of spontaneous CNS metastases. In contrast, the overexpression of other highlighted genes, such as BCL2A1, did not affect the incidence of CNS metastases but nevertheless appears to facilitate intracranial tumor growth. The prometastatic effect in the CNS associated with EDNRB appears to be mediated by the interaction with its ligands resulting in enhanced tumor cell proliferation and thus intracranial melanoma growth. That EDNRB contributes to melanoma metastasis is underscored by the fact that its therapeutic inhibition by the EDNRB-specific inhibitor A192621 translated into improved outcomes when treating mice with either visceral metastases or intracranial tumors. The identification of an influential role of EDNRB in CNS melanoma spontaneous metastasis may provide both a target for therapeutic intervention as well as a potential prognostic marker for patients having an increased predisposition for incidence of CNS melanoma metastases.

Engineering and therapeutic application of single-chain bivalent TGF-ß family traps.

Deregulation of TGF-ß superfamily signaling is a causative factor in many diseases. Here we describe a protein engineering strategy for the generation of single-chain bivalent receptor traps for TGF-ß superfamily ligands. Traps were assembled using the intrinsically disordered regions flanking the structured binding domain of each receptor as "native linkers" between two binding domains. This yields traps that are approximately threefold smaller than antibodies and consists entirely of native receptor sequences. Two TGF-ß type II receptor-based, single-chain traps were designed, termed (TßRII)2 and (TßRIIb)2, that have native linker lengths of 35 and 60 amino acids, respectively. Both single-chain traps exhibit a 100 to 1,000 fold higher in vitro ligand binding and neutralization activity compared with the monovalent ectodomain (TßRII-ED), and a similar or slightly better potency than pan-TGF-ß-neutralizing antibody 1D11 or an Fc-fused receptor trap (TßRII-Fc). Despite its short in vivo half-life (<1 hour), which is primarily due to kidney clearance, daily injections of the (TßRII)2 trap reduced the growth of 4T1 tumors in BALB/c mice by 50%, an efficacy that is comparable with 1D11 (dosed thrice weekly). In addition, (TßRII)2 treatment of mice with established 4T1 tumors (100 mm(3)) significantly inhibited further tumor growth, whereas the 1D11 antibody did not. Overall, our results indicate that our rationally designed bivalent, single-chain traps have promising therapeutic potential.

Molecular imaging of breast tumors using a near-infrared fluorescently labeled clusterin binding peptide.

Several reports have shown that secreted clusterin (sCLU) plays multiple roles in tumor development and metastasis. Here, we report on a 12-mer sCLU binding peptide (designated P3378) that was identified by screening a phage-display peptide library against purified human sCLU. Differential resonance perturbation nuclear magnetic resonance using P3378 and a scrambled control peptide (designated P3378R) confirmed the P3378-sCLU interaction and demonstrated that it was sequence specific. P3378 and P3378R peptides were conjugated to an Alexa680 near infrared fluorophore (NIRF) and assessed for their tumor homing abilities in in vivo time-domain fluorescence optical imaging experiments using living 4T1 tumor bearing BALB/c mice. When injected in separate animals, both peptides accumulated at the tumor site, however the NIRF-labeled P3378 peptide was retained for a significant longer period of time than the P3378R peptide. Similar observations were made after simultaneously injecting the same tumor-bearing animal with a peptide mixture of P3378 DyLight (DL)680 and the P3378R-DL800. Coinjection of P3378-DL680 with excess unlabeled P3378 blocked tumor accumulation of fluorescent signal while excess P3378R control peptide did not confirming the sequence specificity of the tumor accumulation. Finally, ex vivo fluorescence microscopy of these tumors confirmed the presence of P3378-DL680 in the tumor and its colocalization with CLU. These results confirm the tumor targeting specificity of the P3378 CLU-binding peptide and suggest its usefulness for the in vivo monitoring of solid tumors secreting detectable levels of CLU.

Structures of TGF-ß receptor complexes: implications for function and therapeutic intervention using ligand traps.

TGF-ß isoforms (TGF-ß1, -ß2, and -ß3) are secreted signaling ligands that stimulate the expression of protein components of the extracellular matrix, regulate the growth and differentiation of epithelial cells, modulate immune cell function, and play roles in the development of several essential organs, including the heart and lungs. The importance of the TGF-ßs is underscored by their conservation among vertebrates and by their demonstrated roles in a variety of human diseases, including tissue fibrosis and cancer. The objective of this review is to highlight recent progress in characterizing the structures of the three TGF-ß isoforms in complex with their receptors, and to compare these with one another as well as with other members of the superfamily. Although the structural information and accompanying biophysical studies emphasize the shared ancestry of TGF-ßs, they also provide insight as to how the TGF-ßs diverged from other members of the superfamily and one another to fulfill distinct roles in vivo. The similarities and differences by which the isoforms bind their receptors present unique opportunities for designing pan-isoform and isoform-specific ligand traps and progress toward developing these is described.

Identification of high-quality cancer prognostic markers and metastasis network modules.

Cancer patients are often overtreated because of a failure to identify low-risk cancer patients. Thus far, no algorithm has been able to successfully generate cancer prognostic gene signatures with high accuracy and robustness in order to identify these patients. In this paper, we developed an algorithm that identifies prognostic markers using tumour gene microarrays focusing on metastasis-driving gene expression signals. Application of the algorithm to breast cancer samples identified prognostic gene signature sets for both estrogen receptor (ER) negative (-) and positive (+) subtypes. A combinatorial use of the signatures allowed the stratification of patients into low-, intermediate- and high-risk groups in both the training set and in eight independent testing sets containing 1,375 samples. The predictive accuracy for the low-risk group reached 87-100%. Integrative network analysis identified modules in which each module contained the genes of a signature and their direct interacting partners that are cancer driver-mutating genes. These modules are recurrent in many breast tumours and contribute to metastasis.

A multi-strategy approach to informative gene identification from gene expression data.

An unsupervised multi-strategy approach has been developed to identify informative genes from high throughput genomic data. Several statistical methods have been used in the field to identify differentially expressed genes. Since different methods generate different lists of genes, it is very challenging to determine the most reliable gene list and the appropriate method. This paper presents a multi-strategy method, in which a combination of several data analysis techniques are applied to a given dataset and a confidence measure is established to select genes from the gene lists generated by these techniques to form the core of our final selection. The remainder of the genes that form the peripheral region are subject to exclusion or inclusion into the final selection. This paper demonstrates this methodology through its application to an in-house cancer genomics dataset and a public dataset. The results indicate that our method provides more reliable list of genes, which are validated using biological knowledge, biological experiments, and literature search. We further evaluated our multi-strategy method by consolidating two pairs of independent datasets, each pair is for the same disease, but generated by different labs using different platforms. The results showed that our method has produced far better results.

TbetaR-II discriminates the high- and low-affinity TGF-beta isoforms via two hydrogen-bonded ion pairs.

The TGF-beta isoforms, TGF-beta1, -beta2, and -beta3, share greater than 70% sequence identity and are almost structurally identical. TGF-beta2 differs from the others, however, in that it binds the TGF-beta type II receptor (TbetaR-II) with much lower affinity than either TGF-beta1 or -beta3. It has been previously shown that three conserved interfacial residues, Arg25, Val92, Arg94, in TGF-beta1 and -beta3 are responsible for their high-affinity interaction with TbetaR-II. In this study, the role of each of these residues was examined by creating single, double, and triple substitutions resulting in both TGF-beta3 loss-of-function and TGF-beta2 gain-of-function variants. One-dimensional 1H NMR spectra of the variants confirmed a lack of large structural perturbations. Affinities, kinetics, and thermodynamics for TbetaR-II binding were determined by surface plasmon resonance biosensor analysis. Double substitutions revealed that nearly all of the high-affinity binding is contributed by Arg25 and Arg94. Single site substitutions showed that Arg94 makes the greatest contribution. Substitution of Arg25 and Arg94 with alanine verified the requirement of the arginine guanidinium functional groups for the highly specific hydrogen-bonded ion pairs formed between Arg25 and Arg94 of TGF-beta1 and -beta3, and Glu119 and Asp32 of TbetaR-II. Further kinetic and thermodynamic analyses confirmed that Arg25 and Arg94 are primarily responsible for high-affinity binding and also revealed that noninterfacial longer range effects emanating from the TGF-beta structural framework contribute slightly to TbetaR-II binding. Growth inhibition assays showed that binding changes generally correlate directly with changes in function; however, a role Val92 in this cellular context was uncovered.

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications.

Heterodimerizing peptides, such as the de novo designed E5/K5 peptide pair, have several applications including as tags for protein purification or immobilization. Recently, we demonstrated that E5-tagged epidermal growth factor (EGF), when bound to a K4 expressing adenovirus, promotes retargeting of the adenovirus to EGFR expressing target cells. In this study, we present the Escherichia coli expression, refolding and purification of human EGF fused with the E5-coil (E5-coil-EGF) or with the K5-coil (K5-coil-EGF). EGF receptor phosphorylation and cell proliferation assays demonstrated that the biological activity of the coil-tagged EGF versions was comparable to that of non-tagged EGF. Additionally, analysis of the binding of E5/K5-coil-EGF to cell surface EGFR or to soluble EGFR ectodomain, as measured by cell-based binding competition assays and by SPR-based biosensor experiments, indicated that the coil-tagged EGF versions bound to EGFR with affinities similar to that of non-tagged EGF. Finally, we show that E-coil-tagged EGF, but not non-tagged EGF, can retarget a K-coil containing adenovirus to EGF receptor expressing glioblastoma tumor cells. Overall these results indicate that E. coli expression offers a practical platform for the reproducible production of fully biologically active E5/K5-coil-tagged EGF, and support applications of heterodimerizing coil-tagged ligands, e.g. the targeting of viruses or other entities such as nanoparticles to tumor cells, or growth factor immobilization on cell culture scaffolds for tissue engineering.

Analysis of the contribution of receptor subdomains to the cooperative binding and internalization of transforming growth factor-beta (TGF-beta) type I and type II receptors.

The mechanistic basis underlying the striking cooperativity observed for the assembly of TGF-beta family ligand/receptor complexes is not well understood. We report here an investigation in which we used a novel ligand sequestration assay, in combination with immunofluorescent light microscopy and flow cytometry analyses, to examine and quantify cooperative assembly of TGF-beta ligand/receptor complexes on the cell surface, as well as ligand/receptor complex internalization. We analyzed the roles played by the ecto/transmembrane (ecto/TM) domains and endodomains of RI and RII TGF-beta receptors in these processes by transfecting 293 or HeLa cells with different combinations of receptor mutants. We found that the ecto/TM domains of RII and RI cooperated together to promote the formation of cell surface receptor/ligand complexes. Furthermore, in agreement with the recently determined structure of the TGF-beta 3/RII ectodomain/RI ectodomain complex [J. Groppe, C.S. Hinck, P. Samavarchi-Tehrani, C. Zubieta, J.P. Schuermann, A.B. Taylor, P.M. Schwarz, J.L. Wrana, A.P. Hinck, Cooperative assembly of TGF-beta superfamily signaling complexes is mediated by two disparate mechanisms and distinct modes of receptor binding, Mol. Cell 29 (2008) 157-168], we observed that the N-terminus of the RII ectodomain was required for full assembly. With respect to endodomains, we found that the RI endodomain enhanced cooperative complex assembly at the cell surface, whereas both the RI and RII endodomains enhanced internalization. Finally, we observed that ligand/receptor internalization, but not complex assembly at the cell surface, was partly raft-dependent. In light of these results, currently proposed mechanisms of cooperative ligand/receptor assembly are discussed.

The crystal structure and dimerization interface of GADD45gamma.

Gadd45 proteins are recognized as tumor and autoimmune suppressors whose expression can be induced by genotoxic stresses. These proteins are involved in cell cycle control, growth arrest, and apoptosis through interactions with a wide variety of binding partners. We report here the crystal structure of Gadd45gamma, which reveals a fold comprising an alphabetaalpha sandwich with a central five-stranded mixed beta-sheet with alpha-helices packed on either side. Based on crystallographic symmetry we identified the dimer interface of Gadd45gamma dimers by generating point mutants that compromised dimerization while leaving the tertiary structure of the monomer intact. The dimer interface comprises a four-helix bundle involving residues that are the most highly conserved among Gadd45 isoforms. Cell-based assays using these point mutants demonstrate that dimerization is essential for growth inhibition. This structural information provides a new context for evaluation of the plethora of protein-protein interactions that govern the many functions of the Gadd45 family of proteins.

A ligand-pseudoreceptor system based on de novo designed peptides for the generation of adenoviral vectors with altered tropism.

BACKGROUND: Delivery of transgenes into specific tissues by adenovirus vectors (AdVs) relies on ablations of their natural tropism and on introduction of a new tropism. If the interaction with its natural receptor is ablated, a new packaging cell line is required to produce the AdV. In the present study, we have used two de novo designed peptides (E-Coil and K-Coil) that interact with each other with high affinity to establish a new receptor-ligand system for the propagation of retargeted AdVs. METHODS: We produced a cell line (293E) expressing on its surface a pseudoreceptor containing the E-Coil. An AdV (AdFK4m/GFP) lacking the interaction with the primary receptor for adenovirus (CAR) and containing the K-Coil inserted at the fiber C-terminus was constructed and tested using two strategies: (1) an RGD motif (Arg-Gly-Asp) was inserted into the HI-loop of the fiber; (2) AdFK4m/GFP was conjugated to a bispecific adaptor for the epidermal growth factor receptor (EGFR). RESULTS: AdFK4m/GFP infected 293E cells more efficiently than cells lacking the pseudoreceptor. The transduction was due to the K-Coil/E-Coil specific interaction since it was competed by addition of soluble K-Coil, but not soluble fiber. We demonstrated that the modified AdV was retargeted toward alpha v integrin by inclusion of the RGD motif, or toward EGFR using the bispecific adaptor. CONCLUSIONS: We have established a new system to produce AdVs ablated of natural tropism. This system should permit the retargeting of AdVs by inserting new ligands within the fiber or through the interaction with bispecific adaptors.

Investigation of three new mouse mammary tumor cell lines as models for transforming growth factor (TGF)-beta and Neu pathway signaling studies: identification of a novel model for TGF-beta-induced epithelial-to-mesenchymal transition.

INTRODUCTION: This report describes the isolation and characterization of three new murine mammary epithelial cell lines derived from mammary tumors from MMTV (mouse mammary tumor virus)/activated Neu + TbetaRII-AS (transforming growth factor [TGF]-beta type II receptor antisense RNA) bigenic mice (BRI-JM01 and BRI-JM05 cell lines) and MMTV/activated Neu transgenic mice (BRI-JM04 cell line). METHODS: The BRI-JM01, BRI-JM04, and BRI-JM05 cell lines were analyzed for transgene expression, their general growth characteristics, and their sensitivities to several growth factors from the epidermal growth factor (EGF) and TGF-beta families (recombinant human EGF, heregulin-beta1 and TGF-beta1). The BRI-JM01 cells were observed to undergo a striking morphologic change in response to TGF-beta1, and they were therefore further investigated for their ability to undergo a TGF-beta-induced epithelial-to-mesenchymal transition (EMT) using motility assays and immunofluorescence microscopy. RESULTS: We found that two of the three cell lines (BRI-JM04 and BRI-JM05) express the Neu transgene, whereas, unexpectedly, both of the cell lines that were established from MMTV/activated Neu + TbetaRII-AS bigenic tumors (BRI-JM01 and BRI-JM05) do not express the TbetaRII-AS transgene. The cuboidal BRI-JM01 cells exhibit a short doubling time and are able to form confluent monolayers. The BRI-JM04 and BRI-JM05 cell lines are morphologically much less uniform, grow at a much slower rate, and do not form confluent monolayers. Only the BRI-JM05 cells can form colonies in soft agar. In contrast, all three cell lines form colonies in Matrigel, although the BRI-JM04 and BRI-JM05 cell lines do so more efficiently than the BRI-JM01 cell line. All three cell lines express the cell surface marker E-cadherin, confirming their epithelial character. Proliferation assays showed that the three cell lines respond differently to recombinant human EGF and heregulin-beta1, and that all are growth inhibited by TGF-beta1, but that only the BRI-JM01 cell line undergoes an EMT and exhibits increased motility upon TGF-beta1 treatment. CONCLUSION: We suggest that the BRI-JM04 and BRI-JM05 cell lines can be used to investigate Neu oncogene driven mammary tumorigenesis, whereas the BRI-JM01 cell line will be useful for studying TGF-beta1-induced EMT.