Anne E.G. Lenferink

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

There has been great interest in attempting to identify gene expression signatures that predict cancer survival. In this study a new algorithm is developed to analyse gene expression datasets that accurately classify both ER+ and ER− breast cancers into low- and high-risk groups.

Glycoproteomic analysis of two mouse mammary cell lines during transforming growth factor (TGF)-β induced epithelial to mesenchymal transition

BackgroundTGF-β acts as an antiproliferative factor in normal epithelial cells and at early stages of oncogenesis. However, later in tumor development TGF-β can become tumor promoting through mechanisms including the induction of epithelial-to-mesenchymal transition (EMT), a process that is thought to contribute to tumor progression, invasion and metastasis. To identify EMT-related breast cancer therapeutic targets and biomarkers, we have used two proteomic approaches to find proteins that change in abundance upon the induction of EMT by TGF-β in two mouse mammary epithelial cell lines, NMuMG and BRI-JM01.ResultsPreliminary experiments based on two-dimensional electrophoresis of a hydrophobic cell fraction identified only 5 differentially expressed proteins from BRI-JM01 cells. Since 3 of these proteins were glycoproteins, we next used the lectin, wheat germ agglutinin (WGA), to enrich for glycoproteins, followed by relative quantification of tryptic peptides using a label-free LC-MS based method. Using these approaches, we identified several proteins that are modulated during the EMT process, including cell adhesion molecules (several members of the Integrin family, Fibronectin, Activated leukocyte cell adhesion molecule, and Neural cell adhesion molecule 1) and regulators of cellular signaling (Tumor-associated calcium signal transducer 2, Basigin).ConclusionInterestingly, despite the fact that TGF-β induces similar EMT phenotypes in NMuMG and BRI-JM01 cells, the proteomic results for the two cell lines showed only minimal overlap. These differences likely result in part from the conservative cut-off values used to define differentially-expressed proteins in these experiments. Alternatively, it is possible that the two cell lines may use different mechanisms to achieve an EMT transition.

Identification of Vascular Breast Tumor Markers by Laser Capture Microdissection and Label-Free LC−MS

Blood vessels in tumors frequently show abnormal characteristics, such as tortuous morphology or leakiness, but very little is known about protein expression in tumor vessels. In this study, we have used laser capture microdissection (LCM) to isolate microvessels from clinical samples of invasive ductal carcinoma (IDC), the most common form of malignant breast cancer, and from patient-matched adjacent nonmalignant tissue. This approach eliminates many of the problems associated with the heterogeneity of clinical tumor tissues by controlling for differences in protein expression between both individual patients and different cell types. Proteins from the microvessels were trypsinized and the resulting peptides were quantified by a label-free nanoLC-MS method. A total of 86 proteins were identified that are overexpressed in tumor vessels relative to vessels isolated from the adjacent nonmalignant tissue. These proteins include well-known breast tumor markers such as Periostin and Tenascin C but also proteins with lesser-known or emerging roles in breast cancer and tumor angiogenesis (i.e., Serpin H1, Clic-1, and Transgelin 2). We also identified 40 proteins that were relatively under-expressed in IDC tumor vessels, including several components of the basement membrane whose lower expression could be responsible for weakening tumor vessels. Lastly, we show that a subset of 29 proteins, derived from our list of differentially expressed proteins, is able to predict survival in three publicly available clinical breast cancer microarray data sets, which suggests that this subset of proteins likely plays a functional role in cancer progression and outcome.

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

IntroductionThis 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 + TβRII-AS (transforming growth factor [TGF]-β type II receptor antisense RNA) bigenic mice (BRI-JM01 and BRI-JM05 cell lines) and MMTV/activated Neu transgenic mice (BRI-JM04 cell line).MethodsThe 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-β families (recombinant human EGF, heregulin-β1 and TGF-β1). The BRI-JM01 cells were observed to undergo a striking morphologic change in response to TGF-β1, and they were therefore further investigated for their ability to undergo a TGF-β-induced epithelial-to-mesenchymal transition (EMT) using motility assays and immunofluorescence microscopy.ResultsWe 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 + TβRII-AS bigenic tumors (BRI-JM01 and BRI-JM05) do not express the TβRII-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-β1, and that all are growth inhibited by TGF-β1, but that only the BRI-JM01 cell line undergoes an EMT and exhibits increased motility upon TGF-β1 treatment.ConclusionWe 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-β1-induced EMT.

Expression of TGF-β type II receptor antisense RNA impairs TGF-β signaling in vitro and promotes mammary gland differentiation in vivo

In order to clarify the role of TGF‐β in mammary development and tumorigenesis, we investigated the efficacy of full‐ or partial‐length TβRII antisense RNA specifically to reduce TβRII levels in both in vitro and in vivo model systems. Here we show that the expression of TβRII antisense RNA in vitro reduced TβRII cell surface expression and inhibited the antiproliferative and transcriptional responses to exogenous TGF‐β. Expression of full‐length TβRII antisense RNA in a transgenic mouse model under control of the mouse mammary tumor virus promotor resulted in precocious lobuloalveolar development of the mammary gland, a phenotype that resembles that of early pregnancy. These data demonstrate that TβRII plays a critical role in maintaining the nondifferentiated character of virgin mammary gland epithelium.

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.

A novel pattern based clustering methodology for time-series microarray data

Identification of co-expressed genes sharing similar biological behaviours is an essential step in functional genomics. Traditional clustering techniques are generally based on overall similarity of expression levels and often generate clusters with mixed profile patterns. A novel pattern recognition method for selecting co-expressed genes based on rate of change and modulation status of gene expression at each time interval is proposed in this paper. This method is capable of identifying gene clusters consisting of highly similar shapes of expression profiles and modulation patterns. Furthermore, we develop a quality index based on the semantic similarity in gene annotations to assess the likelihood of a cluster being a co-regulated group. The effectiveness of the proposed methodology is demonstrated by applying it to the well-known yeast sporulation dataset and an in-house cancer genomics dataset.

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.

Abstract 1467: AB-16B5, a therapeutic monoclonal antibody against human clusterin that blocks the epithelial-to-mesenchymal transition

Studies are increasingly implicating clusterin (CLU) as an important contributing factor in cancer promotion and invasion. Although there is evidence that secreted (s) CLU plays a pro-survival role in tumors, recently published results demonstrated that sCLU is also a potent stimulator of the epithelial-to-mesenchymal transition (EMT). A family of monoclonal antibodies (mAbs) specific for human sCLU was generated and a subset of these was found to inhibit the migration and invasion of several types of cancer cells. Importantly, this EMT-inhibiting subset of mAbs all bind to a specific amino acid sequence in sCLU and inhibit metastasis in vivo. The variable regions of the lead candidate mAb were modified using an in silico molecular modeling approach to generate a humanized IgG2, designated AB-16B5, which exhibited almost identical binding parameters compared to the original mouse antibody, with an apparent KD of 2 − 5 nM for recombinant human CLU. Treatment of 4T1 carcinoma cells with AB-16B5 inhibited TGFβ-induced EMT indicating that the biological activity in vitro was maintained in the humanized antibody. Moreover, AB-16B5 inhibited the motility of EMT6 cells in scratch assays and reduced the invasion of DU145 and PC-3 hormone-insensitive human prostate cancer cells when cultured in Matrigel. In animal studies, DU145 prostate cancer cells were implanted in SCID mice and treated with 5 mg/kg AB-16B5 twice per week as a monotherapy or in combination with the anti-mitotic drug, docetaxel (TxT). These experiments showed that the tumors in the AB-16B5 treated animals were 55% smaller than those in the control group. Furthermore, combining the AB-16B5 treatment with that of TxT caused a reduction of tumor size by 40% compared to the TxT-only group. PC-3-derived tumors in SCID mice treated with AB-16B5 showed a similar degree of tumor growth inhibition. Importantly, in both prostate cancer models, the mice exposed to AB-16B5 exhibited a marked increase in their overall survival. Furthermore, AB-16B5 treatment of Nude mice with intra-cardiac implantations of MDA-231 breast cancer cells resulted in a reduction of the number of metastatic bone lesions. The pharmacokinetic parameters of AB-16B5 are comparable to other IgG2 antibodies with its half-life being approximately 15 days following a bolus intravenous injection in mice. Finally, mice exposed to AB-16B5 at 10-times the therapeutic dose displayed no observable signs of toxicity and no major changes in serum biochemistry were detected. In conclusion, AB-16B5 is one of the rare therapeutic mAbs that directly targets EMT to reduce the invasion of tumors. Its therapeutic effects hold much promise to control metastasis from breast and prostate tumors, in addition to enhancing the response to chemotherapeutic drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1467.

Abstract B058: Development of AVID200, a novel and highly potent TGF-beta neutralizing immunotherapy

Introduction: Elevated TGF-beta ligand markedly augments cancer progression primarily by suppressing the immune system in the tumor microenvironment, in particular by suppressing T-cell recruitment and/or activation. We developed AVID200, a novel decoy receptor trap that potently blocks TGF-beta and induces T-cell infiltration into tumors. This promotes the “T-cell-inflamed” tumor state, which is expected to render tumors sensitive to immune checkpoint inhibitors and other immunotherapeutics. Experimental Procedures: AVID200 is a computationally designed novel class of avidity-enhanced receptor-ectodomain-based trap that binds and neutralizes TGF-beta 1 and 3 with low pM potency. Several trap formats have been produced and tested, with each format exhibiting varying characteristics, including differing circulating half-lives and in vitro blocking potencies. AVID200 was evaluated for efficacy in in vivo studies using the syngeneic 4T1 triple negative breast cancer (TNBC) tumor model. Additionally, ex vivo studies were performed on CD4+ and CD8+ T-cells harvested from the draining lymph nodes of treated animals. Results: In efficacy studies using the syngeneic 4T1 TNBC model, novel TGF-beta traps were shown to promote significant T-cell infiltration into tumors. This infiltration resulted in reduced primary tumor growth as well as significant reductions in metastatic lesions. Additionally, ex vivo studies revealed that trap treatment decreased T-cell apoptosis, promoted T-cell proliferation in response to tumor cell lysates in the presence of dendritic cells, as well as increased the capacity of T-cells to specifically lyse 4T1 tumor cells. Conclusion: AVID200 is a novel computationally-designed TGF-beta trap that neutralizes TGF-beta 1 and 3 with pM potency and markedly promotes the “T-cell-inflamed” tumor state. Combination studies with immune checkpoint inhibitors are ongoing. Citation Format: Maureen D. O9Connor-McCourt, Anne E.G. Lenferink, John Zwaagstra, Traian Sulea, Jason Baardsnes, Catherine Collins, Christiane Cantin, Lucie Couture, Limei Tao, Yves Durocher, Renu Singh, James Koropatnick. Development of AVID200, a novel and highly potent TGF-beta neutralizing immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B058.