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SummaryThe extracellular matrix (ECM) contributes to the biological and clinical heterogeneity of breast cancer, and different prognostic groups can be identified according to specific ECM signatures. In high-grade, but not low-grade, tumors, an ECM signature characterized by high SPARC expression (ECM3) identifies tumors with increased epithelial-to-mesenchymal transition (EMT), reduced treatment response, and poor prognosis. To better understand how this ECM3 signature is contributing to tumorigenesis, we expressed SPARC in isogenic cell lines and found that SPARC overexpression in tumor cells reduces their growth rate and induces EMT. SPARC expression also results in the formation of a highly immunosuppressive microenvironment, composed by infiltrating T regulatory cells, mast cells, and myeloid-derived suppressor cells (MDSCs). The ability of SPARC to induce EMT depended on the localization and suppressive function of myeloid cells, and inhibition of the suppressive function MDSCs by administration of aminobisphosphonates could revert EMT, rendering SPARC-overexpressing tumor cells sensitive to Doxil. We conclude that that SPARC is regulating the interplay between MDSCs and the ECM to drive the induction of EMT in tumor cells.

The recent dramatic increase in breast cancer incidence across China with progressive urbanization and economic development has signaled the urgent need for molecular and clinical detailing of breast cancer in the Chinese population. Our analyses of a unique transethnic collection of breast cancer frozen specimens from Shanghai Fudan Cancer Center (Chinese Han) profiled simultaneously with an analogous Caucasian Italian series revealed consistent transcriptomic data lacking in batch effects. The prevalence of Luminal A subtype was significantly lower in Chinese series, impacting the overall prevalence of estrogen receptor (ER)-positive disease in a large cohort of Chinese/Caucasian patients. Unsupervised and supervised comparison of gene and microRNA (miRNA) profiles of Chinese and Caucasian samples revealed extensive similarity in the comprehensive taxonomy of transcriptional elements regulating breast cancer biology. Partition of gene expression data using gene lists relevant to breast cancer as “intrinsic” and “extracellular matrix” genes identified Chinese and Caucasian subgroups with equivalent global gene and miRNA profiles. These findings indicate that in the Chinese and Caucasian groups, breast neoplasia and the surrounding stromal characteristics undergo the same differentiation and molecular processes. Transcriptional similarity across transethnic cohorts may simplify translational medicine approaches and clinical management of breast cancer patients worldwide.

Background Currently used CA15–3 and CEA have found their clinical application particularly in the follow-up of patients with advanced disease. Novel biomarkers are urgent, especially for improving early diagnosis as well as for discriminating between benign and malignant disease. Patients and methods In the present study, we used a proteomic approach based on surface-enhanced laser desorption/ionization–time of flight–mass spectrometry screening with the aim of identifying differentially expressed 2–30 kDa proteins in plasma of patients with malignant (65 cases) and benign (88 cases) breast lesions with respect to 121 healthy controls. Results We found that the most promising SELDI peaks were those corresponding to hepcidin-25 and ferritin light chain. We evaluated the capability of these peaks in predicting malignant and benign breast lesions using the area under the receiver operating characteristic curve (AUC). The results showed a good capacity to predict malignant breast lesions for hepcidin-25 [AUC: 0.82; 95% confidence interval (CI) 0.75–0.90] and ferritin light chain (AUC: 0.86; 95% CI 0.79–0.92). Conversely, a weak and satisfactory capability to predict benign breast lesion was observed for hepcidin-25 (AUC: 0.63; 95% CI 0.41–0.85) and ferritin light chain (AUC: 0.73; 95% CI 0.49–0.97). A significant association between HER2 status and hepcidin-25 was observed and the distribution of transferrin and ferritin were found significantly different in patients with breast cancer when compared with that of controls. Conclusions This study provides evidence that hepcidin and ferritin light chain level in plasma may be of clinical usefulness to predict malignant and benign disease with respect to healthy controls.

We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related proteins also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA) or reverse-phase protein array (RPPA), were used to measure the expression of 20 proteins linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012). The univariate analysis of RPPA data highlighted the following seven proteins differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5), signal transducer and activator of transcription 3 (STAT3), bone morphogenetic protein 6 (BMP6), cluster of differentiation 74 (CD74), transferrin receptor (TFRC), inhibin alpha (INHA), and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related 10-protein panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88), CD74, iron exporter ferroportin (FPN), high mobility group box 1 (HMGB1), STAT3_pS727, TFRC, ferritin heavy chain (FTH), and ferritin light chain (FTL). Our results showed an association between some iron-related proteins and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer.

Objective: Hepcidin-25 production is stimulated by systemic inflammation, and it interferes with iron utilization, leading to anemia. This study aimed to investigate the relationships between the plasma levels of hepcidin, interleukin-6 (IL-6), erythropoietin (EPO) and erythroferrone (ERFE) in patients with benign breast disease or cancer. Methods: Plasma samples from a cohort of 131 patients (47 with benign breast disease and 84 with breast cancer) were subjected to the evaluation of hepcidin, IL-6, EPO and ERFE using SELDI-TOF-MS or immunoassays. Results: An elevated hepcidin was observed in malignant breast tumors compared to benign ones. No correlation was observed between hepcidin and IL-6, EPO or ERFE. Conclusion: Since the study included a cohort of patients (87%) with breast cancers smaller than 2 cm, these results may support our previous evidence about the potential role of hepcidin in breast cancer disease.

The ectopic expression of the gene SEL1L in the human breast carcinoma cell line MCF-7 resulted in a reduction of the aggressive behaviour of these cells in vitro. In addition, in vivo analysis on a series of primary breast carcinomas revealed an association between the SEL1L protein levels and the patient's overall survival. We aimed to find those proteins, associated with SEL1L, which may be involved in modulating the aggressive or invasive behaviour of breast cancer cells. For this purpose, we used both the proteomic and microarray approaches. Image analysis of two-dimensional electropherograms revealed the presence of 27 qualitative and 35 quantitative variations between the MCF7-SEL1L expressing cells compared to control. Mass spectrometry identified 32 changing proteins mostly involved in cytoskeletal and metabolic activities, stress response and protein folding, selenoprotein synthesis and cellular proliferation. Five of these also showed changes in transcript levels, as assessed by Affymetrix microarray analysis. Interestingly, seven proteins: carbonic anhydrase (CA) II, ovarian/breast septin, S100A16 calcium binding protein, 14-3-3 protein sigma, proteasome subunit beta type 6, Hsp60 and protein disulphide-isomerase A3 merit particular attention since they are known to be involved in cancer, in response to cellular stress and in protein folding.

In this work, we explored the existence of genetic variants within the SEL1L transcriptional regulatory region by direct sequencing of the basal promoter. SEL1L is the human ortholog of the Caenorhabditis elegans gene sel-1, a negative regulator of LIN-12/NOTCH receptor proteins. To understand the relation in SEL1L transcription pattern observed in different epithelial cells, we analysed its promoter activity. We found it to be considerably higher only in pancreatic cells. We then looked for the presence of genetic variability within this region by sequencing the minimal promoter of 63 individuals (126 alleles); two new and associated polymorphic variants were found only in few lung carcinoma bearing patients. The functional effects of this polymorphism was analysed by transient transfection assay which resulted in a significant increase in the transcriptional activity of the gene.

Screening a random peptide library displayed on phage as fusion to the major capsid protein pVIII identified a ligand binding the human epidermal growth factor receptor 2 (HER2) specifically. By mutating the sequence of this ligand, a “secondary” library was generated, whose panning on HER2-positive cells isolated a phage-borne peptide with increased specific binding to HER2 (phage NL1.1). The same peptide recognised HER2 specifically when expressed as an N-terminal fusion to the minor coat protein pIII. Phage NL1.1 was engineered to include a mammalian expression cassette for a reporter gene within its genome. This modified phage transduced HER2-expressing cells with very high specificity (more than 1000-fold that of parental HER2-negative cells) and with an efficiency comparable to that of chemical transfection protocols. The gene delivery process was remarkably fast, requiring less than 15 minutes incubation of phage with target cells to generate detectable levels of gene expression.

To determine whether p185HER2 overexpression per se triggers p185HER2 cellular signaling or whether an extracellular signal is required, we transfected PC12 cells with the human erbB-2 proto-oncogene, and established a cell line that overexpresses p185HER2. PC12-HER2 cells, maintained in suspension culture or plated on a collagen layer, showed the same morphology and growth rate as PC12 and PC12 mock-transfected control cells. When treated with monoclonal antibody (MAb) MGr6 or other anti-p185HER2 MAbs, PC12-HER2 cells specifically underwent neuronal differentiation comparable to that induced by nerve growth factor (NGF), and the differentiation-inducing effect of the MAb was dramatically enhanced by the addition of a second anti-mouse IgG. MAb-induced cell differentiation correlated with p185HER2 phosphorylation, recruitment of Shc and Grb-2 transducer molecules into complexes, and MAPK phosphorylation. These data indicate the requirement for a specific binding-induced activation of the overexpressed p185HER2 receptor in inducing PC12 cell differentiation. PC12-HER2 cells represent a suitable system for selection of p185HER2-activating ligands (peptides, phage-displayed peptides or proteins) or specific inhibitors of its tyrosine kinase activity.

Valproic acid (VPA), an histone deacetylase inhibitor, is emerging as a promising therapeutic agent for the treatments of gliomas by virtue of its ability to reactivate the expression of epigenetically silenced genes. VPA induces the unfolded protein response (UPR), an adaptive pathway displaying a dichotomic yin yang characteristic; it initially contributes in safeguarding the malignant cell survival, whereas long-lasting activation favors a proapoptotic response. By triggering UPR, VPA might tip the balance between cellular adaptation and programmed cell death via the deregulation of protein homeostasis and induction of proteotoxicity. Here we aimed to investigate the impact of proteostasis on glioma stem cells (GSC) using VPA treatment combined with subversion of SEL1L, a crucial protein involved in homeostatic pathways, cancer aggressiveness, and stem cell state maintenance. We investigated the global expression of GSC lines untreated and treated with VPA, SEL1L interference, and GSC line response to VPA treatment by analyzing cell viability via MTT assay, neurosphere formation, and endoplasmic reticulum stress/UPR-responsive proteins. Moreover, SEL1L immunohistochemistry was performed on primary glial tumors. The results show that (i) VPA affects GSC lines viability and anchorage-dependent growth by inducing differentiative programs and cell cycle progression, (ii) SEL1L down-modulation synergy enhances VPA cytotoxic effects by influencing GSCs proliferation and self-renewal properties, and (iii) SEL1L expression is indicative of glioma proliferation rate, malignancy, and endoplasmic reticulum stress statuses. Targeting the proteostasis network in association to VPA treatment may provide an alternative approach to deplete GSC and improve glioma treatments.

Hybridoma technology has enabled the production of large quantities of murine monoclonal antibodies (mAbs) directed to specific antigens. However, the therapeutic usefulness of these reagents in humans has been limited by the unwanted immune response induced in patients. Although human mAbs are difficult to obtain with conventional techniques, murine mAbs can be ‘humanised’ and tailored to the particular clinical application by antibody engineering. Human antibody fragments can be derived from repertoires of associated immunoglobulin heavy and light chains displayed on the surface of bacteriophages, and are readily diversified by random point mutation or by chain shuffling. Here we review strategies in the development of therapeutic recombinant human antibodies.

Tumor vascularization is a fundamental step in solid tumor progression and is orchestrated by different pathways of vasculogenesis. In malignant tumors, neoplastic cells can differentiate into endothelial-like cells acquiring the expression of endothelial markers (i.e. CD31 and CD34) and participating in the formation of vascular-like structures that functionally deliver oxygen and nutrients to the tumor site. We recently identified PDGFRβ as an important player of this process in triple negative breast cancer (TNBC). Interestingly, increasing evidence supported a connection between PDGFRβ and epithelial to mesenchymal transition (EMT), important step for the endothelial trans-differentiation process. PDGFR signaling arose as a promising target for TNBC; thus a better understanding of this new role of PDGFRβ could be relevant in the biology and treatment of TNBC. We then aimed at investigating microRNAs able to regulate tumor vascularization via PDGFRβ-mediated endothelial differentiation in TNBC, focusing on miR-9 and miR-200 family in light of their known relation with PDGFRβ and EMT. Tube formation assay showed that in MDA-MB-231 and MDA-MB-157 TNBC cell lines transfection of miR-9 and miR-200 family members (miR-200b and miR-200c) promoted or inhibited, respectively, the ability of cells to form vascular-like structures when seeded on matrigel. Stimulation of PDGFRβ with PDGF-BB ligand induced miR-9 expression and enhanced the loop formation ability of treated cells. This advantage was, however, almost completely abrogated by the concomitant inhibition of miR-9, thus demonstrating that miR-9 contributed to PDGFRβ-mediated vasculogenic properties of TNBC. Moreover, we found that silencing of STARD13, direct target of miR-9 validated by luciferase reporter assay, improved the vasculogenic potential. Ectopic expression of miR-200 members, instead, suppressed PDGFRβ at protein level in both TNBC cell lines. Furthermore, the silencing of ZEB1, known target of miR-200 family, induced downregulation of PDGFRβ at protein and mRNA level; interestingly, the mRNA levels of ZEB1 and PDGFRβ strongly correlated in the TNBC subset of the TGCA dataset. The in vivo effect of miR-9 and miR-200 family on vasculogenic properties of TNBC was then validated first through the generation of MDA-MB-231 clones for stable inhibition of miR-9 and restoration of miR-200c; the resulting miR-9 sponge and miR-200c xenografted tumors showed lower number of vascular lacunae than controls, identified through immunohistochemical (IHC) staining for CD31. These results were validated in orthotopic MDA-MB-231 breast tumors treated with miR-9 inhibitors or miR-200c mimics by peritumoral injection. We finally evaluated PDGFRβ, by IHC, and miR-9 and miR-200c, by Real-time PCR, in a cohort of TNBC. IHC analysis demonstrated that PDGFRβ staining identified tumor cells participating in vascular lacunae, data further confirmed by immunofluorescence co-localization of PDGFRβ and CD31. Interestingly, unlike miR-9, the expression of miR-200c negatively associated with both the presence of vascular lacunae and tumor nests positive for PDGFRβ. Finally, in situ hybridization analysis revealed that miR-200c was mainly expressed by tumor cells. In conclusion, our results suggest that miR-9 and miR-200 family play an opposite role in the regulation of the vasculogenic aptitude of TNBC. MiR-9 is induced by PDGFRβ and enhances the vasculogenic properties of tumor cells in part through the negative regulation of STARD13. MiR-200, instead, inhibits this aggressive phenotype indirectly suppressing PDGFRβ through targeting of ZEB1. Citation Format: Elvira D'Ippolito, Ilaria Plantamura, Lucia Bongiovanni, Patrizia Casalini, Sara Baroni, Claudia Piovan, Rosaria Orlandi, Ambra V. Gualeni, Annunziata Gloghini, Anna Rossini, Filippo De Braud, Elda Tagliabue, Claudio Tripodo, Marilena V. Iorio. miR-9 and miR-200 regulate PDGFRβ-mediated endothelial differentiation of neoplastic cells in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A18.

SEL1L-A, a transmembrane glycoprotein residing in the endoplasmic reticulum (ER), is a component of the ER-associated degradation (ERAD) pathway. Alternative splicing generates two smaller SEL1L isoforms, -B and -C, that lack the SEL1L-A membrane-spanning region but retain some sel-1-like repeats, known to be involved in multi-protein interactions and signal transduction. In this study the functional characteristics of SEL1L-B and -C were investigated in human cell models. We show that these two isoforms are induced upon ER stress and activation of the unfolded protein response, together with SEL1L-A. Using transient transfection experiments (based on wild-type and mutant SEL1L constructs) combined with several biochemical tests we show that SEL1L-B and, more prominently, SEL1L-C are secreted glycoproteins. Although SEL1L-C is in monomeric form, SEL1L-B is engaged in intramolecular/intermolecular disulfide bonds. Both isoforms localize in secretory and degradative cellular compartments and in areas of cell-cell contact. However, whereas SEL1L-B is mainly associated with membranes, SEL1L-C shows the typical intralumenal localization of soluble proteins and is present in intercellular spaces. Furthermore, because of its peroxisomal domain, SEL1L-C localizes to peroxisomes. Both SEL1L-B and -C are involved in sorting and exporting unassembled Ig-μs but do not affect two other ERAD substrates, the null Hong Kong variant of α1-antitrypsin, and mutant α1-AT Z. Overall these findings suggest that SEL1L-B and -C participate to novel molecular pathways that, in parallel with ERAD, contribute to the disposure of misfolded/unfolded or orphan proteins through degradation or secretion.

Prediction of the clinical outcome of breast cancer is multi-faceted and challenging. There is growing evidence that the complexity of the tumour micro-environment, consisting of several cell types and a complex mixture of proteins, plays an important role in development, progression, and response to therapy. In the current study, we investigated whether invasive breast tumours can be classified on the basis of the expression of extracellular matrix (ECM) components and whether such classification is representative of different clinical outcomes. We first examined the matrix composition of 28 primary breast carcinomas by morphology and gene expression profiling using 22K oligonucleotide Agilent microarrays. Hierarchical clustering of the gene expression profile of 278 ECM-related genes derived from the literature divided the tumours into four main groups (ECM1-4). A set of selected differentially expressed genes was validated by immunohistochemistry. The robustness of the ECM classification was confirmed by studying the four ECM groups in a previously published gene expression data set of 114 early-stage primary breast carcinomas profiled using cDNA arrays. Univariate survival analysis showed significant differences in clinical outcome among the various ECM subclasses. One set of tumours, designated ECM4, had a favourable outcome and was defined by the overexpression of a set of protease inhibitors belonging to the serpin family, while tumours with an ECM1 signature had a poorer prognosis and showed high expression of integrins and metallopeptidases, and low expression of several laminin chains. Furthermore, we identified three surrogate markers of ECM1 tumours: MARCO, PUNC, and SPARC, whose expression levels were associated with breast cancer survival and risk of recurrence. Our findings suggest that primary breast tumours can be classified based upon ECM composition and that this classification provides relevant information on the biology of breast carcinomas, further supporting the hypothesis that clinical outcome is strongly related to stromal characteristics.

Since the cloning in 1997 of SEL1L, the human ortholog of the sel-1 gene of C. elegans, most studies have focused on its role in cancer progression and have provided significant evidences to link its increased expression to a decrease in tumor aggressiveness. SEL1L resides on a “Genome Desert area” on chromosome 14q24.3-31 and is highly conserved in evolution. The function of the SEL1L encoded protein is still very elusive although, several evidences from lower organisms indicate that it plays a major role in protein degradation using the ubiquitin-proteosome system. SEL1L has a very complex structure made up of modules: genomically it consists of 21 exons featuring several alternative transcripts encoding for putative protein isoforms. This structural complexity ensures protein flexibility and specificity, indeed the protein was found in different sub-cellular compartments and may turn on a particular transcript in response to specific stimuli. The overall architecture of SEL1L guarantees an exquisite regulation in the expression of the gene. © 2005 Wiley-Liss, Inc.

Recent data suggest that SEL1L may play an important role in pancreatic carcinoma, similar to breast cancer, where the expression of SEL1L has been associated with a reduction in both proliferative activity in vitro and clinical tumor aggressiveness. To investigate this possibility, we examined the expression of Sel1L in a series of primary pancreatic carcinomas by immunohistochemistry and characterized the effects of Sel1L overexpression both in vitro and in vivo. In 74 pancreatic cancers analysed, 36% lacked Sel1L expression, although there was no significant correlation between the expression of Sel1L and any clinicopathologic parameter, including survival. However, immunohistochemical reactivity for Sel1L and Dpc4/Smad4 was concordant in 69% of cases (chi(2) test P0.004). Overexpression of SEL1L in stably transfected pancreatic cancer cells caused both a decrease in clonogenicity and anchorage-independent growth as well as a significant increase in the levels of activin A and SMAD4. When implanted in nude mice, Suit-2-SEL1L-overexpressing clones displayed a considerably reduced rate of tumor growth. Thus, it can be hypothesized that Sel1L plays an important function in the growth and aggressiveness of pancreatic carcinoma. Moreover, our data provide evidence that SEL1L has an impact on the expression of genes involved in regulation of cellular growth, possibly through the TGF-beta signaling pathway.

SEL1L, highly similar to the C. elegans sel-1 gene, is a recently cloned human gene whose function is under investigation. SEL1L is differentially expressed in tumors and normal tissues and seems to play a role in tumor growth and aggressiveness. We used the recombinant N-terminus of the SEL1L protein to immunize a Balb/c mouse and produce a monoclonal antibody. A hybridoma secreting an antibody specifically reacting on the SEL1L recombinant fragment was selected. This monoclonal antibody, named MSel1, recognizes the SEL1L protein by Western blotting, immunofluorescence and immunohistochemistry on normal and tumor cells. MSel1 is able to recognize SEL1L even on archival tumor specimens and is therefore particularly appropriate to study SEL1L involvement in tumor progression.

Based on a previous finding that amplification of the c-erbB-2 oncogene and alteration of p53 are strongly associated in most aggressive breast tumours, the present study investigated whether microsatellite instability (MI) might also be associated with this tumour phenotype. Nine polymorphic microsatellite markers, including six dinucleotide, one trinucleotide, and two tetranucleotide repeats, were amplified from paired normal and tumour DNA samples of 15 breast tumours that overexpressed both c-erbB-2 and p53 and of 15 control breast tumours that overexpressed neither protein. All 30 breast tumours analysed exhibited a replication error-negative phenotype, with only one sample showing MI in one of the nine loci. This suggests that the genetic events underlying MI, which are critical in colorectal and gastric tumours, are not involved in the pathogenesis of c-erbB-2/p53 double-altered breast tumours and do not play a central role in breast tumour formation.

The triple negative breast cancers (TNBC) is an extremely aggressive cancer subtype that although account for only 10-17% of breast cancers carries the higher rate of distant recurrence and death than women with other breast cancer subtypes and, unfortunately has limited treatment options. The TNBC pattern of recurrence present a distant recurrence peak at approximately 3 years and then declines rapidly thereafter. It's now well known that growth-factors released during the healing process can accelerate the early recurrences in breast cancer patients. We speculate that wound healing drainages (WHD) stimulation may specifically contribute to TNBC early relapse. To this aim, a panel of TNBC cell lines (BT-549; HCC1937, MDA-MB-157; MDA-MB-231; MDA-MB-468; SUM159; SUM149) and the luminal cell lines MCF7 were treated with WHD. All breast carcinoma cell lines are induced to proliferate in response to more than 20 drainage fluids obtained from breast cancer patients, but median proliferation level was 3-folds higher in TNBC- compared to luminal-cell line. To identify which receptors/pathways can be activated and play a driving role in TNBC progression, a reverse phase protein microarray (RPMA) experiment on TNBC cells WHD stimulated was performed. We revealed a specific activation of PDGFR, VEGFR and their downstream pathways, whereas no significant changes were observed in other receptors, such as EGFR, IRS, Met and ERB3. Conversely, PDGFR and VEGFR pathways were not activated in MCF7. The type of activated receptors suggested the involvement of angiogenic properties, besides canonical proliferation pathways, upon WHD stimulation. To prove whether the receptors found activated by WHD play a key role in in vitro and in vivo TNBC progression, we targeted PDGFR, VEGFR and other receptors possibly involved in proliferation and angiogenic capabilities with sunitinib (targeting PDGFR, VEGFR, FGF and c-kit), anti-bFGF antibody (targeting the ligand bFGF) and bevacizumab (targeting the VEGF) in TNBC cells WHD-stimulated. Sunitinib and anti-bFGF antibody halved the proliferation of TNBC cell lines, whereas bevacizumab modestly affect proliferation. Notably, sunitinib and anti-bFGF antibody strongly inhibited MDA-MB-231 and MDA-MB-468 xenografts tumor growth (sunitinib: 80%, and 70% Growth Index (GI), respectively; anti-bFGF antibody 70% and 60% GI, respectively) whereas bevacizumab determined no more than 30% decrease of tumor volume. In conclusion, WHD promotes TNBC progression probably by sustaining both proliferation and angiogenic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5642. doi:1538-7445.AM2012-5642

To recover peptides that antigenically and immunogenically mimic the p185HER2 oncoprotein, we selected the phage-peptide libraries pVIII-9aa and pVIII-9aa. Cys using murine monoclonal antibodies (mAb) MGr2 and MGr6, directed against two distinct epitopes of the p185HER2 extracellular domain. Phage-displayed peptides containing consensus amino acid motifs were recovered and shown to compete specifically for mAb binding on tumor cells that overexpress p185HER2. The deduced amino acid sequence of the peptides suggests that both epitopes defined by the mAb on p185HER2 are discontinuous and that hydrophobic interactions are involved in binding with the mAb. A phage clone displaying the GPLDSLFAQ peptide elicited a specific immune response against the p185HER2 in BALB/c mice, demonstrating that this phage-displayed peptide represents an immunological equivalent of the MGr2 epitope on p185HER2 and might be used as a substitute for this oncoprotein in in vitro and in vivo immunological studies.

Three murine monoclonal antibodies (MoAbs), MBrl and MOv2 of IgM isotype and MOv8 of IgG isotype, with restricted reactivity for breast or ovarian carcinomas, were labelled with 125I in the perspective of obtaining specific and stable radioimmunopharmaceutical reagents. The radiolabeled MoAbs were analyzed for their « in vitro » stability in human blood. They were incubated at 37 °C for various lengths of time in human or, as a control, in murine blood and their binding capacity was evaluated by solid-phase RIA and compared with that obtained after incubation with buffer. In human blood, serum and plasma, but not with other components such as erythrocytes, leukocytes, HSA and IgG, the MoAbs revealed a loss of binding reactivity which was marked and constant for the IgM MoAbs, and only occasional for the IgG MoAb. In murine serum the decrease was not so rapid. The same change in the binding capacity was observed when the MoAbs were labelled with 3H or 35S, excluding the involvement of dehalogenating mechanisms. In the perspective of using MoAbs for intracavity therapy the effect of ascitic or pleural fluids on their binding activity was also evaluated. The inhibition of the binding reactivity was not as evident and was not related to the MoAb isotype.

Ricin A chain was coupled to murine monoclonal antibodies MBr1 and MOv2 respectively raised against human breast and ovarian carcinomas. Inhibition of protein synthesis only occurred in those cultured human tumor cells bearing the appropriate target antigens, demonstrating that both components of the conjugate were unchanged in regards to specificity and toxicity. Conjugates were 125-200 times more efficient in inhibiting [3H]proline incorporation than the uncoupled ricin A chain. They were however unable to kill the entire population of the appropriate cells even after repeated treatment. Although the two monoclonal antibodies had similar binding kinetics, the conjugates differed in their cytotoxicity kinetics. The MBr1-ricin A chain conjugate had slow kinetics, and about 20 hours were needed to obtain a protein synthesis inhibition above 50% on the appropriate line (mammary carcinoma MCF-7). In contrast, the MOv2-ricin A chain conjugate showed very fast kinetics, reaching 50% inhibition after only 30 minutes of treatment on both appropriate cell lines SW626 and HT-29 from ovarian and colon carcinomas, respectively. Growth conditions of cell lines, i.e., adherent cells versus suspended cells, and plating time were found to greatly influence the conjugates' killing efficiencies. These studies confirm the possibility of preparing ricin A chain-antibody conjugates, which retain specific cytotoxicity against tumor cells; but they also underline the need for further in vitro studies of various parameters before one considers a therapeutic use of such conjugates.

The protein toxin restrictocin, isolated from the mould Aspergillus restrictus, inactivates protein synthesis in eukaryotic cells by blocking the ribosome elongation cycle. This protein acts as a specific nuclease that cuts off a small fragment from the 28-S rRNA. Biochemical and biological characterization of this toxin indicated that it is a non-glycosylated polypeptide of Mr 16836, exhibiting in cell-free systems a protein synthesis inhibition capacity similar to that of the ricin A chain. This polypeptide seemed unable to penetrate most of the cancer cell lines tested, as measured by its low in vitro cytotoxicity. In addition in vivo studies in BALB/c mice demonstrated that restrictocin toxicity was very low and that in rabbits, after intravenous injection 15% of the toxin was still present in the blood stream 24 h later. After derivatization with N-succinimidyl 3-(2-pyridyldithio)propionate and reduction by dithiothreitol, the restrictocin maintained its protein synthesis inhibitory activity, as assayed in a cell-free system. This derivatized toxin was then coupled to monoclonal antibodies (MBr1, MLuC1, MLuC2, MOv17, MOv18, MOv19) which exhibited a restricted spectrum of reactivity against human carcinomas. The biochemical and biological characterization of the immunoconjugates indicated that (a) when restrictocin was coupled to monoclonal antibodies with an average molar ratio of about 2, the immunoconjugates maintained the binding activity of the antibody and protein synthesis inhibition activity of the toxin; (b) four immunoconjugates were tested for cytotoxicity and three of them obtained with the MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited a good level of cytotoxicity for relevant target cells and low or no toxicity for the irrelevant cell lines. The MLuC2 monoclonal antibody which gave rise to a completely ineffective immunoconjugate, induced internalization of less than one tenth of the antigenic sites whereas the MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited about one third of the antigenic sites internalized. From these data it is concluded that, providing an appropriate target antigen and coupling procedure are selected, restrictocin can be considered a suitable toxin for immunoconjugate generation.

In the perspective of therapeutic approaches the monoclonal antibody, MBrl, with a quite restricted spectrum of reactivity for human breast carcinoma, was coupled to restrictocin (Res), a ribosome inactivating protein produced by Aspergillus restrictus. In a cell-free system this toxin was found to have an activity comparable to that of other plant toxins, but its in vitro toxicity was shown to be low on different cell lines. Three batches of MBr1-Res conjugate were prepared and their specificity, efficiency, and maximum level of cytotoxicity were analyzed on the cell line MCF-7 expressing the relevant antigen, on several irrelevant tumor cell lines, and on normal cells. Conjugates were from 600 to 1500 times more efficient than the uncoupled derivatized Res towards MCF-7 cells and were completely ineffective on the other target cells. The antigen-driven cytotoxicity was confirmed by the nontoxicity of an irrelevant conjugate on MCF-7 cells. The cytotoxic efficiency of MBr1-Res was low when compared to the binding level of MBr1 at the same concentration and a portion of treated cells (from 10% to 30%) survived the treatment. The heterogeneity of expression of the relevant antigen, together with its only partial internalization, could account for these limitations. The lysosomotropic agent ammonium chloride and the carboxylic ionophore monensin were tested as potentiating agents but in both cases the cytotoxicity remained unmodified. A neutralization assay performed on a xenogenic model indicated that the MBr1-Res conjugate was capable of reducing the tumor take. These data indicate the possibility of using the Res to prepare a reproducible and highly selective breast cancer conjugate. However, there are still a number of problems which must first be solved before we can consider its clinical application.

In order to investigate in vivo clinical applications of murine monoclonal antibodies directed against human ovarian carcinoma a preclinical in vivo model was developed using BALB/c athymic mice. Three human carcinoma cell lines (MCF7, HT29, and SW626) were injected into the peritoneal cavity of pristane-primed animals and the biological and antigenic characteristics of the i.p. grown tumors were studied. The animals were killed when moribund or 6-8 weeks after tumor injection. At autopsy tumor take was observed in 85% of the injected animals, whereas palpable nodules were evident in only 83%. Examination of the peritoneal cavity revealed intraabdominal carcinomatosis with tumor masses varying in size between 0.2 and 0.5 cm in diameter and tumor sheets. The most frequently affected organs were the diaphragm, the liver, and the reproductive system. Ascitic fluid formation was rare and no animal developed tumors outside the peritoneal cavity. To determine whether the in vivo tumors retained the same antigenic characteristics as the in vitro cell lines, four monoclonal antibodies (MBr1, MOv2, MOv8, and MOv15) directed against ovarian carcinoma-associated antigens and two different experimental approaches (immunofluorescence and immunoblotting) were used. Variations at either a quantitative or a qualitative level were observed for some antigens, whereas no evident changes were apparent for others. In particular, the antigens detected by MBr1 and MOv15 on the MCF7 line both maintained high levels of expression and immunoblotting staining pattern, whereas the antigens detected by MOv2 on the HT29 and SW626 lines, although present at a high level, clearly changed their staining pattern. As regards the antigens recognized by MOv8 and MOv15 on the HT29 and SW626 lines, we observed a drastic decrease in the level of their expression and in many cases a drop below the threshold of detectability of the test. The intraabdominal carcinomatosis described partially mimics the growth characteristics of human ovarian cancer and maintains the expression of some antigenic markers associated with epithelial tumors of the ovary and may therefore be useful in devising immunodiagnostic and/or immunotherapeutic strategies for ovarian carcinoma.

We have designed a set of oligonucleotide primers to amplify the cDNA of mouse immunoglobulin heavy and light chain variable domains by the polymerase chain reaction. The primers incorporate restriction sites that allow the cDNA of the variable domains to be force-cloned for sequencing and expression. Here we have applied the technique to clone and sequence the variable domains of five hybridoma antibodies and to express a mouse-human chimeric antibody that binds to the human mammary carcinoma line MCF-7. The technique should also lead to the cloning of antigen-binding specificities directly from immunoglobulin genes.

Retinoic acid (RA)-resistance in breast cancer cells has been associated with irreversible loss of retinoic acid receptor beta, RARbeta, gene expression. Search of the causes affecting RARbeta gene activity has been oriented at identifying possible differences either at the level of one of the RARbeta promoters, RARbeta2, or at regulatory factors. We hypothesized that loss of RARbeta2 activity occurs as a result of multiple factors, including epigenetic modifications, which can pattern RARbeta2 chromatin state. Using methylation-specific PCR, we found hypermethylation at RARbeta2 in a significant proportion of both breast cancer cell lines and primary breast tumors. Treatment of cells with a methylated RARbeta2 promoter, by means of the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR), led to demethylation within RARbeta2 and expression of RARbeta indicating that DNA methylation is at least one factor, contributing to RARbeta inactivity. However, identically methylated promoters can differentially respond to RA, suggesting that RARbeta2 activity may be associated to different repressive chromatin states. This supposition is supported by the finding that the more stable repressive RARbeta2 state in the RA-resistant MDA-MB-231 cell line can be alleviated by the HDAC inhibitor, trichostatin A (TSA), with restoration of RA-induced RARbeta transcription. Thus, chromatin-remodeling drugs might provide a strategy to restore RARbeta activity, and help to overcome the hurdle of RA-resistance in breast cancer.

We have previously reported on the isolation and chromosomal mapping of a novel human gene (SEL1L), which shows sequence similarity to sel-1, an extragenic suppressor of C. elegans. sel-1 functions as a negative regulator of lin-12 activity, the latter being implicated in the control of diverse cellular differentiation events. In the present study we compare the expression patterns of SEL1L and TAN-1, the human ortholog of lin-12 in normal and neoplastic cells. We found that, whereas both genes are expressed in fetal tissues at similar levels, they are differentially expressed in normal adult and neoplastic cells. In normal adult cells SEL1L is generally present at very low levels; only in the cells of the pancreas does it show maximum expression. By contrast, SEL1L is generally well represented in most neoplastic cells but not in those of pancreatic and gastric carcinomas, where transcription is either downregulated or completely repressed. TAN-1 on the other hand is well represented in almost all normal and neoplastic cells, with very few exceptions. Our observations suggest that SEL1L is presumably implicated in pancreatic and gastric carcinogenesis and that, along with TAN-1, it is very important for normal cell function. Alterations in the expression of SEL1L may be used as a prognostic marker for gastric and pancreatic cancers.

The use of monoclonal antibodies for in vivo therapeutic approaches depends largely on their specificity. During the characterization of ricin A-chain-murine monoclonal antibody conjugates we found that the binding specificity of a monoclonal antibody raised against human ovarian carcinoma (MOv2) seemed altered. Therefore, the binding reactivity of the unmodified antibody (MOv2), the conjugation intermediate (MOv2-PDP) and the conjugate (MOv2-A chain) was titrated by solid-phase radioimmunoassay on 11 human tumor cell lines belonging to seven different histotypes. The three reagents bound with the two reference cell lines (SW626:ovary carcinoma and HT-29:colon carcinoma). The MOv2-PDP and the Mov2-A chain also reacted with seven other cell lines which were unreactive with the unmodified MOv2. In addition MOv2-PDP exhibited reactivity on all normal cells tested (peripheral blood lymphocytes and skin fibroblasts). To elucidate the significance of these findings the following experiments were performed: cross inhibitions between the unmodified and modified monoclonal antibodies; comparative absorption tests with different cell lines; and immunoblotting analysis of the target antigens. The results suggest that after chemical modification with SPDP the monoclonal antibody MOv2 increases its binding activity, so that even a low number of antigenic sites can be detected. This study underlines the need to redefine the specificity of a conjugate before considering therapeutic applications.

Point mutations affecting the NTRK1/TRKA gene, encoding one of the receptors for the nerve growth factor (NGF), have been detected in congenital insensitivity to pain with anhidrosis (CIPA), a human hereditary sensory neuropathy characterized by absence of reaction to noxious stimuli and anhidrosis. To define the defect of NTRK1 in CIPA patients, we have introduced one of the previously reported mutations (Gly571Arg) into both the NTRK1 and the TRK-T3 oncogene cDNAs. The expression of the mutated constructs into COS1 cells revealed that the introduced mutation, while not affecting its correct membrane localization, rendered the NTRK1 protein unable to undergo activation upon stimulation with NGF. Similarly, the mutation abolished the constitutive activation of the TRK-T3 oncogene. Transfection into NIH3T3 and PC12 cells showed the loss of transforming and differentiating activity by the mutated constructs. Our results demonstrate clearly that the CIPA mutations cause the inactivation of the NTRK1 receptor, thus exerting a loss of function effect, and provide an experimental approach to distinguish functional mutations from genetic polymorphisms. J. Cell. Physiol. 182:127–133, 2000. © 2000 Wiley-Liss, Inc.