Expression pattern, subcellular localization, and functional implications of ODAM in ameloblasts, odontoblasts, osteoblasts, and various cancer cells

During tooth development and tumorigenesis, the odontogenic ameloblast-associated protein (ODAM) is involved in cellular differentiation and matrix protein production. However, the precise function of ODAM remains largely unknown. To suggest new functional roles of ODAM, we investigated the cellular expression and subcellular localization of ODAM in tooth and cancer cells. ODAM was expressed in ameloblasts, odontoblasts, and osteoblasts in vivo and in vitro. Furthermore, ODAM was localized in both the nucleus and cytoplasm of MMP-20 expressing ameloblasts and odontoblasts, but only in the cytoplasm of non-MMP-20 expressing osteoblasts. The extracellular secretion of ODAM was not observed in odontoblasts and osteoblasts, but was seen in ameloblasts. In addition, ODAM was discovered in the nucleus, cytoplasm, and extracellular matrix of various cancer cells. These results suggest that the expression pattern and subcellular localization of ODAM is highly variable and dependent on cell types and their differentiation states, and that functional correlations exist between ODAM and MMP-20. This study provides the first evidence for ODAM in multiple cellular compartments of differentiating odontogenic and cancer cell lines with important functional implications.     

Odontogenic ameloblasts-associated protein (ODAM), via phosphorylation by bone morphogenetic protein receptor type IB (BMPR-IB), is implicated in ameloblast differentiation

To elucidate the function of the odontogenic ameloblast-associated protein (ODAM) in ameloblasts, we identified more than 74 proteins that interact with ODAM using protoarray. Of the identified proteins, bone morphogenetic protein receptor type-IB (BMPR-IB) was physiologically relevant in differentiating ameloblasts. ODAM and BMPR-IB exhibited similar patterns of expression in vitro, during ameloblast differentiation. ODAM and BMPR-IB interacted through the C-terminus of ODAM, which resulted in increased ODAM phosphorylation in the presence of bone morphogenetic protein 2 (BMP-2). Immunoprecipitation assays using Ser-Xaa-Glu (SXE) mutants of ODAM demonstrated that the phosphorylation of ODAM by BMPR-IB occurs at this motif, and this phosphorylation is required for the activation of MAPKs. ODAM phosphorylation was detected in ameloblasts during ameloblast differentiation and enamel mineralization in vitro and involved in the activation of downstream factors of MAPKs. Therefore, the BMP-2-BMPR-IB-ODAM-MAPK signaling cascade has important roles in ameloblast differentiation and enamel mineralization. Our data suggest that ODAM facilitates the progression of tooth development in cooperation with BMPR-IB through distinct domains of ODAM.     

Odontogenic Ameloblast-associated Protein (ODAM) Mediates Junctional Epithelium Attachment to Teeth via Integrin-ODAM-Rho Guanine Nucleotide Exchange Factor 5 (ARHGEF5)-RhoA Signaling

Adhesion of the junctional epithelium (JE) to the tooth surface is crucial for maintaining periodontal health. Although odontogenic ameloblast-associated protein (ODAM) is expressed in the JE, its molecular functions remain unknown. We investigated ODAM function during JE development and regeneration and its functional significance in the initiation and progression of periodontitis and peri-implantitis. ODAM was expressed in the normal JE of healthy teeth but absent in the pathologic pocket epithelium of diseased periodontium. In periodontitis and peri-implantitis, ODAM was extruded from the JE following onset with JE attachment loss and detected in gingival crevicular fluid. ODAM induced RhoA activity and the expression of downstream factors, including ROCK (Rho-associated kinase), by interacting with Rho guanine nucleotide exchange factor 5 (ARHGEF5). ODAM-mediated RhoA signaling resulted in actin filament rearrangement. Reduced ODAM and RhoA expression in integrin β₃- and β₆-knockout mice revealed that cytoskeleton reorganization in the JE occurred via integrin-ODAM-ARHGEF5-RhoA signaling. Fibronectin and laminin activated RhoA signaling via the integrin-ODAM pathway. Finally, ODAM was re-expressed with RhoA in regenerating JE after gingivectomy in vivo. These results suggest that ODAM expression in the JE reflects a healthy periodontium and that JE adhesion to the tooth surface is regulated via fibronectin/laminin-integrin-ODAM-ARHGEF5-RhoA signaling. We also propose that ODAM could be used as a biomarker of periodontitis and peri-implantitis.     

Neoplastic odontogenic epithelial cells express bone sialoprotein

Bone sialoprotein (BSP) is synthesized and secreted by bone-, dentine- and cementum-forming cells and has been implicated in de novo bone formation and mineralization. In this study, we used histological sections of odontogenic neoplasms and performed immunohi stochemical and in situ hybridization analyses. In ameloblastoma, BSP mRNA signals were seen in the neoplastic epithelial cells forming nests, strips and islands. BSP deposition was also seen in the stellate reticulum of the tumour masses revealed by immunohistochemistry using human BSP antibodies. In calcifying epithelial odontogenic tumour, the calcified masses demonstrated positive immunoreactivity to the human BSP antibodies, and the hybridization signals for BSP were located in the cells near the calcified particles. In the calcifying odontogenic cyst, strong BSP signals were seen in cells surrounding the characteristic nests of ghost cells, which often calcify subsequently. BSP protein was also found in these cells by immunohistochemistry. The active expression of BSP in the epithelial elements of the odontogenic tumours of adult patients suggests the activation of this matrix protein gene in the neoplastic process, and that BSP may play an important role in tumour formation and differentiation with respect to pathological calcification. © Chapman & Hall     

ODAM inhibits RhoA‐dependent invasion in breast cancer

Odontogenic ameloblast‐associated protein (ODAM) contributes to cell adhesion. In human cancer, ODAM is down‐regulated, and the overexpression of ODAM results in a favourable prognosis; however, the molecular mechanisms underlying ODAM‐mediated inhibition of cancer invasion and metastasis remain unclear. Here, we identify a critical role for ODAM in inducing cancer cell adhesion. ODAM induced RhoA activity and the expression of downstream factors, including Rho‐associated kinase (ROCK). ODAM‐mediated RhoA signalling resulted in actin filament rearrangement by activating PTEN and inhibiting the phosphorylation of AKT. When ODAM is overexpressed in MCF7 breast cancer cells and AGS gastric cancer cells that activate RhoA at high levels, it decreases motility, increases adhesion and inhibits the metastasis of MCF7 cells. Conversely, depletion of ODAM in cancer cells inhibits Rho GTPase activation, resulting in increased cancer migration and invasion. These results suggest that ODAM expression in cells maintains their adhesion, resulting in the prevention of their metastasis via the regulation of RhoA signalling in breast cancer cells. Copyright © 2015 John Wiley & Sons, Ltd. SIGNIFICANCE Breast cancer represents the first most frequent cancer, and the ratio of mortality is high in women. Of utmost importance for reducing risk by breast cancer are their anti‐invasion mechanisms, particularly in the non‐invasive cancer cells because metastasis is the principal cause of death among cancer patients. ODAM induced RhoA activity. ODAM‐mediated RhoA signalling resulted in actin filament rearrangement, increased cell adhesion and inhibited the migration/invasion of MCF7 cells. These results suggest that ODAM expression maintains their adhesion, resulting in the prevention of their metastasis via the regulation of RhoA signalling in breast cancer cells.     

15-prostaglandin dehydrogenase expression alone or in combination with ACSM1 defines a subgroup of the apocrine molecular subtype of breast carcinoma

Established histopathological criteria divide invasive breast carcinomas into defined groups. Ductal of no specific type and lobular are the two major subtypes accounting for around 75 and 15% of all cases, respectively. The remaining 10% include rarer types such as tubular, cribriform, mucinous, papillary, medullary, metaplastic, and apocrine breast carcinomas. Molecular profiling technologies, on the other hand, subdivide breast tumors into five subtypes, basal-like, luminal A, luminal B, normal breast tissue-like, and ERBB2-positive, that have different prognostic characteristics. An additional subclass termed "molecular apocrine" has recently been described, but these lesions did not exhibit all the histopathological features of classical invasive apocrine carcinomas (IACs). IACs make up 0.5-3% of the invasive ductal carcinomas, and despite the fact that they are morphologically distinct from other breast lesions, there are presently no standard molecular criteria available for their diagnosis and as a result no precise information as to their prognosis. Toward this goal our laboratories have embarked in a systematic proteomics endeavor aimed at identifying biomarkers that may characterize and subtype these lesions as well as targets that may lead to the development of novel targeted therapies and chemoprevention strategies. By comparing the protein expression profiles of apocrine macrocysts and non-malignant breast epithelial tissue we have previously reported the identification of a few proteins that are specifically expressed by benign apocrine lesions as well as by the few IACs that were available to us at the time. Here we reiterate our strategy to reveal apocrine cell markers and present novel data, based on the analysis of a considerably larger number of samples, establishing that IACs correspond to a distinct molecular subtype of breast carcinomas characterized by the expression of 15-prostaglandin dehydrogenase alone or in combination with a novel form of acyl-CoA synthetase medium-chain family member 1 (ACSM1). Moreover we show that 15-prostaglandin dehydrogenase is not expressed by other breast cancer types as determined by gel-based proteomics and immunohistochemistry analysis and that antibodies against this protein can identify IACs in an unbiased manner in a large breast cancer tissue microarray making them potentially useful as a diagnostic aid.     

Epithelial tight junction proteins as potential antibody targets for pancarcinoma therapy

Recombinant monoclonal antibodies are beginning to revolutionize cancer therapy. In combination with standard chemotherapy, high response rates have been reported with antibodies of the human IgG1 isotype for treatment of non-Hodgkins lymphoma and breast cancer. It is becoming apparent that targets for antibody-based therapies do not necessarily need to be absent from normal tissues but can be present there either in low copy numbers or with binding epitopes shielded from the therapeutic antibody. Here, we studied whether claudin proteins that form tight junctions in normal epithelia are still expressed on carcinoma cells and whether their extracellular domains can be recognized by antibodies. We show that mRNAs of claudins 1, 3, 4, and 7 are all expressed in different human carcinoma cell lines, while claudin 8 was selectively expressed in breast and pancreas cancer lines. Chicken polyclonal antibodies were raised against peptides contained within predicted extracellular domains of claudins 1, 3, and 4. Affinity-purified IgG fractions for claudins 3 and 4 were monospecific and bound to human breast and colon carcinoma lines, but not to a line of monocytic origin. Claudin 3 antibodies also homogeneously stained human renal cell carcinoma tissue and micrometastatic tumor cells as identified by cytokeratin staining in bone marrow biopsies of breast cancer patients. Fluorescence-activated cell sorting and immunocytochemistry indicated that claudin antibodies bound to the surface of tumor cells. By analogy to other tumor-associated antigens that are differentially accessible to antibodies on tumor vs normal tissue, we propose that certain claudin proteins have potential as targets for novel antibody-based therapies of carcinomas.     

Expression and targeting of the tight junction protein CLDN1 in CLDN1-negative human breast tumor cells

Claudins and occludin constitute the major transmembrane proteins of tight junctions (TJs). We have previously identified the human homologue of the murine Cldn1, CLDN1 (SEMP1) that is expressed in normal, mammary gland-derived epithelial cells but is absent in most human breast cancer cell lines. To investigate the potential functions of CLDN1 protein in tumor and normal epithelial cells, we developed an I-NGFR retroviral vector and monoclonal anti-CLDN1 antibody. In subconfluent and confluent breast cancer cells, MDA-MB-435 and MDA-MB-361, endogenous CLDN1 expression was not detected by an anti-CLDN1 monoclonal antibody by Western blot analysis or quantitative RT-PCR. When CLDN1-negative breast cancer cell lines were transduced with a CLDN1 retrovirus the cells express CLDN1 mRNA constitutively as shown by quantitative RT-PCR. Immunofluorescence analyses of the CLDN1 retroviral transduced breast tumor cells using monoclonal antibodies against CLDN1 reveals a subcellular distribution at cell-cell contact sites similar to the CLDN1 homing pattern in T47-D cells, which express endogenous CLDN1. This cell-cell contact co-localization of CLDN1 was evident in CLDN1-transduced breast tumor cells which fail to express occludin protein (MDA-MB-361 and MDA-MB-435) and express relatively little ZO-1 protein (MDA-MB-435), suggesting that other proteins may be responsible for targeting of CLDN1 to cell-cell contact sites. The re-expression of CLDN1 decreases the paracellular flux of 3 and 40 kDa dextran despite the absence of occludin in the MDA-MB-361 tumor cells. Our findings indicate that in CLDN1-negative breast tumor cells, the basal protein partner requirements for physiological homing of the CLDN1 protein are intact, and that CLDN1 gene transfer and protein expression itself might be sufficient to exert a TJ-mediate gate function in metastatic tumor cells even in the absence of other TJ-associated proteins, such as occludin.     

MUC1 and cancer

The MUC1 membrane mucin was first identified as the molecule recognised by mouse monoclonal antibodies directed to epithelial cells, and the cancers which develop from them. Cloning the gene showed that the extracellular domain is made up of highly conserved repeats of 20 amino acids, the actual number varying between 25 and 100 depending on the allele. Each tandem repeat contains five potential glycosylation sites, and between doublets of threonines and serines lies an immunodominant region which contains the epitopes recognised by most of the mouse monoclonal antibodies. The O-glycans added to the mucin produced by the normal breast are core 2 based and can be complex, while the O-glycans added to the breast cancer mucin are mainly core 1 based. This means that some core protein epitopes in the tandem repeat which are masked in the normal mucin are exposed in the cancer associated mucin. Since novel carbohydrate epitopes are also carried on the breast cancer mucin, the molecule is antigenically distinct from the normal breast mucin. (Changes in glycosylation in other epithelial cancers have been observed but are not so well documented.) Immune responses to MUC1 have been seen in breast and ovarian cancer patients and clinical studies have been initiated to evaluate the use of antibodies to MUC1 and of immunogens based on MUC1 for immunotherapy of these patients. The role of the carbohydrates in the immune response and in other interactions with the effector cells of the immune system is of particular interest and is discussed.     

The odontogenic ameloblast‐associated protein (ODAM) cooperates with RUNX2 and modulates enamel mineralization via regulation of MMP‐20

We have previously reported that the odontogenic ameloblast‐associated protein (ODAM) plays important roles in enamel mineralization through the regulation of matrix metalloproteinase‐20 (MMP‐20). However, the precise function of ODAM in MMP‐20 regulation remains largely unknown. The aim of the present study was to uncover the molecular mechanisms responsible for MMP‐20 regulation. The subcellular localization of ODAM varies in a stage‐specific fashion during ameloblast differentiation. During the secretory stage of amelogenesis ODAM was localized to both the nucleus and cytoplasm of ameloblasts. However, during the maturation stage of amelogenesis, ODAM was observed in the cytoplasm and at the interface between ameloblasts and the enamel layer, but not in the nucleus. Secreted ODAM was detected in the conditioned medium of ameloblast‐lineage cell line (ALC) from days 14 to 21, which coincided with the maturation stage of amelogenesis. Interestingly, the expression of Runx2 and nuclear ODAM correlated with MMP‐20 expression in ALC. We therefore examined whether ODAM cooperates with Runx2 to regulate MMP‐20 and modulate enamel mineralization. Increased expression of ODAM and Runx2 augmented MMP‐20 expression, and Runx2 expression enhanced expression of ODAM, although overexpression of ODAM did not influence Runx2 expression. Conversely, loss of Runx2 in ALC decreased ODAM expression, resulting in down‐regulation of MMP‐20 expression. Increased MMP‐20 expression accelerated amelogenin processing during enamel mineralization. Our data suggest that Runx2 regulates the expression of ODAM and that nuclear ODAM serves an important regulatory function in the mineralization of enamel through the regulation of MMP‐20 apart from a different, currently unidentified, function of extracellular ODAM. J. Cell. Biochem. 111: 755–767, 2010. © 2010 Wiley‐Liss, Inc.     

Expression of glial filament protein (GFP) in nerve sheaths and non-neural cells re-examined using monoclonal antibodies, with special emphasis on the co-expression of GFP and cytokeratins in epithelial cells of human salivary gland and pleomorphic adenomas

We describe two novel monoclonal antibodies specific for glial filament protein (GFP), i.e., GF12.23 and GF12.24 (both IgG2a]. These cross-react over a broad range of species with epitopes located in the alpha-helical rod domain typical of all intermediate filament (IF) proteins. These monoclonal antibodies were used, in conjunction with other monoclonal GFP antibodies, rabbit antiserum to GFP, and various antibodies to other cytoskeletal proteins, to examine the occurrence of GFP in cells outside of the central nervous system of rodents, cows, and humans. We detected some scattered GFP-containing cells in the neural sheaths in some species but not in others, and we obtained different results when comparing the rabbit antisera with the monoclonal GFP antibodies. In the enteric glia of rats, we observed GFP-positive cells with all of the antibodies used, whereas in human intestine, the various monoclonal antibodies showed no reaction with any intestinal cells. Similarly, no GFP was detected in surface cells of the lens of cows and rats using any of the GFP antibodies, whereas some reaction was seen in murine lens tissue. We were also unable to detect GFP-positive cells in human, bovine, or rat liver with any of the monoclonal antibodies, which is in contrast to the reactivity of the rabbit GFP antisera with some stellate perisinusoidal cells of rat but not bovine or human liver. The possible reasons for the discrepancies between the different species and the different antibody preparations used are discussed. In addition, using double-label immunofluorescence microscopy, we showed that normal human parotid glands contain a certain type of epithelial cell that co-expresses cytokeratins and desmosomal proteins with GFP. The histological distribution of these GFP-positive cells suggests that they represent a subset of the myoepithelial cells present in this tissue. Cells co-expressing cytokeratins and GFP - in some cases, apparently together with vimentin as the third IF protein present - were also identified in tumors derived from this salivary-gland epithelium, i.e., pleomorphic adenomas, in which GFP-positive cells were relatively frequent in the myxoid and chondroid components, thus confirming the work of other investigators. Possible implications for the concept of histogenesis of these tumor cells are discussed, as are possible mechanisms resulting in the co-expression of IF proteins.     

Human kallikrein 10 expression in normal tissues by immunohistochemistry.

The normal epithelial cell-specific 1 (NES1) gene (official name kallikrein gene 10, KLK10) was recently cloned and encodes for a putative secreted serine protease (human kallikrein 10, hK10). Several studies have confirmed that hK10 shares many similarities with the other kallikrein members at the DNA, mRNA, and protein levels. The enzyme was found in biological fluids, tissue extracts, and serum. Here we report the first detailed immunohistochemical (IHC) localization of hK10 in normal human tissues. We used the streptavidin-biotin method with two hK10-specific antibodies, a polyclonal rabbit and a monoclonal mouse antibody, developed in house. We analyzed 184 paraffin blocks from archival, current, and autopsy material, prepared from almost every normal human tissue. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. Previously, we reported the expression of another novel human kallikrein, hK6, by using similar techniques. The IHC expression of hK10 was generally cytoplasmic and not organ-specific. A variety of normal human tissues expressed the protein. Glandular epithelia constituted the main immunoexpression sites, with representative organs being the breast, prostate, kidney, epididymis, endometrium, fallopian tubes, gastrointestinal tract, bronchus, salivary glands, bile ducts, and gallbladder. The choroid plexus epithelium, the peripheral nerves, and some neuroendocrine organs (including the islets of Langerhans, cells of the adenohypophysis, the adrenal medulla, and Leydig cells) expressed the protein strongly and diffusely. The spermatic epithelium of the testis expressed the protein moderately. A characteristic immunostaining was observed in Hassall's corpuscles of the thymus, oxyphilic cells of the thyroid and parathyroid glands, and chondrocytes. Comparing these results with those of hK6, we observed that both kallikreins had a similar IHC expression pattern.     

Steroid receptors and proliferation in the human breast

Despite recent gains in our knowledge of the hormonal control of proliferation and differentiation in the rodent mammary gland, the factors regulating these processes in the human are poorly understood. We have developed a model in which intact normal human breast tissue is grafted subcutaneously into adult female athymic nude mice and treated with oestrogen (E) and/or progesterone (P) at human physiological serum levels. We have shown that (i) E and not P is the major epithelial cell mitogen in the adult non-pregnant, non-lactating breast, (ii) E induces progesterone receptor (PR) expression and (iii) PR expression is maximally induced at low E concentrations while a higher amount of E is required to stimulate proliferation. These data raised the question of whether one cell type demonstrated two different responses to the two different E concentrations or whether PR expression and proliferation occurred in separate cell populations. Using dual label immunofluorescence, we showed that steroid receptor expression and proliferation (Ki67 antigen) are detected in separate cell populations in normal human breast epithelium, and that cells expressing the oestrogen receptor-alpha (ERalpha) invariably contained the PR. We also reported that this separation between steroid receptor expression and proliferation observed in the normal human epithelium is disrupted at an early stage in breast tumourigenesis. One interpretation supported by our recent findings is that some ERalpha/PR-positive epithelial cells are quiescent breast stem cells that act as "steroid hormone sensors". Such hormone sensor cells might secrete positive or negative paracrine/juxtacrine factors dependent on the prevailing E or P concentration to influence the proliferative activity of adjacent ERalpha/PR-negative epithelial cells.     

Expression Profiling during Mammary Epithelial Cell Three-Dimensional Morphogenesis Identifies PTPRO as a Novel Regulator of Morphogenesis and ErbB2-Mediated Transformation

Identification of genes that are upregulated during mammary epithelial cell morphogenesis may reveal novel regulators of tumorigenesis. We have demonstrated that gene expression programs in mammary epithelial cells grown in monolayer cultures differ significantly from those in three-dimensional (3D) cultures. We identify a protein tyrosine phosphate, PTPRO, that was upregulated in mature MCF-10A mammary epithelial 3D structures but had low to undetectable levels in monolayer cultures. Downregulation of PTPRO by RNA interference inhibited proliferation arrest during morphogenesis. Low levels of PTPRO expression correlated with reduced survival for breast cancer patients, suggesting a tumor suppressor function. Furthermore, we showed that the receptor tyrosine kinase ErbB2/HER2 is a direct substrate of PTPRO and that loss of PTPRO increased ErbB2-induced cell proliferation and transformation, together with tyrosine phosphorylation of ErbB2. Moreover, in patients with ErbB2-positive breast tumors, low PTPRO expression correlated with poor clinical prognosis compared to ErbB2-positive patients with high levels of PTPRO. Thus, PTPRO is a novel regulator of ErbB2 signaling, a potential tumor suppressor, and a novel prognostic marker for patients with ErbB2-positive breast cancers. We have identified the protein tyrosine phosphatase PTPRO as a regulator of three-dimensional epithelial morphogenesis of mammary epithelial cells and as a regulator of ErbB2-mediated transformation. In addition, we demonstrated that ErbB2 is a direct substrate of PTPRO and that decreased expression of PTPRO predicts poor prognosis for ErbB2-positive breast cancer patients. Thus, our results identify PTPRO as a novel regulator of mammary epithelial transformation, a potential tumor suppressor, and a predictive biomarker for breast cancer.     

Identification of annexin II as a novel secretory biomarker for breast cancer

Early prediction of metastatic breast cancer is important for improvement of prognosis and survival rate. The present study aimed to identify secreted protein biomarkers for detection of invasive breast cancer. To this end, we performed a comparative proteomic analysis by a combination of 2DE and MALDI‐TOF MS analysis of conditioned media from invasive H‐Ras MCF10A human breast epithelial cells and noninvasive MCF10A and N‐Ras MCF10A cells. We identified a list of 25 proteins that were strongly detected in media of H‐Ras MCF10A and focused on annexin II, which was shown to be involved in cell motility. Invasive triple‐negative human breast carcinoma cells, Hs578T, and MDA‐MB‐231, showed increased levels of annexin II in media, demonstrating that secretion of annexin II correlated well with the invasive phenotype of cells. We demonstrated a crucial role of annexin II in breast cell invasion/migration and actin cytoskeleton reorganization required for filopodia formation. Annexin II levels in the plasma samples and breast cancer tissues of breast cancer patients were significantly higher than those of normal groups, providing a clinical relevance to our in vitro findings. Taken together, we identified annexin II as a novel secretory biomarker candidate for invasive breast cancer, especially estrogen receptor‐negative breast cancer.     

Characterization of cell surface antigens of human mammary epithelial cells with monoclonal antibodies prepared against human milk fat globule

Hybridomas have been prepared that secrete monoclonal antibodies against three different surface antigens of normal human mammary epithelial cells by fusion of mouse myeloma cells with spleen cells from mice and rats immunized with delipidated human milk fat globules. Using a novel method for molecular weight determination, the three different monoclonal antibodies, BLMRL-HMFG-Mc3, BLMRL-HMFG-McR2, and BLMRL-HMFG-Mc5, were found to identify molecules with apparent molecular weights of 46,000, 70,000, and 400,000 daltons, respectively. The latter is a mucin-like glycoprotein with a high sugar content and has not previously been described as a component of the human milk fat globule or of human mammary epithelial cell membranes. Single-cell quantitation of binding of monoclonal BLMRL-HMFG-Mc5 to three breast tumor cell lines using a Microscope Spectrum Analyzer and indirect immunofluorescence revealed a heterogeneous expression. Further, using a competitive radioimmunoassay, it was found that breast tumor cell lines differed by at least 10-fold in the 400,000-molecular-weight antigen content. None of the three antigens are detectable on several nonbreast cell lines, including normal breast fibroblasts.     

Reconstruction of human mammary tissues in a mouse model

Establishing a model system that more accurately recapitulates both normal and neoplastic breast epithelial development in rodents is central to studying human breast carcinogenesis. However, the inability of human breast epithelial cells to colonize mouse mammary fat pads is problematic. Considering that the human breast is a more fibrous tissue than is the adipose-rich stroma of the murine mammary gland, our group sought to bypass the effects of the rodent microenvironment through incorporation of human stromal fibroblasts. We have been successful in reproducibly recreating functionally normal breast tissues from reduction mammoplasty tissues, in what we term the human-in-mouse (HIM) model. Here we describe our relatively simple and inexpensive techniques for generating this orthotopic xenograft model. Whether the model is to be applied for understanding normal human breast development or tumorigenesis, investigators with minimal animal surgery skills, basic cell culture techniques and access to human breast tissue will be able to generate humanized mouse glands within 3 months. Clearing the mouse of its endogenous epithelium with subsequent stromal humanization takes 1 month. The subsequent implantation of co-mixed human epithelial cells and stromal cells occurs 2 weeks after humanization, so investigators should expect to observe the desired outgrowths 2 months afterward. As a whole, this model system has the potential to improve the understanding of crosstalk between tissue stroma and the epithelium as well as factors involved in breast stem cell biology tumor initiation and progression.     

Comparative Analysis of Evolutionarily Conserved Motifs of Epidermal Growth Factor Receptor 2 (HER2) Predicts Novel Potential Therapeutic Epitopes

Overexpression of human epidermal growth factor receptor 2 (HER2) is associated with tumor aggressiveness and poor prognosis in breast cancer. With the availability of therapeutic antibodies against HER2, great strides have been made in the clinical management of HER2 overexpressing breast cancer. However, de novo and acquired resistance to these antibodies presents a serious limitation to successful HER2 targeting treatment. The identification of novel epitopes of HER2 that can be used for functional/region-specific blockade could represent a central step in the development of new clinically relevant anti-HER2 antibodies. In the present study, we present a novel computational approach as an auxiliary tool for identification of novel HER2 epitopes. We hypothesized that the structurally and linearly evolutionarily conserved motifs of the extracellular domain of HER2 (ECD HER2) contain potential druggable epitopes/targets. We employed the PROSITE Scan to detect structurally conserved motifs and PRINTS to search for linearly conserved motifs of ECD HER2. We found that the epitopes recognized by trastuzumab and pertuzumab are located in the predicted conserved motifs of ECD HER2, supporting our initial hypothesis. Considering that structurally and linearly conserved motifs can provide functional specific configurations, we propose that by comparing the two types of conserved motifs, additional druggable epitopes/targets in the ECD HER2 protein can be identified, which can be further modified for potential therapeutic application. Thus, this novel computational process for predicting or searching for potential epitopes or key target sites may contribute to epitope-based vaccine and function-selected drug design, especially when x-ray crystal structure protein data is not available.