Richard I. Near

Expression of the aryl hydrocarbon receptor/transcription factor (AhR) and AhR-regulated CYP1

Exposure to ubiquitous environmental chemicals, such as polycyclic aromatic hydrocarbons (PAH), may contribute to human breast cancer. In animals, PAH induce tumors in part by activating the aryl hydrocarbon receptor (AhR)/transcription factor. Historically, investigations into AhR-regulated carcinogenesis have focused on AhR-dependent transcriptional regulation of cytochrome P450 (CYP) enzymes which oxidize PAH to mutagenic intermediates. However, recent studies suggest that the AhR directly regulates cell growth. Given the postulated role of the AhR in carcinogenesis, we predicted that: (1) tissue predisposed to PAH tumorigenesis would express the AhR and (2) aberrant AhR and/or AhR-regulated gene expression would accompany malignant transformation. To test these hypotheses, AhR and CYP1 protein and/or mRNA levels were evaluated in rat mammary tumors induced with 7, 12-dimethylbenz[a]anthracene (DMBA), a prototypic PAH and AhR ligand. Results indicate modest AhR expression in normal mammary myoepithelial and ductal epithelial cells. In contrast, high AhR levels were detected in DMBA-induced tumors. Nuclear AhR localization in tumors suggested constitutive AhR activation. In situ hybridization and quantitative RT-PCR assays indicated high AhR mRNA levels in neoplastic epithelial cells. While both AhR-regulated CYP1A1 and CYP1B1 mRNAs were induced in breast tissue within 6 h of DMBA gavage, only CYP1B1 mRNA remained elevated in tumors. These results: (1) help explain targeting of breast tissue by carcinogenic PAH, (2) imply that AhR and CYP1B1 hyper-expression represent molecular biomarkers for, at least, PAH-induced mammary cell transformation, and (3) suggest mechanisms through which the AhR may contribute to carcinogenesis well after exogenous AhR ligands have been eliminated.

The GDP Exchange Factor AND-34 Is Expressed in B Cells, Associates With HEF1, and Activates Cdc42

AND-34, a novel GDP exchange factor, is expressed constitutively at significant levels in murine splenic B cells, but not in murine splenic T cells or thymocytes. In B cell lines, anti-IgM treatment up-regulates AND-34 transcript levels. B cell AND-34 associates with both the docking molecules p130Cas and HEF1. AND-34 binds by its GDP exchange factor domain to the C terminus of HEF1, a region of HEF1 previously implicated in apoptotic, adhesion, and cell cycle-regulated signaling. Overexpression of AND-34 in murine B cell lines activates the Rho family GTPase Cdc42, but not Rac, Rho, RalA, or Rap1. Consistent with this, a subpopulation of AND-34 overexpressing B cells have long filamentous actin-containing cellular extensions. AND-34 overexpression augments both autophosphorylation and kinase activity of the Cdc42/Rac-responsive serine/threonine kinase PAK1. As previously reported for lymphoid cells transfected with constitutively active Cdc42, AND-34 overexpression inhibits SDF-1α-induced B cell polarization. These studies suggest that p130Cas and HEF1-associated AND-34 may regulate B cell adhesion and motility through a Cdc42-mediated signaling pathway.

AND-34/BCAR3 differs from other NSP homologs in induction of anti-estrogen resistance, cyclin D1 promoter activation and altered breast cancer cell morphology

Over‐expression of AND‐34/BCAR3/NSP2 (BCAR3) or its binding‐partner p130Cas/BCAR1 generates anti‐estrogen resistance in human breast cancer lines. Here, we have compared BCAR3 to two related homologs, NSP1 and NSP3/CHAT/SHEP, with regards to expression, anti‐estrogen resistance, and signaling. BCAR3 is expressed at higher levels in ERα‐negative, mesenchymal, than in ERα‐positive, epithelial, breast cancer cell lines. Characterization of “intermediate” epithelial‐like cell lines with variable ER‐α expression reveals that BCAR3 expression correlates with both mesenchymal and ERα‐negative phenotypes. Levels of the BCAR3/p130Cas complex correlate more strongly with the ERα‐negative, mesenchymal phenotype than levels of either protein alone. NSP1 and NSP3 are expressed at lower levels than BCAR3 and without correlation to ERα/mesenchymal status. Among NSP‐transfectants, only BCAR3 transfectants induce anti‐estrogen resistance and augment transcription of cyclin D1 promoter constructs. Over‐expression of all homologs results in activation of Rac, Cdc42 and Akt, suggesting that these signals are insufficient to induce anti‐estrogen resistance. BCAR3 but not NSP1 nor NSP3 transfectants show altered morphology, transitioning from polygonal cell groups to rounded, single cells with numerous blebs. Whereas stable over‐expression of BCAR3 in MCF‐7 cells does not lead to classic epithelial‐to‐mesenchymal transition, it does result in down‐regulation of cadherin‐mediated adhesion and augmentation of fibronectin expression. These studies suggest that BCARs ability to induce anti‐estrogen resistance is greater than that of other NSP homologs and may result from altered interaction of breast cancer cells with each other and the extracellular matrix. J. Cell. Physiol. 212:655–665, 2007.

Regulation of PreB Cell Apoptosis by Aryl Hydrocarbon Receptor/Transcription Factor‐Expressing Stromal/Adherent Cells

Polycyclic aromatic hydrocarbons (PAH) are environmental chemicals that mediate immunosuppression. In long-term bone marrow B-cell lymphopoiesis models, PAH induce apoptosis in immature (preB) lymphocytes. Since the biologic function of PAH is often mediated by the aryl hydrocarbon receptor/transcription factor (AhR), the role of the AhR or AhR-regulated genes was assessed in preB cell apoptosis. Specifically, a bone marrow-derived preB cell line (BU-11) was cultured on monolayers of the AhR + bone marrow-derived stromal cell line BMS2, hepatoma sublines that express various levels of AhR activity (Hepa-1c1c7 and variants), AhR+ thymic epithelial cells, and primary bone marrow stromal cells from wildtype or AhR-/- mice. Cultures were treated with one of two prototypic PAH, 7,12-dimethylbenz[a] anthracene (DMBA) or benz[a]pyrene (B[a]P), and the percentage of cells undergoing apoptosis measured. The data demonstrated that: 1) bone marrow- and hepatic-derived stromal/adherent cells support preB cell growth and regulate apoptosis induced by DMBA or B[a]P; 2) B[a]P is more effective than DMBA when preB cells are maintained on Hepa-1c1c7 monolayers than when maintained on BMS2 monolayers; 3) DMBA is more effective than B[a]P when preB cells are cultured on BMS2 monolayers; 4) alpha-naphthoflavone, an AhR antagonist and cytochrome P-450 inhibitor, blocks preB cell apoptosis in both BU-11/Hepa-1c1c7 and BU-11/BMS2 cultures; 5) although preB cells grow well in Hepa-1c1c7 or BMS2 supernatants, addition of PAH in the absence of hepatic- or bone marrow-derived adherent cells does not result in preB cell apoptosis; 6) preB cell apoptosis is dependent on AhR activity in adherent hepatic- or bone marrow-derived stromal cells; and 7) apoptosis is induced by DMBA when preB cells are maintained on primary bone marrow stromal cell monolayers from wildtype but not from AhR-/- mice. Collectively, the data indicated that AhR-regulated activities in the hematopoietic microenvironment influence the susceptibility of immature lymphocytes to low-dose PAH exposure.

AND-34/BCAR3 Regulates Adhesion-Dependent p130Cas Serine Phosphorylation and Breast Cancer Cell Growth Pattern

NSP protein family members associate with p130Cas, a focal adhesion adapter protein best known as a Src substrate that integrates adhesion-related signaling. Over-expression of AND-34/BCAR3/NSP2 (BCAR3), but not NSP1 or NSP3, induces anti-estrogen resistance in human breast cancer cell lines. BCAR3 over-expression in epithelial MCF-7 cells augments levels of a phosphorylated p130Cas species that migrates more slowly on SDS-PAGE while NSP1 and NSP3 induce modest or no phosphorylation, respectively. Conversely, reduction in BCAR3 expression in mesenchymal MDA-231 cells by inducible shRNA results in loss of such p130Cas phosphorylation. Replacement of NSP3s serine/proline-rich domain with that of AND-34/BCAR3 instills the ability to induce p130Cas phosphorylation. Phospho-amino acid analysis demonstrates that BCAR3 induces p130Cas serine phosphorylation. Mass spectrometry identified phosphorylation at p130Cas serines 139, 437 and 639. p130Cas serine phosphorylation accumulates for several hours after adhesion of MDA-231 cells to fibronectin and is dependent upon BCAR3 expression. BCAR3 knockdown alters p130Cas localization and converts MDA-231 growth to an epithelioid pattern characterized by striking cohesiveness and lack of cellular projections at colony borders. These studies demonstrate that BCAR3 regulates p130Cas serine phosphorylation that is adhesion-dependent, temporally distinct from previously well-characterized rapid Fak and Src kinase-mediated p130Cas tyrosine phosphorylation and that correlates with invasive phenotype.

BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130Cas

BCAR3 binds to the carboxy-terminus of p130Cas, a focal adhesion adapter protein. Both BCAR3 and p130Cas have been linked to resistance to anti-estrogens in breast cancer, Rac activation and cell motility. Using R743A BCAR3, a point mutant that has lost the ability to bind p130Cas, we find that BCAR3-p130Cas complex formation is not required for BCAR3-mediated anti-estrogen resistance, Rac activation or discohesion of epithelial breast cancer cells. Complex formation was also not required for BCAR3-induced lamellipodia formation in BALB/c-3T3 fibroblasts but was required for optimal BCAR3-induced motility. Although both wildtype and R743A BCAR3 induced phosphorylation of p130Cas and the related adapter protein HEF1/NEDD9, chimeric NSP3:BCAR3 experiments demonstrate that such phosphorylation does not correlate with BCAR3-induced anti-estrogen resistance or lamellipodia formation. Wildtype but not R743A BCAR3 induced lamellipodia formation and augmented cell motility in p130Cas(-/-) murine embryonic fibroblasts (MEFs), suggesting that while p130Cas itself is not strictly required for these endpoints, complex formation with other CAS family members is, at least in cells lacking p130Cas. Overall, our work suggests that many, but not all, BCAR3-mediated signaling events in epithelial and mesenchymal cells are independent of p130Cas association. These studies also indicate that disruption of the BCAR3-p130Cas complex is unlikely to reverse BCAR3-mediated anti-estrogen resistance.

Breast Cancer Anti-estrogen Resistance 3 (BCAR3) Protein Augments Binding of the c-Src SH3 Domain to Crk-associated Substrate (p130cas)

Background: BCAR3 binds to p130cas, a known substrate of Src. Results: BCAR3 augments binding of the Src SH3 domain to p130cas as well as p130cas tyrosine phosphorylation in a BCAR3-p130cas complex-dependent manner. Conclusion: Formation of a BCAR-p130cas complex enhances Src SH3 domain binding to p130cas and p130cas substrate domain tyrosine phosphorylation. Significance: These results suggest a molecular basis for BCAR3-mediated enhancement of cell motility. The focal adhesion adapter protein p130cas regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130cas. AND-34/BCAR3, one of three NSP family members, binds the p130cas carboxyl terminus, adjacent to a bipartite p130cas Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130cas. Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130cas complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130cas to bind the Src SH3 domain through an RPLPSPP motif in the p130cas SBD. Although our prior work identified phosphorylation of the serine within the p130cas RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130cas. The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130cas complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130cas substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130cas. Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130cas and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130cas complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130cas SBD.

Abstract C136: An analysis of BCAR3‐mediated regulation of p130Cas localization and focal adhesion formation in breast cancer cells

BCAR3 and p130Cas, two focal adhesion proteins that bind to one another and that were previously identified in a screen for genes that confer anti‐estrogen resistance in ER+ breast cancer cells, were analyzed in breast cancer cell lines to determine the effect of BCAR3 expression on cell morphology and protein localization including the Src‐mediated redistribution of p130Cas. The effect of altered BCAR3 expression and role of BCAR3 and p130Cas association was studied in MCF7 cells stably over‐expressing wildtype BCAR3 or BCAR3 containing an R743A missense mutation that prevents p130Cas association. Protein localization and cellular morphology were examined by immunofluorescence and Western analyses of cells were conducted to observe p130Cas post‐translational modifications and the potential involvement of the TGFβ and β‐catenin signaling pathways. Stable over‐expression of BCAR3 in MCF7 epithelial cells induces the loss of cohesive association between cells and the formation of membrane projections as well as large focal adhesions that contain abundant p130Cas. This BCAR3‐mediated increase in p130Cas localization to focal adhesions is independent of the association of BCAR3 and p130Cas. Src, which is known to phosphorylate p130Cas, also induces augmented localization of p130Cas and this effect is also further enhanced by BCAR3 expression. Once again, physical interaction between BCAR3 and p130Cas is not necessary for the ability of BCAR3 to increase Src‐mediated redistribution of p130Cas to focal adhesions. In spite of inducing a loss of epithelial morphology in MCF7 cells, BCAR3 over‐expression did not induce SMAD2 phosphorylation or nuclear translocation of β‐catenin, suggesting that neither the TGFβ or β‐catenin signaling pathways are activated in response to BCAR3 over‐expression in epithelial breast cancer cells. Consistent with these results, shRNA‐mediated reduction of BCAR3 expression in MDA‐231 mesenchymal cells ablates basal p130Cas phosphorylation, alters p130Cas and focal adhesion localization and induces an epithelial‐like growth pattern with attenuated stress fiber formation and decreased tensin expression. The inhibition of stress fiber formation in MDA‐231 cells prevents p130Cas phosphorylation. BCAR3 expression regulates the morphology and cytoskeleton of epithelial and mesenchymal breast cancer cells by affecting focal adhesion formation, p130Cas localization and the cytoskeletal response to Src signaling. The effect of BCAR3 appears not to involve either the TGFβ or β‐catenin signaling pathways and is largely independent of BCAR39s direct physical interaction with p130Cas. Our current studies are seeking to examine the role of BCAR39s SH2 domain and serine‐proline‐rich region in the regulation of focal adhesion biology. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C136.