The ERa signaling pathway is one of the most important and most studied pathways in human breast cancer, yet numerous questions still exist such as how hormonally responsive cancers progress to a more aggressive and hormonally independent phenotype. We have noted that human breast cancers exhibit a strong direct correlation between ERa and E-cadherin expression by immunohistochemistry, suggesting that ERa signaling might regulate E-cadherin and implying that this regulation might influence epithelial-mesenchymal transition (EMT) and tumor progression. To investigate this hypothesis and the mechanisms behind it, we studied the effects of ERa signaling in ERa-transfected ERa-negative breast carcinoma cell lines, the MDA-MB-468 and the MDA-MB-231 and the effects of ERa knockdown in naturally expressing ERa-positive lines, MCF-7 and T47D. When ERa was overexpressed in the ERa-negative lines, 17b-estradiol (E2) decreased slug and increased E-cadherin. Clones maximally exhibiting these changes grew more in clumps and became less invasive in Matrigel. When ERa was knocked down in the ERapositive lines, slug increased, E-cadherin decreased, cells became spindly and exhibited increased Matrigel invasion. ERa signaling decreased slug expression by two different mechanisms: directly, by repression of slug transcription by the formation of a corepressor complex of ligand-activated ERa, HDAC inhibitor (HDAC1), and nuclear receptor corepressor (N-CoR) that bound the slug promoter in three half-site estrogen response elements (EREs); indirectly by phosphorylation and inactivation of GSK-3b through phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt). The GSK-3b inactivation, in turn, repressed slug expression and increased E-cadherin. In human breast cancer cases, there was a strong inverse correlation between slug and ERa and E-cadherin immunoreactivity. Our findings indicate that ERa signaling through slug regulates E-cadherin and EMT.

Inflammatory breast carcinoma (IBC) is a particularly lethal form of breast cancer characterized by exaggerated lymphovascular invasion, which is a phenotype recapitulated in our human xenograft MARY-X. MARY-X generated spheroids in vitro that resemble the embryonal blastocyst. Because of the resemblance of the spheroids to the embryonal blastocyst and their resistance to traditional chemotherapy/radiotherapy, we hypothesized that the spheroids expressed a stem cell-like phenotype. MARY-X spheroids expressed embryonal stem cell markers including stellar, rex-1, nestin, H19, and potent transcriptional factors, oct-4, nanog, and sox-2, which are associated with stem cell self-renewal and developmental potential. Most importantly, MARY-X spheroids expressed a cancer stem cell profile characterized by CD44 Inflammatory breast cancer (IBC) is an aggressive form of human breast cancer characterized by florid lymphovascular invasion (LVI) and early metastasis.1,2 LVI is considered an important rate-limiting step in the metastatic process and is characterized by the formation of tumor emboli within lymphovascular channels.3,4 These emboli are relatively resistant to radiotherapy and chemotherapy because the tight aggregates of tumor cells exert autocrine and paracrine cytoprotective effects from these therapies by unknown mechanisms. 5,6 These emboli are also efficient at escaping local organ confinement with subsequent distant implantation. They therefore exhibit a strong penchant for both distant metastasis as well as local recurrence and confound attempts at both local as well as systemic control. The tumor emboli bear a strong resemblance to the human embryonal blastocyst, a structure also efficient at escaping local organ confinement with subsequent distant implantation. The IBC phenotype has been successfully recapitulated in a human xenograft model of IBC termed MARY-X.3-7 This model exhibits florid LVI in vivo, which generates tight aggregates of tumor cells (spheroids) in vitro. These spheroids also bear a strong resemblance to the blastocyst. Because the blastocyst is the source of human embryonal stem cells (ESs), cells capable of self renewal, proliferation and differentiation along multiple lineages and because tumor stem cells have been implicated in the properties of local recurrence, distant metastasis, and drug resistance that human cancers exhibit, we hypothesized that the tumor lymphovascular emboli of MARY-X and its derived spheroids might, in fact, express a stem cell-like phenotype reflected by both stem cell markers and stem cell biology. In addition we could test this hypothesis in human cases of IBC and, if confirmed, might explain some of the aggressive behavior of IBC.

Two of the most common signalling pathways in breast cancer are the ER (oestrogen receptor) ligand activation pathway and the E-cadherin snai1 slug EMT (epithelial–mesenchymal transition) pathway. Although these pathways have been thought to interact indirectly, the present study is the first to observe direct interactions between these pathways that involves the regulation of slug expression. Specifically we report that ligand-activated ERα suppressed slug expression directly by repression of transcription and that knockdown of ERα with RNA interference increased slug expression. More specifically, slug expression was down-regulated in ERα-negative MDA-MB-468 cells transfected with ERα after treatment with E2 (17β-oestradiol). The down-regulation of slug in the ERα-positive MCF-7 cell line was mediated by direct repression of slug transcription by the formation of a co-repressor complex involving ligand-activated ERα protein, HDAC1 (histone deacetylase 1) and N-CoR (nuclear receptor co-repressor). This finding was confirmed by sequential ChIP (chromatin immunoprecipitation) studies. In the MCF-7 cell line, slug expression normally was low. In addition, knockdown of ERα with RNA interference in this cell line increased slug expression. This effect could be partially reversed by treatment of the cells with E2. The efficacy of the effect of ERα on slug repression was dependent on the overall level of ERα. These observations confirmed that slug was an E2-responsive gene.

The genesis and unique properties of the lymphovascular tumor embolus are poorly understood largely because of the absence of an experimental model that specifically reflects this important step of tumor progression. The lymphovascular tumor embolus is a blastocyst-like structure resistant to chemotherapy, efficient at metastasis and overexpressing E-cadherin (E-cad). Conventional dogma has regarded E-cad as a metastasis-suppressor gene involved in epithelial-mesenchymal transition. However, within the lymphovascular embolus, E-cad and its proteolytic processing by calpain and other proteases have a dominant oncogenic rather than suppressive role in metastasis formation and tumor cell survival. Studies using a human xenograft model of inflammatory breast cancer, MARY-X, demonstrated the equivalence of xenograft-generated spheroids with lymphovascular emboli in vivo with both structures demonstrating E-cad overexpression and specific proteolytic processing. Western blot revealed full-length (FL) E-cad (120 kDa) and four fragments: E-cad/NTF1 (100 kDa), E-cad/NTF2 (95 kDa), E-cad/NTF3 (85 kDa) and E-cad/NTF4 (80 kDa). Compared with MARY-X, only E-cad/NTF1 was present in the spheroids. E-cad/NTF1 was produced by calpain, E-cad/NTF2 by γ-secretase and E-cad/NTF3 by a matrix metalloproteinase (MMP). Spheroidgenesis and lymphovascular emboli formation are the direct result of calpain-mediated cleavage of E-cad and the generation of E-cad/NTF1 from membrane-associated E-cad rather than the de novo presence of either E-cad/NTF1 or E-cad/CTF1. E-cad/NTF1 retained the p120ctn-binding site but lost both the β-catenin and α-binding sites, facilitating its disassembly from traditional cadherin-based adherens junctions and its 360° distribution around the embolus. This calpain-mediated proteolysis of E-cad generates the formation of the lymphovascular embolus and is responsible for its unique properties of increased homotypic adhesion, apoptosis resistance and budding.

No abstract

SNF2L, a chromatin remodeling gene expressed in diverse tissues, cancers, and derived cell lines, contributes to the chromatin remodeling complex that facilitates transcription. Because of this wide expression, it has not been exploited as a cancer therapeutic target. However, based on our present studies, we find that cancer cells, although expressing SNF2L at similar levels as their normal counterparts, are sensitive to its knockdown. This is not observed when its imitation SWI ortholog, SNF2H, is inhibited. SNF2L siRNA inhibition using two different siRNAs separately reduced SNF2L transcript levels and protein in both normal and cancer lines, but only the cancer lines showed significant growth inhibition, DNA damage, a DNA damage response, and phosphorylation of checkpoint proteins and marked apoptosis. DNA damage and the damage response preceded apoptosis rather than being consequences of it. The damage response consisted of increased phosphorylation of multiple substrates including ATR, BRCA1, CHK1, CHK2, and H2AX. Both the total and phosphorylated levels of p53 increased. The downstream targets of p53, p21, GADD45A, and 14-3-3σ, were also upregulated. The alterations in checkpoint proteins included increased phosphorylated cdc2 but not Rb, which resulted in a modest G 2 -M arrest. Although apoptosis may be mediated by Apaf-1/caspase 9, other caspases could be involved. Other members of the chromatin remodeling or SWI/SNF gene families exhibited overall reduced levels of expression in the cancer lines compared with the normal lines. This raised the hypothesis that cancers are sensitive to SNF2L knockdown because, unlike their normal counterparts, they lack sufficient compensation from other family members.

A standard microscope was reconfigured as a virtual slide generator by adding a Prior Scientific H101 robotic stage with H29 controller and 0.1-m linear scales and a Hitachi HV-C20 3CCD camera. Media Cybernetics Image Pro Plus version 4 (IP4) software controlled stage movement in the X-, Y-, and Z-axis, whereas a Media Cybernetics Pro-Series Capture Kit captured images at 640 ؋ 480 pixels. Stage calibration, scanning algorithms, storage requirements, and viewing modes were standardized. IP4 was used to montage the captured images into a large virtual slide image that was subsequently saved in TIF or JPEG format. Virtual slides were viewed at the workstation using the IP4 viewer as well as Adobe Photoshop and Kodak Imaging. MGI Zoom Server delivered the virtual slides to the Internet, and MicroBrightField's Neuroinformatica viewing software provided a browser-based virtual microscope interface together with labeling tools for annotating virtual slides. The images were served from a Windows 2000 platform with 2 GB RAM, 500 GB of disk storage, and a 1.0 GHz P4 processor. To conserve disk space on the image server, TIF files were converted to the FlashPix (FPX) file format using a compression ratio of 10:1. By using 4؋, 10؋, 20؋, and 40؋ objectives, very large gigapixel images of tissue whole-mounts and tissue arrays with high quality and morphologic detail are now being generated for teaching, publication, research, and morphometric analysis. Technical details and a demonstration of our system can be found on the Web at http://virtualmicroscope.osu.edu. Anat Rec (Part B: New Anat) 272B:91-97, 2003.

Discerning the pathologic significance of cutaneous T-cell infiltrates can pose a diagnostic challenge for dermatopathologists. Reactive conditions such as drug-associated lymphomatoid hypersensitivity and lymphomatoid lupus erythematosus can demonstrate lymphoid atypia and a phenotype resembling cutaneous T-cell lymphoma (CTCL). Further, lymphoid dyscrasias such as pityriasis lichenoides chronica, large plaque parapsoriasis, and atypical pigmentary purpura confuse the picture because they not only mimic CTCL but also represent prelymphomatous states with inherent malignant potential. Although the emergence of a dominant clone has been considered a clue indicative of a T-cell dyscrasia, there are reports concerning the identification of monoclonality in biopsies of reactive lymphoid infiltrates. We have conducted a modified single-stranded DNA conformational polymorphism (SSCP) assay using paraffin-embedded, formalin-fixed tissue on 92 T-cell-rich biopsies to determine the relative specificity and sensitivity of this methodology. In addition, laser capture microdissection (LCM) was performed on 22 of the 92 samples to isolate the area of interest and to compare its specificity and sensitivity with those SSCP assays performed without LCM. We found that monoclonality or oligoclonality is 86% specific for preneoplastic and neoplastic states, whereas the finding of polyclonality appears to be relatively specific for a reactive process. Some cases of reversible T-cell dyscrasia produced a molecular profile mimicking lymphoma or prelymphomatous states by virtue of monoclonality or oligoclonality. Although LCM appears to improve the sensitivity for detecting preneoplastic conditions, the relative specificity appears to be the same as that encountered with routine SSCP.