The genesis and unique properties of the lymphovascular tumor embolus are because of calpain-regulated proteolysis of E-cadherin.

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 ß-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.

The lymphovascular embolus of inflammatory breast cancer exhibits a Notch 3 addiction.

Inflammatory breast carcinoma (IBC) is characterized by exaggerated lymphovascular invasion (LVI), recapitulated in our human xenograft, MARY-X. This model exhibited lymphovascular emboli in vivo and corresponding spheroids in vitro. Owing to the morphological and gene profile resemblance of these spheroids to embryonal blastocysts, we wondered whether they might exhibit embryonic stem cell signaling. Specifically we investigated Notch and observed selective Notch 3 activation by expression profiling, reverse transcriptase- and real-time PCR, western blot and immunofluorescence in vitro, and immunohistochemistry in vivo. Notch 3 intracellular domain (N3icd) and six target genes, HES-5, HEY-1, c-Myc, Deltex-1, NRARP and PBX1, markedly increased in MARY-X. In addition, a significant percentage of MARY-X cells expressed aldehyde dehydrogenase (ALDH), a stem cell marker. Only the ALDH(+) cells were capable of secondary spheroidgenesis, tumorigenicity and self-renewal. Inhibiting Notch 3 activation in vitro with γ-secretase inhibitors (GSIs) or small interfering RNA resulted in a downregulation of Notch target genes, including CD133, and an induction of caspase 3-mediated apoptosis. Transfection of N3icd but not Notch 1 intracellular domain into normal human mammary epithelial cells resulted in increased expression of Notch target genes and induction of spheroidgenesis. GSI in vivo resulted in inhibitory but diffusion-limited effects on Notch 3 signaling, resulting in xenograft growth reduction. The lymphovascular emboli of human IBC exhibited dual N3icd and ALDH1 immunoreactivities independently of molecular subtype. This Notch 3 addiction of lymphovascular emboli might be exploited in future therapeutic strategies.

ERalpha signaling through slug regulates E-cadherin and EMT.

The ERalpha 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 ERalpha and E-cadherin expression by immunohistochemistry, suggesting that ERalpha 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 ERalpha signaling in ERalpha-transfected ERalpha-negative breast carcinoma cell lines, the MDA-MB-468 and the MDA-MB-231 and the effects of ERalpha knockdown in naturally expressing ERalpha-positive lines, MCF-7 and T47D. When ERalpha was overexpressed in the ERalpha-negative lines, 17beta-estradiol (E2) decreased slug and increased E-cadherin. Clones maximally exhibiting these changes grew more in clumps and became less invasive in Matrigel. When ERalpha was knocked down in the ERalpha-positive lines, slug increased, E-cadherin decreased, cells became spindly and exhibited increased Matrigel invasion. ERalpha signaling decreased slug expression by two different mechanisms: directly, by repression of slug transcription by the formation of a corepressor complex of ligand-activated ERalpha, 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-3beta through phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt). The GSK-3beta inactivation, in turn, repressed slug expression and increased E-cadherin. In human breast cancer cases, there was a strong inverse correlation between slug and ERalpha and E-cadherin immunoreactivity. Our findings indicate that ERalpha signaling through slug regulates E-cadherin and EMT.

Proton pump inhibitor therapy is a risk factor for Clostridium difficile-associated diarrhoea.

BACKGROUND: Inhibition of gastric acid removes a defence against ingested bacteria and spores, increasing the risk of some forms of gastroenteritis. Previous studies investigating a possible link between acid suppression therapy and Clostridium difficile-associated diarrhoea have reported conflicting results. AIM: To investigate whether acid suppression therapy is associated with an increased risk of C. difficile-associated diarrhoea. Prospective case-control study of 155 consecutive in-patients with C. difficile-associated diarrhoea. RESULTS: Antibiotics had been received by 143 (92%) of the C. difficile-associated diarrhoea group and 76 (50%) of the controls during the preceding 3 months. Among those receiving antibiotics, 59 (41%) of the C. difficile-associated diarrhoea group had also received acid suppression, compared with 21 (28%) of controls (OR 1.84, CI 1.01, 3.36, chi(2) = 4.0, P = 0.046). Among the entire C. difficile-associated diarrhoea group 64 (41%) had received acid suppression compared with 40 (26%) of controls (OR 1.99, CI 1.19, 3.31, chi(2) = 7.9, P = 0.005). Logistic regression analyses found that C. difficile-associated diarrhoea was independently associated with: antibiotic use (OR 13.1, 95% CI: 6.6, 26.1); acid suppression therapy (OR 1.90, 95% CI: 1.10, 3.29); and female sex (OR 1.79, 95% CI: 1.06, 3.04). CONCLUSIONS: The risk of C. difficile-associated diarrhoea in hospitalized patients receiving antibiotics may be compounded by exposure to proton pump inhibitor therapy.

Detection of antibody responses and delayed dermal hypersensitivity with Blastomyces dermatitidis yeast and mycelial lysate antigens.

Yeast cell lysate and mycelial lysate antigens prepared from one strain (T-58) of Blastomyces dermatitidis were evaluated with respect to the detection of antibodies and delayed dermal hypersensitivity. Comparable ELISA sensitivity values were evidenced with the two antigens when assayed against serum specimens from dogs with blastomycosis, sera from non-infected dogs residing in endemic and non-endemic areas for blastomycosis and sera from rabbits that were hyperimmunized with B. dermatitidis antigens. Specificity determinations with anti-Histoplasma capsulatum rabbit sera indicated that both reagents exhibited only minimal cross-reactivity; the mycelial antigen was slightly more specific than the yeast phase reagent. Similar sensitivity and specificity results were experienced when the two antigens were used to detect delayed dermal hypersensitivity in guinea pigs previously sensitized with B. dermatitidis or H. capsulatum.