Background:Human breast carcinoma cells secrete an adenosine 5′-diphosphate transphosphorylase (sNDPK) known to induce endothelial cell tubulogenesis in a P2Y receptor-dependent manner. We examined sNDPK secretion and its effects on human endothelial cells.Methods:Nucleoside diphosphate kinase (NDPK) secretion was measured by western blot and enzyme-linked immunosorbent assay, while transphosphorylase activity was measured using the luciferin-luciferase ATP assay. Activation of MAPK was determined by western blot analysis, immunofluorescence and endothelial cell proliferation and migration.Results:A panel of breast cancer cell lines with origin as ductal carcinoma, adenocarcinoma or medullary carcinoma, secrete sNDPK-A/B. Addition of purified NDPK-B to endothelial cultures activated VEGFR-2 and Erk1/2, both of which were blocked by inhibitors of NDPK and P2Y receptors. Activation of VEGFR-2 and ErK1/2 by 2-methylthio-ATP (2MeS-ATP) was blocked by pretreatment with the P2Y1-specific antagonist MRS2179, the proto-oncogene non-receptor tyrosine kinase (Src) inhibitor PP2 or the VEGFR-2 antagonist SU1498. Nucleoside diphosphate kinase-B stimulates cell growth and migration in a concentration-dependent manner comparable to the effect of vascular endothelial growth factor. Treatment of endothelial cells with either NDPK-B or 2MeS-ATP induced migration, blocked by P2Y1, Src or VEGFR-2 antagonists.Conclusion:sNDPK supports angiogenesis. Understanding the mechanism of action of sNDPK and P2Y1 nucleotide signalling in metastasis and angiogenesis represent new therapeutic targets for anti-angiogenic therapies to benefit patients.

• Bortezomib ameliorates sclerodermatous cGVHD responses by inhibiting germinal center B cells while maintaining GVT effects in murine models.• Bortezomib provides therapeutic benefits for patients with active steroidrefractory cGVHD.Chronic graft-versus-host disease (cGVHD) following allogeneic hematopoietic stem cell transplantation (HSCT) has emerged as a predominant complication following HSCT and has a distinct etiology. We and others have previously demonstrated that bortezomib, a proteasome inhibitor, can prevent but not treat acute GVHD in mice. To assess the effects of bortezomib on cGVHD, a mouse minor histocompatibility antigen-mismatched strain combination was used to mimic clinical cGVHD sclerodermatous pathogenesis and phenotype. Treatment of ongoing cGVHD with bortezomib ameliorated cutaneous lesions, which were also associated with a reduction in total numbers of germinal center B cells and lower B-cell activating factor gene expression levels in cutaneous tissues. Importantly, lymphoma-bearing mice receiving allogeneic HSCT with bortezomib preserved graftversus-tumor (GVT) effects. Based on these animal studies, we initiated an intrapatient dose escalation clinical trial in patients with extensive steroid-intolerant, dependent, or resistant cGVHD. Marked clinical improvement was observed in patients, which was also associated with reductions of peripheral B cells and minimal toxicity. These results indicate that bortezomib can be of significant use in the treatment of cGVHD and may also allow for maintenance of GVT. This trial was registered at

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LRIG1, a member of the LRIG family of transmembrane leucine rich repeat-containing proteins, is a negative regulator of receptor tyrosine kinase signaling and a tumor suppressor. LRIG1 expression is broadly decreased in human cancer and in breast cancer, low expression of LRIG1 has been linked to decreased relapse-free survival. Recently, low expression of LRIG1 was revealed to be an independent risk factor for breast cancer metastasis and death. These findings suggest that LRIG1 may oppose breast cancer cell motility and invasion, cellular processes which are fundamental to metastasis. However, very little is known of LRIG1 function in this regard. In this study, we demonstrate that LRIG1 is down-regulated during epithelial to mesenchymal transition (EMT) of human mammary epithelial cells, suggesting that LRIG1 expression may represent a barrier to EMT. Indeed, depletion of endogenous LRIG1 in human mammary epithelial cells expands the stem cell population, augments mammosphere formation and accelerates EMT. Conversely, expression of LRIG1 in highly invasive Basal B breast cancer cells provokes a mesenchymal to epithelial transition accompanied by a dramatic suppression of tumorsphere formation and a striking loss of invasive growth in three-dimensional culture. LRIG1 expression perturbs multiple signaling pathways and represses markers and effectors of the mesenchymal state. Furthermore, LRIG1 expression in MDA-MB-231 breast cancer cells significantly slows their growth as tumors, providing the first in vivo evidence that LRIG1 functions as a growth suppressor in breast cancer.

Bortezomib, a proteasome inhibitor capable of direct anti-tumor effects, has been shown to prevent acute graft-versus-host disease (aGVHD) when administered in a short course immediately after bone marrow transplantation (BMT) in mice. However, when given continuously, CD4+ T cell mediated gastrointestinal tract damages increase GVHD mortality. To investigate the protective effects of bortezomib on other organs, we have used a CD8 dependent aGVHD model of C3H.SW donor T cells engrafted into irradiated C57BL/6 recipients (minor MHC mismatch), which lack significant gut GVHD. Our data in this model show that bortezomib can be given continuously to prevent and treat aGVHD mediated by CD8+ T cells, but this effect is organ-specific such that only skin, but not liver, protection was observed. Despite the lack of hepatic protection, bortezomib still significantly improved survival primarily due to its skin protection. Reduced skin GVHD by bortezomib was correlated with reduced serum and skin IL-6 levels. Administration of a blocking IL-6 antibody in this model also resulted in similar cutaneous GVHD protection. These results indicate that bortezomib or blockade of IL-6 may prevent CD8+ T cell mediated cutaneous aGVHD.