A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion

Aberrant display of the truncated core1 O-glycan T-antigen is a common feature of human cancer cells that correlates with metastasis. Here we show that T-antigen in Drosophila melanogaster macrophages is involved in their developmentally programmed tissue invasion. Higher macrophage T-antigen levels require an atypical major facilitator superfamily (MFS) member that we named Minerva which enables macrophage dissemination and invasion. We characterize for the first time the T and Tn glycoform O-glycoproteome of the Drosophila melanogaster embryo, and determine that Minerva increases the presence of T-antigen on proteins in pathways previously linked to cancer, most strongly on the sulfhydryl oxidase Qsox1 which we show is required for macrophage tissue entry. Minerva’s vertebrate ortholog, MFSD1, rescues the minerva mutant’s migration and T-antigen glycosylation defects. We thus identify a key conserved regulator that orchestrates O-glycosylation on a protein subset to activate a program governing migration steps important for both development and cancer metastasis

Mechanism of Nm23/NDPK in Breast Cancer; Heterologous Signaling in Support of Tumor Angiogenesis and Metastasis

Human breast carcinoma cells (MDA-MB-435) secrete an adenosine 5'-diphosphate trans-phosphorylase identified as shed or secreted (s) human (h) nucleoside diphosphate kinase (shNDPK-B) known to induce endothelial cell tubulogenesis in a P2Y-receptor dependent manner. Here, we examined whether sNDPK-A/B secretion is a common feature of breast cancer cell lines and what affects sNDPK has on endothelial cell growth and migration. We demonstrated for the first time that shNDPK-B can be detected specifically with high sensitivity in an ELISA assay of our own design. Our ELISA assay also revealed that a panel of breast cancer cell lines with origins in ductal carcinoma, adenocarcinoma and medullary carcinoma secreted/shed NDPK-A/B into their growth media, while sNDPK-A/B cannot be detected in the media of a control breast cell line (MCF-12f) by western blot. Secreted NDPK-A/B acted as a transphosphorylase converting ADP to ATP in the presence of a phosphoryl donor and promoted endothelial proliferation and cell migration through activation of P2Y purine nucleotide receptors (P2YRs). This finding is consistent with the hypothesis that human breast tumor cell-secreted NDPK-A/B increases extracellular ATP levels in the immediate environment of metastatic tumors. We also extended our studies of purinergic regulation of angiogenesis. We hypothesized that in the absence of growth factors, activated P2Y1 receptors transactivate the proto-oncogene Src kinase and vascular endothelial growth factor receptor-2 (VEGFR-2) in angiogenic signaling. The Immunoprecipitation and direct western blots results showed that P2Y1 receptors activated by the P2Y1 specific agonist 2-methylthio-ATP (2MeS-ATP) induce phosphorylation of VEGFR-2 (Tyr 1059 and Tyr 1175). The transactivation of VEGFR-2 by P2Y1 activation was blocked by pretreatment with the P2Y1 specific antagonist, Src or the VEGFR-2. Further, the results showed that the addition of extracellular NDPK to human endothelial cultures resulted in transactivation of VEGFR-2 and transactivation was blocked by the NDPK inhibitor, EA. Activation of the endothelial nucleotide P2Y1 receptor by its specific agonist 2MeS-ATP or NDPK, transactivates VEGFR-2 in the absence of VEGF. We suggest that sNDPK and activated P2Y1R support tumor angiogenesis via VEGFR-2. Next, we examined nucleotide regulation of angiogenesis linking sNDPK to MAPK activation using western blot analysis, immunofluorescence and measurement of human endothelial cell migration. Our results demonstrated that the addition of extracellular NDPK to HEC cultures activated Erk1/2 and induced cell migration, both of which were blocked by inhibitors of NDPK and P2Y receptors. Activation of pErk1/2 by 2MeS-ATP was blocked by pretreatment with the P2Y1 specific antagonist, the proto-oncogene non-receptor tyrosine kinase (Src) inhibitor, or the VEGFR-2 antagonist. The ability of ATP-P2 receptor stimulation to transactivate the endothelial VEGF receptor and downstream Erk1/2 activation in the absence of VEGF suggests the possibility that cancer cell-secreted NDPK-A/B, endothelial P2Y receptors, and VEGF receptors interact as partners in tumor angiogenesis in vitro. We extended our studies of purinergic regulation of angiogenesis in vivo in a breast cancer metastasis model in mice carrying luciferase-tagged human breast carcinoma cells (MDA-MB-23-Luc2) to permit tracking of primary tumor development and tumor metastases. We hypothesized that orthotopic-human tumors growing in SCID mice modulate extracellular sNDPK-A/B levels to attract angiogenic growth, development and metastasis; and that inhibition of NDPK-A/B's extracellular action as transphosphorylase and that inhibition of P2Y1 receptors prevents/delays the growth of metastatic breast tumor in a mouse model. Examination of serum using Western blot and an ELISA assay of our own design revealed that tumors secreted sNDPK-A/B in large amounts two weeks after subcutaneous injection of cancer cells in the mammary fat pad. The antitumor therapy showed that NDPK-A/B and activation of P2Y1R promotes tumor growth in a mouse model of human breast cancer and that the inhibition of NDPK activity by EA and inhibition of P2Y1R activation by MRS2179 inhibited primary tumor growth and reduced metastases in MRS2179 or EA alone and completely eradicated the metastatic lesions when combined. Therefore, we proposed that cancer cell-secreted sNDPK-A/B positively regulates angiogenesis through its regeneration of extracellular ATP levels at the surface of capillary blood vessels in an animal model of breast cancer, and that these data support the hypothesis that shNDPK-A/B may be responsible for the early events in angiogenesis supporting both primary and metastatic tumor growth and development

LRIG1 opposes epithelial-to-mesenchymal transition and inhibits invasion of basal-like breast cancer cells.

LRIG1 (leucine-rich repeat and immunoglobulin-like domain containing), 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 and 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 that are fundamental to metastasis. However, very little is known of LRIG1 function in this regard. In this study, we demonstrate that LRIG1 is downregulated 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

LRIG1 opposes epithelial-to-mesenchymal transition and inhibits invasion of basal-like breast cancer cells.

LRIG1 (leucine-rich repeat and immunoglobulin-like domain containing), 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 and 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 that are fundamental to metastasis. However, very little is known of LRIG1 function in this regard. In this study, we demonstrate that LRIG1 is downregulated 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

Extracellular NM23 Signaling in Breast Cancer: Incommodus Verum

The notion that breast cancers can survive in an individual patient in a dormant state only to grow as metastatic disease in the future, is in our view incontrovertibly established. Convincing too is the evidence that surgery to remove the primary tumor often terminates dormancy resulting in accelerated relapses. Accepting that many deaths due to breast cancer might be averted were we to understand the cellular mechanisms underlying escape from dormancy, we have examined the extracellular signals produced by breast cancers derived from women with metastatic breast disease. In this perspective, we explore the role of extracellular nucleotide signaling that we have proposed constitutes a pathological axis from the transformed tumor cell to the endothelium in the service of intravasation, dissemination, extravasation and angiogenesis. A role for the dinucleotide kinase NM23/NDPK (nucleoside diphosphate kinase) secreted by breast tumor cells in the generation of signals that stimulate vascular leakiness, anti-thrombosis, endothelial migration and growth, constitutes a mechanistic basis for escape from latency and offers putative therapeutic targets for breast cancer management not previously appreciated