Mannose supplements induce embryonic lethality and blindness in phosphomannose isomerase hypomorphic mice.

Patients with congenital disorder of glycosylation (CDG), type Ib (MPI-CDG or CDG-Ib) have mutations in phosphomannose isomerase (MPI) that impair glycosylation and lead to stunted growth, liver dysfunction, coagulopathy, hypoglycemia, and intestinal abnormalities. Mannose supplements correct hypoglycosylation and most symptoms by providing mannose-6-P (Man-6-P) via hexokinase. We generated viable Mpi hypomorphic mice with residual enzymatic activity comparable to that of patients, but surprisingly, these mice appeared completely normal except for modest (~15%) embryonic lethality. To overcome this lethality, pregnant dams were provided 1-2% mannose in their drinking water. However, mannose further reduced litter size and survival to weaning by 40 and 66%, respectively. Moreover, ~50% of survivors developed eye defects beginning around midgestation. Mannose started at birth also led to eye defects but had no effect when started after eye development was complete. Man-6-P and related metabolites accumulated in the affected adult eye and in developing embryos and placentas. Our results demonstrate that disturbing mannose metabolic flux in mice, especially during embryonic development, induces a highly specific, unanticipated pathological state. It is unknown whether mannose is harmful to human fetuses during gestation; however, mothers who are at risk for having MPI-CDG children and who consume mannose during pregnancy hoping to benefit an affected fetus in utero should be cautious.

A decreased ratio of laminin-332 beta3 to gamma2 subunit mRNA is associated with poor prognosis in colon cancer.

Laminin-332 (Ln-332) is a heterotrimeric glycoprotein (alpha3beta3gamma2) unique to epithelial cells with crucial roles in signaling, adhesion, and migration. Altered localization or expression levels of Ln-332, particularly its gamma2 subunit, are of prognostic value in a variety of cancers. However, the lack of standardized methodology and the limited quantification of previous study results have left unanswered questions, including the role of gamma2 transcript variants and whether differential expression of this chain represents dysregulation of the whole heterotrimer. Herein, we test the hypothesis that mRNA changes in one or more Ln-332 encoding genes can be used to distinguish between early- and advanced-stage cancer specimens and shed light on mechanistic questions raised by previous studies. Statistical analyses of human microarray data from the publicly available expression project in Oncology (expO) dataset, including examination of the distributions of Ln-332 subunit mRNA levels, identified a significant decrease in the Ln-332 beta3:gamma2 mRNA ratio between normal (n = 10) and early-stage colon cancer (n = 29) specimens. The beta3:gamma2 ratio was further decreased in metastatic colon cancer (n = 41) compared with early-stage samples. Our findings raise the possibility that Ln-332 gamma2 may be a therapeutic target against metastatic colon cancer because a lowered beta3:gamma2 ratio would reduce expression of heterotrimeric Ln-332 and increase monomeric gamma2 secretion. Further, standardized, quantitative methods for patient prognosis and therapeutic choice could be developed based upon the Ln-332 mRNA changes we uncovered.

A novel circular invasion assay mimics in vivo invasive behavior of cancer cell lines and distinguishes single-cell motility in vitro.

BACKGROUND: Classical in vitro wound-healing assays and other techniques designed to study cell migration and invasion have been used for many years to elucidate the various mechanisms associated with metastasis. However, many of these methods are limited in their ability to achieve reproducible, quantitative results that translate well in vivo. Such techniques are also commonly unable to elucidate single-cell motility mechanisms, an important factor to be considered when studying dissemination. Therefore, we developed and applied a novel in vitro circular invasion assay (CIA) in order to bridge the translational gap between in vitro and in vivo findings, and to distinguish between different modes of invasion. METHOD: Our method is a modified version of a standard circular wound-healing assay with an added matrix barrier component (Matrigel), which better mimics those physiological conditions present in vivo. We examined 3 cancer cell lines (MCF-7, SCOV-3, and MDA-MB-231), each with a different established degree of aggressiveness, to test our assay's ability to detect diverse levels of invasiveness. Percent wound closure (or invasion) was measured using time-lapse microscopy and advanced image analysis techniques. We also applied the CIA technique to DLD-1 cells in the presence of lysophosphatidic acid (LPA), a bioactive lipid that was recently shown to stimulate cancer cell colony dispersal into single migratory cells, in order to validate our method's ability to detect collective and individual motility. RESULTS: CIA method was found to be highly reproducible, with negligible levels of variance measured. It successfully detected the anticipated low, moderate, and high levels of invasion that correspond to in vivo findings for cell lines tested. It also captured that DLD-1 cells exhibit individual migration upon LPA stimulation, and collective behavior in its absence. CONCLUSION: Given its ability to both determine pseudo-realistic invasive cell behavior in vitro and capture subtle differences in cell motility, we propose that our CIA method may shed some light on the cellular mechanisms underlying cancer invasion and deserves inclusion in further studies. The broad implication of this work is the development of a reproducible, quantifiable, high-resolution method that can be applied to various models, to include an unlimited number of parameters and/or agents that may influence invasion.

Defining the role of laminin-332 in carcinoma.

The deadly feature of cancer, metastasis, requires invasion of cells through basement membranes (BM), which normally act as barriers between tissue compartments. In the case of many epithelially-derived cancers (carcinomas), laminin-332 (Ln-332) is a key component of the BM barrier. This review provides a historical examination of Ln-332 from its discovery through identification of its functions in BM and possible role in carcinomas. Current understanding points to distinct roles for the three Ln-332 subunits (alpha3, beta3, gamma2) in cell adhesion, extracellular matrix stability, and cell signaling processes in cancer. Given the large number of studies linking Ln-332 gamma2 subunit with cancer prognosis, particular attention is given to the crucial role of this subunit in cancer invasion and to the unanswered questions in this area.

Dispersal of epithelial cancer cell colonies by lysophosphatidic acid (LPA).

We describe a model system in which cancer cell colonies disperse into single, highly migratory cells in response to lysophosphatidic acid (LPA). Though LPA is known to stimulate chemotaxis and chemokinesis, a colony dispersal effect has not been reported, to our knowledge. Cancer colony dispersal by LPA is comprised of an ordered sequence of events: (1) stimulation of membrane ruffling and formation of lamellipodia, (2) dissolution of adherens junctions, (3) single cell migration in a mesenchymal-like morphology we term "ginkgo-leaf." The net result is dispersal of carcinoma cells from a compact colony. We analyzed these three steps using live-cell imaging and computer-assisted quantification and measured the following parameters: onset of lamellipodia formation, lamellipodia velocity, colony dispersal, trans-epithelial resistance, migrating cell number and speed. Because hepatocyte growth factor (HGF) was described as an epithelial scatter factor, we compared it to LPA in our system and found that HGF has no epithelial colony dispersal properties and that this effect is strictly related to LPA. Given its striking similarity to tumor cell budding observed in patients, we propose that LPA-colony dispersal may provide a cellular mechanism underlying cancer invasion and as such deserves further studies.