Principal Investigator: Kazuyuki Kitatani
Grant Title: Involvement of acid b-glucosidase (GBA1) in cancer metastasis.
Abstract
Objective: Sphingolipids characterized with sphingobase backbone are an
essential class of membrane lipids. Sphingolipid metabolism involves
multiple metabolic pathways such as de novo pathway, salvage pathway
and sphingomyelinase pathway etc. and a number of enzymes are involved.
Ceramide, a central sphingolipid in the metabolism, has been emerging as abioactive lipid that serves as a regulatory molecule in anti-inflammatoryresponses, apoptosis, autophagy, and cell motility of cancer cells. Veryrecently, we discovered that ceramide limits phosphatidylinositol-3-kinase C2β-controlled cell
motility in ovarian cancer and proposed that ceramide serves as a tumor metastasis suppressor.
Considering those bioactivities of ceramides, down-regulation of anti-metastatic ceramides isthought to promote the metastatic potential. Possibly, significant alternations in expression of
sphingolipid metabolizing enzymes affect metastatic potential by down- or up-regulating cellar
levels of ceramides. In the present studies, we characterized highly metastatic ovarian cancer cellswith ceramide synthesis and identified anti-metastatic ceramide-generating enzyme responsible forsuppressing cell motility and metastatic potential of ovarian cancer.
Methods: To establish highly metastatic cancer cells, in vivo screening was conducted innude mice. SKOV3 ovarian cancer cells were inoculated into peritoneal of five-week-old female
nude mice. Mice were sacrificed at 4 weeks after inoculation, and then four metastatic nodules werecollected and cultured to establish metastasis-prone ovarian cancer cells. Analysis ofsphingolipids in lipid extracts was performed by liquid chromatography-tandem mass spectrometry.
Acid-b-glucosidase (GBA1) proteins were detected by the Western blotting.
Results: To define roles of ceramide in ovarian cancer, we tested effects of ceramide liposomes on
the growth of ovarian cancer cells. Treatment of ovarian cancer cells with ceramide liposomes
decreased the number of living cells through necroptosis but not apoptosis. Mechanistically, dying
SKOV3 ovarian cancer cells exhibit activation of pseudokinase mixed lineage kinase domain-like asevidenced by oligomerization, showing necroptotic characteristics. Moreover, systemic ceramide
liposomes administration suppressed metastatic growth in an ovarian cancer cell xenograft model.
Therefore, ceramide is suggested to serve as a tumor suppressive lipid. To characterize metastatic
cancer cells, we established metastasis-prone SKOV3 cells (M1-1, M1-2, M1-3, M1-4). Those cells
showed decreased ceramide and higher invasiveness compared to the parental SKOV3 cells.
Importantly, GBA1 expression was upregulated in all metastasis-prone cells. Considering that GBA1cleaves glucosylceramide to form ceramide, GBA1-ceramide pathway is thought to limit cell growthand metastasis in ovarian cancer cells. In order to assess the clinical relevance of GBA1 as a tumor
suppressor in ovarian cancer, an online analysis using ovarian cancer microarray datasets wasperformed. Patients were divided into 2 groups according to the GBA1 mRNA expression, giving
high and low expression groups. In individual groups, patients-matched survival periods were plottedto generate Kaplan-Meier curves. Expression of LARP4 mRNA in ovarian cancer tissues ispositively correlated with patient prognosis. Taken together, GBA1 is thought to serve as a tumor
suppressor in ovarian cancer.