Principal Investigator: Alberto Passi
Grant Title: O-GlcNAcylation regulates expression of HAS2 and CD44
ABSTRACT:The O-GlcNAcylation of the proteins is a dynamic posttranslational
modification, which is involved in several biological processes and in humandiseases. The O-GlcNAcylation is a reaction able to link N-acetylglucosamine(OGlcNAc) to the side chain hydroxyl group of a serine or threonine residue. In
recent years, in several human malignant tumors the metastatic potential waslinked to O-GlcNAcylation. Hyaluronan (HA) is an unbranched and unsulphatedglycosaminoglycan synthesized in the cells by specific membrane synthases (HAS1, 2 and 3). HA synthesis is often related to cancer progression and metastaticpotential. We found that HAS2 could be O-GlcNAcylated at serine 221 increasing
its stability in the cell membrane and its capacity to produce HA (1). CD44 is a
HA receptor and represents a signaling platform that integrates cellular microenvironmental cues withgrowth factor and cytokine action transducing signals. Accumulating evidence indicates that CD44,especially CD44v isoforms, are cancer stem cell (CSC) markers and critical players in regulating theproperties of CSCs.
Objectives:
In the study, we would like to demonstrate that O-GlcNAcylation triggers the HAS2 expression at
epigenetic level (2) and increases the expression of its antisense HAS2-AS1 and this can be coupledwith CD44 expression.
Methods:
For the study, we used human smooth muscle cells and breast cancer MCF7 (low malignant) andMDA-MB-231 (triple negative highly malignant) in vitro. The cell have been transfected by using siRNAs coding for different genes and lncRNA HAS2-AS1, a long non coding RNA that we foundinvolved in our model. Cell viability was assessed by MTT and the cell migration with scratch test
and matrigel has been used for cell invasion test. Gene expression was measured by quantitative RTPCR with specific TaqMan probes.
Results:
After the O-GlcNAcylation induction by glucosamine incubation, we previously found that HAS2
antisense (HAS2-AS1) expression was highly increased due to the p65 O-GlcNAcylation, indicatingthat the antisense was induced by NFkB pathway (3). In breast cancer cells HAS2-AS1 is expressed
as long and short forms, both highly expressed in tumours correlating with cell aggressiveness. The
modulation of HAS2-AS1 affects the cancer cell behaviour and its knock down increased the cancer
cell invasion and viability. On the other hand, the overexpression of the two isoforms of HAS2-AS1(HAS2-AS1 long being the more effective) decreased cell viability and their ability to penetrate
Matrigel. The abrogation of HAS2-AS1 induced a higher expression of HAS2 and HAS3 mRNA, as
well as increased the hyaluronidase 2 transcript levels. The treatment brought to an increase of CD44mRNA levels. Although we observed an increase of CD44 transcript, there was not an activation of
the p44/42 pathway (ERK1/2 pathway). These data indicate that HAS2-AS1 could globally control
HA metabolism, not only through the expression of its synthases but also through its receptor anddegrading enzymes.
1: Vigetti D et al. Role of UDP-N-acetylglucosamine (GlcNAc) and O-GlcNAcylation of hyaluronan synthase 2 in thecontrol of chondroitin sulfate and hyaluronan synthesis. J Biol Chem. 2012 Oct 12; 287(42):35544-55.
2: Vigetti D et al. Epigenetics in extracellular matrix remodeling and hyaluronan metabolism. FEBS J. 2014 Nov;
281(22):4980-92
3: Vigetti et al. Natural antisense transcript for hyaluronan synthase 2 (HAS2-AS1) induces transcription of HAS2 via
protein O-GlcNAcylation. JBC 289, 28816-28826