Downregulation Of SPINT2 Expression In Stromal Cells From Myelodysplastic Syndrome Patients Increases HGF and SDF-1 Secretion and Promotes Cell-Cell Adhesion By Alteration In Integrin Expression

Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells (HSC) characterized by ineffective hematopoiesis. In addition to HSC defects, an important role is also played by the hematopoietic microenvironment niche that has as key component the mesenchymal stromal cells (MSC). The MSC of MDS patients have morphological and functional abnormalities. Recently, our group identified new possible target genes involved in MDS pathophysiology through microarray analysis of MSC from MDS patients. An interesting underexpressed gene found was SPINT2, a gene that encodes a transmembrane protein which inhibits the hepatocyte growth factor activator (HGFA), the enzyme responsible for the conversion of hepatocyte growth factor (HGF) into its active form. SPINT2 is downregulated in some types of solid cancer and correlated with their prognostic and progression; however, the functional role of SPINT2 in MDS remains unknown. We herein investigated the role of SPINT2 in MSC, studying HGF and SDF1 secretion and cell adhesion with normal CD34⁺, P39 and U937 cells by silencing SPINT2 gene in HS5 and HS-27a stromal cell lines. We also investigated, using qPCR, SPINT2 and HGF mRNA expression in MSC and total bone marrow (BM) cells from 56 untreated MDS patients (WHO 35 low-risk, 21 high-risk) and 28 healthy donors. To inhibit SPINT2, specific shRNA expressing lentiviral vectors targeting SPINT2 gene or no specific sequence were used. The HGF and SDF1 secretion in cell supernatant from the cells silenced or not for SPINT2 was measured by BioPlex after 6, 12, 24 and 48h. The cell-cell adhesion of CD34⁺, P39 or U937 cells onto transduced stromal cells and the adhesion molecule profile were analyzed by flow cytometry. We observed a significant decrease in SPINT2 mRNA expression of MDS MSC (P=0.006) and MDS BM cells (P=0.03) compared to normal cells. Further, HGF mRNA expression of MDS MSC was significantly increased (P=0.01) compared to normal cells. Spearman analysis showed a negative significant correlation between SPINT2 and HGF expressions (P=0.01;r²=0.60). In both stromal cell lines, SPINT2 inhibition resulted in a significant increase in HGF secretion after 24 and 48h and a significant increased secretion of SDF1 after 48h. Moreover, SPINT2 silencing induced a significant increased adherence of CD34⁺, P39 and U937 cells onto stromal cells probably due to the alteration in integrin expression, since an increase in CD49b and CD49d and decrease in CD49e expressions were also observed in cells silenced for SPINT2. Considering that SPINT2 limits signaling via HGF pathway by inhibiting HGF activation through HGFA, the SPINT2 underexpression in MDS would allow the conversion of the inactive HGF monomer to an active heterodimer. Interestingly, a significant decrease of SPINT2 and increase of HGF expressions were observed in MSC of MDS patients, compared to normal cells. Moreover, recent studies have demonstrated that HGF serum levels are significantly increased in MDS patients and dependent on MDS severity. In addition, SPINT2 inhibition in stromal cells significantly increased HGF secretion by these cells. The increased HGF secretion can result in an autocrine regulation which induces the production and secretion of SDF1 by stromal cells themselves. Cytokines secretions provided by MSC are required for adhesion, survival and proliferation of HSC cells. In this way, in MDS MSC, SPINT2 underexpression and, consequently, increased HGF and SDF1 secretion, may lead to an increased adhesion between MSC and normal or malignant HSC cells. Corroborating our hypotheses, we found overexpression of integrins CD49b and CD49d, which mediate cell-MSC interaction, and a decrease in CD49e expression, an integrin that promotes interaction with extracellular matrix. The interaction onto MSC contributes to the maintenance of the stem and malignant cell properties, such as self-renewal, survival and proliferation. Cytokine secretion and cell adhesion onto MSC is important for MDS physiology. Hence, we demonstrate for the first time that, in MDS, SPINT2 plays a role in the HGF and SDF1 secretion by MSC, resulting in an alteration in cell-cell adhesion and molecule adhesion profile. In view of these data, the SPINT2 expression alteration in MDS MSC may constitute a particular mechanism of MDS pathophysiology and maintenance of self-renewal, homing and proliferation of HSC and malignant clones in MDS.Support: FAPESP, CNPq