Matrix metalloproteinases (MMPs) belong to a unique family of zinc dependent endopeptidases, they are strictly regulated by their endogenous inhibitors called tissue inhibitor of MMPs (TIMPs). MMPs have been associated with tumorigenesis. In addition to their role in extracellular matrix turnover and cancer cell migration, MMPs regulate signaling pathways that control cell growth, inflammation, or angiogenesis and may even work in nonproteolytic manner. MMPs play a key role during invasion and metastasizing of cancer cells and they have been shown to be associated to invasive phenotype and poor prognosis in several solid tumors. Little is known about their role in MDS. A total of 23 different human MMPs and 4 TIMPs have been identified. To better understand the role of MMPs in MDS, we performed an expression profile screen study by QPCR to detect the expression level of all MMP and TIMP family members, except MMP23, in CD34+ cells from a cohort (N=10) of newly diagnosed, untreated patients with MDS, and compared the expression level with CD34+ cells from 5 normal donors. Of these 26 genes, we identified MMP2, MMP8, MMP9, MMP25, MMP28, TIMP1, TIMP2 and TIMP3 as aberrantly up-regulated genes in MDS and expanded the study to a larger cohort of patients to correlate with clinical features and clinical outcomes.
CD34+ cells from 98 newly diagnosed patients with MDS and 5 normal donors were evaluated for MMP2, MMP8, MMP9, MMP25, MMP28, TIMP1, TIMP2 and TIMP3 expression profiling. CD34+ cells were sorted from patients and normal donor bone marrow. Total cellular RNA was isolated using Trizol, cDNA was obtained by using TaqMan reverse transcription reagent (Applied Biosystems). For real-time PCR, all assays were purchased from Applied Biosystems and analyzed with an Applied Biosystems Prism 7500 Sequencing detection system. GAPDH was used as internal control. Immunohistochemistry was used to detect MMP9 protein level in cytospin from CD34+ cells. MMP9 abtibody was obtained from R&D systems, MMP9 ELISA kit (R&D systems) was used to detect the MMP9 protein level in bone marrow plasma.
For the 98 MDS patients studied, 78% were older than 60 years; by IPSS score, 17 (17.3%) low risk, 35 (35.7%) INT-1, 24 (24.5%) INT-2, 10 (10.2%) high risk, 12 (12.2%) not available. By Cytogenetics, diploid 44 (45%), 20q- 7(7%), −5/5q- 7 (7%), −7/7q- 7 (7%), −5/5q- and −7/7q- 6 (6%), 8+ 6 (6%), IM 6 (6%), MISC 14 (14%). By QPCR and comparing MDS CD34+ cells with normal CD34+ cells, aberrant up-regulation (fold>2) of MMP2, MMP8, MMP9, MMP25, MMP28, TIMP1, TIMP2 and TIMP3 was detected in 40%, 93.8%, 94%, 84%, 15.6%, 45%, 21% and 27% of patients respectively. Up-regulation of MMP8, MMP9 and MMP25 were significant with a mean fold value 350.7 (0–3363.7), 1112.4 (0–15641) and 143.8 (0–1017.9) respectively. We performed MMP9 immunohistochemistry of MDS CD34+ cytospins from 16 pts with higher and lower MMP9 RNA relative expression level and found consistent protein expression level in cytoplasm. No elevated MMP9 protein levels were detected in bone marrow plasma. We then performed an analysis of associations with clinical variables and observed that relative expression value of MMP9 was associated with lower bone marrow blast (p=0.001) and longer survival (p=0.02) (figure 1). We did not found association between survival and the other 7 up-regulated genes.
Bone marrow CD34+ cells from patients with MDS have abnormal up-regulation of MMP2, MMP8, MMP9, MMP25, MMP28, TIMP1, TIMP2 and TIMP3. MMP9 up-regulation is associated with longer survival. Our results suggest that MMP-9 could be a useful prognostic indicator for MDS and that this family of proteins needs further study in MDS.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.