Matrix Metalloproteinase and Tissue Inhibitor of Metalloproteinases Is Associated with Multiple Myeloma Progression, Prognosis and Extramedullary Plasmacytoma
Authors: Ishihara Rei , Murakami Yuki , Homma Kazuki , Watanabe Saki , Oda Tsukasa , Sunaga Masanobu , Yamane Eiko , Kobayashi Nobuhiko , Osaki Yohei , Ishizaki Takuma , Shimizu Hiroaki , Iriuchishima Hirono , Koiso Hiromi , Tsukamoto Norifumi , Yokohama Akihiko , Nanami Gotoh , Ino Rumi , Saitoh Takayuki , Murakami Hirokazu , Handa Hiroshi 
Published: October 2019

Abstract
Backgrounds and Aims: Matrix metalloproteinase (MMP) is endopeptidase enzyme degrading extracellular matrix, and tissue inhibitor of metalloproteinases (TIMP) is negative regulator of MMP. MMP is well known to be involved in metastatic mechanism of cancer cell and oncogenesis. However expression and role of MMP and TIMP has not been well established in multiple myeloma (MM). Therefore we examined whether expression of MMP and TIMP was involved in progression and prognosis of MM and extramedullary plasmacytoma (EMP) formation.
Materials and Methods: Purified bone marrow plasma cells by using anti-CD138 antibody and magnetic beads obtained from 151 MM, 64 MGUS, 18 control and 5 EMP were subjected to the study after informed consent. The study was approved by IRB following Declaration of Helsinki. Whole transcriptome by next generation sequencer (NGS) using Illumina Next Seq 500 was performed in part of the samples to select genes to be studied, then expression level of MMP and TIMP determined by RQ-PCR Delta Ct value normalized with ACTB was used for analysis. MM cell lines KMS11, KMS12PE, KMS12BM, KMM1, RPMI8226, MM1S were used for the in vitro study.
Results: We selected TIMP-1, 2and MMP14, 24 based on transcriptome analysis data comparing MM and EMP. The expression level of TIMP1 and MMP24 was significantly higher in MM (median delta Ct: 0.033 for TIMP1, 0.00025 for MMP24) than in MGUS (median delta Ct: 0.013 for TIMP1, 0.00006 for MMP24) (p=0.005, p=0.001), however TIMP2, MMP14 level did not differ in between MM and MGUS. Interestingly, TIMP1, 2 and MMP14, MMP24 were expressed with strikingly higher levels in EMP than in MM with 20 times, 60 times, 300 times, 500 times respectively (p=0.01, p=0.02, p=0.004, p=0.004). Both TIMP1 and 2 expression were higher in the MM patients with high risk cytogenetic karyotype t(4;14), t(14;16), del 17p than in the patients without such karyotype (p=0.006, p=0.008), however the levels of MMP14 and 24 did not differ in between cytogenetic risk groups. Positive correlations were found in between TIMP1 and 2, MMP14 and 24 in both MM and MGUS group respectively (r=0.34, p<0.001, r=0.49, p<0.001 in MM, r=0.32, p=0.008, r=0.63, p<0.001 in MGUS). Since TIMP2 and MMP14 were higher in EMP, the effect of recombinant TIMP2, siRNA-TIMP2 and MMP14 inhibitors marimastat/ilomastat were tested in MM cell lines KMM1, KMS11, KMS12PE and RPMI8226. However, these interventions did not change proliferation rates of these cell lines. The cell lines were treated with doxorubicin or bortezomib to study if these agent can change TIMP and MMP expression. Doxorubicin significantly increased expression level of MMP14, 24 and TIMP1, 2, but bortezomib increased only MMP14, 24. In the newly diagnosed MM (NDMM) patients (n=77), median time of overall survival (OS) of the patients with high TIMP1 (more than median value) was significantly shorter (2.7 years vs not reached) and 3 year OS rate was inferior (40% vs 65%) (p=0.0095). Progression free survival (PFS) tended to be inferior for high TIMP1 group, but the difference did not reach statistical significance (p=0.221). OS and PFS were not different according to either TIMP2, MMP14 or MMP24.
Conclusions: Our results suggest that TIMP1, 2 and MMP14, 24 were associated with EMP formation. Among those factors, TIMP1 is the one which may play a key role for MM progression and chemo-resistance based on the results revealing its upregulation by antineoplastic agents and association with poor prognosis of MM patients. Our results is consistent with a previous report describing that high serum TIMP1 concentration was associated with poor prognosis of MM. TIMP is recently shown to play another role besides negative regulator for MMP, so further study to elucidate its specific role for chemo-resistance contributes to develop novel therapy targeting TIMP and MMP pathway.

Disclosures
Tsukamoto: Kyowa-Kirin: Research Funding; Chugai: Research Funding; Eisai: Research Funding; Pfizer: Research Funding. Handa:Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau.