Identification of NOTCH-driven matrisome-associated genes as prognostic indicators of multiple myeloma patient survival

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Multiple myeloma (MM) is a rarely curable plasma cell malignancy of the bone marrow (BM) that provides a permissive tumor microenvironment (TME), supporting tumor cell growth and dissemination and conferring therapy resistance.The TME contains cellular components, specifically stromal cells, osteoclasts, osteoblasts, osteocytes, endothelial and immune cells, and a non-cellular component, the extracellular matrix (ECM).In cancer, the ECM is an important determiner of cell fate and composition of the TME.Recent research has coined the term "matrisome" for the ensemble of genes encoding ECM proteins and ECM-associated proteins and defined gene sets for core matrisome (approximately 274 genes) and matrisome-associated genes (approximately 1027 genes), including secreted modifiers [1].Specified cancer matrisomes regulate proliferation, migration, and survival [1].Hence, changes in ECM composition, integrity, abundance, biomechanical properties, and related signal transduction contribute to tumor progression and outcome in patients [1].In MM, the ECM bidirectionally interacts with MM cells and coinhabitants of tumor cell/metastatic niches.Notably, expression levels of genes in MM cells involved in the interaction with the TME have been linked to better (BMP6 [2]) or worse (ANXA2, LGALS1 [3,4]) survival of patients.We and others have shown that NOTCH signaling alters the TME through juxtacrine signaling between signal-sending cells such as MM or stromal cells expressing the ligands, and signal-receiving cells expressing the receptors [5,6].Whether deregulated NOTCH signaling in MM cells controls the expression of genes that dysregulate ECM composition in the BM niche and have prognostic significance, is unknown.Here, we correlated transcriptome profiles of NOTCHdepleted MM cells with recently published matrisome libraries, to identify NOTCH-regulated genes that belong to the matrisome and are related to patient survival.
These findings prompted the systematic search for NOTCHdriven matrisome genes within the entire gene expression data set.To this end, we used the MatrisomeDB database that provides live cross-referencing to gene and protein databases for every ECM and ECM-associated gene [1].GO analysis revealed that N1 and N2 regulate both core matrisome genes and a series of matrisome-associated genes in RPMI 8226 (Table 1) and MM.1S cells (Supplementary Table 2).Overall comparison between up-or downregulated genes showed that expression of 14 and 34 matrisome genes is commonly regulated by N1 and N2 in both cell lines (Supplementary Table 1).QPCR analysis or immunoblotting demonstrated that lower levels of N1 or N2 correlate with lower levels of HPSE in NOTCH-depleted RPMI 8226 and MM.1S cells (Supplementary Fig. 2).The same trend of low HPSE expression can be found in the MM cell line AMO-1, which is characterized by low NOTCH levels.Similarly, lower levels of MMP13, S100A6, IGF1 correspond to decreased NOTCH levels in RPMI 8226 cells, and low levels of MMP13 to low NOTCH levels in AMO-1 cells.In contrast, lower levels of N1 and N2 are associated with higher protein levels of EMID1, TGFBI, and C1QA in RPMI 8226 cells (Supplementary Fig. 2).These data confirm the reliability of the transcriptome analysis and the regulation of matrisome genes through N1 and N2 in MM cells.
To correlate expression levels of ECM genes and patient survival, we first analyzed gene expression in samples of BM plasma cells (BMPC) from healthy donors, in patient samples from monoclonal gammopathy of undetermined significance (MGUS), smoldering (sMM), untreated MM, and in samples human MM cell lines (HMCL) [9].Next, we determined their association with survival in MM patients (Fig. 1).We focused the analysis on the 64 matrisome genes regulated in RPMI 8226 cells (Table 1), since we found the same classes of ECM glycoproteins, regulators, or secreted factors regulated in MM.1 S cells (Supplementary Table 2).Seven out of the 64 matrisome genes, TGFBI, C1QA, S100A6, IGF1, HPSE, CXCL12, and CXCL8, showed an association with progression-free and overall survival (Fig. 1, Supplementary Fig. 4) in a previously published cohort of MM patients (n = 387) [9].TGFBI is an N2-driven target gene with low expression being associated with adverse overall survival (Fig. 1).Accordingly, a global DNA hypermethylation analysis linked the methylation status of TGFBI to an unfavorable prognosis [10].We further identified C1QA as a novel N2-regulated ECM gene.High levels of C1QA were associated with better prognosis of MM patients (Fig. 1).C1QA encodes the A-chain polypeptide of serum complement subcomponent C1q binding to immunoglobulins complexed to antigen and initiating the complement cascade [11].In skin cutaneous melanoma, C1QA is a novel prognostic biomarker that has a function as a core TME-related gene [11].Similarly, high levels of NOTCH-driven EMI domain containing 1 (EMID1) correlate with tumor-infiltrating immune cells and are associated with a favorable prognosis in lung adenocarcinoma [12].S100A6 is a Ca 2+ -binding protein that belongs to the S100 family controlling cell growth, differentiation, and survival in cancer and cancer stem cells [13].S100A6 binds to ECM-associated proteins such as LUM, PRELP, IGFBP-1, and high serum levels are positively correlated with cancer progression of gastric, non-small cell lung, ovarian, and urinary bladder cancer [13].We showed that S100A6 is downregulated after N2 knockdown in MM cells, and high levels were associated with adverse prognosis of MM patients (Fig. 1).Moreover, S100 proteins are classical binding partners of ANXA2, and in pancreatic cancer the interaction between S1006 and ANXA2 promotes motility and invasiveness of cancer cells [14].In addition, N2 controls the expression of the ECM regulator HPSE that cleaves heparan sulfate glycosaminoglycans from proteoglycan core proteins to small oligosaccharides [15].HPSE promotes shedding of syndecan-1 from the MM cell surface, modulates the expression of proteases, alters histone acetylation and gene expression, and promotes tumor growth, angiogenesis, and metastasis of MM cells [15].
Taken together, our data show that both NOTCH receptors participate in the transcriptional control of ECM glycoproteins (TGFBI), ECM-affiliated proteins (C1QA), ECM regulators (HPSE) and secreted factors (S100A6, IGF1) in MM cells in vitro, proofing to be of prognostic significance in clinical settings.Our data confirm that the TME and ECM represented by a tumorassociated matrisome contain potential biomarkers and support findings in omental metastasis of ovarian cancer, in which a 22matrisome gene and protein signature has been identified, predicting overall survival in solid cancers such as breast, head, and neck squamous cell carcinoma, non-small-cell lung adenoma, kidney clear cell carcinoma, hepatocellular carcinoma, colon cancer or pancreatic ductal adenocarcinoma [1].In MM, similar signatures with prognostic significance should be refined and may confer impact in diagnostic/prognostic classification and the characterization of therapeutic targets as in colorectal cancer [1].However, further studies are required to comprehensively answer the question how expression changes in NOTCHdriven matrisome-associated proteins in the BM niche promote MM growth and dissemination.

Table 1 .
Expression of matrisome genes in RPMI 8226 cells after N1 and N2 knockdown.