Insulin Like Growth Factor Binding Protein 7 (IGFBP7) Is Downregulated in Multiple Myeloma with Consequneces for Myeloma Cell Growth and Bone Disease

Insulin like growth factor binding protein 7 (IGFBP7) has been described as a secreted tumor suppressor and inducer of apoptotic and senescence pathways, with downregulation in lung cancer, hepatocellular carcinoma, pancreatic carcinoma and other solid tumors, linked to poor prognosis. Recently, a more complex picture has emerged, with IGFBP7 shown to regulate leukemia-stromal cell interactions and to contribute to chemotherapy resistance and leukemia cell survival. IGFBP7 possesses high affinity binding sites for activin A, VEGF-A and insulin and – with lower affinity – to insulin-like growth factor 1 (IGF-1). IGFBP7 has so far not been investigated in multiple myeloma (MM). Our initial gene-expression studies focussing on microenvironmental factors revealed a significant downregulation of IGFBP7 in the MM microenvironment. We therefore aimed to further characterize the role of IGFBP7 in the pathophysiology of MM.

IGFBP7 expression was analyzed by gene expression profiling and quantitative PCR. MMCLs were treated with 5-aza-2x-deoxycytidine, Trichostatin A and recombinant human IGFBP7 for 96 hours. Viability was assessed by the proliferation kit 8. The percentage of apoptotic cells was analyzed by Annexin V/7-AAD staining. Pyrosequencing was performed by Varionostic^® GmbH. Immortalized human bone-marrow stromal cells were co-cultured with MMCLs for 72 h. Primary human BMSCs were kept in osteogenic differentiation medium for 7–14 days. Alkaline Phosphatase activity was determined using the AttoPhos^® AP Fluorescent Substrate System.

Microarray analysis in a large set of MGUS (n = 22) and MM patient samples (n = 329) as well as MM cell lines (MMCLs) (n = 17) demonstrated a significant downregulation of IGFBP7 in each entity compared to normal plasma cells (n = 10). Analyzing the mechanism of IGFBP7 silencing in MM revealed a median upregulation of 2.8 fold after treatment with 5-aza-2x-deoxycytidine and Trichostatin A in 5 of 7 MMCLs (range: 1.8 – 30.2, P < 0.05), suggesting that IGFBP7 is controlled via methylation. This was confirmed by pyrosequencing of the IGFBP7 promoter region in 3 primary MM cell samples and 2 MMCLs. We subsequently studied possible functional consequences of IGFBP7 alterations and found that treatment with recIGFBP7 for 96 hours significantly decreased viable cell numbers in 7 of 7 MMCLs tested (relative viability compared to control: 0.68 – 0.92; P < 0.05). This effect was due to an impairment of proliferation, as no increase in apoptosis could be detected. Initial data suggest upregulation of cell cycle regulator p21 by IGFBP7 as possibly underlying this effect. Studying the role of IGFBP7 in the MM microenvironment we observed a significant downregulation of IGFBP7 in BMSCs after co-culture with 4 of 5 MMCLs (relative expression compared to control: 0.06 – 0.50; P < 0.05). We also studied osteoblast development in vitro and found that IGFBP7 expression is significantly increased during osteogenesis. Treatment with recIGFBP7 further stimulated osteoblast (OB) activity up to 1.8 fold at day 14 (P < 0.05). Complementary to these in vitro data, IGFBP7 expression level was associated with the presence of myeloma bone disease in an independent set of bone-marrow CD138+ sorted cells from myeloma patients (n = 62) (P < 0.05; see Fig.).

Taken together, these results demonstrate that IGFBP7 is downregulated in MM cells by methylation, which likely contributes to loss of cell cycle control and proliferation of malignant plasma cells. IGFBP7 expression seems also to be suppressed in stromal cells in the vicinity of MM cells, which might be involved in the impairment of osteoblast development and contribute to myeloma bone disease. Upregulation of IGFBP7 might be a useful therapeutic intervention in the treatment of MM.

Figure:

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IGFBP7 expression in MM cells of newly diagnosed myeloma patients (n=62) is significantly decreased in patients with osteolytic lesions. Patients were grouped according to the absence (n = 16) or presence (n = 46) of osteolytic lesions at diagnosis. * P < 0.05 compared to control.

Figure:

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IGFBP7 expression in MM cells of newly diagnosed myeloma patients (n=62) is significantly decreased in patients with osteolytic lesions. Patients were grouped according to the absence (n = 16) or presence (n = 46) of osteolytic lesions at diagnosis. * P < 0.05 compared to control.

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No relevant conflicts of interest to declare.