Decreased LIMD1 Gene Expression Is Associated with HIF-1α Over-Expression and Angiogenesis in Multiple Myeloma

Enhanced angiogenesis in multiple myeloma (MM) is an important step in its tumor growth, invasion, and metastasis into the bone marrow. The bone marrow is a complex and dynamic microenvironment with multiple cell types contributing to niches that support myeloma tumor cell proliferation. One of the important microenvironmental factors, hypoxia-inducible factor 1α (HIF-1α), has been known to be associated with increased angiogenesis and metastatic potential as well as poor prognosis in MM. However, the role of HIF-1α in the etiology, pathogenesis and possible treatment of MM remains unclear. LIM domain-containing protein (LIMD1), a tumor suppressor molecular links prolyl hydroxylases (PHD1, 2 or 3) to von Hippel–Lindau (VHL) as a molecular scaffold, PHDs-LIMD1-VHL. This results in down-regulation of HIF-1α.

Following informed consent, ten bone marrow aspirates and five biopsies were obtained from MM patients and healthy subjects, respectively. Bone marrow mononuclear cells (BMMCs) were isolated by using density-gradient centrifugation with Histopaque-1077 (Sigma, St Louis). For gene expression studies, total RNA was isolated from BMMCs. RNA was reverse-transcribed into cDNA and amplified using the Thermo-Script RT-PCR System and PCR performed again using the GeneAmp PCR System 9700. Protein expression in BMMCs from MM and healthy subjects was determined using Western blot analysis and immunohistochemical (IHC) staining.

The results showed that LIMD1 gene expression in BMMCs as assessed using RT-PCR in MM patients was reduced compared to healthy subjects. In addition, PHD1 gene expression in BMMCs from MM patients was markedly reduced whereas PHD2 and PHD3 expression was similar to healthy subjects' BMMCs. The gene expression of VHL1, VHL2, and VHL3 in BMMCs was similar in MM patients compared to healthy subjects. Notably, LIMD1 gene expression in BMMCs from patients with monoclonal gammopathy of undetermined significance and primary amyloidosis patients was lover than among patients with progressive MM. Furthermore, the results of IHC staining showed LIMD1 protein expression in biopsy samples from MM patients with progressive disease was deceased and associated with high expression of HIF-1α. We also determined LIMD1 gene expression in MM patients treated with or without bortezomib. The results showed that this proteasome inhibitor did not affect LIMD1 expression.

LIMD1 expression is reduced with down-regulation of PHD1 and up-regulation of HIF-1α in MM patients. The proteasome inhibitor did not inhibit LIMD1 gene expression in our experiments although it has reported that bortezomib inhibits HIF-1α levels in MM. The results from these studies suggest that LIMD1 may be an important key regulator of HIF1-1a and angiogenesis in MM. It has been reported that knocking out the LIMD1 gene will lead to over-expression of a single gene K-Ras and result in the development of lung adenocarcinoma. We are currently investigating the other pathways that are impacted by the reduction of LIMD1 expression, and how this may lead to the pathogenesis of MM.

No relevant conflicts of interest to declare.