Timothy N. Trotter

Heparanase inhibits osteoblastogenesis and shifts bone marrow progenitor cell fate in myeloma bone disease.

A major cause of morbidity in patients with multiple myeloma is the development and progression of bone disease. Myeloma bone disease is characterized by rampant osteolysis in the presence of absent or diminished bone formation. Heparanase, an enzyme that acts both at the cell-surface and within the extracellular matrix to degrade polymeric heparan sulfate chains, is upregulated in a variety of human cancers including multiple myeloma. We and others have shown that heparanase enhances osteoclastogenesis and bone loss. However, increased osteolysis is only one element of the spectrum of myeloma bone disease. In the present study, we hypothesized that heparanase would also affect mesenchymal cells in the bone microenvironment and investigated the effect of heparanase on the differentiation of osteoblast/stromal lineage cells. Using a combination of molecular, biochemical, cellular and in vivo approaches, we demonstrated that heparanase significantly inhibited osteoblast differentiation and mineralization, and reduced bone formation in vivo. In addition, heparanase shifts the differentiation potential of osteoblast progenitors from osteoblastogenesis to adipogenesis. Mechanistically, this shift in cell fate is due, at least in part, to heparanase-enhanced production and secretion of the Wnt signaling pathway inhibitor DKK1 by both osteoblast progenitors and myeloma cells. Collectively, these data provide important new insights into the role of heparanase in all aspects of myeloma bone disease and strongly support the use of heparanase inhibitors in the treatment of multiple myeloma.

Myeloma cell–derived Runx2 promotes myeloma progression in bone

The progression of multiple myeloma (MM) is governed by a network of molecular signals, the majority of which remain to be identified. Recent studies suggest that Runt-related transcription factor 2 (Runx2), a well-known bone-specific transcription factor, is also expressed in solid tumors, where expression promotes both bone metastasis and osteolysis. However, the function of Runx2 in MM remains unknown. The current study demonstrated that (1) Runx2 expression in primary human MM cells is significantly greater than in plasma cells from healthy donors and patients with monoclonal gammopathy of undetermined significance; (2) high levels of Runx2 expression in MM cells are associated with a high-risk population of MM patients; and (3) overexpression of Runx2 in MM cells enhanced tumor growth and disease progression in vivo. Additional studies demonstrated that MM cell-derived Runx2 promotes tumor progression through a mechanism involving the upregulation of Akt/β-catenin/Survivin signaling and enhanced expression of multiple metastatic genes/proteins, as well as the induction of a bone-resident cell-like phenotype in MM cells. Thus, Runx2 expression supports the aggressive phenotype of MM and is correlated with poor prognosis. These data implicate Runx2 expression as a major regulator of MM progression in bone and myeloma bone disease.

Matricellular proteins as regulators of cancer metastasis to bone

Metastasis is the major cause of death in cancer patients, and a frequent site of metastasis for many cancers is the bone marrow. Therefore, understanding the mechanisms underlying the metastatic process is necessary for future prevention and treatment. The tumor microenvironment is now known to play a role in the metastatic cascade, both at the primary tumor and in metastatic sites, and includes both cellular and non-cellular components. The extracellular matrix (ECM) provides structural support and signaling cues to cells. One particular group of molecules associated with the ECM, known as matricellular proteins, modulate multiple aspects of tumor biology, including growth, migration, invasion, angiogenesis and metastasis. These proteins are also important for normal function in the bone by regulating bone formation and bone resorption. Recent studies have described a link between some of these proteins and metastasis of various tumors to the bone. The aim of this review is to summarize what is currently known about matricellular protein influence on bone metastasis. Particular attention to the contribution of both tumor cells and non-malignant cells in the bone has been given.

Heparanase promotes myeloma progression by inducing mesenchymal features and motility of myeloma cells

Bone dissemination and bone disease occur in approximately 80% of patients with multiple myeloma (MM) and are a major cause of patient mortality. We previously demonstrated that MM cell-derived heparanase (HPSE) is a major driver of MM dissemination to and progression in new bone sites. However the mechanism(s) by which HPSE promotes MM progression remains unclear. In the present study, we investigated the involvement of mesenchymal features in HPSE-promoted MM progression in bone. Using a combination of molecular, biochemical, cellular, and in vivo approaches, we demonstrated that (1) HPSE enhanced the expression of mesenchymal markers in both MM and vascular endothelial cells; (2) HPSE expression in patient myeloma cells positively correlated with the expression of the mesenchymal markers vimentin and fibronectin. Additional mechanistic studies revealed that the enhanced mesenchymal-like phenotype induced by HPSE in MM cells is due, at least in part, to the stimulation of the ERK signaling pathway. Finally, knockdown of vimentin in HPSE expressing MM cells resulted in significantly attenuated MM cell dissemination and tumor growth in vivo. Collectively, these data demonstrate that the mesenchymal features induced by HPSE in MM cells contribute to enhanced tumor cell motility and bone-dissemination.

Runx2 Suppression by miR-342 and miR-363 Inhibits Multiple Myeloma Progression

In multiple myeloma, abnormal plasma cells accumulate and proliferate in the bone marrow. Recently, we observed that Runx2, a bone-specific transcription factor, is highly expressed in multiple myeloma cells and is a major driver of multiple myeloma progression in bone. The primary goal of the present study was to identify Runx2-targeting miRNAs that can reduce tumor growth. Expression analysis of a panel of miRNAs in multiple myeloma patient specimens, compared with healthy control specimens, revealed that metastatic multiple myeloma cells express low levels of miR-342 and miR-363 but high levels of Runx2. Reconstituting multiple myeloma cells (CAG) with miR-342 and miR-363 reduced the abundance of Runx2 and the expression of metastasis-promoting Runx2 target genes RANKL and DKK1, and suppressed Runx2 downstream signaling pathways Akt/β-catenin/survivin, which are required for multiple myeloma tumor progression. Intravenous injection of multiple myeloma cells (5TGM1), stably overexpressing miR-342 and miR-363 alone or together, into syngeneic C57Bl/KaLwRij mice resulted in a significant suppression of 5TGM1 cell growth, decreased osteoclasts and increased osteoblasts, and increased antitumor immunity in the bone marrow, compared with mice injected with 5TGM1 cells expressing a miR-Scramble control. In summary, these results demonstrate that enhanced expression of miR-342 and miR-363 in multiple myeloma cells inhibits Runx2 expression and multiple myeloma growth, decreases osteolysis, and enhances antitumor immunity. Thus, restoring the function of Runx2-targeting by miR-342 and miR-363 in multiple myeloma cells may afford a therapeutic benefit by preventing multiple myeloma progression. Implications: miR-342 and miR-363–mediated downregulation of Runx2 expression in multiple myeloma cells prevents multiple myeloma progression. Mol Cancer Res; 16(7); 1138–48. ©2018 AACR.

Abstract 2525: Targeting myeloma cell-derived runx2 by miRNAs suppresses multiple myeloma growth and progression

In Multiple myeloma (MM), abnormal plasma cells accumulate in the bone marrow and spread to new bone sites. However, the mechanisms underlying this spread of MM cells remain unclear. Runx2 is a bone specific transcription factor upregulated in various human tumors, including MM. Our studies have previously demonstrated that Runx2 is a major driver of MM progression in bone. In the present study, our goal was to identify miRNAs targeting Runx2 to reduce tumor growth and dissemination of MM to new bone sites and asses their therapeutic potential. Expression analysis of a panel of miRNA9s regulating Runx2 revealed an inverse relationship between Runx2 expression and two miRNAs: miR-342 and miR-363. miRNA expression analysis using the Gene Expression Omnibus database showed that miR-342 and miR-363 are highly expressed in plasma cells of normal donors (n= 3), while Runx2 is detected at very low levels. In contrast, MM cells of patients with newly diagnosed (n=23) and relapsed MM (n=17) expressed very little miR-342 and miR-363 but showed high levels of Runx2 expression. Reconstituting CAG human MM cells with synthetic miR-342, miR-363 or miR-342+miR-363 in combination reduced the expression of Runx2 and the metastasis-promoting Runx2 target genes RANKL, DKK1 and DMP1. Tumor cell viability and migration were also decreased compared to control in vitro. We then transfected miR-342, miR-363 or miR-342+miR-363 in mouse 5TGM1 MM cells expressing high levels of Runx2 and tested bone-homing and growth in syngenic C57Bl/KaLwRij mice by intravenous injection. Mouse sera were collected at week 2 and 4 after tumor cell injection and the levels of IgG2bκ, a marker of 5TGM1 cells, were measured by ELISA. The results showed significantly decreased IgG2bκ levels in mice bearing miR-342 or miR-363 transfected tumors compared to mice bearing control miR-scramble tumors. Additionally, miR-342+miR-363 co-transfection produced a synergistic effect on reducing tumor growth. Mechanistic studies demonstrated that miR-342 and miR-363 induced Runx2 suppression, results in inhibition of Runx2-downstream signaling pathways Akt/β-catenin/survivin, which are required for MM tumor progression. In conclusion, we have identified two novel miRNAs, miR-342 and miR-363, that negatively regulate Runx2 expression in MM cells. We further demonstrated that enhanced expression of miR-342 or miR-363 in MM cells inhibits MM growth in vivo. Thus, miR-342 and miR-363 are potential novel markers of MM prognosis and can be developed as new therapeutic drugs for MM treatment. Citation Format: Pramod S. Gowda, Mohammad Q. Hassan, Timothy N. Trotter, Yang Yang. Targeting myeloma cell-derived runx2 by miRNAs suppresses multiple myeloma growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2525. doi:10.1158/1538-7445.AM2017-2525

Abstract 4102: The involvement of EMT in heparanase promoted bone-metastasis in multiple myeloma

Multiple myeloma (MM), a hematologic malignancy, preferentially grows in bone marrow and frequently metastasizes between bones. Our previous studies have demonstrated that heparanase (HPSE), an enzyme overexpressed in MM cells, promotes MM bone-metastasis and progression. However, the mechanism of HPSE action is unclear. Epithelial-mesenchymal-transition (EMT) is a process giving epithelial cells the features of mesenchymal cells which has been shown to be critical in the metastatic process of epithelial-origin tumors. Although MM is not an epithelial-derived malignancy, MM cells indeed express epithelial marker E-cadherin and mesenchymal marker Vimentin, and have the ability to adhere to and interact with the extracellular matrix (ECM). In the present study, we determined the role of EMT in HPSE-induced MM bone metastasis via: (1) Using Western blot to evaluate the expression of EMT markers E-cadherin, Vimentin and Fibronectin in MM cells that were transfected with empty vector (HPSE-low) or HPSE cDNA (HPSE-high), as well as MM cells treated with recombinant human HPSE (rHPSE) for 48hrs; (2) staining HPSE and EMT markers on tumors established by HPSE-low or HPSE-high cells in SCID mice (n = 10), and on bone marrow biopsies of 35 MM patients; (3) knocking down Vimentin in HPSE-high-luc MM cells by Vimentin shRNA (VIM k/d-luc cells) and testing bone homing of VIM k/d-luc cells in SCID mice by i.v. and s.c. injection of these cells. Tumor growth and bone-metastasis was determined by measuring human kappa levels (a soluble marker of MM cells) in mouse sera and weekly bioluminescent imaging. Western blots demonstrated HPSE-high and rHPSE treated MM cells expressed significantly higher levels of the mesenchymal markers Vimentin and Fibronectin, and lower levels of the epithelial marker E-cadherin, compared to HPSE-low or PBS treated MM cells. Significantly increased expression of Vimentin and decreased expression of E-cadherin was revealed in tumors formed by HPSE-high cells compared with the tumors formed by HPSE-low cells. Immunostaining of bone marrow biopsies of MM patients indicated HPSE and Vimentin expression in MM cells have a significant positive correlation (rs = 0.414, p = 0.014). Finally, both SCID-i.v. injection and SCID-s.c. injection models showed that knocking down mesenchymal marker Vimentin in HPSE-high MM cells successfully blocked HPSE-induced bone metastasis and inhibited tumor growth, compared with mice bearing shRNA control HPSE-high MM cells. In conclusion, our studies demonstrated (1) EMT-like features are a novel biomarker of poor prognosis in MM; (2) HPSE promotes MM metastasis to bone and disease progression, at least in part, through inducing EMT-like features in MM cells; (3) Inhibition of Vimentin expression in MM cells impairs HPSE-induced tumor growth and bone metastasis. Note: This abstract was not presented at the meeting. Citation Format: Qianying Pan, Juan Li, Patrick D. Rowan, Timothy N. Trotter, Yang Yang. The involvement of EMT in heparanase promoted bone-metastasis in multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4102. doi:10.1158/1538-7445.AM2015-4102

Abstract 2387: Pre-adipocytes promote myeloma homing to and growth in bone by secretion of soluble molecules

Multiple myeloma (MM) is a hematologic malignancy of plasma cells that thrives in and metastasizes throughout the bone marrow microenvironment. This microenvironment is host to a variety of cell types, including osteoblasts, osteoclasts and stromal cells, that become altered both at the primary tumor site and distant bone sites to support the growth and spread of MM cells. We recently reported that under certain conditions, such as when MM cells overexpress heparanase, MM cells redirect osteoblast progenitors from osteoblastogenesis towards adipogenesis. In addition, adipocytes naturally accumulate in aging marrow and adipocytes/adipose tissues have endocrine functions, secreting soluble factors such as adiponectin and leptin. Thus, adipocytes have the potential to be influential on MM cell behavior. Here, we hypothesized that adipocytes play an active role in MM progression through the secretion of soluble molecules that promote MM growth and metastasis to bone. To test this we used a co-culture system in which 3T3-L1 mouse pre-adipocytes or mature adipocytes were separated by a porous membrane from 5TGM1 mouse MM cells, allowing cross-talk by secreted molecules but not direct cell-cell contact. We then tested the motility of these “educated” 5TGM1 MM cells by migration assay. Somewhat surprisingly, we found that it was not mature adipocyte but pre-adipocyte education that enhanced the migration ability of MM cells compared to MM cells cultured alone. In a separate experiment, we injected 5TGM1 MM cells cultured alone, with pre-adipocytes, or with mature adipocytes into syngeneic C57BL/KaLwRij mice via tail vein and monitored progression by bioluminescent imaging. Total tumor burden was evaluated by IgG2bκ (a soluble marker of 5TGM1 MM cells) levels in mouse serum. Compared to MM cells cultured alone, those cultured with pre-adipocytes homed to the bone sooner and grew faster whereas those cultured with mature adipocytes showed no difference in homing or growth. To identify factors that may contribute to this apparent difference, pre-adipocyte or mature adipocyte conditioned media (CM) was analyzed using a cytokines/chemokines array. The results showed that pre-adipocytes secrete significantly larger quantities of molecules HGF, MCP-1 and IL-6 than mature adipocytes. Additional migration assays also confirmed that MM cells migrate more towards pre-adipocyte CM than to fresh media alone or mature adipocyte CM. In conclusion, this study demonstrates that pre-adipocytes, through the secretion of a variety of soluble molecules, promote a more aggressive phenotype in MM cells and contribute to MM growth and bone homing. Citation Format: Timothy N. Trotter, Patrick D. Rowan, Qianying Pan, Yang Yang. Pre-adipocytes promote myeloma homing to and growth in bone by secretion of soluble molecules. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2387. doi:10.1158/1538-7445.AM2015-2387