Tetraspanin 7 (TSPAN7) expression is upregulated in multiple myeloma patients and inhibits myeloma tumour development in vivo.

BACKGROUND: Increased expression of the tetraspanin TSPAN7 has been observed in a number of cancers; however, it is unclear how TSPAN7 plays a role in cancer progression. METHODS: We investigated the expression of TSPAN7 in the haematological malignancy multiple myleoma (MM) and assessed the consequences of TSPAN7 expression in the adhesion, migration and growth of MM plasma cells (PC) in vitro and in bone marrow (BM) homing and tumour growth in vivo. Finally, we characterised the association of TSPAN7 with cell surface partner molecules in vitro. RESULTS: TSPAN7 was found to be highly expressed at the RNA and protein level in CD138(+) MM PC from approximately 50% of MM patients. TSPAN7 overexpression in the murine myeloma cell line 5TGM1 significantly reduced tumour burden in 5TGM1/KaLwRij mice 4 weeks after intravenous adminstration of 5TGM1 cells. While TSPAN7 overexpression did not affect cell proliferation in vitro, TSPAN7 increased 5TGM1 cell adhesion to BM stromal cells and transendothelial migration. In addition, TSPAN7 was found to associate with the molecular chaperone calnexin on the cell surface. CONCLUSION: These results suggest that elevated TSPAN7 may be associated with better outcomes for up to 50% of MM patients.

Myeloma plasma cells alter the bone marrow microenvironment by stimulating the proliferation of mesenchymal stromal cells.

Multiple myeloma is an incurable hematologic cancer characterized by the clonal proliferation of malignant plasma cells within the bone marrow. Numerous studies suggest that the myeloma plasma cells occupy and alter the stromal tissue of the bone marrow as a means of enhancing their survival and growth. However, the nature and magnitude of the changes to the stromal cell tissue remain to be determined. In this study, we used mesenchymal stromal cell and osteoblast-related cell surface marker expression (STRO-1 and alkaline phosphatase, respectively) and flow cytometry to enumerate mesenchymal stromal cell and osteoblast numbers in bone marrow recovered from myeloma patients at the time of diagnosis. Using this approach, we identified an increase in the number of STRO-1 positive colony forming mesenchymal stromal cells and a concomitant decrease in alkaline phophatase osteoblasts. Notably, this increase in mesenchymal stromal cell numbers correlated closely with plasma cell burden at the time of diagnosis. In addition, in comparison with the osteoblast population, the STRO-1+ mesenchymal stromal cell population was found to express higher levels of plasma cell- and osteoclast-activating factors, including RANKL and IL-6, providing a mechanism by which an increase in mesenchymal stromal cells may promote and aid the progression of myeloma. Importantly, these findings were faithfully replicated in the C57BL/KaLwRij murine model of myeloma, suggesting that this model may present a unique and clinically relevant system in which to identify and therapeutically modulate the bone microenvironment and, in turn, alter the progression of myeloma disease.

Circulating N-cadherin levels are a negative prognostic indicator in patients with multiple myeloma.

N-cadherin (cadherin 2, type 1, N-cadherin (neuronal); CDN2) is a homotypic adhesion molecule that is upregulated in breast, prostate and bladder cancer. Here we investigated the prognostic significance of upregulated N-cadherin expression in multiple myeloma (MM). Our results indicate that N-cadherin protein and gene expression is abnormally increased in trephine biopsies and CD38(++) /CD138(+) plasma cells from MM patients, when compared with those of normal donors. In addition, levels of circulating N-cadherin were elevated in a subset of patients with MM (n = 81; mean: 14·50 ng/ml, range: 0-146·78 ng/ml), relative to age-matched controls (n = 27; mean: 2·66 ng/ml, range: 0-5·96 ng/ml), although this did not reach statistical significance. Notably, patients with abnormally high levels of N-cadherin (>6 ng/ml) had decreased progression-free survival (P = 0·036; hazard ratio: 1·94) and overall survival (P = 0·002; hazard ratio: 3·15), when compared with patients with normal N-cadherin levels (≤6 ng/ml). Furthermore, multivariate analyses revealed that the combination of N-cadherin levels and International Staging System (ISS) was a more powerful prognostic indicator than using ISS alone. Collectively, our studies demonstrate that circulating N-cadherin levels are a viable prognostic marker for high-risk MM patients.

Multiple functions of CXCL12 in a syngeneic model of breast cancer.

BACKGROUND: A growing body of work implicates chemokines, in particular CXCL12 and its receptors, in the progression and site-specific metastasis of various cancers, including breast cancer. Various agents have been used to block the CXCL12-CXCR4 interaction as a means of inhibiting cancer metastasis. However, as a potent chemotactic factor for leukocytes, CXCL12 also has the potential to enhance anti-cancer immunity. To further elucidate its role in breast cancer progression, CXCL12 and its antagonist CXCL12(P2G) were overexpressed in the syngeneic 4T1.2 mouse model of breast carcinoma. RESULTS: While expression of CXCL12(P2G) significantly inhibited metastasis, expression of wild-type CXCL12 potently inhibited both metastasis and primary tumor growth. The effects of wild-type CXCL12 were attributed to an immune response characterized by the induction of CD8+ T cell activity, enhanced cell-mediated cytotoxicity, increased numbers of CD11c+ cells in the tumor-draining lymph nodes and reduced accumulation of myeloid-derived suppressor cells in the spleen. CONCLUSIONS: This study highlights the need to consider carefully therapeutic strategies that block CXCL12 signaling. Therapies that boost CXCL12 levels at the primary tumor site may prove more effective in the treatment of metastatic breast cancer.

Hypoxia-inducible factor-2 is a novel regulator of aberrant CXCL12 expression in multiple myeloma plasma cells.

BACKGROUND: Multiple myeloma is an incurable malignancy of bone marrow plasma cells. Progression of multiple myeloma is accompanied by an increase in bone marrow angiogenesis. Studies from our laboratory suggest a role for the CXCL12 chemokine in this process, with circulating levels of CXCL12 correlating with bone marrow angiogenesis in patients with multiple myeloma. While the mechanisms responsible for aberrant plasma cell expression of CXCL12 remain to be determined, studies in other systems suggest a role for hypoxia and hypoxia-inducible transcription factors. DESIGN AND METHODS: The expression of hypoxia-inducible factor protein was examined in patients' bone marrow biopsy specimens using immunohistochemistry. The hypoxic regulation of CXCL12 was examined in multiple myeloma plasma cell lines using polymerase chain reaction and western blotting. The role of hypoxia-inducible factors-1 and -2 in the regulation of CXCL12 expression was examined using over-expression and short hairpin RNA knockdown constructs, electrophoretic mobility shift assays and chromatin immunoprecipitation. The contribution of CXCL12 to hypoxia-induced angiogenesis was examined in vivo using a subcutaneous murine model of neovascularization. RESULTS: Strong hypoxia-inducible factor-2 protein expression was detected in CD138(+) multiple myeloma plasma cells in patients' biopsy specimens. Prolonged exposure to hypoxia strongly up-regulated CXCL12 expression in multiple myeloma plasma cells and hypoxia-inducible factor-2 was found to play a key role in this response. Promoter analyses revealed increased hypoxia-inducible factor-2 binding to the CXCL12 promoter under hypoxic conditions. Over-expression of hypoxia-inducible factor in multiple myeloma plasma cells strongly induced in vivo angiogenesis, and administration of a CXCL12 antagonist decreased hypoxia-inducible factor-induced angiogenesis. CONCLUSIONS: Hypoxia-inducible factor-2 is a newly identified regulator of CXCL12 expression in multiple myeloma plasma cells and a major contributor to multiple myeloma plasma cell-induced angiogenesis. Targeting the hypoxic niche, and more specifically hypoxia-inducible factor-2, may represent a viable strategy to inhibit angiogenesis in multiple myeloma and progression of this disease.

PTTG1 expression is associated with hyperproliferative disease and poor prognosis in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is an incurable haematological malignancy characterised by the clonal proliferation of malignant plasma cells within the bone marrow. We have previously identified pituitary tumour transforming gene 1 (Pttg1) as a gene that is significantly upregulated in the haematopoietic compartment of the myeloma-susceptible C57BL/KaLwRij mouse strain, when compared with the myeloma-resistant C57BL/6 mouse. Over-expression of PTTG1 has previously been associated with malignant progression and an enhanced proliferative capacity in solid tumours. METHODS: In this study, we investigated PTTG1 gene and protein expression in MM plasma cells from newly diagnosed MM patients. Gene expression profiling was used to identify gene signatures associated with high PTTG1 expression in MM patients. Additionally, we investigated the effect of short hairpin ribonucleic acid (shRNA)-mediated PTTG1 knockdown on the proliferation of the murine myeloma plasma cell line 5TGM1 in vitro and in vivo. RESULTS: PTTG1 was found to be over-expressed in 36-70 % of MM patients, relative to normal controls, with high PTTG1 expression being associated with poor patient outcomes (hazard ratio 2.49; 95 % CI 1.28 to 4.86; p = 0.0075; log-rank test). In addition, patients with high PTTG1 expression exhibited increased expression of cell proliferation-associated genes including CCNB1, CCNB2, CDK1, AURKA, BIRC5 and DEPDC1. Knockdown of Pttg1 in 5TGM1 cells decreased cellular proliferation, without affecting cell cycle distribution or viability, and decreased expression of Ccnb1, Birc5 and Depdc1 in vitro. Notably, Pttg1 knockdown significantly reduced MM tumour development in vivo, with an 83.2 % reduction in tumour burden at 4 weeks (p < 0.0001, two-way ANOVA). CONCLUSIONS: This study supports a role for increased PTTG1 expression in augmenting tumour development in a subset of MM patients.

SAMSN1 is a tumor suppressor gene in multiple myeloma.

Multiple myeloma (MM), a hematological malignancy characterized by the clonal growth of malignant plasma cells (PCs) in the bone marrow, is preceded by the benign asymptomatic condition, monoclonal gammopathy of undetermined significance (MGUS). Several genetic abnormalities have been identified as critical for the development of MM; however, a number of these abnormalities are also found in patients with MGUS, indicating that there are other, as yet unidentified, factors that contribute to the onset of MM disease. In this study, we identify a Samsn1 gene deletion in the 5TGM1/C57BL/KaLwRij murine model of myeloma. In addition, SAMSN1 expression is reduced in the malignant CD138+ PCs of patients with MM and this reduced expression correlates to total PC burden. We identify promoter methylation as a potential mechanism through which SAMSN1 expression is modulated in human myeloma cell lines. Notably, re-expression of Samsn1 in the 5TGM1 murine PC line resulted in complete inhibition of MM disease development in vivo and decreased proliferation in stromal cell-PC co-cultures in vitro. This is the first study to identify deletion of a key gene in the C57BL/KaLwRij mice that also displays reduced gene expression in patients with MM and is therefore likely to play an integral role in MM disease development.