Laurence Blavier

Modifying the soil to affect the seed: role of stromal-derived matrix metalloproteinases in cancer progression

In the 1980s, as the importance of matrix metalloproteinases (MMPs) in cancer progression was discovered, it was recognized that in most tumors these proteases were abundantly and sometimes exclusively expressed not by tumor cells, but by normal host-derived cells like fibroblasts, vascular endothelial cells, myofibroblasts, pericytes or inflammatory cells that contribute to the tumor microenvironment. Later experiments in mice deficient in specific MMPs revealed that host-derived MMPs play a critical role not only in tumor cell invasion, but also in carcinogenesis, angiogenesis, vasculogenesis and metastasis. Tumor cells secrete many factors, cytokines and chemokines that directly or indirectly increase the expression of these MMPs in the tumor microenvironment where they exert extracellular matrix (ECM) degrading and sheddase activities. The knowledge of the complex role that stromal-derived MMPs play in the interaction between tumor cells and stromal cells should allow us to consider specific windows in cancer treatment when MMP inhibition could have a valuable therapeutic effect.

The Contribution of Bone Marrow-Derived Cells to the Tumor Vasculature in Neuroblastoma Is Matrix Metalloproteinase-9 Dependent

The contribution of the tumor stroma to cancer progression has been increasingly recognized. We had previously shown that in human neuroblastoma tumors orthotopically implanted in immunodeficient mice, stromal-derived matrix metalloproteinase-9 (MMP-9) contributes to the formation of a mature vasculature by promoting pericyte recruitment along endothelial cells. Here we show that MMP-9 is predominantly expressed by bone marrow-derived CD45-positive leukocytes. Using a series of bone marrow transplantation experiments in MMP-9(+/+) and MMP-9(-/-) mice xenotransplanted with human neuroblastoma tumors, we show that bone marrow-derived MMP-9 is critical for the recruitment of leukocytes from bone marrow into the tumor stroma and for the integration of bone marrow-derived endothelial cells into the tumor vasculature. Expression of MMP-9 by bone marrow-derived cells in the tumor stroma is also critical for the formation of a mature vasculature and coverage of endothelial cells with pericytes. Furthermore, in primary human neuroblastoma tumor specimens of unfavorable histology, we observed a higher level of tumor infiltration with MMP-9 expressing phagocytic cells and a higher degree of coverage of endothelial cells by pericytes when compared with tumor specimens with a favorable histology. Taken together, the data show that in neuroblastoma, MMP-9 plays a critical role in the recruitment of bone marrow-derived cells to the tumor microenvironment where they positively contribute to angiogenesis and tumor progression.

Tissue inhibitors of metalloproteinases (TIMP) in invasion and proliferation.

Tissue inhibitors of matrix metalloproteinases (TIMPs) are a family of natural inhibitors that control the activity of matrix metalloproteinases (MMPs) in the extracellular matrix (ECM). Four members of this family have been so far characterized in a variety of species. These inhibitors share a similar structural feature characterized by the presence of 12 cysteine residues involved in disulfide bonds and a similar function by their ability to form inhibitory complexes with MMPs. The role of TIMPs in cancer has been the subject of conflicting reports with an antitumor activity reported by some investigators and a growth stimulation activity reported by others. Here we will discuss a series of data obtained in our laboratory supporting a role of TIMPs not only as inhibitors of invasion but also as regulators of cell growth. Using placental development as an example of a regulated invasive process, we have observed that the levels of TIMP‐2 and TIMP‐3 steadily increase between day 14.5 and 17.5 post‐coitus. TIMPs are selectively expressed by spongiotrophoblastic cells that separate the labyrinthine zone, rich in fetal blood vessels and maternal blood sinuses, from the zone of giant cells forming the border between fetal and maternal tissues. TIMPs are also potent inhibitors of tumor growth in vivo. In melanoma cells, we have previously reported that over‐expression of TIMP‐2 inhibits the growth of tumors implanted in the skin of scid mice. This growth inhibition seems independent of angiogenesis but dependent on the collagen matrix. We observed that in the presence of fibrillar type I collagen, melanoma cells undergo a growth arrest at the G1 to S interphase transition of the cell cycle. This arrest is specific to the fibrillar structure of collagen because it is not observed in the presence of non‐fibrillar collagen or other ECM components. It is associated with a specific upregulation of the cyclin inhibitor p27KIPI. The data therefore indicate that anchorage independent cells remain sensitive to growth regulatory signals that originate from the ECM and that these signals can specifically block tumor cell cycle. Thus our concept of the role of protease inhibitors such as TIMPs in cancer has substantially changed from an initial focus on inhibition of tumor invasion and metastasis to a broader focus on being molecules that‐via their function as regulators of the ECM homeostasis‐can control tumor cell growth.

Tissue inhibitors of matrix metalloproteinases in cancer.

ABSTRACT: Tissue inhibitors of metalloproteinases (TIMPs) play a key regulatory role in the homeostasis of the extracellular matrix (ECM) by controlling the activity of matrix metalloproteinases (MMPs). Some TIMPs have a second function as well, unrelated to their antiMMP activity, which affects cell proliferation and survival. The role of these inhibitors in cancer has been the subject of extensive investigations that have examined their biological activity in tumor growth, invasion, metastasis and angiogenesis, as well as their potential use in the diagnosis and treatment of human cancer.

Matrix metalloproteinases play an active role in Wnt1-induced mammary tumorigenesis.

The Wnt signaling transduction pathway plays a critical role in the pathogenesis of several murine and human epithelial cancers. Here, we have used mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice, which develop spontaneous mammary adenocarcinoma, to examine whether matrix metalloproteinases (MMPs)--a family of extracellular proteases implicated in multiple steps of cancer progression--contributed to Wnt1-induced tumorigenesis. An analysis of the expression of several MMPs by RT-PCR and in situ hybridization revealed an increase in the expression of MMP-2, MMP-3, MMP-9, MMP-13, and MT1-MMP (MMP-14) in hyperplastic glands and in mammary tumors of MMTV-Wnt1 transgenic mice. Interestingly, whereas MMP-2, MMP-3, and MMP-9 were exclusively expressed by stromal cells in mammary tumors, MMP-13 and MT1-MMP were expressed by transformed epithelial cells in addition to the tumor stroma. To determine whether these MMPs contributed to tumorigenesis, MMTV-Wnt1 mice were crossed with transgenic mice overexpressing tissue inhibitor of metalloproteinase-2-a natural MMP inhibitor-in the mammary gland. In the double MMTV-Wnt1/tissue inhibitor of metalloproteinases-2 transgenic mice, we observed an increase in tumor latency and a 26.3% reduction in tumor formation. Furthermore, these tumors grew at a slower rate, exhibited an 18% decrease in proliferative rate, and a 12.2% increase in apoptotic rate of the tumor cells in association with a deficit in angiogenesis when compared with tumors from MMTV-Wnt1 mice. Thus, for the first time, the data provides evidence for the active role of MMPs in Wnt1-induced mammary tumorigenesis.

Bone Marrow–Derived Mesenchymal Stromal Cells Promote Survival and Drug Resistance in Tumor Cells

Bone marrow mesenchymal stromal cells (BMMSC) have antitumorigenic activities. Here, we hypothesized that circulating BMMSC are incorporated into tumors and protect tumor cells from therapy-induced apoptosis. Adherent cells harvested from murine bone marrow and expressing phenotypic and functional characteristics of BMMSC were tested for their antitumor activity against murine 4T1 mammary adenocarcinoma and LL/2 Lewis lung carcinoma cells. BMMSC but not NIH3T3 or murine skin fibroblasts stimulated the expansion of 4T1 cells in three-dimensional (3D) cocultures, and conditioned medium (CM) from these cells increased the viability of 4T1 and LL/2 cells in two-dimensional (2D) cultures. 4T1 cells exposed to BMMSC CM exhibited a 2-fold reduction in apoptosis under low serum concentrations (0.5% to 1%). Furthermore, exposure of 4T1 and LL/2 cells to BMMSC CM increased their viability in the presence of paclitaxel or doxorubicin at therapeutic concentrations. This effect was accompanied by reductions in caspase-3 activity and Annexin V expression. When coinjected with 4T1 cells in the mammary fat pad of mice subsequently treated with doxorubicin, BMMSC (and not fibroblasts) also inhibited drug-induced apoptosis in tumor cells by 44%. We demonstrated that BMMSC were attracted by 4T1 and LL/2 cells but not by NIH3T3 cells in vitro and that when injected intravenously in 4T1 tumor-bearing mice, these cells (and not NIH3T3) were specifically detected in tumors within 12 to 18 days in which they preferentially localized at the invasive front. Overall, our data identify BMMSC as an important mediator of tumor cell survival and treatment resistance in primary tumors. Mol Cancer Ther; 13(4); 962–75. ©2014 AACR.

Stromelysin-1 (MMP-3) is a target and a regulator of Wnt1-induced epithelial-mesenchymal transition (EMT)

Matrix metalloproteinases (MMPs) play a well-defined role in later stages of tumor progression. However, there has been evidence that they also contribute to earlier stages of malignant transformation. The Wnt signaling transduction pathway plays a critical role in development and in the pathogenesis of many epithelial cancers. Here we have used Wnt1-induced epithelial-mesenchymal transition (EMT) in C57MG murine mammary epithelial cells to study the role of MMPs in this early step of malignant progression. Overexpression of Wnt1 in C57MG cells promoted EMT, the translocation of b-catenin from the cell membrane to the nucleus and its transcriptional activity, cell proliferation and cell motility. Simultaneously, we observed an increased expression of stromelysin-1 (MMP-3) and a 5.5-fold increase in MMP-3 promoter activity in C57MG cells expressing Wnt1 compared with C57MG cells. Treatment of Wnt-overexpressing cells with MMP inhibitor AG3340 decreased MMP-3 expression. We also found evidence that MMP-3 and Wnt3a cooperate in enhancing the transcriptional activity of β-catenin in C57MG cells. Consistently, the effects of Wnt1 on EMT, proliferation, and migration were inhibited by MMP inhibitors, or upon downregulation of MMP-3 by siRNA. These results suggest that MMP-3 is both a direct transcriptional target and a necessary contributor of the Wnt/b-catenin signaling pathway.

Interaction between bone marrow stromal cells and neuroblastoma cells leads to a VEGFA-mediated osteoblastogenesis.

The potential role of osteoblasts in bone and bone marrow (BM) metastases in neuroblastoma (NBL) remains unclear. In this study, we examined the effect of NBL cells on the osteoblastic differentiation of BM‐derived mesenchymal stromal cells (BMMSC). We show that the presence of NBL cells enhanced the osteoblastic differentiation of BMMSC driven by bone morphogenetic protein (BMP)‐4, in the absence of any effect on NBL cell proliferation. Expression profiles of BMMSC driven toward osteoblastic differentiation revealed an increase in vascular endothelial growth factor A (Vegfa) expression in the presence of NBL cells. We demonstrated that NBL cells increased BMMSC‐derived VEGFA mRNA and protein and that this was enhanced by BMP‐4. However, in similar conditions, neither the addition of an mVEGFA blocking antibody nor exogenous recombinant (r) mVEGFA affected osteoblastic differentiation. In contrast, siRNA‐ mediated knock‐down of VEGFA in BMMSC prevented osteoblastic differentiation in BMP‐4‐treated cocultures, an effect that was not reversed in the presence of rmVEGFA. An analysis of murine bones injected with hNBL cells revealed an increase of mVEGFA producing cells near tumor cells concomitantly with an increase in Vegfa and Runx2 mRNA. This coincided with an increase in osteoclasts, in Rankl/Opg mRNA ratio and with the formation of osteolytic lesions. Thus NBL cells promote osteoblastogenesis in the BM by increasing VEGFA expression in BMMSC. Our study provides a new insight into the role of VEGFA in NBL metastases by pointing to the role of stroma‐derived intracrine VEGFA in osteoblastogenesis.

Abstract 2601: Interaction between bone marrow mesenchymal stem cells and neuroblastoma cells leads to osteoblastogenesis and VEGFA expression.

Bone metastasis in patients with neuroblastoma often heralds a poor prognosis despite intensive therapy. We previously reported that neuroblastoma cells interact with osteoclasts and bone marrow mesenchymal stem cells (BMSC), and that the nature of this interaction is both osteolytic and pro-tumorigenic. In many cancers, the suppression of osteoblastogenesis plays a part in increased osteolysis. However, little is known about the interaction between neuroblastoma cells and osteoblasts within the bone marrow microenvironment. We have examined this interaction within the context of BMSC differentiation to osteoblasts by bone morphogenetic protein 4 (BMP4). To examine the effect of secreted factors from neuroblastoma cells on the BMP4 induced differentiation of murine BMSC, we cultured them in the presence or absence of neuroblastoma cells using culture inserts. We found that the mRNA encoding the osteoblast marker alkaline phosphatase (ALP) was increased by 10 fold and ALP protein level and activity were 1.5 fold higher in the presence versus absence of neuroblastoma cells. The mRNAs encoding the osteoblast master regulator Runx2 and its target osteocalcin were increased by 4 and 1.5 fold (p 2 fold. Together, these data indicate that neuroblastoma cells synergise with BMP4 to promote osteoblast growth and differentiation from BMSC. To understand the mechanism by which neuroblastoma cells enhance osteoblastogenesis, we compared the mRNA profile of murine MSC driven to differentiate into osteoblasts by doxycycline-induced Runx2 expression (ST2 Rx2dox ) in the presence or absence of neuroblastoma cells. Triplicate RNA samples were labeled and hybridized to Illumina BeadChips containing probes for >19,100 genes. In silico analysis identified 9 genes upregulated when co-cultured with neuroblastoma cells. Of these, two (Spondin2 and VEGFA) encode proteins with established roles in osteoblastogenesis. To validate these findings in primary murine BMSC, we showed by RT-qPCR a 2 fold increase in VEGFA mRNA in BMSC treated with BMP4 in the presence versus absence of neuroblastoma cells. ELISA analysis demonstrated a parallel 2 fold increase in intra- and extracellular VEGFA protein levels. We then found that the treatment of BMSC with BMP4 and VEGFA together led to a 1.5 fold increase (p Our data suggest that in contrast to many cancers that suppress osteoblastogenesis, neuroblastoma may promote osteoblastogenesis in part by enhancing the expression of VEGFA in BMSC. We are currently investigating the consequence of this osteoblastogenic activity on neuroblastoma bone metastasis. Citation Format: Josephine H. HaDuong, Laurence Blavier, Sanjeev K. Baniwal, Baruch Frenkel, Yves A. DeClerck. Interaction between bone marrow mesenchymal stem cells and neuroblastoma cells leads to osteoblastogenesis and VEGFA expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2601. doi:10.1158/1538-7445.AM2013-2601

Abstract 1478: Bone marrow mesenchymal stem cells suppress tumor cell apoptosis and are selectively recruited to early-stage tumors

Bone marrow mesenchymal stem cells (BMMSC) are recruited to primary tumors and have been reported to display pro-tumorigenic as well as anti-tumorigenic activities. However, the mechanisms behind their effect on tumor development are still unclear. We have isolated murine bone marrow aspirates and plated them on fibronectin/collagen I, which allows expansion of an adherent stromal cell population in 3-6 weeks. These cells express the CD44 hyaluronic acid receptor in the absence of hematopoietic (CD34), myeloid (CD45), and endothelial (CD31/VEGFR2) markers and display both spontaneous and inducible adipogenesis/ osteoblastogenesis in culture. 3D matrigel cultures of murine 4T1 mammary carcinoma cells revealed enhanced generation of tumor spheroids in the presence of BMMSC or their conditioned media. Conditioned media from BMMSC also increased 4T1 cell expansion in 2D monolayer cultures, with loss of this phenomenon after heat inactivation suggesting the presence of protein-based signaling. Cell cycle analysis of 4T1 cells treated with BMMSC conditioned media revealed no statistically significant change in mitotic potential in either growth (serum present) or growth-arrest (serum depleted) phase, as determined by PI staining and BrdU uptake. However, analysis of apoptosis in 4T1 cells treated with BMMSC conditioned media showed increased percentages of live cells by PI uptake and AnnexinV staining, as well as a reduction in caspase-3 and caspase-9 activation by colorimetric assay, as the cells entered growth arrest phase. Furthermore, treatment with BMMSC conditioned media preserved viability in 4T1 cells exposed to paclitaxel under no-serum conditions. Complementary studies of BMMSC tumor-directed migration indicate that BMMSC show preferential in vitro recruitment by murine tumor cell lines which generate reactive stroma (4T1, LL/2 lung carcinoma) vs. no stroma (B16 melanoma). In vivo recruitment of BMMSC by early-stage/slow-growing subcutaneous 4T1 tumors was also visualized by bioluminescent imaging, red fluorescence, and magnetic resonance imaging of BMMSC labeled with iron-nanoparticles. These results were confirmed by immunohistochemical evaluation of iron deposits by Prussian blue stain and by immunofluorescent detection of PKH26-labeled BMMSC. Overall, our data identify BMMSC as an important mediator of tumor cell survival/treatment resistance and reveal an enhanced tropism of BMMSC toward early 4T1 tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1478. doi:1538-7445.AM2012-1478