Inhibition of AQP1 Hampers Osteosarcoma and Hepatocellular Carcinoma Progression Mediated by Bone Marrow-Derived Mesenchymal Stem Cells

Abstract

The complex cross-talk between tumor cells and their surrounding stromal environment plays a key role in the pathogenesis of cancer. Among several cell types that constitute the tumor stroma, bone marrow-derived mesenchymal stem cells (BM-MSCs) selectively migrate toward the tumor microenvironment and contribute to the active formation of tumor-associated stroma. Therefore, here we elucidate the involvement of BM-MSCs to promote osteosarcoma (OS) and hepatocellular carcinoma (HCC) cells migration and invasion and deepening the role of specific pathways. We analyzed the function of aquaporin 1 (AQP1), a water channel known to promote metastasis and neoangiogenes. AQP1 protein levels were analyzed in OS (U2OS) and HCC (SNU-398) cells exposed to conditioned medium from BM-MSCs. Tumor cell migration and invasion in response to BM-MSC conditioned medium were evaluated through a wound healing assay and Boyden chamber, respectively. The results showed that the AQP1 level was increased in both tumor cell lines after treatment with BM-MSC conditioned medium. Moreover, BM-MSCs-mediated tumor cell migration and invasion were hampered after treatment with AQP1 inhibitor. These data suggest that the recruitment of human BM-MSCs into the tumor microenvironment might cause OS and HCC cell migration and invasion through involvement of AQP1.

Keywords: AQP1; BM-MSCs; hepatocellular carcinoma; invasion; migration; osteosarcoma.

Figure 1

The primary tumor microenvironment. Cancer cells in primary tumor are surrounded by a complex microenvironment including multiple stromal cell types that converge to promote tumor growth and dormancy, invasion and metastasis development and resistance to therapy. Key role of bone-marrow derived mesenchymal stem cells (BM-MSCs) in tumor cell migration and invasion. Cancer stem cell (CSC); carcinoma associated fibroblasts (CAFs); dendritic cell (DC); myeloid-derived-suppressor cells (MDSCs); natural killer (NK); tumor associated macrophages (TAM).

Figure 2

Conditioned medium (CM) from bone-marrow-derived mesenchymal stem cells (BM-MSC) increases aquaporin 1 (AQP1) levels in SNU-398 and osteosarcoma cells (U2OS) tumor cell lines. (A) Western blot analysis of AQP1 expression in U2OS and SNU-398 after treatment with BM-MSC-CM for 24 h. The equal protein loading was evaluated by using actin. Representative images from three independent studies; (B) AQP1 levels were represented as fold change respect to 10% fetal bovine serum (FBS), which was arbitrarily determined to be 1. Results are expressed as means ± SD.

Figure 3

AQP1 inhibitor hampered BM-MSC-CM-mediated wound closure in U2OS and SNU-398 cells. (A,B) Scratch wounds were performed when both tumor cells were confluent and images of wounds area were captured at T = 0 and 24 h using a digital camera attached to phase contrast microscopy. The experiments were performed in the presence or absence of 100 µM tetraethylammnium chloride (TEA) used as the AQP1 inhibitor. Wound closure was measured using Image J (National Institutes of Health, Bethesda, MD, USA) (%) = 1 − (wound width tx/wound width t0) × 100. Results are expressed as means ± SD. * p < 0.001 and # p < 0.001. Scale bar = 600 µm.

Figure 4

AQP1 inhibitor prevented BM-MSC-CM-mediated invasion of U2OS and SNU-398 cells. (A,B) Invasion assay was performed using a Boyden chamber pre-coated with matrigel. Tumor cells were seeded in the upper chamber whereas 1% FBS (negative control), 10% FBS (positive control) and BM-MSC-CM were added to the lower chamber as chemoattractants. Tumor cells were treated or not treated with AQP1 inhibitor TEA (100 µM). Results are expressed as means ± SD. * p < 0.001 and # p < 0.001. Scale bar = 600 µm.