title: An endogenous inhibitor of angiogenesis inversely correlates with side population phenotype and function in human lung cancer cells.
abstract: The side population (SP) in human lung cancer cell lines and tumors is enriched with cancer stem cells. An endogenous inhibitor of angiogenesis known as tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), characterized for its ability to inhibit matrix metalloproteinases (MMPs), has been shown by several laboratories to impede tumor progression through MMP-dependent or -independent mechanisms. We recently reported that forced expression of TIMP-2, as well as the modified form Ala+TIMP-2 (that lacks MMP inhibitory activity) significantly blocks growth of A549 human lung cancer cells in vivo. However, the mechanisms underlying TIMP-2 antitumor effects are not fully characterized. Here, we examine the hypothesis that the TIMP-2 antitumor activity may involve regulation of the SP in human lung cancer cells. Indeed, using Hoechst dye efflux assay and flow cytometry, as well as quantitative reverse transcriptase-PCR analysis, we found that endogenous TIMP-2 mRNA levels showed a significant inverse correlation with SP fraction size in six non-small cell lung cancer cell lines. In A549 cells expressing increased levels of TIMP-2, a significant decrease in SP was observed, and this decrease was associated with lowered gene expression of ABCG2, ABCB1 and AKR1C1. Functional analysis of A549 cells showed that TIMP-2 overexpression increased chemosensitivity to cytotoxic drugs. The SP isolated from TIMP-2-overexpressing A549 cells also demonstrated impaired migratory capacity compared with the SP from empty vector control. More importantly, our data provide strong evidence that these TIMP-2 functions occur independent of MMP inhibition, as A549 cells overexpressing Ala+TIMP-2 exhibited identical behavior to those overexpressing TIMP-2 alone. Our findings provide the first indication that TIMP-2 modulates SP phenotype and function, and suggests that TIMP-2 may act as an endogenous suppressor of the SP in human lung cancer cells.
authors.affiliation: Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, the National Institutes of Health, Advanced Technology Center, Bethesda, MD, USA.
authors.forename: H
authors.initials: H
authors.lastname: Han
authors.affiliation: Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, the National Institutes of Health, Advanced Technology Center, Bethesda, MD, USA.
authors.forename: D
authors.initials: D
authors.lastname: Bourboulia
authors.affiliation: Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, the National Institutes of Health, Advanced Technology Center, Bethesda, MD, USA.
authors.forename: S
authors.initials: S
authors.lastname: Jensen-Taubman
authors.affiliation: Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, the National Institutes of Health, Advanced Technology Center, Bethesda, MD, USA.
authors.forename: B
authors.initials: B
authors.lastname: Isaac
authors.affiliation: Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, the National Institutes of Health, Advanced Technology Center, Bethesda, MD, USA.
authors.forename: B
authors.initials: B
authors.lastname: Wei
authors.affiliation: Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, the National Institutes of Health, Advanced Technology Center, Bethesda, MD, USA.
authors.forename: W G
authors.initials: WG
authors.lastname: Stetler-Stevenson
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002465:Cell Movement / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D061965:Matrix Metalloproteinase Inhibitors / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000235:genetics* / Q000473:pathology; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics; D058985:Side-Population Cells / Q000378:metabolism*; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D061965:Matrix Metalloproteinase Inhibitors; D009363:Neoplasm Proteins; D012333:RNA, Messenger; C560823:TIMP2 protein, human; D019716:Tissue Inhibitor of Metalloproteinase-2; D020782:Matrix Metalloproteinases