Characteristics of endometrial cancer patients used for the study.

<p>Characteristics of endometrial cancer patients used for the study.</p

Correlation between miR-205 and PTEN expression in endometrial cancer patients.

<p>(A) Correlation of miR-205 and PTEN expression was analyzed by two-tailed Spearman nonparametric correlation test (P = 0.034, Spearman correlation coefficient = −0.502). PTEN protein expression accompanied by miR-205 expression (in triplicate) is illustrated in individual cases; undifferentiated carcinoma (B), and clear cell carcinoma (C).</p

Sequences of PCR primers and probes used in qRT-PCR of PTEN mRNA.

<p>Sequences of PCR primers and probes used in qRT-PCR of PTEN mRNA.</p

miRNA expression in normal and endometrial cancer tissue specimens.

<p>Relative quantification of miRNAs was expressed as normalized with an internal control RNU6B gene. (A) miR-26a expression (P = 0.742). (B) let-7g expression (p = 0.91). (C) miR-21 expression (P = 0.641). (D) miR-181b expression (P = 0.313). (E) miR-192 expression (P = 0.106). (F) miR-215 expression (P = 0.336). (G) miR-200c expression (P<0.0001). (H) miR-205 expression (P<0.0001). Statistical significance was calculated by a paired Student's t-test.</p

SK-3rd TICs possess properties of EMT.

<p>A–B) Differential expression of EMT markers in SK-3rd TICs: SK-BR3 and mammosphere forming SK-3rd cells were examined by real time RT-PCR (A) and Western blotting (B) for expression of EMT markers. E-cad: E-cadherin; Vim: Vimentin; Cytok: cytokeratin. C–E) Upregulation of Twist-1 in SK-3rd TICs: The total RNA of SK-BR3 and SK-3rd cells was examined for expression of Twist-1 and Snail using real time RT-PCR (C). Housekeeping genes were used to normalize Twist-1 and Snail mRNAs in all samples. At the end of real time RT-PCR, the samples were examined in a 1.2% agarose gel (D). The cell lysates from nuclear fractions were examined by Western blotting using anti-Twist-1 antibodies (E, Upper panel). Histone 3 was used as a loading control for nuclear preparation. F) Effect of Twist-1 on MT1-MMP expression: SK-3rd TICs silenced for Twist-1 or GFP control were examined by Western blotting using antibodies as indicated for expression of MT1-MMP and Twist-1. β-actin was used as a loading control.</p

Hypoxia induces intracellular MT1-MMP trafficking to the cell surface, resulting in enhanced invasiveness of SK-3rd cells.

<p>A) Determination of MT1-MMP expression in SK-3rd cells: Total cell lysates from SK-3rd cells either overexpressing of MT1-GFP or silencing of endogenous MT1-MMP were analyzed by Western blotting using anti-MT1-MMP antibodies. β-Actin was used as a loading control. B) Examination of the role of MT1-MMP in SK-3rd TIC invasion: SK-3rd TICs with overexpression or downregulation of MT1-MMP as indicated were pretreated with or without CoCl₂ (250 µM) for 24 hours followed by evaluation of cell invasive ability in the 3-D invasion assay (Left panel). The invaded cells were microscopically counted (Right panel). Hypoxia promotes SK-3rd TIC invasion and this process requires expression of MT1-MMP in the cells. C) Expression of MT1-MMP in TICs: Total RNAs from breast cancer cells (SK-BR3 parental cells and SK-3rd TICs) and colon cancer cells (HCT116 parental cells and HCT116 TICs) were examined for the expression of MT1-MMP using a real time RT-PCR approach. Housekeeping genes (GAPDH and HPRT) were used to normalize MT1-MMP mRNA in all samples. D) Determination of functional MT1-MMP in SK-3rd TICs: Top panel: Cell surface MT1-MMP in SK-BR3 and SK-3rd cells treated with or without CoCl₂ for 24 hours were biotinylated and precipitated with streptavidin-beads followed by Western blotting using anti-MT1-MMP antibodies. Densitometry analysis was performed and ratios between MT1-MMP latent and active forms over β-actin were given. Middle panel: An aliquot of total cell lysates of cell surface biotinylated SK-BR3 and SK-3rd cells were examined by Western blotting using anti-MT1-MMP antibodies. β-actin was used as a loading control. Densitometry analysis was performed and ratios between MT1-MMP latent and active forms over β-actin were given. Bottom panel: The conditioned medium in the presence of recombinant proMMP-2 was examined by gelatin zymogram to determine functional MT1-MMP. E) Effect of CoCl₂ on MT1-MMP expression: SK-BR3 and SK-3rd cells treated with or without CoCl₂ (250 µM) for 24 hours were examined by real time RT-PCR using MT1-MMP specific primers and housekeeping genes for normalization. F–G) Correlation of cell surface expression of MT1-MMP with proMMP-2 activation: SK-3rd TICs pretreated with CoCl₂ (Pre CoCl₂; 250 µM) for 24 hours were switched to complete medium in the absence of CoCl₂ (Post CoCl₂) for indicated dates followed by flow cytometry analysis using anti-MT1-MMP antibody (F). The cells were also examined by cell surface biotinylation experiment using antibodies for MT1-MMP (G, Upper panel) and β-actin (G, loading control, Middle panel), and gelatin zymogram (G, Lower panel) and input of MT1-MMP (Bottom panel). Densitometry analysis was performed and ratios between MT1-MMP active form over β-actin was given.</p

In Vivo Tumorigenicity and Spontaneous Metastasis Assays.

<p>“n.d.” = not done.</p

Effect of hypoxia-induced cell surface MT1-MMP on cell surface CD44 and mammosphere formation.

<p>A–B) Cell surface CD44 inversely correlates with hypoxia-induced cell surface MT1-MMP: SK-3rd TICs were cultured in the presence of CoCl₂ (Pre CoCl₂; 250 µM) for 24 hours and then switched to CoCl₂-free medium (Post CoCl₂) for indicated times. Flow cytometry analysis employing APC-anti-CD44 antibodies and anti-MT1-MMP antibodies in conjunction with FITC-labeled secondary antibodies (A). Cell surface proteins were biotinylated and examined for CD44 appearance (B, Upper panel). Shed CD44 in the conditioned medium was examined by Western blotting using anti-CD44 antibodies (B, Middle panel). β-actin was used as a loading control (B, Lower panel). Ponceau staining of the membrane with samples from the conditioned medium was employed as a loading control. C) Effect on cell adhesion by hypoxia using a hyaluronic acid adhesion assay: SK-3rd TICs treated with or without CoCl₂ (250 µM) for 24 hours were examined for their ability to adhere to hyaluronic acid-coated 96-well tissue culture plates. The adherent cells were enumerated based on calcein staining. D) Dissociation of mammospheres by low oxygen (Upper panel) or CoCl₂ treatment: Mammospheres from SK-3rd TICs were cultured under either 21% O₂ or 1% O₂ or treated with CoCl₂ (250 µM) for indicated times. The cells were microscopically examined daily. Bar, 100 µm. E) Effect of MT1-MMP on mammosphere forming ability and cell surface CD44 expression: SK-3rd TICs were infected with retrovirus containing cDNAs for GFP or MT1- GFP and shRNAs against luciferase (shRNA-Luc) or MT1-MMP (shRNA-MT1). The cells were cultured in ultra-low attachment culture dishes for their mammosphere forming abilities (Upper panel) and cell surface expression of CD44 and CD24 were examined by flow cytometry using corresponding antibodies (Lower panel). F) Reversal of hypoxia-induced MT1-MMP under normoxic conditions: SK-3rd TICs were cultured under 1% O₂ for 24 hours and then switched to normoxic conditions for indicated time. Flow cytometry analysis was performed using anti-CD44 and anti-MT1-MMP antibodies. NT refers to non-hypoxia-cultured cells.</p

Hypoxia Enhances TIC Invasion.

<p>A) Characterization of SK-3rd TICs: SK-3rd and SK-BR3 cells were cultured on ultra-low attachment culture dishes for 3 days and mammosphere formation was examined by microscopy (Upper panel). Bar, 100 µm. These cells were collected and incubated with APC-conjugated anti-CD44 antibodies and PE-conjugated anti-CD24 antibodies followed by flow cytometry analysis (Lower panel). B–C) Determination of invasive ability of SK-3rd TICs: SK-BR3 and SK-3rd cells were pretreated with or without CoCl₂ (250 µM) or cultured under hypoxia (1% O₂) for 24 hours followed by examination of invasive ability of the cells using the 3-D invasion assay (B). After 24 hours, the cells were fixed and invaded cells in six different fields in the invasion zone were microscopically counted (C). D) Evaluation of hypoxic conditions by examining HIF-1α in SK-3rd cells: Total cell lysate from SK-3rd TICs pretreated with or without CoCl₂ (250 µM) or under 1% O₂ for 24 hours were examined by Western blotting using anti-HIF-1α antibody. NT refers to cells without pre-hypoxia culture.</p