A p27(kip1)-binding protein, p27RF-Rho, promotes cancer metastasis via activation of RhoA and RhoC

Abstract

Rho family proteins regulate multiple cellular functions including motility and invasion through regulation of the actin cytoskeleton and gene expression. Activation of Rho proteins is controlled precisely by multiple regulators in a spatiotemporal manner. RhoA and/or RhoC are key players that regulate the metastatic activity of malignant tumor cells, and it is therefore of particular interest to understand how activation of these Rho proteins is controlled. We recently identified an upstream regulator of RhoA activation, p27RF-Rho (p27(kip1) releasing factor from RhoA) that acts by freeing RhoA from inhibition by p27(kip1). p27(kip1) is a cell cycle regulator when it is localized to the nucleus, but it binds RhoA and inhibits activation of the latter when it is localized to the cytoplasm. Here, we show that a metastatic variant of mouse melanoma B16 cells (F10) exhibits greater expression of p27RF-Rho, RhoA, and RhoC than the nonmetastatic parental cells (F0). Injection of F10 cells into mouse tail vein resulted in the formation of metastatic lung colonies, whereas prior knockdown of expression of either one of the three proteins using specific shRNA sequences decreased metastasis markedly. p27RF-Rho regulated the activation of RhoA and RhoC and thereby modulated cellular adhesion and motility, in addition to pericellular proteolysis. The Rho activities enhanced by p27RF-Rho had a marked effect upon efficiency of lodging of F10 cells in the lung, which represents an early step of metastasis. p27RF-Rho also regulated metastasis of human melanoma and fibrosarcoma cells. Thus, p27RF-Rho is a key upstream regulator of RhoA and RhoC that controls spreading of tumor cells.

FIGURE 1.

Analysis of Rho mRNA and protein in B16 melanoma cells. a, quantitative PCR analysis of p27RF-Rho, RhoA, RhoB, and RhoC in B16F0 and B16F10 cells. Gene expression was normalized to that of GAPDH and presented as the ratio of expression in B16F0 relative to B16F10 cells. The data are presented as the means ± standard deviation (n = 3). *, p < 0.05. **, not detected. b, protein levels of p27RF-Rho, RhoA, and RhoC in B16F0 and B16F10 cells. c, quantification of Fig. 1B. The data are presented as the means ± standard deviation (n = 3). *, p < 0.05.

FIGURE 2.

The p27RF-Rho-Rho axis is required for melanoma cell metastasis in vivo. a, the efficiency of knockdown of p27RF-Rho (sh-p27RF1 and sh-p27RF2), RhoA (sh-RhoA1, 2), or RhoC (sh-RhoC1, 2) in B16F10 cells was analyzed by Western blot analysis. b, appearance of murine lungs 14 days following injection of control or p27RF-Rho-, RhoA-, or RhoC-depleted cells into the tail veins of 7–8-week-old mice. The number of surface tumor nodules is shown in c. The data represent the means ± standard deviation (n = 7).

FIGURE 3.

p27RF-Rho modulates RhoA and RhoC activation via inhibition of the p27^kip1 pathway. a, regulation of Rho proteins by p27RF-Rho. B16F0 cells expressing an empty vector (mock) or p27RF-Rho fused to a V5 tag (p27RF-V5, lanes 1 and 2) and B16F10 cells expressing a control shRNA (sh-LacZ, lane 3) or either one of two shRNA sequences targeted to the p27RF-Rho mRNA (sh-p27RF1 and sh-p27RF2, lanes 3–5) were used for the assay. The cell lysates were analyzed by Western blot using antibodies that recognize p27RF-Rho, RhoA, or RhoC. Active Rho proteins were pulled down using the Rhotekin fragment and analyzed similarly. The ratio of active to total protein is indicated below the blots (n = 3). b, subcellular localization of p27^kip1 in B16F0 and B16F10 cells. After cells were lysed and separated into cytoplasmic and nuclear fractions, p27kip1 was detected by Western blot analysis. Tubulin and lamin B1 are markers for cytoplasmic and nuclear proteins, respectively. T, total lysate; C, cytoplasmic fraction; N, nuclear fraction. c, p27RF-Rho suppresses p27^kip1 so as to inhibit activation of RhoA and RhoC. An empty vector (mock, lane 1), p27^kip1 fused to an mVenus tag (mVkip1, lanes 2–4), and mVkip1 with additional empty vector (mVkip1-mock, lane 3) or mVkip1 with p27RF-Rho fused to an mCherry tag (mVkip1-p27RFmC, lane 4) were expressed in B16F10 cells. Total and active Rho proteins were analyzed as in a. The ratio of active to total protein is indicated below the blots (n = 3).

FIGURE 4.

p27RF-Rho regulates gelatin degradation activity in F10 cells. a, degradation of Oregon Green-labeled gelatin (middle column) and actin immunohistochemistry (red, left column) in control (sh-LacZ) and p27RF-Rho-depleted (sh-p27RF1 and sh-p27RF2) cells cultured on glass coverslips (scale bar, 20 μm). Higher magnification views of the boxed areas are shown underneath each image. b, quantification of cells with invadopodia. The cells were scored as invadopodia-positive if they contained punctate structures positive for both actin filaments and gelatin degradation. c, quantification of gelatin degradation area. The data are presented as the means ± standard errors of means (n = 5). The number of cells analyzed in each data set is indicated at the bottom of the data bars in the graphs. *, p < 0.05.

FIGURE 5.

p27RF-Rho is required for cell migration, adhesion, and spreading. a, the number of migratory cells analyzed using a Transwell chamber equipped with a fibronectin-coated filter. The data are presented as the means ± standard deviation (n = 3). b and c, B16F10 cells depleted of p27RF-Rho or RhoA were plated on fibronectin (b) or vitronectin (c), and adhesion was quantified by counting cells 30 min after plating. The data are presented as the means ± standard deviation (n = 3). *, p < 0.05. d, quantification of cell spreading. The data are presented as the means ± standard errors of means (n = 3). The number of cells analyzed in each data set is indicated at the bottom of the graphs.

FIGURE 6.

p27RF-Rho is required for murine melanoma lung colonization in vivo. Localization of tumor cells to the lung 1 or 24 h following injection into the tail vein of mice is shown. a, localization of a mixture of B16F0 (labeled with CellTracker Green) and B16F10 cells (labeled with CellTracker Orange). c, localization of a mixture of control B16F10 cells (expressing sh-LacZ, labeled with CellTracker Green) and B16F10 cells depleted for p27RF-Rho (expressing sh-p27RF1, labeled with CellTracker Orange). b and d, the numbers of cells in a and c are presented in b and d, respectively.

FIGURE 7.

p27RF-Rho is required for malignant human tumor cells lung colonization in vivo. Knockdown efficiency of p27RF-Rho (sh-p27RF) in A375 (a), Mum2B (b), and HT1080 (c) cells was evaluated by Western blot analysis. Localization of a mixture of control A375, Mum2B, and HT1080 cells (expressing sh-LacZ, labeled with CellTracker Green) and corresponding cells depleted for p27RF-Rho (expressing sh-p27RF1, labeled with CellTracker Orange) is shown. The numbers of cells shown in d–f are presented in g–i. The data represent the means ± standard deviation (n = 4).