Transducin-like enhancer of split 3 regulates proliferation of melanoma cells via histone deacetylase activity

Abstract

Melanoma, one of the most aggressive neoplasms, is characterized by rapid cell proliferation. Transducin-like Enhancer of Split (TLE) is an important regulator of cell proliferation via Histone deacetylase (HDAC) recruitment. Given that HDAC activity is associated with melanoma progression, we examined the relationship between TLE3, a TLE family member, and melanoma. TLE3 expression was increased during the progression of human patient melanoma (p < 0.05). Overexpression of Tle3 in B16 murine melanoma cells led to an increase in cell proliferation (p < 0.01) as well as the number of cyclinD1-positive cells. in vivo injection of mice with B16 cells overexpressing Tle3 resulted in larger tumor formation than in mice injected with control cells (p < 0.05). In contrast, siRNA-mediated knockdown of Tle3 in B16 cells or TLE3 in HMV-II human melanoma cells decreased proliferation (p < 0.01). Treatment of B16 cells with trichostatin A (2.5 μM), a class I and II HDAC inhibitor, prevented the effect s of Tle3 on proliferation. In conclusion, these data indicate that Tle3 is required, at least in part, for proliferation in the B16 mouse melanoma model.

Keywords: HDAC inhibitors; malignant melanoma; transcriptional co-repressor; trichostatin A.

Figure 1. The expression levels of TLE3 are increased in human malignant melanoma

The expression of TLE3 in normal skin, benign nevi, and malignant melanoma of patients (GSE3189 dataset) [19]. Expression levels of TLE3 are presented as boxplots and means were compared using unpaired ANOVA with Tukey-Kramer post-hoc test and Wilcoxon’s signed rank test (A). HMV-II cells were stained with TLE3 antibody, rhodamine phalloidin (phalloidin), or DAPI (B). Skin from 12-week-old C57BL/6J male mice was immunostained with anti-Tle3 antibody. The boxed areas in the left panel are shown as magnified images of hair follicles in the right panel. Scale bars indicate 500 μm (left panel) and 100 μm (right panel) respectively. Representative images of several sections are shown (C). B16 cells were stained with Tle3 antibody, phalloidin, or DAPI (D). Representative images are several experimental repeats shown. Scale bar corresponds to 100 μm (B and D).

Figure 2. Overexpression of Tle3 increases proliferation in B16 melanoma cells

(A-F) B16 cells stably expressing Myc-tagged Tle3 or empty vector were generated after positive selection with G418. The messenger RNA levels of Tle3 (A), CyclinD1 (B), CyclinD2 (C), or CyclinA2 (D) were determined by qPCR on day 2. B16 cells with high expression of Tle3 co-expressed CyclinD1 in the nuclei. Scale bar corresponds to 40 μm (E). Protein levels of CyclinD1, Myc-tagged Tle3, or β-actin were determined by western-blot analysis on day 2 (F). Overexpression of Tle3 increased the proliferation of B16 cells assessed by water-soluble tetrazolium salt (WST) -8 assay. Proliferation was quantified on day 2 by spectrophotometric absorbance measurement at 450 nm (G). Data are expressed as the mean ± SD (n = 3). ^**, p < 0.01 versus control (A-D, G). Representative images were shown (E and F).

Figure 3. Overexpression of Myc-Tle3 in subcutaneously injected B16 melanoma cells increases tumor size in vivo

BALB/cA Jcl-nu/nu mice (n=5) were injected subcutaneously with 1 × 10⁵ control B16 cells (left side; arrow heads) or cells stably expressed Myc-tagged Tle3 (right side; arrows). Representative photograph of a mouse (A) and resected tumors (B) 3 weeks after injection of with B16 cells. Scale bar corresponds to 10 mm (B). Resected tumors were immunostained with anti-Tle3 antibody. Scale bars indicate 25 μm (C). Representative images of several sections are shown. The volume of resected tumors was quantified. The data are expressed as the mean ± SD (n = 10). ^*, p < 0.05 versus control (D).

Figure 4. Knockdown of Tle3 (TLE3) in melanoma cells decreases proliferation

(A-C) B16 cells were transfected with scramble siRNA, or siRNA against murine Tle3 (siTle3-1, siTle3-2). Protein levels of Tle3, cyclinD1, or β-actin were assessed by western blotting analysis (A). The numbers of cyclinD1 positive cells were decreased in the Tle3 knockdown B16 cells (B). In cells Tle3 knockdown cells, proliferation ability on day 2 and day 3 was decreased in comparison to scramble siRNA cells (C). (D-F) HMV-II cells were transfected with scrambled siRNA or siRNA against human TLE3 (siTLE3-1, siTLE3-2). Protein levels of TLE3, CYCLIN A2, or β-ACTIN were assessed by western blotting analysis (D). The numbers of KI67 positive cells were decreased in the TLE3 knockdown HMV-II cells (E). In TLE3 knockdown HMV-II cells, proliferation on day 4 was decreased in comparison to scrambled siRNA cells (F). Scale bar corresponds to 100 mm (B and E). ^**, p < 0.01 versus scramble (C and F).

Figure 5. Knockdown of Tle3 in subcutaneously injected B16 melanoma cells decreases tumor size in vivo

B16 cells, stably expressing control shRNA (Control) or shRNA against Tle3 (sh-Tle3) were generated by blasticidin selection. Protein levels of Tle3 were decreased in cells stably expressing sh-Tle3 (A). BALB/cA Jcl-nu/nu mice (n=5) were injected subcutaneously with 1 × 10⁵ control cells (left side; arrow heads) or sh-Tle3 (right side; arrows). Representative pictures of subcutaneous tumors (B) and resected tumors (C) 3 weeks after injection of B16 cells are shown. Scale bar corresponds to 10 mm (C). Resected tumors were immunostained with anti -Tle3 antibody. Scale bars indicate 25 μm. Representative images of several sections are shown (D). The volume of resected tumors were quantified (E). The data are expressed as the mean ± SD (n = 10). ^*, p < 0.05 versus control (E).

Figure 6. HDACs are involved in the enhancement of the proliferation of B16 cells by Tle3

(A-C) B16 cells were transiently transfected with empty vector (Mock) or Myc-tagged Tle3 and then treated with DMSO, or the indicated concentration of trichostatin A. Cell proliferation was evaluated on day 2 by water-soluble tetrazolium salt (WST) assay and absorbance measurement at 450 nm (A). Schematic of the C-terminally truncated forms of the Myc-tagged Tle3 plasmids used in these experiments. Q; glutamine rich domain, GP; glycine/proline rich domain, CcN; CcN domain, SP; serine/proline rich domain, WD, tryptophan/aspartic acid repeat domain (B). C-terminally truncated forms of Tle3 were transfected in B16 cells and proliferation ability measured on day 2 by WST assay (C). The data are expressed as the mean ± SD (n = 3). ^**, p < 0.01 versus Mock transfection (A and C). B16 cells were transfected with empty vector (Mock), Myc-tagged Tle3 (1-782), or Myc-tagged Tle3 (1-140). Protein levels of cyclinD1, Myc (tagged) or β-actin were assessed by western blotting analysis on day 2 (D). (E and F) B16 cells stably expressing Myc-tagged Tle3 (1-782), Myc-tagged Tle3 (1-140), or empty vector were generated after positive selection with G418. BALB/cA Jcl-nu/nu mice (n=5) were injected subcutaneously with 1 × 10⁵ mock B16 cells (a), cells stably expressing Myc-tagged Tle3 (1-782) (b), or cells stably expressing Myc-tagged Tle3 (1-140) (c). Representative photograph of a mouse 3 weeks after injection with B16 cells (E). The volume of resected tumors was quantified. The data are expressed as the mean ± SD (n = 5). ^*, p < 0.05 (F).