Tudor Domain Containing Protein 3 Promotes Tumorigenesis and Invasive Capacity of Breast Cancer Cells

Abstract

Tudor domain containing protein 3 (TDRD3) is a modular protein identified based on its ability to recognize methylated arginine motifs through its Tudor domain. We have previously shown that TDRD3 localizes to cytoplasmic stress granules, a structure shown to promote survival upon treatment with chemotherapeutic drugs in cancer cells. Here, we report TDRD3 as a novel regulator of cell proliferation and invasion in breast cancer cells. Our study also demonstrates that TDRD3 depletion inhibits tumor formation and metastasis to the lung in vivo. Furthermore, we show that TDRD3 regulates the expression of a number of key genes associated with promotion of breast cancer tumorigenesis and disease progression. Strikingly, we report that TDRD3 regulates some of these key targets at the level of translation. These findings provide the first experimental demonstration of a functional role for TDRD3 in promoting breast cancer development and progression, and identify TDRD3 as a potential new therapeutic target for breast cancer.

Figure 1

TDRD3 Regulates Cell Proliferation In Vitro and In Vivo. (a) MTT assay were performed on MCF7 shControl (•), shTDRD3-1 (▪), and shTDRD3-2 (▴) cells over a 8 day time course. Data is the mean +/− SEM normalized to 0 h (n = 6, ***p < 0.0001). Significance was determined using a one-way ANOVA. (b) MTT assays were performed on MCF7 GFP-tagged empty vector (•) or TDRD3 (▪) cells over a 8 day time course. Data is the mean +/− SEM normalized to 0 h (n = 6, ***p < 0.0001). Significance was determined using a two-tailed t-test. MTT assays were performed on (c) MDA MB 231 (n = 3, **p = 0.0041) and (d) Hs578T (n = 4, ***p < 0.0001) shControl (•), shTDRD3-1 (▪), and shTDRD3-2 (▴) cells over a 8 day time course. Data is the mean +/− SEM normalized to 0 h. Significance was determined using a one-way ANOVA. (e) Images of six-week old Crl:SHO-Prkdc ^scid Hr ^hr (SHO) and CB17.Cg-Prkdc ^scid Hr ^hr/IcrCrl (SHC) mice injected with MDA MB 231 shControl or shTDRD3 cells in their flank. Tumor volume (mm³) of (f) SHO (n = 3, Days Post Injection D31 *p = 0.0371) and (g) SHC (n = 3, Days Post Injection D38 **p = 0.0070) mice. Tumor volume measurements are the mean +/− SEM. Significance was determined using a two-tailed t-test.

Figure 2

TDRD3 Promotes Cell Invasion in Breast Cancer Cells. (a) MCF7 cells were transiently transfected with Myc-tagged TDRD3 or empty vector (left panel) and GFP-tagged TDRD3 or empty vector (right panel) for 48 h and Western blotting was performed (b) Invasive cells/field for Myc-tagged TDRD3 or empty vector (n = 7, ***p = 0.0008) and GFP-tagged TDRD3 or empty vector (n = 7, ***p = 0.0001) cells placed in Transwell chambers for 72 h. Data is the mean +/− SEM. Significance was determined using a two-tailed t-test. (c) Images of Transwell chambers transiently transfected with Myc-tagged TDRD3 or empty vector and GFP-tagged TDRD3 or empty vector (Magnification, 40X). Invasive cells/field of (d) MDA MB 231 (n = 5, ***p < 0.0001) or (e) Hs578T (n = 6, ***p < 0.0001) shControl, shTDRD3-1 or shTDRD3-2 stable cell lines placed in Transwell chambers for 24 h. Data is the mean +/− SEM. Images of invasive cells (Magnification, 40X) are depicted below graph. Significance was determined using a one-way ANOVA.

Figure 3

Full Length TDRD3 is Required to Promote Cell Invasion in Breast Cancer Cells. (a) Schematic depiction of Myc-tagged TDRD3 wild type or deletion constructs used in rescue experiments. (b) Representative Western blot of stably expressing MDA MB 231 shTDRD3 cells transfected with Myc-tagged TDRD3 wild type or deletion mutants for 48 h. Endogenous Myc was detected in some experiments and is indicated by an asterisk. (c) Stably expressing MDA MB 231 shTDRD3 cells transfected with Myc-tagged TDRD3 wild type or deletion constructs for 48 h and placed in Transwell chambers for 24 h. Data is the mean +/− SEM (n = 3, *p < 0.05). Data was normalized to protein expression of each construct (n = 3). Myc-tagged TDRD3 full length was set to 1 and the relative invasive potential of each TDRD3 construct was determined. Significance was determined using a one-way ANOVA.

Figure 4

TDRD3 Promotes Metastasis, In Vivo. Six-week-old female NOD.CB17-Prkdcscid/JSCID mice were injected with MDA MB 231 shTDRD3 and luciferase or shControl and luciferase expressing cells. (a) TDRD3 protein levels, mean ± SEM (n = 3, *p = 0.0274) (b) Luciferase intensity was measured using the IVIS spectrum imaging system. Representative images show bioluminescence from shControl and shTDRD3 cells. (c) Average radiance (p/s/cm²/sr) of shControl (n = 6) and shTDRD3 (n = 4) injected mice (*p = 0.018). (d) Lung tumor nodule formation depicting the mean ± SEM for shControl (n = 6) and shTDRD3 (n = 4) mice (*p = 0.0198). (e) Images showing the anterior (upper image) and posterior (lower image) lung surface in shControl and shTDRD3 mice. Significance was determined using a two-tailed t-test.

Figure 5

TDRD3 Regulates Epithelial to Mesenchymal Markers in Breast Cancer Cells. (a) MDA MB 231 cells were infected with shControl or shTDRD3 expressing lentivirus for 96 h, RNA was isolated, and qPCR was performed from cDNA synthesized from the RNA. Data was normalized to GAPDH and is the mean +/− SEM (n = 6). Significance (**p < 0.01, ***p < 0.001) was determined using a two-tailed t-test. (b) MDA MB 231 cells were infected with shControl or shTDRD3 expressing lentivirus for 96 h, protein was isolated, and Western blotting was performed. (c) Chromatin immunoprecipitation showing MDA MB 231 cells transfected with Myc-tagged TDRD3 for 48 h. (d) RNA immunoprecipitation depicting MDA MB 231 transiently transfected with Myc-tagged TDRD3 transfected cells for 48 h (left panel, RNA immunoprecipitation; right panel, Western blot showing myc-tagged TDRD3 immunoprecipitation).

Figure 6

TDRD3 Regulates Translation in Breast Cancer Cells. Cytoplasmic extracts from MDA MB 231 cells infected with shControl or shTDRD3 for 96 h were subjected to fractionation on a 10–45% sucrose gradient. RNA was isolated from each fraction and RT-qPCR analysis was performed determining (a,b) Slug, (c,d) Snail, (e,f) β-catenin, and (g,h) Vimentin mRNA distribution. The line graph is a representative result of one independent trial, while the bar graph represents the pooled mRNA association from each monosome and polysome fraction. Significance (n = 4, *p < 0.05, **p < 0.01) was determined using a two-tailed t-test.