TIG3 Tumor Suppressor-Dependent Organelle Redistribution and Apoptosis in Skin Cancer Cells

TIG3 is a tumor suppressor protein that limits keratinocyte survival during normal differentiation. It is also important in cancer, as TIG3 level is reduced in tumors and in skin cancer cell lines, suggesting that loss of expression may be required for cancer cell survival. An important goal is identifying how TIG3 limits cell survival. In the present study we show that TIG3 expression in epidermal squamous cell carcinoma SCC-13 cells reduces cell proliferation and promotes morphological and biochemical apoptosis. To identify the mechanism that drives these changes, we demonstrate that TIG3 localizes near the centrosome and that pericentrosomal accumulation of TIG3 alters microtubule and microfilament organization and organelle distribution. Organelle accumulation at the centrosome is a hallmark of apoptosis and we demonstrate that TIG3 promotes pericentrosomal organelle accumulation. These changes are associated with reduced cyclin D1, cyclin E and cyclin A, and increased p21 level. In addition, Bax level is increased and Bcl-XL level is reduced, and cleavage of procaspase 3, procaspase 9 and PARP is enhanced. We propose that pericentrosomal localization of TIG3 is a key event that results in microtubule and microfilament redistribution and pericentrosomal organelle clustering and that leads to cancer cell apoptosis

TIG3: An Important Regulator of Keratinocyte Proliferation and Survival

Tazarotene-induced gene 3 (TIG3) is a tumor suppressor protein. In normal human epidermis, TIG3 is present in the differentiated, suprabasal layers, and it regulates terminal differentiation. TIG3 level is reduced in hyperproliferative diseases, including psoriasis and skin cancer, suggesting that loss of TIG3 is associated with enhanced cell proliferation. Moreover, transient expression of TIG3 leads to terminal differentiation in normal keratinocytes and apoptosis in skin cancer cells. In both cell types, TIG3 distributes to the cell membrane and to the centrosome. At the cell membrane, TIG3 interacts with and activates type I transglutaminase to enhance keratinocyte terminal differentiation. TIG3 at the centrosome acts to inhibit centrosome separation during mitosis and to alter microtubule function. These findings argue that TIG3 is involved in the control of keratinocyte differentiation and that loss of TIG3 in transformed cells contributes to the malignant phenotype

Pericentrosomal Localization of the TIG3 Tumor Suppressor Requires an N-Terminal Hydrophilic Region Motif

Tazarotene-induced gene 3 (TIG3) is a tumor suppressor protein that has a key role in controlling cell proliferation. TIG3 is observed at reduced levels in epidermal squamous cell carcinoma, and the restoration of expression in skin cancer cells reduces cell survival. TIG3 suppresses cell survival through mechanisms that involve localization at the plasma membrane and at the centrosome. TIG3 interacts at the plasma membrane to activate enzymes involved in keratinocyte terminal differentiation, and at the centrosome to inhibit daughter centrosome separation during mitosis leading to cessation of cell proliferation and induction of apoptosis. An important goal is identifying the motifs required for TIG3 localization at these intracellular sites as a method to understand the function of TIG3 at each location. TIG3 encodes an N-terminal hydrophilic region (amino acids 1–135) and a C-terminal membrane-anchoring domain (amino acids 135–164). We show that the C-terminal hydrophobic domain targets intact TIG3 to the plasma membrane, but when isolated as an independent element localizes at the mitochondria. We further demonstrate that a segment of the N-terminal hydrophilic region targets the centrosome. These studies provide important insights regarding the mechanisms that guide subcellular localization of this keratinocyte survival regulator

TIG3 reduces cell cycle progression.

<p><b>A</b> SCC-13 cells grown on coverslips were infected with 10 MOI of EV or TIG3-encoding virus and after 24 h treated with 10 µM BrdU for 2 h and then fixed and stained with anti-BrdU (red) and anti-TIG3 (green). BrdU incorporation is a marker of the synthesis phase of the cell cycle. The number of BrdU positive cells as a percentage of total cell number is presented beneath each panel. The values are mean ± SEM (n = 3) and the values are significantly different as determined by Student's t-test (p<0.001). Bars = 10 µm. <b>B</b> SCC-13 cells were collected for flow cytometry at 24 h post-infection with EV or TIG3-encoding virus. Cells were stained with 50 µg/ml propidium iodide prior to analysis. TIG3 reduces events in G1 and increases subG1 events. <b>C</b> At 24 h post-infection, cells were harvested and extracts prepared for detection of cell cycle regulatory proteins. Molecular weights are indicated to the left of the blot in kDa. <b>D</b> Cells were treated as above and then harvested for detection of p21 encoding mRNA by real time-PCR. A similar result was observed in each of three experiments.</p

TIG3 alters microtubule distribution.

<p><b>A</b> SCC-13 cells were infected with 10 MOI tAd5-EV or tAd5-TIG3 and at 24 h post-infection cells were fixed and stained with anti-TIG3 (green stain) and anti-β-tubulin (red stain). TIG3 accumulates at the expected perinuclear location (black arrow). β-tubulin accumulates in an atypical ring at the cell periphery (red arrows). The normal β-tubulin network in the TIG3-negative cell is indicated by a white arrow pointing to the centrosome. Nuclei were Hoechst stained (blue). The bottom panel is identical to the top, except that only the β-tubulin (red) signal is indicated. Bars = 10 µM. The graph shows the number of tAd-EV and tAd5-TIG3 infected cells with centrosome-originated microtubule arrays. Cells were counted in twenty independent microscope fields and a minimum of one-hundred cells were counted per treatment group. The values are mean ± SEM. Paired Student's t-test analysis reveals that the means are significantly different (p<0.001). To assess the impact of TIG3 on tubulin solubility, cells were infected with tAd5-EV or tAd5-TIG3 and after 24 h total extract, soluble and pellet fractions were prepared and electrophoresed followed by immunostaining to detect β-tubulin and β-actin. The presence of the majority of β-actin in the soluble fraction indicates that the fractionation was successful. <b>B</b> TIG3 expression causes actin filament collapse around the nucleus. SCC-13 cells were infected with adenovirus as above and after 24 h stained with anti-β-actin (red stain) and anti-TIG3 (green stain). Nuclei were Hoechst stained (blue). For the TIG3-positive cells, the left panel shows the TIG3 and β-actin signals (red and green), while the right panel shows only the β-actin (red) channel. The black arrow indicates TIG3 accumulation at the centrosome and the red arrow indicates the β-actin microfilament nuclear ring. Bars = 10 µm. The plot shows the number of tAd-EV and tAd5-TIG3 infected cells displaying actin microfilament rings surrounding the nucleus. Cells were counted in eighteen independent microscope fields and a minimum of one-hundred cells were counted per treatment group. The values are mean ± SEM. Paired Student's t-test analysis reveals that the means are significantly different (p<0.001).</p

TIG3 localizes in the vicinity of pericentrin.

<p><b>A</b> SCC-13 cells were infected with 10 MOI tAd5-EV or tAd5-TIG3 and at 24 h post-infection were fixed and stained with anti-TIG3 (green). Arrows indicate pericentrosomal and arrowheads indicate membrane localization. No TIG3 is detected in empty vector-infected cells. <b>B</b> Cells, infected as above, were fixed and stained with TIG3 (green) and pericentrin (red). The arrows indicate pericentrin staining of the centrosome and n indicates the nuclei. Bars = 10 µm.</p

TIG3 decreases cell survival.

<p>Subconfluent cultures of SCC-13 cells, growing in 3.8 cm² wells, were infected with 10 MOI tAd5-EV or tAd5-TIG3. At 0, 24, 48, and 72 h post-infection, cells were counted and lysates prepared. <b>A</b> TIG3 expression decreases cell number. Values are mean ± SEM, n = 3. Those comparisons marked by an asterisk are significantly different as determined by Student's t-test (p<0.001). <b>B</b> TIG3 is detected by immunoblot in tAd5-TIG3 infected SCC-13 cells. The monomer is visible at 18 kDa and the bracket indicates higher molecular weight crosslinked TIG3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023230#pone.0023230-Sturniolo1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023230#pone.0023230-Sturniolo2" target="_blank">[11]</a>. Molecular weights are indicated to the left of the blot in kDa.</p