STK31 Is a Cell-Cycle Regulated Protein That Contributes to the Tumorigenicity of Epithelial Cancer Cells

<div><p>Serine/threonine kinase 31 (STK31) is one of the novel cancer/testis antigens for which its biological functions remain largely unclear. Here, we demonstrate that STK31 is overexpressed in many human colorectal cancer cell lines and tissues. STK31 co-localizes with pericentrin in the centrosomal region throughout all phases of the cell cycle. Interestingly, when cells undergo mitosis, STK31 also localizes to the centromeres, central spindle, and midbody. This localization behavior is similar to that of chromosomal passenger proteins, which are known to be the important players of the spindle assembly checkpoint. The expression of STK31 is cell cycle-dependent through the regulation of a putative D-box near its C-terminal region. Ectopically-expressed STK31-GFP increases cell migration and invasive ability without altering the proliferation rate of cancer cells, whereas the knockdown expression of endogenous STK31 by lentivirus-derived shRNA results in microtubule assembly defects that prolong the duration of mitosis and lead to apoptosis. Taken together, our results suggest that the aberrant expression of STK31 contributes to tumorigenicity in somatic cancer cells. STK31 might therefore act as a potential therapeutic target in human somatic cancers.</p></div

Knockdown expression of hSTK31 results in cell apoptosis.

<p>(<b>A</b>) After 72 h infection of lentiviral-based <i>STK31</i> shRNA (sh<i>STK31</i>) or control shRNA (shCtrl), AZ521 cells were harvested to determine the STK31 expression by IB. (<b>B–D</b>) Control (shCtrl) or sh<i>STK31</i>-infected cells (sh<i>STK31</i>) were collected to analyze the cell cycle population by flow cytometry (B), to detect the expression of active caspase-3 by IB (C), and to perform the TUNEL assay (D). Cells treated with DNase I were used as positive controls in C and D. Cells without treatment were the negative controls for the TUNEL assay. The red signal in 6D represents the apoptotic cells. ***, <i>P</i> value <0.001. (<b>E</b>) Time-lapse images of cells transfected with <i>STK31</i> shRNA tagged with EGFP (sh<i>STK31</i>-GFP) (A–H). The cells were synchronized with nocodazole (150 ng/ml) for 16 h. After release from nocodazole, cells were observed with time-lapse microscopy. Neighboring untransfected cells (indicated by the white arrow) successfully underwent mitosis and complete cytokinesis, whereas the knockdown cell (indicated by the yellow arrow) remained at a prolonged mitotic-like stage. We timed the duration of mitosis from nuclear envelope breakdown (at 0 min, a) and found that at 272 min the knockdown cell ended up with an apoptotic-like feature (h).</p

Overexpression of hSTK31 does not promote cell proliferation, but increases cell migration and invasive abilities.

<p>(<b>A</b>) Equal amount of AZ521 or HCT116 cells with GFP or STK31-GFP expression were seeded, and cell growth was then monitored by calculating viable cell numbers at the indicated time. Three independent experiments were performed and their quantitative results are shown. The expression of GFP or STK31-GFP at Day 1 and Day 5 was detected by IB using anti-GFP antibody. α-tubulin is an internal control. (<b>B</b>) AZ521 cells with GFP or STK31-GFP expression were collected to analyze cell population distribution by flow cytometry. (<b>C</b>) AZ521 or HCT116 cells with GFP or STK31-GFP expression were used to perform the wound-healing migration assay. The interval of cell migration at different time (0–24 h) was measured and is shown. Quantitative results are shown below. (<b>D</b>) AZ521 (upper) or HCT116 (lower) cells with GFP or STK31-GFP expression were seeded to type II collagen-coated or matrigel-coated trans-wells to perform cell migration or invasion assays. After stained with DAPI, a DNA specific dye, cells were counted and the quantitative results are shown. Three independent experiments were done. (<b>E</b>) Cells transfected with STK31-GFP or infected with lentiviral-based <i>STK31</i> shRNA (sh<i>STK31</i>) were collected to detect the expressions of <i>MMP-2</i> by real-time PCR (right). Overexpressed STK31-GFP was detected by IB analysis (left). The expression of <i>STK31</i> in sh<i>STK31</i> cells was determined by real-time PCR and normalized by <i>β-actin</i> (middle).</p

Expressions of <i>STK31</i> in human cancer cell lines and colorectal cancer.

<p>(<b>A</b>) Total RNA from four human cancer cell lines of different tissues, AZ521 (a human colorectal cancer cell line), A549 (a human lung cancer cell line), HeLa (a human cervical cancer cell line), A375 (a human melanoma cancer cell line), and one normal human mammary epithelial cell (Normal) that was isolated for real-time PCR to determine <i>STK31</i> mRNA levels. <i>STK31</i> was significantly overexpressed in the human gastric cancer cell line, AZ521, compared to other human cancer cell lines. (<b>B</b>) Total RNA prepared from six gastrointestinal cancer cell lines were used to detect <i>STK31</i> mRNA expression level by real-time PCR as described in A. (<b>C</b>) Paired human colorectal cancer tissues (N, normal tissue; T, tumor tissue) were collected to isolate total RNA for detecting the expression levels of <i>STK31</i> by RT-PCR. <i>Actin</i> was used as an internal control. Eight representative paired specimens were shown.</p

Cell cycle expression pattern of STK31.

<p>(<b>A</b>) Asynchronized or nocodazole (NZ)-treated AZ521 cells were collected to analyze their cell cycle population by flow cytometry. The percentages of G1, S, and G2/M are shown below. The round-up cells after NZ treatment were shake-off and defined as M phase cells, and the attached cells remaining after shake-off were considered to be the G2 phase. (<b>B</b>) Total AZ521 lysates from asynchronized (Asy), M phase (M), and G2 stage of cells were collected for Western blot analysis with anti-STK31 polyclonal antibodies. Phospho-histone H3 serine 10 (P-H3/S10) is a marker for mitotic cells. (<b>C</b>) AZ521 cells released from nocodazole treatment with different time points (from 0 to 360 min) were collected to analyze the expression pattern of STK31 by IB analysis. Anti-Cyclin-B and anti-phospho-histone H3 (p-H3/S10) antibodies were used as mitotic markers. (<b>D</b>) AZ521 cells ectopically expressing GFP or STK31-GFP were treated with nocodazole. M phase round-up cells were collected by shake-off, and remaining attached cells collected as the G2 phase. Asynchronized cells (Asyn) are shown. α-tubulin or GAPDH was used as the internal control for IB. (<b>E</b>) Total RNA from different cell cycle stages of AZ521 was harvested to analyze the expression of <i>STK31</i> mRNA by real-time PCR. Three independent experiments were performed (N = 3), and means with standard errors are shown.</p

STK31 contains a putative D-box motif.

<p>(<b>A</b>) A putative D-box motif is located within the amino acids 643–651 of STK31. The conserved position, Arg643 (R643), within the D-box was shown. (<b>B</b>) Asynchronized (Asy), M phase, and G2 stage AZ521 cells with STK31-GFP or STK31-GFP/D-box mutant expression were collected to perform the IB analysis using anti-GFP antibody. Expression levels of STK31-GFP are displayed as ratios. Phospho-histone H3 serine 10 (P-H3/S10) is a marker for mitotic cells. GAPDH is a loading control.</p

Subcellular localization of STK31.

<p>(<b>A</b>) Schematic representation of the STK31 polypeptide. Two epitopes, amino acids 231–350 and 982–996, were used as antigens to generate the monoclonal antibody, 1G10, and polyclonal antibodies, 19717, respectively. After purification, the specificity of 19717 (lanes 4–6) and 1G10 (lanes 7–9) was confirmed by immunoblot (IB) analysis using AZ521 total lysate (lanes 1, 4, and 7), STK31-GFP (contains the human form STK31 lanes 2, 5, and 8), or Stk31-GFP (contains the mouse form Stk31 lanes 3, 6, and 9) transfected AZ521 cell lysates. Anti-GFP was used as a positive control for IB to detect the expression of STK31-GFP and Stk31-GFP (lanes 1–3). α-tubulin was used as a loading control. (<b>B</b>) AZ521 cells were doubly stained with 1G10 (green) and pericentrin (red, a known centrosomal protein). DNA was visualized using DAPI (blue). Prophase, a–d; metaphase, e–h; anaphase, i–l; and telophase, m–p. Merged images are shown on the right (d, h, l, and p). (<b>C</b>) AZ521 cells were co-immunostained with 1G10 (green, a–e) and anti-Aurora B (red, f–j) antibodies. The merged images are shown (k–o). (<b>D</b>) Cells were doubly stained with STK31 (green), Aurora-B (red), and DNA (blue). Only merged images were shown. Arrows indicated the colocalization of STK31 and Aurora-B throughout the cell cycle.</p

Early Phthalates Exposure in Pregnant Women Is Associated with Alteration of Thyroid Hormones

<div><p>Introduction</p><p>Previous studies revealed that phthalate exposure could alter thyroid hormones during the last trimester of pregnancy. However, thyroid hormones are crucial for fetal development during the first trimester. We aimed to clarify the effect of phthalate exposure on thyroid hormones during early pregnancy.</p><p>Method</p><p>We recruited 97 pregnant women who were offered an amniocentesis during the early trimester from an obstetrics clinic in southern Taiwan from 2013 to 2014. After signing an informed consent form, we collected amniotic fluid and urine samples from pregnant women to analyze 11 metabolites, including mono-ethyl phthalate (MEP), mono-(2-ethyl-5-carboxypentyl) phthalate (MECPP), mono-(2-ethylhexyl) phthalate (MEHP), mono-butyl phthalate (MnBP), of 9 phthalates using liquid chromatography/ tandem mass spectrometry. We collected blood samples from each subject to analyze serum thyroid hormones including thyroxine (T₄), free T₄, and thyroid-binding globulin (TBG).</p><p>Results</p><p>Three phthalate metabolites were discovered to be >80% in the urine samples of the pregnant women: MEP (88%), MnBP (81%) and MECPP (86%). Median MnBP and MECPP levels in pregnant Taiwanese women were 21.5 and 17.6 μg/g-creatinine, respectively, that decreased after the 2011 Taiwan DEHP scandal. Results of principal component analysis suggested two major sources (DEHP and other phthalates) of phthalates exposure in pregnant women. After adjusting for age, gestational age, TBG, urinary creatinine, and other phthalate metabolites, we found a significantly negative association between urinary MnBP levels and serum T₄ (β = –5.41; p-value = 0.012; n = 97) in pregnant women using Bonferroni correction.</p><p>Conclusion</p><p>We observed a potential change in the thyroid hormones of pregnant women during early pregnancy after DnBP exposure. Additional study is necessitated to clarify these associations.</p></div

Pearson correlation between thyroid hormone, age, duration of gestation, time to pregnancy and urinary phthalate metabolite levels (ng/ mL, N = 97)^a.

<p>Pearson correlation between thyroid hormone, age, duration of gestation, time to pregnancy and urinary phthalate metabolite levels (ng/ mL, N = 97)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159398#t004fn001" target="_blank">^a</a>.</p

Comparison of urinary phthalate metabolites (ng/mL) at the first trimester in pregnant women in Taiwan, Unite States, Canada and Puerto Rico.

<p>TBC: Tainan Birth Cohort; TIDES: The Infant Development and the Environment Study; MIREC: Maternal-Infant Research on Environmental Chemicals; PROTECT: Puerto Rico Testsite for Exploring Contamination Threats.</p