LATS1/2 kinases trigger self-renewal of cancer stem cells in aggressive oral cancer

Cancer stem cells (CSCs), which play important roles in tumor initiation and progression, are resistant to many types of therapies. However, the regulatory mechanisms underlying CSC-specific properties, including self-renewal, are poorly understood. Here, we found that LATS1/2, the core Hippo pathway-kinases, were highly expressed in the oral squamous cell carcinoma line SAS, which exhibits high capacity of CSCs, and that depletion of these kinases prevented SAS cells from forming spheres under serum-free conditions. Detailed examination of the expression and activation of LATS kinases and related proteins over a time course of sphere formation revealed that LATS1/2 were more highly expressed and markedly activated before initiation of self-renewal. Moreover, TAZ, SNAIL, CHK1/2, and Aurora-A were expressed in hierarchical, oscillating patterns during sphere formation, suggesting that the process consists of four sequential steps. Our results indicate that LATS1/2 trigger self-renewal of CSCs by regulating the Hippo pathway, the EMT, and cell division

Late cornified envelope 1C (LCE1C), a transcriptional target of TAp63 phosphorylated at T46/T281, interacts with PRMT5

Abstract p63, a transcriptional factor that belongs to the p53 family, regulates epidermal differentiation, stemness, cell death, tumorigenesis, metastasis, and senescence. However, its molecular mechanism remains elusive. We report here that TAp63 phosphorylated at T46/T281 specifically upregulates the late cornified envelope 1C (LCE1C) gene that is essential at a relatively late stage of epithelial development. We identified these phosphorylation sites during a search for the targets of Cyclin G-associated kinase (GAK) in vitro. LCE1C was drastically upregulated by doxycycline-dependent expression of Myc-TAp63 wild-type protein. Luciferase reporter assays using the promoter region of the LCE1C gene confirmed that the phosphorylations of TAp63-T46/T281 contributed to full transcriptional activation of the LCE1C gene. LCE1C interacted with protein arginine methyltransferase 5 (PRMT5) and translocated it from the nucleus to the cytoplasm. Mass spectrometry and co-immunoprecipitation identified importin-α as one of the association partners of LCE1C. In summary, we propose that the GAK_TAp63-pT46/pT281_LCE1C axis plays an important role in preventing the nuclear function of PRMT5

Gefitinib and Luteolin Cause Growth Arrest of Human Prostate Cancer PC-3 Cells via Inhibition of Cyclin G-Associated Kinase and Induction of miR-630

<div><p>Cyclin G-associated kinase (GAK), a key player in clathrin-mediated membrane trafficking, is overexpressed in various cancer cells. Here, we report that GAK expression is positively correlated with the Gleason score in surgical specimens from prostate cancer patients. Embryonic fibroblasts from knockout mice expressing a kinase-dead (KD) form of GAK showed constitutive hyper-phosphorylation of the epidermal growth factor receptor (EGFR). In addition to the well-known EGFR inhibitors gefitinib and erlotinib, the dietary flavonoid luteolin was a potent inhibitor of the Ser/Thr kinase activity of GAK <i>in vitro</i>. Co-administration of luteolin and gefitinib to PC-3 cells had a greater effect on cell viability than administration of either compound alone; this decrease in viability was associated with drastic down-regulation of GAK protein expression. A comprehensive microRNA array analysis revealed increased expression of miR-630 and miR-5703 following treatment of PC-3 cells with luteolin and/or gefitinib, and exogenous overexpression of miR-630 caused growth arrest of these cells. GAK appears to be essential for cell death because co-administration of gefitinib and luteolin to EGFR-deficient U2OS osteosarcoma cells also had a greater effect on cell viability than administration of either compound alone. Taken together, these findings suggest that GAK may be a new therapeutic target for prostate cancer and osteosarcoma.</p></div

Luteolin and gefitinib induce death of PC-3 cells.

<p>A, the numbers of viable PC-3 cells 0, 24, 48, and 72 h after the addition of solvent alone (DMSO), 60 µM luteolin, 60 µM gefitinib, or 60 µM luteolin plus 60 µM gefitinib to the culture medium. The data are represented as the mean ± SEM of n = 3 independent experiments at each concentration. B, immunoblot analyses of GAK, AR, caspase-3, and α-tubulin (control) protein levels in PC-3 cells exposed to solvent alone (DMSO), 60 µM luteolin, 60 µM gefitinib, or 60 µM luteolin plus 60 µM gefitinib for 0–72 h. The arrowheads indicate reduced expression levels of GAK and AR in cells exposed to the drugs for 72 h. The arrows indicate the emergence of activated caspase-3, which suggested that gefitinib and luteolin induced apoptosis. C, FACS analysis of PC-3 cells PC-3 cells exposed to solvent alone (DMSO), 60 µM luteolin, 60 µM gefitinib, or 60 µM luteolin plus 60 µM gefitinib for 72 h. The arrowheads indicate the subG1 populations. D, the percentages of PC-3 cells in the indicated phases of the cell cycle after exposure to the same treatments for 72 h.</p

GAK is overexpressed in the nuclei of cancer cells.

<p>Western blot analysis of GAK, lamin A/C, and α-tubulin (control) in cytoplasmic (Cyt) and nuclear (Nuc) extracts from prostate cancer (PC-3 and LNCaP), breast cancer (MDA-MB231 and MCF-7) and cervical cancer (HeLaS3) cell lines. Lamin A/C was used as a nuclear marker. Two different anti-GAK antibodies were used. The arrows and arrowheads indicate the GAK bands identified primarily in the nuclear and cytoplasmic fractions, respectively.</p

Loss of GAK activity leads to constitutive hyper-phosphorylation of the EGFR.

<p>A, western blot analysis of EGFR expression in WT (GAK-kd^+/+) and mutant (GAK-kd^−/−) MEFs. B, the effects of a Tyr-phosphatase inhibitor (50 mM Na₃VO₄) and Ser/Thr phosphatase inhibitors (50 mM NaF and 50 mM β-glycerophosphate, or 2.5 µM okadaic acid) on the inhibition of EGFR phosphorylation by λ-phosphatase (λPPase; 200 U). A, B, the arrows indicate the phosphorylated EGFR protein. Alpha-tubulin (α-Tub) was used as a loading control. (C, D) Western blot analysis of expression levels of the EGFR and ERK1/2 in WT (+/+) and GAK-kd (−/−) MEFs following EGF stimulation for the indicated times. Cycloheximide (50 µg/ml) was added to the culture medium 1 h prior to EGF (10 µg/ml) to inhibit novel protein synthesis. The tilted and horizontal arrows indicate the phosphorylated and hyper-phosphorylated EGFR bands, respectively. GAPDH was used as a loading control. C, the arrowheads indicate differential expression of phosphorylated ERK1/2 in WT and GAK-kd MEFs. D, NT, non-treated. E, immunostaining of the EGFR protein in WT (+/+) and mutant (−/−) MEFs treated with or without the proteasome inhibitor MG132 (50 µg/ml). Notable panels are encircled by turquoise and green lines. F, the numbers of EGFR-positive cells in WT and GAK-kd (Homo) cells in the presence or absence of MG132. The data are represented as the mean ± SEM of n = 3 independent experiments at each time point.</p

Statistical analysis of the relationship between the Gleason score and GAK immunostaining in the prostatectomy specimens.

<p><i>P</i><0.01.</p

Luteolin and erlotinib inhibit GAK activity similarly to gefitinib and SB203580.

<p>A–C, representative SDS-PAGE analysis of proteins subjected to <i>in vitro</i> kinase assays using ³²P-γATP, GAK (∼100 µg/ml) as the enzyme, PP2A B′γ (∼10 µg/ml) as the substrate, and the indicated concentrations of gefitinib, erlotinib, and SB203580 as inhibitors. The signals incorporated were detected by autoradiography and the amounts of proteins loaded were assessed by Coomassie Brilliant Blue (CBB) staining. The graphs show the intensity ratios of phosphorylated PP2A B′γ and GAK relative to that of non-treated samples. The data are represented as the mean ± SEM of n = 3 independent experiments at each concentration.</p

Working model showing the molecular mechanisms involved in the induction of growth arrest and apoptosis of PC-3 cells by luteolin and gefitinib.

<p>The arrows and T-shaped lines signify positive and negative actions, respectively. The thickness of each line is proportional to the strength of the denoted action. The dashed green line and arrow indicate that these actions were verified by another group <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100124#pone.0100124-Lee1" target="_blank">[10]</a>. The red and blue arrows indicate up-regulation and down-regulation, respectively. Down-regulation of GAK leads to hyper-phosphorylation of tyrosine residues (pY) in EGFR.</p

GAK is overexpressed in radical prostatectomy specimens from prostate cancer patients.

<p>A–D, hematoxylin and eosin (H-E) staining (A, B) and anti-GAK immunostaining (C, D) of representative radical prostatectomy sections of cancerous (A, C) and normal (B, D) tissues from prostate cancer patients (n = 42). Scale bar = 50 µm. E, higher power magnification of the boxed area shown in C. Scale bar = 50 µm.</p