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

GAK is phosphorylated by c-Src and translocated from the centrosome to chromatin at the end of telophase

<p>Cyclin G-associated kinase (GAK) harbors a consensus phosphorylation motif (Y412) for c-Src; however, its physiological significance remains elusive. Here, we show that GAK is phosphorylated by c-Src not only at Y412 but also at Y1149. An anti-GAK-pY412 antibody recognized the shifted band of GAK during M phase. Immunofluorescence (IF) showed that GAK-pY412/pY1149 signals were present in the nucleus during interphase, translocated to chromosomes at prophase and prometaphase, moved to centrosomes at metaphase, and finally translocated to chromosomes at the end of telophase, when nuclear membrane formation was almost complete. These subcellular movements of GAK resemble those of DNA licensing factors. Indeed, mass spectrometry identified mini-chromosome maintenance (MCM) 3, an essential component of the DNA licensing system, as one of the association partners of GAK; immunoprecipitation-mediated Western blotting confirmed their association <i>in vivo</i>. These results suggest that the c-Src_GAK_MCM axis plays an important role in cell cycle progression through control of the DNA replication licensing system.</p

Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells

<div><p>Withaferin A (WA), a major bioactive component of the Indian herb <i>Withania somnifera</i>, induces cell death (apoptosis/necrosis) in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD). WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs) in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L). Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS) in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L) suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death.</p></div

WA induces BAG3-mediated autophagy in PC-3 cells.

<p>A) Observation of EGFP and EGFP-LC3 signals by immunofluorescence microscopy. Bar, 10 μm. (B) Bar graphs showing the percentage of cells containing punctate EGFP-LC3; arrows show that the values gradually increased under the indicated conditions. Data are represented as the means ± SEM of three independent experiments; green arrows indicate statistically significant increases (**, <i>P</i> < 0.01). (C) Western blot analysis to detect LC-3 and GAPDH (loading control) in PC-3 under the indicated conditions. (D) Bar graphs showing cell viability (%) under the indicated conditions. (E) Western blot analysis to detect BAG3, LC-3, and GAPDH (loading control) in PC-3 cells in the presence of the indicated conditions of siBAG3 or siGL2 (negative control). (F) Bar graphs showing cell viability (%) at the indicated conditions. (D, F) Data are represented as means ± SEM of three independent experiments; red arrows indicate statistically significant reductions in cell viability (**, <i>P</i> < 0.01). (A–F) Samples were collected at 4 h after WA treatment. (G) Expression profiles of the autophagy-related proteins BAG3 and LC3 in PC-3 cells at 4, 8, and 24 h after treatment with 4 μM WA. NT: non-treated.</p

c-Fos and FLIP play a role in induction of cell death.

<p>(A, B) Western blot analysis to detect c-Fos (FosB) in TIG-1, LNCaP, DU-145, and PC-3 cells at 4, 8, and 24 h after treatment with 4 μM (A) or 2 μM (B) WA. NT, non-treated. (C) Western blot analysis to detect c-Fos, PARP, FLIP and GAPDH in PC-3 cells at 12 h after 4 μM WA treatment in the presence (+) or absence (-) of three different siRNAs (X–Z) from OriGene. (D) Viability of PC-3 cells after siFos treatment. Data are represented as means ± SEM of three independent experiments; pink arrows indicate a statistically significant reduction in cell number following siFos treatment (**, <i>P</i> < 0.01). (E) Population of apoptotic, necrotic, and live cells distinctly stained with Annexin V–EnzoGold, 7-AAD-Red, and GFP. Data are represented as means ± SEM of three independent experiments; red, blue, and green arrows indicate statistically significant changes (**, <i>P</i> < 0.01). (F) Western blot analysis of c-Fos, PARP, FLIP, and GAPDH in DU-145 at 12 h after 4 μM WA treatment in the presence (+) or absence (-) of siRNAs; siFos or siGL2 (siControl). (G) Viability of DU-145 cells after exogenous overexpression of pcDNA3-FLIP or vector alone in the presence of DMSO (solvent) or 4 μM WA.</p

Expression of HSP genes is up-regulated after WA treatment.

<p>(A) Western blot analysis to detect HSPA6, HSP40, HSPA70, EGR-1, PAR-4, and GAPDH (loading control) in TIG-1, LNCaP, DU-145, and PC-3 cells at 0, 4, 8 and 24 h after 4 μM WA treatment. Arrow indicates the band for bona fide EGR-1. (B) Influence of siHSPA6 expression on PC-3 cell growth. (i) Schematic for this experiment. (ii) Western blot analysis to confirm the knockdown of HSPA6 protein by siHSPA6, relative to cells treated with siGL2 (negative control). (iii) Influence of siHSPA6 and siGL2 on PC-3 cell growth, with or without WA treatment. Arrow highlights the reduction in PC-3 cell growth. (C) Influence of siHSF-1 expression on PC-3 cell growth. (i) Schematics for this experiment. (ii) Western blot analysis to confirm the knockdown of HSP protein by siHSF-1, relative to cells treated with siGL2 (negative control), and to determine whether HSF-1 knockdown affected HSP protein levels by detection of HSPA6, HSP40, HSPA70, and GAPDH in PC-3 cells. (iii) Influence of siHSF-1 and siGL2 on PC-3 cell growth, with or without WA treatment. Arrow highlights the reduction in PC-3 cell growth. Data are represented as means ± SEM of three independent experiments; pink, green, and blue arrows indicate significant decreases in cell viabilities (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01).</p

Sphere-forming PC3 and SAS cells had higher resistance to cisplatin, but not to WA, than adherent cells.

<p>(A) Frequency of spheres larger than 100 μm (i) and typical images (ii) after incubation of TIG-1, LNCaP, PC-3, DU-145, and SAS cells in sphere-formation medium for 10 days. (B) Viability of SAS cells after WA treatment. Bars represent means ± SEM of three independent experiments. Cell viability was measured at 4, 8, and 24 h after treatment with 2 μM (blue bars) or 4 μM (red bars) WA. NT, non-treated. Data are represented as means ± SEM of three independent experiments; red arrows indicate statistically significant reductions in cell viability (**, <i>P</i> < 0.01). (C) Western blot analysis to detect c-Fos, HSPA6, HSP40, HSP70, PARP, and GAPDH (loading control) in SAS cells at 4, 8, and 24 h after treatment with 2 μM or 4 μM WA. NT: non-treated. (D) Comparison of sensitivity to cisplatin between parental cells and spheres of PC-3 (i and ii) and SAS (iii and iv) after 48 h of incubation following treatment with indicated cisplatin concentrations.</p

Cell viability of TIG-1, LNCaP, DU-145, and PC-3 cells after WA treatment.

<p><b>(A, B)</b> Cell viability was measured at 4, 8, and 24 h after 2 μM (A) or 4 μM (B) WA treatment. NT, non-treated. Green and blue arrows indicate bars for surviving cells, whereas pink and red arrows indicate bars for cells that died under the same conditions. Yellow arrows indicate the samples used for DNA microarray analysis. Bars represent means ± SEM for three independent experiments. Purple arrows indicate significant reductions in cell viabilities (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01).</p