PH domain-only protein PHLDA3 is a p53-regulated repressor of Akt.

p53 And Akt are critical players regulating tumorigenesis with opposite effects: whereas p53 transactivates target genes to exert its function as a tumor suppressor, Akt phosphorylates its substrates and transduces downstream survival signals. In addition, p53 and Akt negatively regulate each other to balance survival and death signals within a cell. We now identify PHLDA3 as a p53 target gene that encodes a PH domain-only protein. We find that PHLDA3 competes with the PH domain of Akt for binding of membrane lipids, thereby inhibiting Akt translocation to the cellular membrane and activation. Ablation of endogenous PHLDA3 results in enhanced Akt activity and decrease of p53-dependent apoptosis. We also demonstrate the suppression of anchorage-independent cell growth by PHLDA3. Loss of the PHLDA3 genomic locus was frequently observed in primary lung cancers, suggesting a role of PHLDA3 in tumor suppression. Our results reveal a new mode of coordination between the p53 and Akt pathways.

TSPAN12 is a critical factor for cancer-fibroblast cell contact-mediated cancer invasion.

Communication between cancer cells and their microenvironment controls cancer progression. Although the tumor suppressor p53 functions in a cell-autonomous manner, it has also recently been shown to function in a non-cell-autonomous fashion. Although functional defects have been reported in p53 in stromal cells surrounding cancer, including mutations in the p53 gene and decreased p53 expression, the role of p53 in stromal cells during cancer progression remains unclear. We herein show that the expression of α-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts (CAFs), was increased by the ablation of p53 in lung fibroblasts. CAFs enhanced the invasion and proliferation of lung cancer cells when cocultured with p53-depleted fibroblasts and required contact between cancer and stromal cells. A comprehensive analysis using a DNA chip revealed that tetraspanin 12 (TSPAN12), which belongs to the tetraspanin protein family, was derepressed by p53 knockdown. TSPAN12 knockdown in p53-depleted fibroblasts inhibited cancer cell proliferation and invasion elicited by coculturing with p53-depleted fibroblasts in vitro, and inhibited tumor growth in vivo. It also decreased CXC chemokine ligand 6 (CXCL6) secretion through the ß-catenin signaling pathway, suggesting that cancer cell contact with TSPAN12 in fibroblasts transduced ß-catenin signaling into fibroblasts, leading to the secretion of CXCL6 to efficiently promote invasion. These results suggest that stroma-derived p53 plays a pivotal role in epithelial cancer progression and that TSPAN12 and CXCL6 are potential targets for lung cancer therapy.

Novel p53 target gene FUCA1 encodes a fucosidase and regulates growth and survival of cancer cells.

The tumor suppressor p53 functions by inducing the transcription of a collection of target genes. We previously attempted to identify p53 target genes by microarray expression and ChIP-sequencing analyses. In this study, we describe a novel p53 target gene, FUCA1, which encodes a fucosidase. Although fucosidase, α-l-1 (FUCA1) has been reported to be a lysosomal protein, we detected it outside of lysosomes and observed that its activity is highest at physiological pH. As there is a reported association between fucosylation and tumorigenesis, we investigated the potential role of FUCA1 in cancer. We found that overexpression of FUCA1, but not a mutant defective in enzyme activity, suppressed the growth of cancer cells and induced cell death. Furthermore, we showed that FUCA1 reduced fucosylation and activation of epidermal growth factor receptor, and concomitantly suppressed epidermal growth factor signaling pathways. FUCA1 loss-of-function mutations are found in several cancers, its expression is reduced in cancers of the large intestine, and low FUCA1 expression is associated with poorer prognosis in several cancers. These results show that protein defucosylation mediated by FUCA1 is involved in tumor suppression.

SRY and OCT4 Are Required for the Acquisition of Cancer Stem Cell-Like Properties and Are Potential Differentiation Therapy Targets.

The acquisition of stemness is a hallmark of aggressive human hepatocellular carcinoma (hHCC). The stem cell marker OCT4 is frequently expressed in HCCs, and its expression correlates with those of putative cancer stem cell (CSC) markers and CSC properties. Here, we describe a novel mechanism of CSC maintenance by SRY through OCT4. We previously reported that Sry is involved in tumor malignancy in rodent HCCs. However, the oncogenic function of SRY in hHCCs is poorly understood. Ectopic expression of SRY increased multiple stem cell factors, including OCT4 and CD13. The OCT4 promoter contained SRY-binding sites that were directly activated by SRY. In HCC-derived cells, SRY knockdown decreased OCT4 expression and cancer stem-like phenotypes such as self-renewal, chemoresistance, and tumorigenicity. Conversely, OCT4 and SRY overexpression promoted cancer stem-like phenotypes. OCT4 knockdown in SRY clones downregulated the self-renewal capacity and chemoresistance. These data suggest that SRY is involved in the maintenance of cancer stem-like characteristics through OCT4. Moreover, CSCs of HCC-derived cells differentiated into Tuj1-positive neuron-like cells by retinoic acid. Noteworthily, SRY was highly expressed in some hHCC patients. Taken together, our findings imply a novel therapeutic strategy against CSCs of hHCCs.

The Arf/p53 protein module, which induces apoptosis, down-regulates histone H2AX to allow normal cells to survive in the presence of anti-cancer drugs.

BACKGROUND: It is unclear how DNA-damaging agents target cancer cells over normal somatic cells. RESULTS: Arf/p53-dependent down-regulation of H2AX enables normal cells to survive after DNA damage. CONCLUSION: Transformed cells, which harbor mutations in either Arf or p53, are more sensitive to DNA-damaging agents. SIGNIFICANCE: Cellular transformation renders cells more susceptible to some DNA-damaging agents. Anti-cancer drugs generally target cancer cells rather than normal somatic cells. However, the factors that determine this differential sensitivity are poorly understood. Here we show that Arf/p53-dependent down-regulation of H2AX induced the selective survival of normal cells after drug treatment, resulting in the preferential targeting of cancer cells. Treatment with camptothecin, a topoisomerase I inhibitor, caused normal cells to down-regulate H2AX and become quiescent, a process mediated by both Arf and p53. In contrast, transformed cells that harbor mutations in either Arf or p53 do not down-regulate H2AX and are more sensitive to drugs unless they have developed drug resistance. Such transformation-associated changes in H2AX expression rendered cancer cells more susceptible to drug-induced damage (by two orders of magnitude). Thus, the expression of H2AX and γH2AX (phosphorylated form of H2AX at Ser-139) is a critical factor that determines drug sensitivity and should be considered when administering chemotherapy.

FBI1/Akirin2 promotes tumorigenicity and metastasis of Lewis lung carcinoma cells.

The 14-3-3 family of proteins regulates various signaling pathways involved in cell cycle, apoptosis, stress response, and malignant transformation. We previously demonstrated that the ß isoform of the 14-3-3 protein promotes cell growth and tumorigenicity of rat K2 hepatocellular carcinoma cells. We identified fourteen-three-three beta interactant 1 (FBI1)/Akirin2 as a binding partner of 14-3-3ß and showed that the complex of these proteins promotes tumorigenicity and metastasis of K2 cells. In addition, we demonstrated that FBI1/Akirin2 downregulation shortened the duration of MAPK activity. Because 14-3-3ß and FBI1/Akirin2 overexpression is observed in various cancer cell lines, 14-3-3ß-FBI1/Akirin2 oncogenic function should be elucidated in different types of cancer. In this study, we used LLC1 Lewis lung carcinoma cells as a model. We established FBI1/Akirin2 knockdown cell clones through transfection of an antisense FBI1/Akirin2 expression vector and assessed the capacity for cell growth in vitro and tumorigenicity and metastasis in vivo. FBI1/Akirin2 downregulation decreased anchorage-independent growth, whereas the growth rate in monolayer culture was not affected. Moreover, an in vivo assay in nude mice showed that FBI1/Akirin2 overexpression is required for LLC1 tumor growth and metastasis. These results suggest that FBI1/Akirin2 plays an important role in oncogenesis of LLC1 lung carcinoma cells, and this protein may also serve as an oncogene in other cancers.

Aralin, a type II ribosome-inactivating protein from Aralia elata, exhibits selective anticancer activity through the processed form of a 110-kDa high-density lipoprotein-binding protein: a promising anticancer drug.

Aralin from Aralia elata is a newly identified type II ribosome- inactivating protein, which preferentially induces apoptosis in cancer cells. In this study, we identified that the aralin receptor is a 110-kDa high-density lipoprotein-binding protein (HDLBP), which functions as a HDL receptor. The sensitivities of tumor cell lines to aralin were dependent on the expression levels of the 110-kDa HDLBP and its forced expression in aralin-resistant Huh7 cells conferred aralin sensitivity. HDLBP-knockdown HeLa cells showed a significant aralin resistance in vitro and in vivo. Conversely, ectopic expression of the 150-kDa HDLBP resulted in increased aralin sensitivity in vivo, accompanying enhanced expression of the 110-kDa HDLBP. Thus, these results showed that the 110-kDa HDLBP in lipid rafts acted as an aralin receptor and that its expression levels determined aralin sensitivity, suggesting that aralin could be a promising anticancer drug for HDLBP-overexpressing tumors.

Arf and p53 act as guardians of a quiescent cellular state by protecting against immortalization of cells with stable genomes.

Normal cells undergo a growth-arrested status that is produced by p53-dependent down-regulation of histone H2AX. Immortality is developed after abrogation of the H2AX-diminished state, which is associated with genomic instability (often with tetraploidy) and the induction of mutations in either the Arf or p53 gene. However, the role of Arf in control of H2AX expression and genome stability is still unclear. Here, we show that both Arf and p53 are required for the down-regulation of H2AX and formation of the growth-arrested state. Wild-type (WT) mouse embryonic fibroblasts (MEFs) subjected to tetraploidization with DNA lesions did not undergo mitotic catastrophe-associated cell death and stayed in a growth-arrested state, until immortality was attained with mutations in the Arf/p53 module and recovery of H2AX expression. Whereas tetraploidization was essential for immortalization of WT MEFs, this event was not required for immortalization of MEFs containing mutations in Arf/p53 and these cells still underwent mitotic catastrophe-associated cell death. Thus, WT MEFs are protected from immortalization with genome stability, which is abrogated with tetraploidization and mutation of either Arf or p53.

Cancer susceptibility polymorphism of p53 at codon 72 affects phosphorylation and degradation of p53 protein.

The common polymorphism of p53 at codon 72, either encoding proline or arginine, has drawn attention as a genetic factor associated with clinical outcome or cancer risk for the last 2 decades. We now show that these two polymorphic variants differ in protein structure, especially within the N-terminal region and, as a consequence, differ in post-translational modification at the N terminus. The arginine form (p53-72R) shows significantly enhanced phosphorylation at Ser-6 and Ser-20 compared with the proline form (p53-72P). We also show diminished Mdm2-mediated degradation of p53-72R compared with p53-72P, which is at least partly brought about by higher levels of phosphorylation at Ser-20 in p53-72R. Furthermore, enhanced p21 expression in p53-72R-expressing cells, which is dependent on phosphorylation at Ser-6, was demonstrated. Differential p21 expression between the variants was also observed upon activation of TGF-ß signaling. Collectively, we demonstrate a novel molecular difference and simultaneously suggest a difference in the tumor-suppressing function of the variants.

Interaction of Hand2 and E2a is important for transcription of Phox2b in sympathetic nervous system neuron differentiation.

Transcription factors play a crucial role in the development of various tissues. In particular, the transcription factors of the basic helix-loop-helix (bHLH) family are crucial regulators of neurodifferentiation. Previous studies suggested that the bHLH transcription factor Hand2 is essential for sympathetic nervous system neuron differentiation in vivo, but the molecular mechanisms involved have not been well elucidated. It is important for understanding their mode of action in cellular differentiation to clarify how these bHLH factors regulate distinct transcriptional targets in a temporally and spatially controlled manner. Recent reports on ES cell differentiation suggested that its molecular mechanism mimics that of in vivo neurogenesis. However, the diverse nature of ES cell populations has prevented efficient analysis. To address this issue, we previously established a cell line in P19 embryonal carcinoma (EC) cells. Efficient sympathetic nervous system (SNS) neuron differentiation is induced in the cell line. Using this cell line, we succeeded in showing that the interaction of bHLH transcription factor Hand2 with E2a is required for transcription of Phox2b, which is essential for autonomic nervous system neuron development, and this binding activates this expression in SNS differentiation. Moreover, we also demonstrated that Hes5 regulated the transcription of Phox2b as a negative regulator and it inhibited the SNS differentiation. These findings have enabled us to determine the novel regulatory mechanism of Phox2b in SNS differentiation.

Implication of Akt-dependent Prp19 alpha/14-3-3beta/Cdc5L complex formation in neuronal differentiation.

PRP19alpha and CDC5L are major components of the active spliceosome. However, their association process is still unknown. Here, we demonstrated that PRP19 alpha/14-3-3beta/CDC5L complex formation is regulated by Akt during nerve growth factor (NGF)-induced neuronal differentiation of PC12 cells. Analysis of PRP19 alpha mutants revealed that the phosphorylation of PRP19 alpha at Thr 193 by Akt was critical for its binding with 14-3-3beta to translocate into the nuclei and for PRP19 alpha/14-3-3beta/CDC5L complex formation in neuronal differentiation. Forced expression of either sense PRP19 alpha or sense 14-3-3beta RNAs promoted NGF-induced neuronal differentiation, whereas down-regulation of these mRNAs showed a suppressive effect. The nonphosphorylation mutant PRP19 alpha T193A lost its binding ability with 14-3-3beta and acted as a dominant-negative mutant in neuronal differentiation. These results imply that Akt-dependent phosphorylation of PRP19 alpha at Thr193 triggers PRP19 alpha/14-3-3beta/CDC5L complex formation in the nuclei, likely to assemble the active spliceosome against neurogenic pre-mRNAs.

The bHLH transcription factor Hand2 regulates the expression of nanog in ANS differentiation.

The basic helix-loop-helix transcription factor Hand2 is induced by bone morphogenetic proteins (BMPs) in neural crest-derived precursor cells during the early stage of development of the autonomic nervous system (ANS). Previous studies showed that Hand2 was essential for the ANS differentiation. However, regulatory mechanism of pluripotent genes has not been elucidated in ANS differentiation. Here, we show that Hand2 regulated nanog expression in ANS differentiation. Our studies demonstrated that the forced expression of Hand2 promoted the ANS differentiation program in P19 embryonal carcinoma (EC) cells without aggregation. Furthermore, our results suggested that Hand2 bound to the promoter of nanog, a gene required for embryonic stem cells self-renewal, and suppressed nanog expression after Hand2 induction. The rapid downregulation of nanog mRNA during ANS differentiation correlated with the Hand2 transcriptional activity and nanog promoter methylation. These findings are evidence for a presence of the novel regulatory mechanism of nanog in ANS differentiation.

Association of p16 homozygous deletions with clinicopathologic characteristics and EGFR/KRAS/p53 mutations in lung adenocarcinoma.

PURPOSE: The p16 gene is frequently inactivated in lung adenocarcinoma. In particular, homozygous deletions (HD) have been frequently detected in cell lines; however, their frequency and specificity is not well-established in primary tumors. The purpose of this study was to elucidate the prevalence and the timing for the occurrence of p16 HDs in lung adenocarcinoma progression in vivo. EXPERIMENTAL DESIGN: Multiple ligation-dependent probe amplification was used for the detection of p16 HDs in 28 primary small-sized lung adenocarcinomas and 22 metastatic lung adenocarcinomas to the brain. Cancer cells were isolated from primary adenocarcinoma specimens by laser capture microdissection. HDs were confirmed by quantitative real-time genomic PCR analysis. RESULTS: HDs were detected in 8 of 28 (29%) primary tumors, including 2 of 8 (25%) noninvasive bronchioloalveolar carcinomas, and 5 of 22 (26%) brain metastases, respectively. No significant associations were observed between p16 HDs and gender, age, smoking history, stage, and prognosis. HDs were detected with similar frequencies (17-29%) among adenocarcinomas with epidermal growth factor receptor (EGFR) mutations, with KRAS mutations, and without EGFR/KRAS mutations, and with similar frequencies (22-28%) between adenocarcinomas with and without p53 mutations. CONCLUSIONS: p16 HDs occur early in the development of lung adenocarcinomas and with similar frequencies among EGFR type, KRAS type, and non-EGFR/KRAS type lung adenocarcinomas. Tobacco carcinogens would not be a major factor inducing p16 HDs in lung adenocarcinoma progression.

Crystallization and preliminary X-ray diffraction data of the rat histone H1(0) globular domain.

The linker histones H1 are a family of lysine-rich proteins that associate with the stretch of DNA that enters and exits the nucleosome. The linker histones facilitate the compaction and condensation of chromatin. The globular domain of histone H1(0), a specific subtype of histone H1, was crystallized at 288 K using the microbatch under silicone oil method with potassium phosphate as a precipitating agent. Diffraction data were collected to a resolution of 1.98 A. The crystal belongs to the trigonal space group P3(1)21, with unit-cell parameters a = 54.13, b = 54.13, c = 71.99 A, and contains one molecule per asymmetric unit. The V(M) value and solvent content were calculated to be 3.04 A3 Da(-1) and 59.6%, respectively.

IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis.

The transcription factors HSF1 and p53 both modulate the stress response, thereby protecting and facilitating the recovery of stressed cells, but both have the potential to promote tumor development. Here we show that a p53 target gene, IER5, encodes an activator of HSF1. IER5 forms a ternary complex with HSF1 and the phosphatase PP2A, and promotes the dephosphorylation of HSF1 at numbers of serine and threonine residues, generating a novel, hypo-phosphorylated active form of HSF1. IER5 is also transcriptionally upregulated in various cancers, although this upregulation is not always p53-dependent. The IER5 locus is associated with a so-called super enhancer, frequently associated with hyperactivated oncogenes in cancer cell lines. Enhanced expression of IER5 induces abnormal HSF1 activation in cancer cells and contributes to the proliferation of these cells under stressed conditions. These results reveal the existence of a novel IER5-mediated cancer regulation pathway that is responsible for the activation of HSF1 observed in various cancers.

SGF29 and Sry pathway in hepatocarcinogenesis.

Deregulated c-Myc expression is a hallmark of many human cancers. We have recently identified a role of mammalian homolog of yeast SPT-ADA-GCN5-acetyltransferas (SAGA) complex component, SAGA-associated factor 29 (SGF29), in regulating the c-Myc overexpression. Here, we discuss the molecular nature of SFG29 in SPT3-TAF9-GCN5-acetyltransferase complex, a counterpart of yeast SAGA complex, and the mechanism through which the elevated SGF29 expression contribute to oncogenic potential of c-Myc in hepatocellularcarcinoma (HCC). We propose that the upstream regulation of SGF29 elicited by sex-determining region Y (Sry) is also augmented in HCC. We hypothesize that c-Myc elevation driven by the deregulated Sry and SGF29 pathway is implicated in the male specific acquisition of human HCCs.

Implication of Trip15/CSN2 in early stage of neuronal differentiation of P19 embryonal carcinoma cells.

Trip15/CSN2 is a transcriptional corepressor/a component of COP9 signalosome (CSN) and participates in various signaling pathways. However, participation of Trip15/CSN2 in neural differentiation is still obscure. Here, we show that Trip15/CSN2 plays a critical role in neuronal differentiation. The expression of Trip15/CSN2 mRNA was induced at an early stage of neuronal differentiation in the retinoic acid (RA)-treated P19 cells, but not in the triiodothyronine (T3)-primed cardiac muscular cell differentiation. The expression of Trip15/CSN2 mRNA in the rat brain was detected at E14 and the protein was localized in the nuclei of neonatal rat CNS neurons. Enforced expression of sense rat Trip15/CSN2 mRNA caused the downregulation of Oct-3/4 mRNA expression and was sufficient to convert P19 cells into neurons, but not glial cells, only after the aggregation without RA. In the presence of RA, exogenous expression of the sense mRNA caused the intense and rapid induction of neurogenic Brn-2 and Mash-1 mRNA expressions accompanying the strong downregulation of Oct-3/4 mRNA expression, and stimulated both neuronal and glial cell differentiations of P19 cells. In contrast, enforced expression of the antisense mRNA suppressed the commitment of RA-treated aggregation form of P19 cells to neuronal lineage. These data strongly suggest that Trip15/CSN2 could implicate in the commitment of multipotent embryonal carcinoma (EC) cells to neuronal fate through the downregulation of Oct-3/4 which suppresses neurogenic genes. Moreover, in addition to Trip15/CSN2, RA-regulated other factor(s) may be required for glial cell differentiation.

Sodium 5, 6-benzylidene-L-ascorbate induces in vitro neuronal cell differentiation accompanying apoptosis and necrosis.

Antiproliferative activity through induction of differentiation by chemotherapeutic agents is required for certain types of cancers. Here, we report that a potent antitumor agent, sodium 5, 6-benzylidene-L-ascorbate (SBA), could induce morphological change of human neuroblastoma IMR-32 cells into a ganglion-like cell aggregate (pseudoganglion) having many neurites and the property of cholinergic neurons. Simultaneously with neuronal differentiation, substantial apoptosis and necrosis/type 2 physiological cell death, which is independent of apoptosis and resistant to a broad-spectrum caspase inhibitor, Z-Asp-CH2-DCB, were also observed. These data indicated that SBA could suppress tumor cell growth through the induction of three different physiological pathways such as differentiation, apoptosis and necrosis by which tissues and organs regulate their own development and maintenance.

Expression of Nephrin Homologue in the Freshwater Planarian, Dugesia japonica.

Excretory organs contain epithelial cells that form a filtration membrane specialized for ultrafiltration to produce primary urine. In vertebrates, the filtration membrane is made up of slit diaphragm (SD) formed by glomerular podocytes. Basal metazoans such as flatworms are also known have filtration epithelial cells, called flame cells, which exhibit SD-like structures. The molecular components of podocyte SD have been studied in detail, while those of the SD-like structures in basal metazoans including flatworms remain to be clarified. To determine whether the SD-like structures in flatworms have molecular components common to the SD in vertebrate podocytes, we examined the expression of gene homologue for mammalian nephrin, which encodes an essential transmembrane protein that participates in the formation of the SD, in a species of flatworms, planarian (Dugesia japonica). Flame cells were distributed throughout the entire body of the planarian, but the nephrin-expressing cells identified by in situ hybridization were mainly detected at body periphery excluding head region. The distribution pattern of nephrin-expressing cells was similar to that of proliferating cell nuclear antigen-expressing neoblasts, which are pluripotent stem cells characteristic to planarians. These findings indicated that the SD-like structures can be formed without the Nephrin protein in planarian flame cells.

TSPAN2 is involved in cell invasion and motility during lung cancer progression.

In lung cancer progression, p53 mutations are more often observed in invasive tumors than in noninvasive tumors, suggesting that p53 is involved in tumor invasion and metastasis. To understand the nature of p53 function as a tumor suppressor, it is crucial to elucidate the detailed mechanism of the alteration in epithelial cells that follow oncogenic KRAS activation and p53 inactivation. Here, we report that KRAS activation induces epithelial-mesenchymal transition and that p53 inactivation is required for cell motility and invasiveness. Furthermore, TSPAN2, a transmembrane protein, is responsible for cell motility and invasiveness elicited by p53 inactivation. TSPAN2 is highly expressed in p53-mutated lung cancer cells, and high expression of TSPAN2 is associated with the poor prognosis of lung adenocarinomas. TSPAN2 knockdown suppresses metastasis to the lungs and liver, enabling prolonged survival. TSPAN2 enhances cell motility and invasiveness by assisting CD44 in scavenging intracellular reactive oxygen species.