Differential regulation of the clusterin gene by Ha-ras and c-myc oncogenes and during apoptosis

Clusterin (ApoJ) is an extracellular glycoprotein expressed during processes of tissue differentiation and regression that involve programmed cell death (apoptosis). Increased clusterin expression has also been found in tumors, however, the mechanism underlying this induction is not known. Apoptotic processes in tumors could be responsible for clusterin gene activation. Alternatively, oncogenic mutations could modulate signal transduction, thereby inducing the gene. We examined the response of the rat clusterin gene to two oncogenes, Ha-ras and c-myc, in transfected Rat1 fibroblasts. While c-myc overexpression did not modify clusterin gene activity, the Ha-ras oncogene produced a seven to tenfold repression of clusterin mRNA; this down-regulation was also observed in the presence of c-myc. Since no induction of the clusterin gene was observed by the two oncogenes, we tested the alternative mechanism involving apoptosis. Growth factor withdrawal induced apoptosis, as shown by DNA degradation and micronuclei formation in the floating cells. Concomittantly we observed a three to tenfold increase in the amount of clusterin mRNA in the adhering cells of Rat1 and the c-myc transformed cell lines, and a weaker induction in the Ha-ras transformed cell line. On the basis of our results, we suggest that clusterin gene induction in the vital cells is produced by signaling molecules that are generated by the apoptotic cells. We conclude that apoptotic processes, not oncogenic mutations, are responsible for increased clusterin expression in tumors.     

Stem cell exhaustion and leukemogenesis

Cancer is the result of a combination of genetic alterations, which aid transformation of cells. However, oncogenic alterations also simultaneously induce some detrimental effects on the cells such as apoptosis, senescence, and differentiation. Such negative effects caused by certain oncogenic events are overcome by other cooperating genetic hits. We propose stem cell exhaustion as a novel detrimental effect that is caused by a wide variety of oncogenic alterations. Interestingly, in most cases, the stem cell exhaustion due to oncogenic alterations is preceded by an abnormal expansion of stem/progenitor cells. This preceding stem/progenitor cell expansion may be a key feature that still promotes cancer development, along with cooperating hits that rescue stem cell exhaustion. This review summarizes current knowledge about hematopoietic stem cell exhaustion and the mechanisms to overcome stem cell exhaustion in cancer development. J. Cell. Biochem. 107: 393–399, 2009. © 2009 Wiley‐Liss, Inc.     

MYC-induced apoptosis in mammary epithelial cells is associated with repression of lineage-specific gene signatures

Apoptosis caused by deregulated MYC expression is a prototype example of intrinsic tumor suppression. However, it is still unclear how supraphysiological MYC expression levels engage specific sets of target genes to promote apoptosis. Recently, we demonstrated that repression of SRF target genes by MYC/MIZ1 complexes limits AKT-dependent survival signaling and contributes to apoptosis induction. Here we report that supraphysiological levels of MYC repress gene sets that include markers of basal-like breast cancer cells, but not luminal cancer cells, in a MIZ1-dependent manner. Furthermore, repressed genes are part of a conserved gene signature characterizing the basal subpopulation of both murine and human mammary gland. These repressed genes play a role in epithelium and mammary gland development and overlap with genes mediating cell adhesion and extracellular matrix organization. Strikingly, acute activation of oncogenic MYC in basal mammary epithelial cells is sufficient to induce luminal cell identity markers. We propose that supraphysiological MYC expression impacts on mammary epithelial cell identity by repressing lineage-specific target genes. Such abrupt cell identity switch could interfere with adhesion-dependent survival signaling and thus promote apoptosis in pre-malignant epithelial tissue.     

Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells

Small cell lung cancer (SCLC) accounts for nearly 15% of human lung cancers and is one of the most aggressive solid tumors. The SCLC cells are thought to derive from self-renewing pulmonary neuroendocrine cells by oncogenic transformation. However, whether the SCLC cells possess stemness and plasticity for differentiation as normal stem cells has not been well understood thus far. In this study, we investigated the expressions of multilineage stem cell markers in the cancer cells of SCLC cell line (NCI-H446) and analyzed their clonogenicity, tumorigenicity, and plasticity for inducing differentiation. It has been found that most cancer cells of the cell line expressed multilineage stem cell markers under the routine culture conditions and generated single-cell clones in anchorage-dependent or -independent conditions. These cancer cells could form subcutaneous xenograft tumors and orthotopic lung xenograft tumors in BALB/C-nude mice. Most cells in xenograft tumors expressed stem cell markers and proliferation cell nuclear antigen Ki67, suggesting that these cancer cells remained stemness and highly proliferative ability in vivo. Intriguingly, the cancer cells could be induced to differentiate into neurons, adipocytes, and osteocytes, respectively, in vitro. During the processes of cellular phenotype-conversions, autophagy and apoptosis were two main metabolic events. There is cross-talking between autophagy and apoptosis in the differentiated cancer cells. In addition, the effects of the inhibitor and agonist for Sirtuin1/2 on the inducing osteogenic differentiation indicated that Sirtuin1/2 had an important role in this process. Taken together, these results indicate that most cancer cells of NCI-H446 cell line possess stemness and plasticity for multilineage differentiation. These findings have potentially some translational applications in treatments of SCLC with inducing differentiation therapy.     

Oncogenic Ras promotes butyrate-induced apoptosis through inhibition of gelsolin expression

Activation of Ras promotes oncogenesis by altering a multiple of cellular processes, such as cell cycle progression, differentiation, and apoptosis. Oncogenic Ras can either promote or inhibit apoptosis, depending on the cell type and the nature of the apoptotic stimuli. The response of normal and transformed colonic epithelial cells to the short chain fatty acid butyrate, a physiological regulator of epithelial cell maturation, is also divergent: normal epithelial cells proliferate, and transformed cells undergo apoptosis in response to butyrate. To investigate the role of k-ras mutations in butyrate-induced apoptosis, we utilized HCT116 cells, which harbor an oncogenic k-ras mutation and two isogenic clones with targeted inactivation of the mutant k-ras allele, Hkh2, and Hke-3. We demonstrated that the targeted deletion of the mutant k-ras allele is sufficient to protect epithelial cells from butyrate-induced apoptosis. Consistent with this, we showed that apigenin, a dietary flavonoid that has been shown to inhibit Ras signaling and to reverse transformation of cancer cell lines, prevented butyrate-induced apoptosis in HCT116 cells. To investigate the mechanism whereby activated k-ras sensitizes colonic cells to butyrate, we performed a genome-wide analysis of Ras target genes in the isogenic cell lines HCT116, Hkh2, and Hke-3. The gene exhibiting the greatest down-regulation by the activating k-ras mutation was gelsolin, an actin-binding protein whose expression is frequently reduced or absent in colorectal cancer cell lines and primary tumors. We demonstrated that silencing of gelsolin expression by small interfering RNA sensitized cells to butyrate-induced apoptosis through amplification of the activation of caspase-9 and caspase-7. These data therefore demonstrate that gelsolin protects cells from butyrate-induced apoptosis and suggest that Ras promotes apoptosis, at least in part, through its ability to down-regulate the expression of gelsolin.     

Notch-dependent downregulation of the homeodomain gene cut is required for the mitotic cycle/endocycle switch and cell differentiation in Drosophila follicle cells

During Drosophila mid-oogenesis, follicular epithelial cells switch from the mitotic cycle to the specialized endocycle in which the M phase is skipped. The switch, along with cell differentiation in follicle cells, is induced by Notch signaling. We show that the homeodomain gene cut functions as a linker between Notch and genes that are involved in cell-cycle progression. Cut was expressed in proliferating follicle cells but not in cells in the endocycle. Downregulation of Cut expression was controlled by the Notch pathway and was essential for follicle cells to differentiate and to enter the endocycle properly. cut-mutant follicle cells entered the endocycle and differentiated prematurely in a cell-autonomous manner. By contrast, prolonged expression of Cut caused defects in the mitotic cycle/endocycle switch. These cells continued to express an essential mitotic cyclin, Cyclin A, which is normally degraded by the Fizzy-related-APC/C ubiquitin proteosome system during the endocycle. Cut promoted Cyclin A expression by negatively regulating Fizzy-related. Our data suggest that Cut functions in regulating both cell differentiation and the cell cycle, and that downregulation of Cut by Notch contributes to the mitotic cycle/endocycle switch and cell differentiation in follicle cells.     

Oncogenic Ras-induced proliferation requires autocrine fibroblast growth factor 2 signaling in skeletal muscle cells

Constitutively activated Ras proteins are associated with a large number of human cancers, including those originating from skeletal muscle tissue. In this study, we show that ectopic expression of oncogenic Ras stimulates proliferation of the MM14 skeletal muscle satellite cell line in the absence of exogenously added fibroblast growth factors (FGFs). MM14 cells express FGF-1, -2, -6, and -7 and produce FGF protein, yet they are dependent on exogenously supplied FGFs to both maintain proliferation and repress terminal differentiation. Thus, the FGFs produced by these cells are either inaccessible or inactive, since the endogenous FGFs elicit no detectable biological response. Oncogenic Ras-induced proliferation is abolished by addition of an anti-FGF-2 blocking antibody, suramin, or treatment with either sodium chlorate or heparitinase, demonstrating an autocrine requirement for FGF-2. Oncogenic Ras does not appear to alter cellular export rates of FGF-2, which does not possess an NH(2)-terminal or internal signal peptide. However, oncogenic Ras does appear to be involved in releasing or activating inactive, extracellularly sequestered FGF-2. Surprisingly, inhibiting the autocrine FGF-2 required for proliferation has no effect on oncogenic Ras-mediated repression of muscle-specific gene expression. We conclude that oncogenic Ras-induced proliferation of skeletal muscle cells is mediated via a unique and novel mechanism that is distinct from Ras-induced repression of terminal differentiation and involves activation of extracellularly localized, inactive FGF-2.     

Inhibition of NEDD4 inhibits cell growth and invasion and induces cell apoptosis in bladder cancer cells

The neural precursor cell expressed developmentally downregulated protein 4 (NEDD4) plays a pivotal oncogenic role in various types of human cancers. However, the function of NEDD4 in bladder cancer has not been fully investigated. In the present study, we aim to explore whether NEDD4 governs cell proliferation, apoptosis, migration, and invasion in bladder cancer cells. Our results showed that downregulation of NEDD4 suppressed cell proliferation in bladder cancer cells. Moreover, we found that inhibition of NEDD4 significantly induced cell apoptosis. Furthermore, downregulation of NEDD4 retarded cell migration and invasion. Notably, overexpression of NEDD4 enhanced cell growth and inhibited apoptosis. Consistently, upregulation of NEDD4 promoted cell migration and invasion in bladder cancer cells. Mechanically, our Western blotting results revealed that downregulation of NEDD4 activated PTEN and inhibited Notch-1 expression, whereas upregulation of NEDD4 reduced PTEN level and increased Notch-1 level in bladder cancer cells. Our findings indicated that NEDD4 exerts its oncogenic function partly due to regulation of PTEN and Notch-1 in bladder cancer cells. These results further revealed that targeting NEDD4 could be a useful approach for the treatment of bladder cancer.     

TRAIL up-regulation must be accompanied by a reciprocal PKCε down-regulation during differentiation of colonic epithelial cell: implications for colorectal cancer cell differentiation

PKC isoenzymes play central roles in various cellular signalling pathways, participating in a variety of protein phosphorylation cascades that regulate/modulate cellular structure and gene expression. It has been firmly established that several isoforms of PKC have a role in the regulation of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) activity. Our interest in probing the role of the epsilon isoform of PKC in the colonic cell differentiation stems from the discovery that PKCε and TRAIL are involved in the differentiation of other cell types like hematopoietic stem cells. Although the role of PKCε and TRAIL in the gastrointestinal system is unclear, it has been observed that PKCε has oncogenic activity in colon epithelial cells (CEC), while TRAIL increases the death of intestinal epithelial cells during inflammation. Here we demonstrate a reciprocal expression of PKCε and TRAIL in human colon mucosa: CECs at the bottom of the colonic crypts show high levels of PKCε, being negative for TRAIL expression. On the contrary, luminal CECs are positive for TRAIL, while negative for PKCε. Indeed, TRAIL- and butyrate-induced differentiation of the human colorectal cancer cell line HT29 requires the decrease of PKCε expression, whose absence in turn increases cell sensitivity to TRAIL-induced apoptosis. Moreover, TRAIL preferentially promotes HT29 differentiation into goblet cells. Taken together, this data demonstrate that TRAIL and PKCε must be reciprocally regulated to ensure physiological CEC differentiation starting from the stem cell pool, and that the down-regulation of PKCε is however critical for the differentiation and apoptosis of cancer cells.     

Prevention of v-Ha-Ras-dependent apoptosis by PDGF coordinates in phosphorylation of ERK and Akt

In some v-Ha-ras-transfected cell lines, serum deprivation results in apoptosis. Clarification of the molecular mechanisms by which oncogenic Ras controls susceptibility to apoptosis may assist in the development of effective therapies against human cancer with oncogenic ras gene. In this report, we established a v-Ha-ras-transfected human fibroblast clone, R1. In R1 cells, induction of v-Ha-Ras enhanced susceptibility to cell death under serum-deprived conditions. Ladders of cellular DNA were identified only when oncogenic ras was induced under serum-deprived conditions. Platelet-derived growth factor (PDGF) precluded DNA fragmentation of serum-deprived v-Ha-ras-transformed cells. Under serum-depleted conditions, the amounts of activated ERK and Akt decreased as compared with those under serum-containing conditions. The decreased levels of activated ERK and Akt were restored by the addition of PDGF. Inhibition of phosphorylated-ERK and Akt resulted in renewed susceptibility to cell death. These results indicate that failure of signal transduction of oncogenic Ras by the deficiency of growth factors such as PDGF causes v-Ha-Ras-dependent apoptosis.     

Clusterin inhibits apoptosis by interacting with activated Bax

Clusterin is an enigmatic glycoprotein that is overexpressed in several human cancers such as prostate and breast cancers, and squamous cell carcinoma. Because the suppression of clusterin expression renders human cancer cells sensitive to chemotherapeutic drug-mediated apoptosis, it is currently an antisense target in clinical trials for prostate cancer. However, the molecular mechanisms by which clusterin inhibits apoptosis in human cancer cells are unknown. Here we report that intracellular clusterin inhibits apoptosis by interfering with Bax activation in mitochondria. Intriguingly, in contrast to other inhibitors of Bax, clusterin specifically interacts with conformation-altered Bax in response to chemotherapeutic drugs. This interaction impedes Bax oligomerization, which leads to the release of cytochrome c from mitochondria and caspase activation. Moreover, we also find that clusterin inhibits oncogenic c-Myc-mediated apoptosis by interacting with conformation-altered Bax. Clusterin promotes c-Myc-mediated transformation in vitro and tumour progression in vivo. Taken together, our results suggest that the elevated level of clusterin in human cancers may promote oncogenic transformation and tumour progression by interfering with Bax pro-apoptotic activities.     

Atypical Protein Kinase Cs Are the Ras Effectors That Mediate Repression of Myogenic Satellite Cell Differentiation

Oncogenic Ha-Ras is a potent inhibitor of skeletal muscle cell differentiation, yet the Ras effector mediating this process remains unidentified. Here we demonstrate that the atypical protein kinases (aPKCs; lambda and/or zeta) are downstream Ras effectors responsible for Ras-dependent inhibition of myogenic differentiation in a satellite cell line. First, ectopic expression of Ha-RasG12V induces translocation of PKClambda from the cytosol to the nucleus, suggesting that aPKCs are activated by Ras in myoblasts. The aPKCs function as downstream Ras effectors since inhibition of aPKCs by expression of a dominant negative PKCzeta mutant or by treatment of cells with an inhibitor, GO6983, promotes myogenesis in skeletal muscle satellite cells expressing oncogenic Ha-Ras. Arresting cell proliferation synergistically enhances myogenic differentiation only when aPKCs are also inhibited. Thus, the repression of myogenic differentiation in a satellite cell line appears to be directly mediated by aPKCs acting as Ras effectors and indirectly mediated via stimulation of cell proliferation.     

Oncogenic Ras inhibits Fas ligand-mediated apoptosis by downregulating the expression of Fas

Tumor growth is the result of deregulated tissue homeostasis which is maintained through the delicate balance of cell growth and apoptosis. One of the most efficient inducers of apoptosis is the death receptor Fas. We report here that oncogenic Ras (H-Ras) downregulates Fas expression and renders cells of fibroblastic and epitheloid origin resistant to Fas ligand-induced apoptosis. In Ras-transformed cells, Fas mRNA is absent. Inhibition of DNA methylation restores Fas expression. H-Ras signals via the PI 3-kinase pathway to downregulate Fas, suggesting that the known anti-apoptotic effect of the downstream PKB/Akt kinase may be mediated, at least in part, by the repression of Fas expression. Thus, the oncogenic potential of H-ras may reside on its capacity not only to promote cellular proliferation, but also to simultaneously inhibit Fas-triggered apoptosis.