Terfenadine induces apoptosis and autophagy in melanoma cells through ROS-dependent and -independent mechanisms

Previously we found that terfenadine, an H1 histamine receptor antagonist, acts as a potent apoptosis inducer in melanoma cells through modulation of Ca(2+) homeostasis. In this report, focusing our attention on the apoptotic mechanisms activated by terfenadine, we show that this drug can potentially activate distinct intrinsic signaling pathways depending on culture conditions. Serum-deprived conditions enhance the cytotoxic effect of terfenadine and caspase-4 and -2 are activated upstream of caspase-9. Moreover, although we found an increase in ROS levels, the apoptosis was ROS independent. Conversely, terfenadine treatment in complete medium induced ROS-dependent apoptosis. Caspase-4, -2, and -9 were simultaneously activated and p73 and Noxa induction were involved. ROS inhibition prevented p73 and Noxa expression but not p53 and p21 expression, suggesting a role for Noxa in p53-independent apoptosis in melanoma cells. Finally, we found that terfenadine induced autophagy, that can promote apoptosis. These findings demonstrate the great potential of terfenadine to kill melanoma cells through different cellular signaling pathways and could contribute to define new therapeutic strategies in melanoma.     

Intact p53-Dependent Responses in miR-34-Deficient Mice

MicroRNAs belonging to the miR-34 family have been proposed as critical modulators of the p53 pathway and potential tumor suppressors in human cancers. To formally test these hypotheses, we have generated mice carrying targeted deletion of all three members of this microRNA family. We show that complete inactivation of miR-34 function is compatible with normal development in mice. Surprisingly, p53 function appears to be intact in miR-34-deficient cells and tissues. Although loss of miR-34 expression leads to a slight increase in cellular proliferation in vitro, it does not impair p53-induced cell cycle arrest or apoptosis. Furthermore, in contrast to p53-deficient mice, miR-34-deficient animals do not display increased susceptibility to spontaneous, irradiation-induced, or c-Myc-initiated tumorigenesis. We also show that expression of members of the miR-34 family is particularly high in the testes, lungs, and brains of mice and that it is largely p53-independent in these tissues. These findings indicate that miR-34 plays a redundant function in the p53 pathway and suggest additional p53-independent functions for this family of miRNAs.     

miR-34a is essential for p19Arf-driven cell cycle arrest

The Arf tumor suppressor gene product, p19^Arf, regulates cell proliferation in incipient cancer cells and during embryo development. Beyond its commonly accepted p53-dependent actions, p19^Arf also acts independently of p53 in both contexts. One such p53-independent effect with in vivo relevance includes its repression of Pdgfrβ, a process that is essential for vision in the mouse. We have utilized cell culture-based and mouse models to define a new role for miR-34a in this process. Ectopic expression of Arf in cultured cells enhanced the expression of several microRNAs predicted to target Pdgfrß synthesis, including the miR-34 family. Because miR-34a has been implicated as a p53-dependent effector, we investigated whether it also contributed to p53-independent effects of p19^Arf. Indeed, in mouse embryo fibroblasts (MEFs) lacking p53, Arf-driven repression of Pdgfrβ and its blockade of Pdgf-B stimulated DNA synthesis were both completely interrupted by anti-microRNA against miR-34a. Ectopic miR-34a directly targeted Pdgfrβ and a plasmid reporter containing wild-type Pdgfrβ 3′UTR sequence, but not one in which the miR-34a target sequence was mutated. Although miR-34a expression has been linked to p53—a well-known effector of p19^Arf—Arf expression and its knockdown correlated with miR-34a level in MEFs lacking p53. Finally, analysis of the mouse embryonic eye demonstrated that Arf controlled expression of miR-34a, and the related miR-34b and c, in vivo during normal mouse development. Our findings indicate that miR-34a provides an essential link between p19^Arf and its p53-independent capacity to block cell proliferation driven by Pdgfrβ. This has ramifications for developmental and tumor suppressor roles of Arf.     

Mdm2 inhibition induces apoptosis in p53 deficient human colon cancer cells by activating p73- and E2F1-mediated expression of PUMA and Siva-1

Camptothecin (CPT) and Nutlin-3 caused apoptosis by increasing p53 protein and its activation in intestinal epithelial cells (IEC-6). We studied the effectiveness of these inducers on apoptosis in human colon cancer cells (Caco2) lacking p53 expression. CPT failed to activate caspase-3 and cause apoptosis in these cells. The absence of p53 expression, higher basal Bcl-xL and lower Bax proteins prevented CPT-induced apoptosis. However, the Mdm2 antagonist Nutlin-3 induced apoptosis in a dose dependent manner by activating caspases-9 and -3. Nutlin-3 prevented the activation of AKT via PTEN-mediated inhibition of the PI3K pathway. Nutlin-3 increased the phosphorylation of retinoblastoma protein causing E2F1 release leading to induction of Siva-1. Nutlin-3-mediated degradation of Mdm2 caused the accumulation of p73, which induced the expression of p53 up-regulated modulator of apoptosis (PUMA). E2F1 and p73 knockdown decreased the expression of Siva and PUMA, respectively and abolished Nutlin-3-induced caspase-3 activation. Cycloheximide (CHX) inhibited Nutlin-3-induced Siva, Noxa, and PUMA expression and inhibited apoptosis in IEC-6 and Caco2 cells. These results indicate that translation of mRNAs induced by Nutlin-3 is critical for apoptosis. In summary, apoptosis in Caco2 cells lacking functional p53 occurred following the disruption of Mdm2 binding with p73 and Rb leading to the expression of pro-apoptotic proteins, PUMA, Noxa, and Siva-1.     

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.     

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.     

Regulation of Caenorhabditis elegans p53/CEP-1-dependent germ cell apoptosis by Ras/MAPK signaling

Maintaining genome stability in the germline is thought to be an evolutionarily ancient role of the p53 family. The sole Caenorhabditis elegans p53 family member CEP-1 is required for apoptosis induction in meiotic, late-stage pachytene germ cells in response to DNA damage and meiotic recombination failure. In an unbiased genetic screen for negative regulators of CEP-1, we found that increased activation of the C. elegans ERK orthologue MPK-1, resulting from either loss of the lip-1 phosphatase or activation of let-60 Ras, results in enhanced cep-1-dependent DNA damage induced apoptosis. We further show that MPK-1 is required for DNA damage-induced germ cell apoptosis. We provide evidence that MPK-1 signaling regulates the apoptotic competency of germ cells by restricting CEP-1 protein expression to cells in late pachytene. Restricting CEP-1 expression to cells in late pachytene is thought to ensure that apoptosis doesn't occur in earlier-stage cells where meiotic recombination occurs. MPK-1 signaling regulates CEP-1 expression in part by regulating the levels of GLD-1, a translational repressor of CEP-1, but also via a GLD-1-independent mechanism. In addition, we show that MPK-1 is phosphorylated and activated upon ionising radiation (IR) in late pachytene germ cells and that MPK-1-dependent CEP-1 activation may be in part direct, as these two proteins interact in a yeast two-hybrid assay. In summary, we report our novel finding that MAP kinase signaling controls CEP-1-dependent apoptosis by several different pathways that converge on CEP-1. Since apoptosis is also restricted to pachytene stage cells in mammalian germlines, analogous mechanisms regulating p53 family members are likely to be conserved throughout evolution.     

P53-dependent miRNAs mediate nitric oxide-induced apoptosis in colonic carcinogenesis

Both miRNAs and nitric oxide (NO) play important roles in colonic inflammation and tumorigenesis. Resistance of colonic epithelial cells to apoptosis may contribute to tumor development. We hypothesized that some miRNAs could increase the resistance of colonic cancer cells to nitric oxide-induced apoptotic cell death. Here we show that NO induced apoptosis and stimulated expression of some miRNAs. Loss of p53 not only blocked NO-induced apoptosis but also dramatically inhibited the expression of NO-related miRNAs, such as miR-34, miR-203, and miR-1301. In addition, blockage of p53-dependent miRNAs significantly reduced NO-induced apoptosis. Furthermore, forced expression of these miRNAs rendered HT-29 cells, which are resistant to apoptosis with mutant p53, more sensitive to NO-induced apoptotic cell death. Most interestingly, in a colitis-associated colon cancer mouse model, the level of miRNAs dropped significantly, accompanied by downregulation of p21, which is a key target gene of p53. In human colorectal cancer samples, the expression of miR-34 significantly correlated with the level of inducible nitric oxide synthase (iNOS). We contend that increased NO production may select cells with low levels of p53-dependent miRNAs which contributes to human colonic carcinogenesis and tumor progression.     

Downregulation of microRNA-144 inhibits proliferation and promotes the apoptosis of myelodysplastic syndrome cells through the activation of the AKAP12-dependent ERK1/2 signaling pathway

BackgroundMyelodysplastic syndromes (MDS) represent a family of hematopoietic stem cell disorders characterized by ineffective hematopoiesis. While the functions of many microRNAs have been identified in MDS, microRNA-144 (miR-144) remains poorly understood. Thus, the aim of the present study was to determine the effects of miR-144 on cell proliferation and apoptosis in MDS cells and mechanism thereof.MethodsMDS-related microarrays were used for screening differentially expressed genes in MDS. The relationship between miR-144 and A-kinase anchoring protein 12 (AKAP12) was determined by a dual luciferase reporter gene assay. Subsequently, gain- and loss-function approaches were used to assess the effects of miR-144 and AKAP12 on cell proliferation, cell cycle and cell apoptosis by MTT assay and flow cytometry. Following the induction of a mouse model with MDS, the tumor tissues were extract for evaluation of apoptosis and the expression of miR-144, AKAP12, and the relevant genes associated with extracellular-regulated protein kinases 1/2 (ERK1/2) signaling pathway and apoptosis.ResultsWe observed significantly diminished expression of AKAP12 in MDS samples. miR-144 directly bound to AKAP12 3′UTR and reduced its expression in hematopoietic cells. Downregulation of miR-144 or upregulation of AKAP12 was observed to prolong cell cycle, inhibit cell proliferation, and induce apoptosis, accompanied by increased expression of AKAP12, p-ERK1/2, caspase-3, caspase-9, Bax, and p53, as well as decreased expression of Bcl-2. The transplanted tumors in mice with down-regulated miR-144 exhibited a lower mean tumor diameter and weight, and increased apoptosis index and expression of AKAP12 and ERK1/2.ConclusionTaken together, these studies demonstrate the stimulative role of miR-144 in MDS progression by regulating AKAP12-dependent ERK1/2 signaling pathway.     

PRIMA-1Met/APR-246 induces wild-type p53-dependent suppression of malignant melanoma tumor growth in 3D culture and in vivo

Disseminating malignant melanoma is a lethal disease highly resistant to radio- and chemotherapy. Therefore, the development of new treatment strategies is strongly needed. Tumor suppressor p53-mediated apoptosis is essential for the response to radio- and chemotherapy. Although p53 is not frequently mutated in melanoma, it is inactivated by integrin αv-mediated signaling, as we previously demonstrated 1, which may account, at least partially, for increased apoptosis resistance of malignant melanoma. In this study we addressed the question whether functional restoration of p53 by APR-246 (PRIMA-1Met), which can reactivate mutant p53 and induce massive apoptosis in cancer cells, is able to restore the function of inactive p53 in melanoma. Using a three-dimensional collagen gel (3D-collagen) to culture melanoma cells carrying wild-type p53, we found that APR-246 treatment resulted in activation of p53, leading to increased expression of p53 pro-apoptotic targets Apaf1 and PUMA and activation of caspase- 9 and -3. Moreover, APR-246 triggered melanoma cell apoptosis that was mediated by p53 and caspase 9. Importantly, APR-246 treatment also suppressed human melanoma xenograft tumors in vivo in a p53-dependent manner. Thus, wild-type p53 reactivation may provide a novel approach for malignant melanoma treatment, with APR-246 as a candidate drug for such a development.     

The MK5/PRAK kinase and Myc form a negative feedback loop that is disrupted during colorectal tumorigenesis

Expression of the Myc oncoprotein is downregulated in response to stress signals to allow cells to cease proliferation and escape apoptosis, but the mechanisms involved in this process are poorly understood. Cell cycle arrest in response to DNA damage requires downregulation of Myc via a p53-independent signaling pathway. Here we have used siRNA?screening of the human kinome to identify MAPKAPK5 (MK5, PRAK) as a negative regulator of Myc expression. MK5 regulates translation of Myc, since it is required for expression of miR-34b and miR-34c that bind to the 3'UTR of MYC. MK5 activates miR-34b/c expression via phosphorylation of FoxO3a, thereby promoting nuclear localization of FoxO3a and enabling it to induce miR-34b/c expression and arrest proliferation. Expression of MK5 in turn is directly activated by Myc, forming a negative feedback loop. MK5 is downregulated in colon carcinomas, arguing that this feedback loop is disrupted during colorectal tumorigenesis.     

CD95 Is Part of a Let-7/p53/miR-34 Regulatory Network

The death receptor CD95 (APO-1/Fas) mediates apoptosis induction upon ligation by its cognate ligand CD95L. Two types of CD95 signaling pathways have been identified, which are characterized by the absence (Type I) or presence (Type II) of mitochondrial involvement. Micro(mi)RNAs are small noncoding RNAs that negatively regulate gene expression. They are important regulators of differentiation processes and are found frequently deregulated in many human cancers. We recently showed that Type I cells express less of the differentiation marker miRNA let-7 and, hence, likely represent more advanced tumor cells than the let-7 high expressing Type II cells. We have now identified miR-34a as a selective marker for cells that are sensitive to CD95-mediated apoptosis. Both CD95 and miR-34a are p53 target genes, and consequently, both the sensitivity of cancer cells to CD95-mediated apoptosis and the ability to respond to p53 mediated DNA genotoxic stress are linked. Interestingly, while miR-34a was found to positively correlate with the ability of cells to respond to genotoxic stress, let-7 was negatively correlated. The expression level of CD95 inversely correlated with the expression of let-7 suggesting regulation of let-7 expression by CD95. To test a link between p53 and miR-34a, we altered the expression of CD95. This affected the ability of cells to activate p53 and to regulate miR-34a. Our data point to a novel regulatory network comprising p53, CD95, let-7, and miR-34a that affects cancer cell survival, differentiation, and sensitivity to apoptotic signals. The possible relevance of this regulatory network for cancer stem cells is discussed.     

Proapoptotic p53-interacting protein 53BP2 is induced by UV irradiation but suppressed by p53

p53 is an important mediator of the cellular stress response with roles in cell cycle control, DNA repair, and apoptosis. 53BP2, a p53-interacting protein, enhances p53 transactivation, impedes cell cycle progression, and promotes apoptosis through unknown mechanisms. We now demonstrate that endogenous 53BP2 levels increase following UV irradiation induced DNA damage in a p53-independent manner. In contrast, we found that the presence of a wild-type (but not mutant) p53 gene suppressed 53BP2 steady-state levels in cell lines with defined p53 genotypes. Likewise, expression of a tetracycline-regulated wild-type p53 cDNA in p53-null fibroblasts caused a reduction in 53BP2 protein levels. However, 53BP2 levels were not reduced if the tetracycline-regulated p53 cDNA was expressed after UV damage in these cells. This suggests that UV damage activates cellular factors that can relieve the p53-mediated suppression of 53BP2 protein. To address the physiologic significance of 53BP2 induction, we utilized stable cell lines with a ponasterone A-regulated 53BP2 cDNA. Conditional expression of 53BP2 cDNA lowered the apoptotic threshold and decreased clonogenic survival following UV irradiation. Conversely, attenuation of endogenous 53BP2 induction with an antisense oligonucleotide resulted in enhanced clonogenic survival following UV irradiation. These results demonstrate that 53BP2 is a DNA damage-inducible protein that promotes DNA damage-induced apoptosis. Furthermore, 53BP2 expression is highly regulated and involves both p53-dependent and p53-independent mechanisms. Our data provide new insight into 53BP2 function and open new avenues for investigation into the cellular response to genotoxic stress.