Natalia V. Oleinik

Cooperation between JNK1 and JNK2 in activation of p53 apoptotic pathway

FDH (10-formyltetrahydrofolate dehydrogenase) is strongly downregulated in tumors while its elevation suppresses proliferation of cancer cells and induces p53-dependent apoptosis. We have previously shown that FDH induces phosphorylation of p53 at Ser6, which is a required step in the activation of apoptosis. In the present study, we report that FDH-induced p53 phosphorylation is carried out by JNK1 and JNK2 (c-Jun N-terminal kinases) working in concert. We have demonstrated that FDH induces phosphorylation of JNK1 and JNK2, while treatment of FDH-expressing cells with JNK inhibitor SP600125, as well as knockdown of JNK1 or JNK2 by siRNA, prevents phosphorylation of p53 at Ser6 and protects cells from apoptosis. Interestingly, the knockdown of JNK1 abolished phosphorylation of JNK2 in response to FDH, while knockdown of JNK2 did not prevent JNK1 phosphorylation. Pull-down assay with the p53-specific antibody has shown that JNK2, but not JNK1, is physically associated with p53. Our studies revealed a novel mechanism in which phosphorylation of JNK2 is mediated by JNK1 before phosphorylation of p53, and then p53 is directly phosphorylated by JNK2 at Ser6.

ALDH1L1 inhibits cell motility via dephosphorylation of cofilin by PP1 and PP2A

Here we report that ALDH1L1 (FDH, a folate enzyme with tumor suppressor-like properties) inhibits cell motility. The underlying mechanism involves F-actin stabilization, re-distribution of cytoplasmic actin toward strong preponderance of filamentous actin and formation of actin stress fibers. A549 cells expressing FDH showed a much slower recovery of green fluorescent protein-actin fluorescence in a fluorescence recovery after photobleaching assay, as well as an increase in G-actin polymerization and a decrease in F-actin depolymerization rates in pyren-actin fluorescence assays indicating the inhibition of actin dynamics. These effects were associated with robust dephosphorylation of the actin depolymerizing factor cofilin by PP1 and PP2A serine/threonine protein phosphatases, but not the cofilin-specific phosphatases slingshot and chronophin. In fact, the PP1/PP2A inhibitor calyculin prevented cofilin dephosphorylation and restored motility. Inhibition of FDH-induced apoptosis by the Jun N-terminal kinase inhibitor SP600125 or the pan-caspase inhibitor zVAD-fmk did not restore motility or levels of phosphor-cofilin, indicating that the observed effects are independent of FDH function in apoptosis. Interestingly, cofilin small interfering RNA or expression of phosphorylation-deficient S3A cofilin mutant resulted in a decrease of G-actin and the actin stress fiber formation, the effects seen upon FDH expression. In contrast, the expression of S3D mutant, mimicking constitutive phosphorylation, prevented these effects further supporting the cofilin-dependent mechanism. Dephosphorylation of cofilin and inhibition of motility in response to FDH can also be prevented by the increased folate in media. Furthermore, folate depletion itself, in the absence of FDH, resulted in cofilin dephosphorylation and inhibition of motility in several cell lines. Our experiments showed that these effects were folate specific and not a general response to nutrient starvation. Overall, this study shows the presence of distinct intracellular signaling pathways regulating motility in response to folate status and points toward mechanisms involving folates in promoting a malignant phenotype.

ALDH1L2 Is the Mitochondrial Homolog of 10-Formyltetrahydrofolate Dehydrogenase

Cytosolic 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an abundant enzyme of folate metabolism. It converts 10-formyltetrahydrofolate to tetrahydrofolate and CO2 in an NADP+-dependent reaction. We have identified a gene at chromosome locus 12q24.11 of the human genome, the product of which has 74% sequence similarity with cytosolic FDH. This protein has an extra N-terminal sequence of 22 amino acid residues, predicted to be a mitochondrial translocation signal. Transfection of COS-7 or A549 cell lines with a construct in which green fluorescent protein was introduced between the leader sequence and the rest of the putative mitochondrial FDH (mtFDH) has demonstrated mitochondrial localization of the fusion protein, suggesting that the identified gene encodes a mitochondrial enzyme. Purified pig liver mtFDH displayed dehydrogenase/hydrolase activities similar to cytosolic FDH. Real-time PCR performed on an array of human tissues has shown that although cytosolic FDH mRNA is highest in liver, kidney, and pancreas, mtFDH mRNA is most highly expressed in pancreas, heart, and brain. In contrast to the cytosolic enzyme, which is not detectable in cancer cells, the presence of mtFDH was demonstrated in several human cancer cell lines by conventional and real-time PCR and by Western blot. Analysis of genomes of different species indicates that the mitochondrial enzyme is a later evolutionary product when compared with the cytosolic enzyme. We propose that this novel mitochondrial enzyme is a likely source of CO2 production from 10-formyltetrahydrofolate in mitochondria and plays an essential role in the distribution of one-carbon groups between the cytosolic and mitochondrial compartments of the cell.

Binding of the sphingolipid S1P to hTERT stabilizes telomerase at the nuclear periphery by allosterically mimicking protein phosphorylation

Sphingosine 1-phosphate binds and stabilizes telomerase, a process that could be targeted to promote senescence and reduce cancer growth. Telomerase stabilized by a sphingolipid In normal adult cells, the structure at the ends of chromosomes, called the telomere, becomes progressively shorter with each replication cycle. When telomeres get too short or damaged, the cell stops dividing and becomes senescent. The enzyme telomerase maintains the integrity of telomeres, and phosphorylation stabilizes the catalytic subunit (called TERT) of this enzyme. In both normal fibroblasts and lung cancer cells, Panneer Selvam et al. found that binding of the phospholipid S1P to TERT mimicked protein phosphorylation and prevented TERT degration. Disrupting the interaction between S1P and TERT by either depleting S1P or mutating the interaction site in TERT impaired telomere maintenance and promoted senescence in cultured cells and decreased the growth of lung cancer cell xenografts in mice. During DNA replication, the enzyme telomerase maintains the ends of chromosomes, called telomeres. Shortened telomeres trigger cell senescence, and cancer cells often have increased telomerase activity to promote their ability to proliferate indefinitely. The catalytic subunit, human telomerase reverse transcriptase (hTERT), is stabilized by phosphorylation. We found that the lysophospholipid sphingosine 1-phosphate (S1P), generated by sphingosine kinase 2 (SK2), bound hTERT at the nuclear periphery in human and mouse fibroblasts. Docking predictions and mutational analyses revealed that binding occurred between a hydroxyl group (C′3-OH) in S1P and Asp684 in hTERT. Inhibiting or depleting SK2 or mutating the S1P binding site decreased the stability of hTERT in cultured cells and promoted senescence and loss of telomere integrity. S1P binding inhibited the interaction of hTERT with makorin ring finger protein 1 (MKRN1), an E3 ubiquitin ligase that tags hTERT for degradation. Murine Lewis lung carcinoma (LLC) cells formed smaller tumors in mice lacking SK2 than in wild-type mice, and knocking down SK2 in LLC cells before implantation into mice suppressed their growth. Pharmacologically inhibiting SK2 decreased the growth of subcutaneous A549 lung cancer cell–derived xenografts in mice, and expression of wild-type hTERT, but not an S1P-binding mutant, restored tumor growth. Thus, our data suggest that S1P binding to hTERT allosterically mimicks phosphorylation, promoting telomerase stability and hence telomere maintenance, cell proliferation, and tumor growth.

Cancer cells activate p53 in response to 10-formyltetrahydrofolate dehydrogenase expression

A folate enzyme, FDH (10-formyltetrahydrofolate dehydrogenase; EC 1.5.1.6), is not a typical tumour suppressor, but it has two basic characteristics of one, i.e. it is down-regulated in tumours and its expression is selectively cytotoxic to cancer cells. We have recently shown that ectopic expression of FDH in A549 lung cancer cells induces G1 arrest and apoptosis that was accompanied by elevation of p53 and its downstream target, p21. It was not known, however, whether FDH-induced apoptosis is p53-dependent or not. In the present study, we report that FDH-induced suppressor effects are strictly p53-dependent in A549 cells. Both knockdown of p53 using an RNAi (RNA interference) approach and disabling of p53 function by dominant-negative inhibition with R175H mutant p53 prevented FDH-induced cytotoxicity in these cells. Ablation of the FDH-suppressor effect is associated with an inability to activate apoptosis in the absence of functional p53. We have also shown that FDH elevation results in p53 phosphorylation at Ser-6 and Ser-20 in the p53 transactivation domain, and Ser-392 in the C-terminal domain, but only Ser-6 is strictly required to mediate FDH effects. Also, translocation of p53 to the nuclei and expression of the pro-apoptotic protein PUMA (Bcl2 binding component 3) was observed after induction of FDH expression. Elevation of FDH in p53 functional HCT116 cells induced strong growth inhibition, while growth of p53-deficient HCT116 cells was unaffected. This implies that activation of p53-dependent pathways is a general downstream mechanism in response to induction of FDH expression in p53 functional cancer cells.

Activation of p21-Dependent G1/G2 Arrest in the Absence of DNA Damage as an Antiapoptotic Response to Metabolic Stress

The folate enzyme, FDH (10-formyltetrahydrofolate dehydrogenase, ALDH1L1), a metabolic regulator of proliferation, activates p53-dependent G1 arrest and apoptosis in A549 cells. In the present study, we have demonstrated that FDH-induced apoptosis is abrogated upon siRNA knockdown of the p53 downstream target PUMA. Conversely, siRNA knockdown of p21 eliminated FDH-dependent G1 arrest and resulted in an early apoptosis onset. The acceleration of FDH-dependent apoptosis was even more profound in another cell line, HCT116, in which the p21 gene was silenced through homologous recombination (p21(-/-) cells). In contrast to A549 cells, FDH caused G2 instead of G1 arrest in HCT116 p21(+/+) cells; such an arrest was not seen in p21-deficient (HCT116 p21(-/-)) cells. In agreement with the cell cycle regulatory function of p21, its strong accumulation in nuclei was seen upon FDH expression. Interestingly, our study did not reveal DNA damage upon FDH elevation in either cell line, as judged by comet assay and the evaluation of histone H2AX phosphorylation. In both A549 and HCT116 cell lines, FDH induced a strong decrease in the intracellular ATP pool (2-fold and 30-fold, respectively), an indication of a decrease in de novo purine biosynthesis as we previously reported. The underlying mechanism for the drop in ATP was the strong decrease in intracellular 10-formyltetrahydrofolate, a substrate in two reactions of the de novo purine pathway. Overall, we have demonstrated that p21 can activate G1 or G2 arrest in the absence of DNA damage as a response to metabolite deprivation. In the case of FDH-related metabolic alterations, this response delays apoptosis but is not sufficient to prevent cell death.

A Novel Tumor Suppressor Function of Glycine N-Methyltransferase Is Independent of Its Catalytic Activity but Requires Nuclear Localization

Glycine N-methyltransferase (GNMT), an abundant cytosolic enzyme, catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to glycine generating S-adenosylhomocysteine and sarcosine (N-methylglycine). This reaction is regulated by 5-methyltetrahydrofolate, which inhibits the enzyme catalysis. In the present study, we observed that GNMT is strongly down regulated in human cancers and is undetectable in cancer cell lines while the transient expression of the protein in cancer cells induces apoptosis and results in the activation of ERK1/2 as an early pro-survival response. The antiproliferative effect of GNMT can be partially reversed by treatment with the pan-caspase inhibitor zVAD-fmk but not by supplementation with high folate or SAM. GNMT exerts the suppressor effect primarily in cells originated from malignant tumors: transformed cell line of non-cancer origin, HEK293, was insensitive to GNMT. Of note, high levels of GNMT, detected in regenerating liver and in NIH3T3 mouse fibroblasts, do not produce cytotoxic effects. Importantly, GNMT, a predominantly cytoplasmic protein, was translocated into nuclei upon transfection of cancer cells. The presence of GNMT in the nuclei was also observed in normal human tissues by immunohistochemical staining. We further demonstrated that the induction of apoptosis is associated with the GNMT nuclear localization but is independent of its catalytic activity or folate binding. GNMT targeted to nuclei, through the fusion with nuclear localization signal, still exerts strong antiproliferative effects while its restriction to cytoplasm, through the fusion with nuclear export signal, prevents these effects (in each case the protein was excluded from cytosol or nuclei, respectively). Overall, our study indicates that GNMT has a secondary function, as a regulator of cellular proliferation, which is independent of its catalytic role.

Acyl Carrier Protein-specific 4′-Phosphopantetheinyl Transferase Activates 10-Formyltetrahydrofolate Dehydrogenase

4′-Phosphopantetheinyl transferases (PPTs) catalyze the transfer of 4′-phosphopantetheine (4-PP) from coenzyme A to a conserved serine residue of their protein substrates. In humans, the number of pathways utilizing the 4-PP post-translational modification is limited and may only require a single broad specificity PPT for all phosphopantetheinylation reactions. Recently, we have shown that one of the enzymes of folate metabolism, 10-formyltetrahydrofolate dehydrogenase (FDH), requires a 4-PP prosthetic group for catalysis. This moiety acts as a swinging arm to couple the activities of the two catalytic domains of FDH and allows the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. In the current study, we demonstrate that the broad specificity human PPT converts apo-FDH to holoenzyme and thus activates FDH catalysis. Silencing PPT by small interfering RNA in A549 cells prevents FDH modification, indicating the lack of alternative enzymes capable of accomplishing this transferase reaction. Interestingly, PPT-silenced cells demonstrate significantly reduced proliferation and undergo strong G1 arrest, suggesting that the enzymatic function of PPT is essential and nonredundant. Our study identifies human PPT as the FDH-modifying enzyme and supports the hypothesis that mammals utilize a single enzyme for all phosphopantetheinylation reactions.

10-Formyltetrahydrofolate Dehydrogenase–Induced c-Jun-NH2-Kinase Pathways Diverge at the c-Jun-NH2-Kinase Substrate Level in Cells with Different p53 Status

10-Formyltetrahydrofolate dehydrogenase (FDH) suppresses cancer cell proliferation through p53-dependent apoptosis but also induces strong cytotoxicity in p53-deficient prostate cells. In the present study, we have shown that FDH induces apoptosis in PC-3 prostate cells through simultaneous activation of the c-Jun-NH2-kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways with JNK phosphorylating c-Jun and ERK1/2 phosphorylating Elk-1. The JNK1/2 inhibitor SP600125 or ERK1/2 inhibitor PD98059 prevented phosphorylation of c-Jun and Elk-1, correspondingly and partially protected PC-3 cells from FDH-induced cytotoxicity. Combination of the two inhibitors produced an additive effect. The contribution from the JNK cascade to FDH-induced apoptosis was significantly stronger than from the ERK pathway. siRNA knockdown of JNK1/2 or “turning off” the downstream target c-Jun by either siRNA or expression of the dominant-negative c-Jun mutant, TAM67, rescued PC-3 cells from FDH-induced apoptosis. The pull-down assays on immobilized c-Jun showed that c-Jun is directly phosphorylated by JNK2 in FDH-expressing cells. Interestingly, the FDH-induced apoptosis in p53-proficient A549 cells also proceeds through activation of JNK1/2, but the down-stream target for JNK2 is p53 instead of c-Jun. Furthermore, in A549 cells, FDH activates caspase 9, whereas in PC-3 cells, it activates caspase 8. Our studies indicate that the JNK pathways are common downstream mechanisms of FDH-induced cytotoxicity in different cell types, whereas the end point target in the cascade is cell type specific. JNK activation in response to FDH was inhibited by high supplementation of reduced folate leucovorin, further indicating a functional connection between folate metabolism and mitogen-activated protein kinase pathways. (Mol Cancer Res 2009;7(1):99–107)

HPV/E7 induces chemotherapy‐mediated tumor suppression by ceramide‐dependent mitophagy

Human papillomavirus (HPV) infection is linked to improved survival in response to chemo‐radiotherapy for patients with oropharynx head and neck squamous cell carcinoma (HNSCC). However, mechanisms involved in increased HNSCC cell death by HPV signaling in response to therapy are largely unknown. Here, using molecular, pharmacologic and genetic tools, we show that HPV early protein 7 (E7) enhances ceramide‐mediated lethal mitophagy in response to chemotherapy‐induced cellular stress in HPV‐positive HNSCC cells by selectively targeting retinoblastoma protein (RB). Inhibition of RB by HPV‐E7 relieves E2F5, which then associates with DRP1, providing a scaffolding platform for Drp1 activation and mitochondrial translocation, leading to mitochondrial fission and increased lethal mitophagy. Ectopic expression of a constitutively active mutant RB, which is not inhibited by HPV‐E7, attenuated ceramide‐dependent mitophagy and cell death in HPV(+) HNSCC cells. Moreover, mutation of E2F5 to prevent Drp1 activation inhibited mitophagy in HPV(+) cells. Activation of Drp1 with E2F5‐mimetic peptide for inducing Drp1 mitochondrial localization enhanced ceramide‐mediated mitophagy and led to tumor suppression in HPV‐negative HNSCC‐derived xenograft tumors in response to cisplatin in SCID mice.