Amyloid Beta Leads to Decreased Acetylcholine Levels and Non-Small Cell Lung Cancer Cell Survival via a Mechanism That Involves p38 Mitogen-Activated Protein Kinase and Protein Kinase C in a p53-Dependent and -Independent Manner.

Several studies have shown an inverse correlation between the likelihood of developing a neurodegenerative disorder and cancer. We previously reported that the levels of amyloid beta (Aß), at the center of Alzheimer's disease pathophysiology, are regulated by acetylcholinesterase (AChE) in non-small cell lung cancer (NSCLC). Here, we examined the effect of Aß or its fragments on the levels of ACh in A549 (p53 wild-type) and H1299 (p53-null) NSCLC cell media. ACh levels were reduced by cell treatment with Aß 1-42, Aß 1-40, Aß 1-28, and Aß 25-35. AChE and p53 activities increased upon A549 cell treatment with Aß, while knockdown of p53 in A549 cells increased ACh levels, decreased AChE activity, and diminished the Aß effects. Aß increased the ratio of phospho/total p38 MAPK and decreased the activity of PKC. Inhibiting p38 MAPK reduced the activity of p53 in A549 cells and increased ACh levels in the media of both cell lines, while opposite effects were found upon inhibiting PKC. ACh decreased the activity of p53 in A549 cells, decreased p38 MAPK activity, increased PKC activity, and diminished the effect of Aß on those activities. Moreover, the negative effect of Aß on cell viability was diminished by cell co-treatment with ACh.

The role of leptin in regulation of the soluble amyloid precursor protein α (sAPPα) levels in lung cancer cell media.

Previously, we found that the levels of soluble amyloid precursor protein α (sAPPα) are regulated, in part, by acetylcholinesterase (AChE) in human A549 (p53 wild-type) and H1299 (p53-null) NSCLC cell lines. In this study, we found regulation of sAPPα levels in the media by leptin, a widely recognized obesity-associated adipokine that has recently been shown to play a possible role in cancer signaling. Increased levels of sAPPα, that were accompanied by lower Aß40/42 levels in the media of A549 and H1299 cells, were detected upon cell incubation with leptin. Conversely, knockdown of leptin or its receptor led to reduced levels of sAPPα and increased levels of Aß40/42 in the media of A549 and H1299 cells, suggesting that leptin likely shifts APP processing toward the non-amyloidogenic pathway. A549 cell treatment with leptin increased acetylcholine levels and blocked the activities of AChE and p53. Treatment with leptin resulted in increased activation of PKC, ERK1/2, PI3K, and the levels of sAPPα, effects that were reversed by treatment with kinase inhibitors and/or upon addition of AChE to A549 and H1299 cell media. Cell viability increased by treatment of A549 and H1299 cells with leptin and decreased upon co-treatment with AChE and/or inhibitors targeting PKC, ERK1/2, and PI3K. This study is significant as it provides evidence for a likely carcinogenic role of leptin in NSCLC cells via upregulation of sAPPα levels in the media, and highlights the importance of targeting leptin as a potential therapeutic strategy for NSCLC treatment.

Regulation of Vascular Endothelial Growth Factor Signaling by Nicotine in a Manner Dependent on Acetylcholine-and/or ß-Adrenergic-Receptors in Human Lung Cancer Cells.

In addition to binding to nicotinic acetylcholine receptors (nAChRs), nicotine is known to regulate the ß-adrenergic receptors (ß-ARs) promoting oncogenic signaling. Using A549 (p53 wild-type) and H1299 (p53-null) lung cancer cells, we show that nicotine treatment led to: increased adrenaline/noradrenaline levels, an effect blocked by treatment with the α7nAChR inhibitor (α-BTX) but not by the ß-blocker (propranolol) or the α4ß2nAChR antagonist (DhßE); decreased GABA levels in A549 and H1299 cell media, an effect blocked by treatment with DhßE; increased VEGF levels and PI3K/AKT activities, an effect diminished by cell co-treatment with α-BTX, propranolol, and/or DhßE; and inhibited p53 activity in A549 cells, that was reversed, upon cell co-treatment with α-BTX, propranolol, and/or DhßE or by VEGF immunodepletion. VEGF levels increased upon cell treatment with nicotine, adrenaline/noradrenaline, and decreased with GABA treatment. On the other hand, the p53 activity decreased in A549 cells treated with nicotine, adrenaline/noradrenaline and increased upon cell incubation with GABA. Knockdown of p53 led to increased VEGF levels in the media of A549 cells. The addition of anti-VEGF antibodies to A549 and H1299 cells decreased cell viability and increased apoptosis; blocked the activities of PI3K, AKT, and NFκB in the absence or presence of nicotine; and resulted in increased p53 activation in A549 cells. We conclude that VEGF can be upregulated via α7nAChR and/or ß-ARs and downregulated via GABA and/or p53 in response to the nicotine treatment of NSCLC cells.

Regulation of Soluble E-Cadherin Signaling in Non-Small-Cell Lung Cancer Cells by Nicotine, BDNF, and ß-Adrenergic Receptor Ligands.

The ectodomain of the transmembrane protein E-cadherin can be cleaved and released in a soluble form referred to as soluble E-cadherin, or sE-cad, accounting for decreased E-cadherin levels at the cell surface. Among the proteases implicated in this cleavage are matrix metalloproteases (MMP), including MMP9. Opposite functions have been reported for full-length E-cadherin and sE-cad. In this study, we found increased MMP9 levels in the media of two non-small cell lung cancer (NSCLC) cell lines, A549 and H1299, treated with BDNF, nicotine, or epinephrine that were decreased upon cell treatment with the ß-adrenergic receptor blocker propranolol. Increased MMP9 levels correlated with increased sE-cad levels in A549 cell media, and knockdown of MMP9 in A549 cells led to downregulation of sE-cad levels in the media. Previously, we reported that A549 and H1299 cell viability increased with nicotine and/or BDNF treatment and decreased upon treatment with propranolol. In investigating the function of sE-cad, we found that immunodepletion of sE-cad from the media of A549 cells untreated or treated with BDNF, nicotine, or epinephrine reduced activation of EGFR and IGF-1R, decreased PI3K and ERK1/2 activities, increased p53 activation, decreased cell viability, and increased apoptosis, while no effects were found using H1299 cells under all conditions tested.

Phosphorylation of IGFBP-3 by Casein Kinase 2 Blocks Its Interaction with Hyaluronan, Enabling HA-CD44 Signaling Leading to Increased NSCLC Cell Survival and Cisplatin Resistance.

Cisplatin is a platinum agent used in the treatment of non-small cell lung cancer (NSCLC). Much remains unknown regarding the basic operative mechanisms underlying cisplatin resistance in NSCLC. In this study, we found that phosphorylation of IGFBP-3 by CK2 (P-IGFBP-3) decreased its binding to hyaluronan (HA) but not to IGF-1 and rendered the protein less effective at reducing cell viability or increasing apoptosis than the non-phosphorylated protein with or without cisplatin in the human NSCLC cell lines, A549 and H1299. Our data suggest that blocking CD44 signaling augmented the effects of cisplatin and that IGFBP-3 was more effective at inhibiting HA-CD44 signaling than P-IGFBP-3. Blocking CK2 activity and HA-CD44 signaling increased cisplatin sensitivity and more effectively blocked the PI3K and AKT activities and the phospho/total NFκB ratio and led to increased p53 activation in A549 cells. Increased cell sensitivity to cisplatin was observed upon co-treatment with inhibitors targeted against PI3K, AKT, and NFκB while blocking p53 activity decreased A549 cell sensitivity to cisplatin. Our findings shed light on a novel mechanism employed by CK2 in phosphorylating IGFBP-3 and increasing cisplatin resistance in NSCLC. Blocking phosphorylation of IGFBP-3 by CK2 may be an effective strategy to increase NSCLC sensitivity to cisplatin.

Opposing Roles of IGFBP-3 and Heparanase in Regulating A549 Lung Cancer Cell Survival.

In this study, we examined the roles of heparanase and IGFBP-3 in regulating A549 and H1299 non-small-cell lung cancer (NSCLC) survival. We found that H1299 cells, known to be p53-null with no expression of IGFBP-3, had higher heparanase levels and activity and higher levels of heparan sulfate (HS) in the media compared to the media of A549 cells. Inhibiting heparanase activity or its expression using siRNA had no effect on the levels of IGFBP-3 in the media of A549 cells, reduced the levels of soluble HS fragments, and led to decreased interactions between IGFBP-3 and HS in the media. HS competed with HA for binding to IGFBP-3 or IGFBP-3 peptide (215-KKGFYKKKQCRPSKGRKR-232) but not the mutant peptide (K228AR230A). HS abolished the cytotoxic effects of IGFBP-3 but not upon blocking HA-CD44 signaling with the anti-CD44 antibody (5F12). Blocking HA-CD44 signaling decreased the levels of heparanase in the media of both A549 and H1299 cell lines and increased p53 activity and the levels of IGFBP-3 in A549 cell media. Knockdown of p53 led to increased heparanase levels and reduced IGFBP-3 levels in A549 cell media while knockdown of IGFBP-3 in A549 cells blocked p53 activity and increased heparanase levels in the media.

Regulation of Cisplatin Resistance in Lung Cancer Cells by Nicotine, BDNF, and a ß-Adrenergic Receptor Blocker.

It is well-recognized that cigarette smoking is a primary risk factor in the development of non-small cell lung cancer (NSCLC), known to account for ~80% of all lung cancers with nicotine recognized as the major addictive component. In investigating the effect of nicotine, brain-derived neurotrophic factor (BDNF), and the ß-adrenergic receptor blocker, propranolol, on sensitivity of NSCLC cell lines, A549 and H1299, to cisplatin, we found increased cell viability, and enhanced cisplatin resistance with nicotine and/or BDNF treatment while opposite effects were found upon treatment with propranolol. Cell treatment with epinephrine or nicotine led to EGFR and IGF-1R activation, effects opposite to those found with propranolol. Blocking EGFR and IGF-1R activation increased cell sensitivity to cisplatin in both cell lines. PI3K and AKT activities were upregulated by nicotine or BDNF and downregulated by cell treatment with inhibitors against EGFR and IGF-1R and by propranolol. Apoptosis and cell sensitivity to cisplatin increased upon co-treatment of cells with cisplatin and inhibitors against PI3K or AKT. Our findings shed light on an interplay between nicotine, BDNF, and ß-Adrenergic receptor signaling in regulating survival of lung cancer cells and chemoresistance which can likely expand therapeutic opportunities that target this regulatory network in the future.

Regulation of the Soluble Amyloid Precursor Protein α (sAPPα) Levels by Acetylcholinesterase and Brain-Derived Neurotrophic Factor in Lung Cancer Cell Media.

In comparing two human lung cancer cells, we previously found lower levels of acetylcholinesterase (AChE) and intact amyloid-ß40/42 (Aß), and higher levels of mature brain-derived neurotrophic factor (mBDNF) in the media of H1299 cells as compared to A549 cell media. In this study, we hypothesized that the levels of soluble amyloid precursor protein α (sAPPα) are regulated by AChE and mBDNF in A549 and H1299 cell media. The levels of sAPPα were higher in the media of H1299 cells. Knockdown of AChE led to increased sAPPα and mBDNF levels and correlated with decreased levels of intact Aß40/42 in A549 cell media. AChE and mBDNF had opposite effects on the levels of Aß and sAPPα and were found to operate through a mechanism involving α-secretase activity. Treatment with AChE decreased sAPPα levels and simultaneously increased the levels of intact Aß40/42 suggesting a role of the protein in shifting APP processing away from the non-amyloidogenic pathway and toward the amyloidogenic pathway, whereas treatment with mBDNF led to opposite effects on those levels. We also show that the levels of sAPPα are regulated by protein kinase C (PKC), extracellular signal-regulated kinase (ERK)1/2, phosphoinositide 3 Kinase (PI3K), but not by protein kinase A (PKA).

Differences in the Relative Abundance of ProBDNF and Mature BDNF in A549 and H1299 Human Lung Cancer Cell Media.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has been linked to several human malignancies and shown to promote tumorigenesis. The purpose of this study was to explore the relative abundance of pro-brain-derived neurotrophic factor (proBDNF) and mature BDNF (mBDNF) in A549 (p53 wild-type) and H1299 (p53-null) lung cancer cell media. Higher levels of proBDNF were detected in the media of A549 cells than in H1299 cell media. Using inhibitors, we found that the levels of proBDNF and mBDNF in the media are likely regulated by PI3K, AKT, and NFκB. However, the largest change in these levels resulted from MMP2/9 inhibition. Blocking p53 function in A549 cells resulted in increased mBDNF and decreased proBDNF, suggesting a role for p53 in regulating these levels. The ratio of proBDNF/mBDNF was not affected by MMP2 knockdown but increased in the media of both cell lines upon knockdown of MMP9. Downregulation of either MMP2 or MMP9 by siRNA showed that MMP9 siRNA treatment of either A549 or H1299 cells resulted in decreased cell viability and increased apoptosis, an effect diminished upon the same treatment with proBDNF immunodepleted media, suggesting that MMP9 regulates the cytotoxic effects induced by proBDNF in lung cancer cells.

Regulation of amyloid-ß levels by matrix metalloproteinase-2/9 (MMP2/9) in the media of lung cancer cells.

In this study, we set out to identify regulators of intact amyloid-ß40/42 (Aß) levels in A549 (p53 wild-type) and H1299 (p53-null) lung cancer cell media. Higher Aß levels were detected in the media of A549 than H1299 cells without or with treatment with 4-methylumbelliferone (4-MU) and/or the anti-CD44 antibody (5F12). Using inhibitors, we found that PI3K, AKT, and NFκB are likely involved in regulating Aß levels in the media. However, increased Aß levels that more closely resembled those found upon 4-MU co-treatment resulted from MMP2/9 inhibition, suggesting that MMP2/9 maybe the main contributors to regulation of Aß levels in the media. Differences in Aß levels might be accounted for, in part, by p53 since blocking p53 function in A549 cells resulted in decreased Aß levels, increased MMP2/9 levels, increased PI3K/AKT activities and the phospho/total NFκB ratio. Using siRNA targeted against MMP2 or MMP9, we found increased Aß levels in the media, however, MMP2 knockdown led to Aß levels closely mimicking those detected by co-treatment with 4-MU. Cell viability or apoptosis upon treatment with either MMP2 or MMP9 siRNA along with Aß immunodepletion, showed that MMP2 is the predominant regulator of the cytotoxic effects induced by Aß in lung cancer cells.

Interaction of amyloid beta with humanin and acetylcholinesterase is modulated by ATP.

Humanin (HN) is known to bind amyloid beta (Aß)-inducing cytoprotective effects, while binding of acetylcholinesterase (AChE) to Aß increases its aggregation and cytotoxicity. Previously, we showed that binding of HN to Aß blocks aggregation induced by AChE and that HN decreases but does not abolish Aß-AChE interactions in A549 cell media. Here, we set out to shed light on factors that modulate the interactions of Aß with HN and AChE. We found that binding of either HN or AChE to Aß is not affected by heparan sulfate, while ATP, thought to reduce misfolding of Aß, weakened interactions between AChE and Aß but strengthened those between Aß and HN. Using media from either A549 or H1299 lung cancer cells, we observed that more HN was bound to Aß upon addition of ATP, while levels of AChE in a complex with Aß were decreased by ATP addition to A549 cell media. Exogenous addition of ATP to either A549 or H1299 cell media increased interactions of endogenous HN with Aß to a comparable extent despite differences in AChE expression in the two cell lines, and this was correlated with decreased binding of exogenously added HN to Aß. Treatment with exogenous ATP had no effect on cell viability under all conditions examined. Exogenously added ATP did not affect viability of cells treated with AChE-immunodepleted media, and there was no apparent protection against the cytotoxicity resulting from immunodepletion of HN. Moreover, exogenously added ATP had no effect on the relative abundance of oligomer versus total Aß in either cell line.

Biochemical determinants of the IGFBP-3-hyaluronan interaction.

IGFBP-3, the most abundant IGFBP and the main carrier of insulin-like growth factor I (IGF-I) in the circulation, can bind IGF-1 with high affinity, which attenuates IGF/IGF-IR interactions, thereby resulting in antiproliferative effects. The C-terminal domain of insulin-like growth factor-binding protein-3 (IGFBP-3) is known to contain an 18-basic amino acid motif capable of interacting with either humanin (HN) or hyaluronan (HA). We previously showed that the 18-amino acid IGFBP-3 peptide is capable of binding either HA or HN with comparable affinities to the full-length IGFBP-3 protein and that IGFBP-3 can compete with the HA receptor, CD44, for binding HA. Blocking the interaction between HA and CD44 reduced viability of A549 human lung cancer cells. In this study, we set out to better characterize IGFBP-3-HA interactions. We show that both stereochemistry and amino acid identity are important determinants of the interaction between the IGFBP-3 peptide and HA and for the peptide's ability to exert its cytotoxic effects. Binding of IGFBP-3 to either HA or HN was unaffected by glycosylation or reduction of IGFBP-3, suggesting that the basic 18-amino acid residue sequence of IGFBP-3 remains accessible for interaction with either HN or HA upon glycosylation or reduction of the full-length protein. Removing N-linked oligosaccharides from CD44 increased its ability to compete with IGFBP-3 for binding HA, while reduction of CD44 rendered the protein relatively ineffective at blocking IGFBP-3-HA interactions. We conclude that both deglycosylation and disulfide bond formation are important for CD44 to compete with IGFBP-3 for binding HA.

Humanin Blocks the Aggregation of Amyloid-ß Induced by Acetylcholinesterase, an Effect Abolished in the Presence of IGFBP-3.

It is known that the humanin (HN) peptide binding to amyloid-ß (Aß) protects against its cytotoxic effects, while acetylcholinesterase (AChE) binding to Aß increases its aggregation and cytotoxicity. HN is also known to bind the insulin-like growth factor binding protein-3 (IGFBP-3). Here, we examined the regulation of Aß conformations by HN, AChE, and IGFBP-3 both in vitro and in the conditioned media from A549 and H1299 lung cancer cells. Our in vitro results showed the following: IGFBP-3 binds HN and blocks it from binding Aß in the absence or presence of AChE; HN and AChE can simultaneously bind Aß but not when in the presence of IGFBP-3; HN is unable to reduce the aggregation of Aß in the presence of IGFBP-3; and HN abolishes the aggregation of Aß induced by the addition of AChE in the absence of IGFBP-3. In the media, AChE and HN can simultaneously bind Aß. While both AChE and HN are detected when using 6E10 Aß antibodies, only AChE is detected when using the Aß 17-24 antibody 4G8, the anti-oligomer A11, and the anti-amyloid fibril LOC antibodies. No signal was observed for IGFBP-3 with any of the anti-amyloid antibodies used. Exogenously added IGFBP-3 reduced the amount of HN found in a complex when using 6E10 antibodies and correlated with a concomitant increase in the amyloid oligomers. Immunodepletion of HN from the media of the A549 and H1299 cells increased the relative abundance of the oligomer vs the total amount of Aß, the A11-positive prefibrillar oligomers, and to a lesser extent the LOC-positive fibrillar oligomers, and was also correlated with diminished cell viability and increased apoptosis.

IGFBP-3 Blocks Hyaluronan-CD44 Signaling, Leading to Increased Acetylcholinesterase Levels in A549 Cell Media and Apoptosis in a p53-Dependent Manner.

Insulin-like growth factor binding protein-3 (IGFBP-3) belongs to a family of six IGF binding proteins. We previously found that IGFBP-3 exerts its cytotoxic effects on A549 (p53 wild-type) cell survival through a mechanism that depends on hyaluronan-CD44 interactions. To shed light on the mechanism employed, we used CD44-negative normal human lung cells (HFL1), A549, and H1299 (p53-null) lung cancer cells. A synthetic IGFBP-3 peptide (215-KKGFYKKKQCRPSKGRKR-232) but not the mutant (K228AR230A), was able to bind hyaluronan more efficiently than the analogous sequences from the other IGFBPs. In a manner comparable to that of the IGFBP-3 protein, the peptide blocked hyaluronan-CD44 signaling, and more effectively inhibited viability of A549 cells than viability of either H1299 or HFL1 cell lines. Treatment with the IGFBP-3 protein or its peptide resulted in increased acetylcholinesterase concentration and activity in the A549 cell media but not in the media of either HFL1 or H1299, an effect that correlated with increased apoptosis and decreased cell viability. These effects were diminished upon the same treatment of A549 cells transfected with either p53 siRNA or acetylcholinesterase siRNA. Taken together, our results show that IGFBP-3 or its peptide blocks hyaluronan-CD44 signaling via a mechanism that depends on both p53 and acetylcholinesterase.

Interaction of Insulin-Like Growth Factor-Binding Protein 3 With Hyaluronan and Its Regulation by Humanin and CD44.

Insulin-like growth factor-binding protein-3 (IGFBP-3) belongs to a family of IGF-binding proteins. Humanin is a peptide known to bind residues 215-232 of mature IGFBP-3 in the C-terminal region of the protein. This region of IGFBP-3 was shown earlier to bind certain glycosaminoglycans including hyaluronan (HA). Here, we characterized the binding affinities of the IGFBP-3 protein and peptide (215-KKGFYKKKQCRPSKGRKR-232) to HA and to humanin and found that HA binds with a weaker affinity to this region than does humanin. Either HA or humanin could bind to this IGFBP-3 segment, but not simultaneously. The HA receptor, CD44, blocked HA binding to IGFBP-3 but had no effect on binding of humanin to either IGFBP-3 or its peptide. Upon incubation of HA with CD44 and either IGFBP-3 protein or peptide, humanin was effective at binding and sequestering IGFBP-3 or peptide, thereby enabling access of CD44 to HA. We show that IGFBP-3 and humanin in the medium of A549 lung cancer cells can immunoprecipitate in a complex. However, the fraction of IGFBP-3 in the medium that is able to bind HA was not complexed with humanin suggesting that HA binding to the 215-232 segment renders it inaccessible for binding to humanin. Moreover, while the cytotoxic effects of IGFBP-3 on cell viability were reversed by humanin, blocking HA-CD44 interaction with an anti-CD44 antibody in combination with IGFBP-3 did not have an additive negative effect on cell viability suggesting that IGFBP-3 exerts its cytotoxic effects on cell survival through a mechanism that depends on HA-CD44 interactions.

D-Amino Acid Analogues of the Antimicrobial Peptide CDT Exhibit Anti- Cancer Properties in A549, a Human Lung Adenocarcinoma Cell Line.

INTRODUCTION: The importance of the antitumor activity of some antimicrobial peptides (AMPs) is being increasingly recognized. The antimicrobial peptide, tachyplesin, has been shown to exhibit anticancer properties and a linear, cysteine deleted analogue (CDT), was found to retain its antibacterial function. OBJECTIVES: The objective was to test CDT and related analogues against normal mammalian, bacterial, and cancer cells to determine their effectiveness and then utilize specific assays to determine a possible mechanism of action. METHODS: We used sequence reversal and D-amino acids to synthesize four CDT analogues by solid phase peptide synthesis. A number of assays were used including liposome dye-leakage, antibacterial activity against both Gram-positive and Gram-negative bacterial strains, hemolytic assays, methyl thiazolyl tetrazolium (MTT), and apoptosis to examine their effectiveness as both AMPs and anti-cancer peptides (ACPs). We then tested the analogues for their ability to inhibit proliferation of the human lung cancer cell line, A549. RESULTS: We found that D-CDT exhibited the best bactericidal properties of those tested and was not damaging to red blood cells. Both D-CDT and reverse D-CDT showed a dose-dependent reduction of cell viability. However, D-CDT was most effective with an IC50 of 9.814 µM, a value 9-fold lower than that calculated for reverse D-CDT (90.16 µM). Apoptosis does not appear to be a mechanism by which D-CDT exerts its anticancer properties since > 100 µM was required to increase activation of caspase 3. Moreover, the ERK1/2 pathway is also unlikely since only a modest (20%) decrease of activity was observed with > 100 µM D-CDT. However, D-CDT was found to operate via a hyaluronan (HA)-dependent mechanism as pretreatment of the cells with hyaluronidase decreased the cytotoxic effects of D-CDT on A549 cells and increased its IC50 29-fold to 283.9 µM. CONCLUSION: D-CDT is both an effective AMP and ACP, and likely exerts its anticancer effects through both membranolytic as well as an HA-mediated mechanism.

FLASH knockdown sensitizes cells to Fas-mediated apoptosis via down-regulation of the anti-apoptotic proteins, MCL-1 and Cflip short.

FLASH (FLICE-associated huge protein or CASP8AP2) is a large multifunctional protein that is involved in many cellular processes associated with cell death and survival. It has been reported to promote apoptosis, but we show here that depletion of FLASH in HT1080 cells by siRNA interference can also accelerate the process. As shown previously, depletion of FLASH halts growth by down-regulating histone biosynthesis and arrests the cell cycle in S-phase. FLASH knockdown followed by stimulating the cells with Fas ligand or anti-Fas antibodies was found to be associated with a more rapid cleavage of PARP, accelerated activation of caspase-8 and the executioner caspase-3 and rapid progression to cellular disintegration. As is the case for most anti-apoptotic proteins, FLASH was degraded soon after the onset of apoptosis. Depletion of FLASH also resulted in the reduced intracellular levels of the anti-apoptotic proteins, MCL-1 and the short isoform of cFLIP. FLASH knockdown in HT1080 mutant cells defective in p53 did not significantly accelerate Fas mediated apoptosis indicating that the effect was dependent on functional p53. Collectively, these results suggest that under some circumstances, FLASH suppresses apoptosis.

FAM129B/MINERVA, a novel adherens junction-associated protein, suppresses apoptosis in HeLa cells.

A recent proteomics study identified FAM129B or MINERVA as a target of the MAP kinase (Erk1/2) signaling cascade in human melanoma cells. Phosphorylation of the protein was found to promote cell invasion and the dissociation of the protein from the cell-cell junctions. Suppression of apoptosis during metastasis is a prerequisite for the survival and spread of cancer cells. During apoptosis, the adherens junctions are disassembled as the dying cell retracts, and new contacts are formed between normal neighboring cells. In this study, we show that FAM129B was cytosolic in exponentially growing HeLa cells but was translocated to the adherens junctions where it colocalized with ß-catenin whenever contact between two or more cells was established. Silencing the FAM129B gene expression by specific siRNAs did not induce apoptosis or inhibit the growth of HeLa cells. However, when apoptosis was induced by exposure to TNFα/cycloheximide or other apoptotic signaling molecules, the onset of apoptosis was accelerated 3-4-fold when FAM129B was depleted. Annexin V binding, the inactivation of the DNA repair enzyme, poly(ADP-ribose) polymerase, and the activation of the caspases occurred more rapidly in the cells lacking FAM129B. The rapid induction of apoptosis in FAM129B knockdown cells was reversed by co-transfection with recombinant FAM129B, indicating that its effect on apoptosis was specific. As apoptosis proceeded, FAM129B was degraded and disappeared from the plasma membrane. Thus, one crucial facet of the mechanism by which FAM129B promotes cancer cell invasion is likely to be the suppression of apoptosis.