Proliferating cell unclear antigen-associated factor (PAF15) : a novel oncogene

Proliferating cell nuclear antigen (PCNA)-Associated Factor (PAF15) is a small protein containing a PCNA interacting motif and sequences for association with ubiquitin enzymes. In interaction with PCNA, PAF15 plays a key role in recruiting DNA replicative polymerase by double monoubiquitination at Lys15 and Lys24. Under DNA damage conditions, PAF15 regulates the switch from DNA replicative polymerase to translesion synthesis polymerase in order to bypass the replication-blocking lesions. Overexpression of PAF15 promotes the repair of ultraviolet-induced DNA damage and prevents cell death, whereas attenuation of PAF15 decreases DNA replication and cell survival. Ectopic expression of PAF15 in mouse fibroblasts increases colony formation and tumourigenicity. PAF15 is aberrantly increased in various human malignancies with poor prognosis. Collectively, PAF15 may contribute to carcinogenesis and represents one of the potential therapeutic targets in the treatment of cancer

Effect of arachidonic acid and its producing enzyme phospholipase A2α on key oncogenic pathways in prostate cancer

The dietary arachidonic acid (AA) is stored at the sn-2 position in the glycerolphospholipid of cell membrane. AA is released via the cleavage by phospholipase A2 in response to stimuli such as cytokines, growth factors and hormones. The freed AA is metabolised typically by cyclooxygenase and lipoxygenase enzymes to form eicosanoids. Of all the phospholipase A2 that catalyse the hydrolysis of fatty acid at sn-2 bond of glycerophospholipids, cytosolic phospholipase A2α (cPLA2α) is preferentially targeting AA-containing phospholipids. Hence, cPLA2α action represents the key step for AA release and eicosanoid synthesis. Stimulation of cells with agents that mobilise intracellular calcium and/or promote the phosphorylation of cPLA2α leads to increased AA release and eicosanoid production

Ubiquitin-conjugating enzyme E2C : a potential cancer biomarker

The ubiquitin-conjugating enzymes 2C (UBE2C) is an integral component of the ubiquitin proteasome system. UBE2C consists of a conserved core domain containing the catalytic Cys residue and an N-terminal extension. The core domain is required for ubiquitin adduct formation by interacting with the ubiquitin-fold domain in the E1 enzyme, and contributes to the E3 enzyme binding. UBE2C N-terminal extension regulates E3 enzyme activity as a part of an intrinsic inhibitory mechanism. UBE2C is required for the destruction of mitotic cyclins and securin, which are essential for spindle assembly checkpoint and mitotic exit. The UBE2C mRNA and/or protein levels are aberrantly increased in many cancer types with poor clinical outcomes. Accumulation of UBE2C stimulates cell proliferation and anchorage-independent growth. UBE2C transgenic mice are prone to develop spontaneous tumors and carcinogen-induced tumor with evidence of chromosome aneuploidy

AKT and cytosolic phospholipase A2α form a positive loop in prostate cancer cells

Aberrant increase in protein kinase B (AKT) phosphorylation (pAKT), due to a gain-of-function mutation of phosphatidylinositol-3-kinase (P13K) or loss-of-function mutation or deletion of phosphatase and tensin homolog (PTEN), is a common alteration in prostate cancer and associated with poor prognosis. Cytosolic phospholipase A₂α (cPLA₂α) is a lipid modifying enzyme that catalyzes the hydrolysis of arachidonic acid from membrane phospholipid. The released arachidonic acid and its metabolites contribute to survival and proliferation of prostate cancer cells. In this mini-review, we summarize the relationship between pAKT and cPLA₂α in prostate cancer cells. There was a concordant increase in pAKT and cPLA₂α levels in prostate tissue of prostate epithelial-specific PTEN-knockout mice compared to PTEN-wild type mice. Restoration of PTEN expression or inhibition of P13K action decreased cPLA₂α protein levels. pAKT had no influence on cPLA₂α expression at mRNA levels but stabilized cPLA₂α at protein levels by protecting it from degradation. Conversely, an induction of cPLA₂α expression led to an increase in pAKT levels in PTEN-mutated or deleted prostate cancer cells while silencing of cPLA₂α expression or pharmacological blocking of cPLA₂α action decreased pAKT levels. The diminishment of pAKT by either genetic silencing or pharmacological blocking of cPLA₂α was mitigated by the addition of arachidonic acid. The stimulatory effect of arachidonic acid on pAKT levels was lessened by inhibiting the production of arachidonic acid metabolites. These studies have revealed a link between an oncogenic pathway and lipid metabolism and provided potential molecular targets for treating prostate cancer

Loss of PTEN stabilizes the lipid modifying enzyme cytosolic phospholipase A₂α via AKT in prostate cancer cells

Aberrant increase in pAKT, due to a gain-of-function mutation of PI3K or lossof-function mutation or deletion of PTEN, occurs in prostate cancer and is associated with poor patient prognosis. Cytosolic phospholipase A2a (cPLA2a) is a lipid modifying enzyme by catalyzing the hydrolysis of membrane arachidonic acid. Arachidonic acid and its metabolites contribute to survival and proliferation of prostate cancer cells. We examined whether AKT plays a role in promoting cPLA2a action in prostate cancer cells. We found a concordant increase in pAKT and cPLA2a levels in prostate tissue of prostate epithelial-specific PTEN-knockout but not PTEN-wide type mice. Restoration of PTEN expression or inhibition of PI3K action decreased cPLA2a expression in PTENmutated or deleted prostate cancer cells. An increase in AKT by Myr-AKT elevated cPLA2a protein levels, which could be diminished by inhibition of AKT phosphorylation without noticeable change in total AKT levels. pAKT levels had no influence on cPLA2a at mRNA levels but reduced cPLA2a protein degradation. Anti-AKT antibody coimmunoprecipitated cPLA2a and vice versa. Hence, AKT plays a role in enhancing cPLA2a protein stability in PTEN-null prostate cancer cells, revealing a link between oncogenic pathway and lipid metabolism

Guttiferone K suppresses cell motility and metastasis of hepatocellular carcinoma by restoring aberrantly reduced profilin 1

Hepatocellular carcinoma (HCC) is an aggressive malignancy and the 5-year survival rate of advanced HCC is < 10%. Guttiferone K (GUTK) isolated from the Garcinia genus inhibited HCC cells migration and invasion in vitro and metastasis in vivo without apparent toxicity. Proteomic analysis revealed that actin-binding protein profilin 1 (PFN1) was markedly increased in the presence of GUTK. Over-expression of PFN1 mimicked the effect of GUTK on HCC cell motility and metastasis. The effect of GUTK on cell motility was diminished when PFN1 was over-expressed or silenced. Over-expression of PFN1 or incubation with GUTK decreased F-actin levels and the expression of proteins involved in actin nucleation, branching and polymerization. Moreover, a reduction of PFN1 protein levels was common in advanced human HCC and associated with poor survival rate. In conclusion, GUTK effectively suppresses the motility and metastasis of HCC cells mainly by restoration of aberrantly reduced PFN1 protein expression

p27Kip1 signaling : transcriptional and post-translational regulation

p27Kip1 is an inhibitor of a broad spectrum of cyclin-dependent kinases (CDKs), and the loss of a single p27Kip1 allele is thereby sufficient to increase tumor incidence via CDK-mediated cell cycle entry. As such, down-regulation of p27Kip1 protein levels, in particular nuclear expressed p27Kip1, is implicated in both disease progression and poor prognosis in a variety of cancers. p27Kip1 expression is positively regulated by the transcription factor MENIN, and inhibited by oncogenic transcription factors MYC and PIM. How-ever, regulation of p27Kip1 protein expression and function is predominantly through post-translational modifications that alter both the cellular localization and the extent of E3 ubiquitin ligase-mediated degradation. Phosphorylation of p27Kip1 at Thr187 and Ser10 is a prerequisite for its degradation via the E3 ubiquitin ligases SKP2 (nuclear) and KPC (cytoplasmic), respectively. Additionally, Ser10 phosphorylated p27Kip1 is predominantly localized in the cytoplasm due to the nuclear export protein CRM1. Another E3 ubiquitin ligase, PIRH2, degrades p27Kip1 in both the cytoplasm and nucleus independent of phosphorylation state. As such, inhibition of cell cycle entry and progression in a variety of cancers may be achieved with therapies designed to correct p27Kip1 localization and/or block its degradation

The effects of 56MESS on mitochondrial and cytoskeletal proteins and the cell cycle in MDCK cells

Background: 56MESS has been shown to be cytotoxic but the mode of this action is unclear. In order to probe the mechanism of action for 56MESS, MDCK cells were utilised to investigate the effect on treated cells. Results: IC 50 values for 56MESS and cisplatin in the MDCK cell line, determined by a SRB assay, were 0.25 ± 0.03 and 18 ± 1.2 µM respectively. In a preliminary study, cells treated with 56MESS displayed no caspase-3/7 activity, suggesting that the mechanism of action is caspase independent. Protein expression studies revealed an increase the expression in the MTC02 protein associated with mitochondria in cells treated with 56MESS and cisplatin. Non-synchronised 56MESS-treated cells caused an arrest in the G2/M phase of the cell cycle, in comparison to the S phase arrest of cisplatin. In G0/G1 synchronised cells, both 56MESS and cisplatin both appeared to arrest within the S phase. Conclusions: these results suggest that 56MESS is capable of causing cell-cycle arrest, and that mitochondrial and cell cycle proteins may be involved in the mode of action of cytotoxicity of 56MESS

Targeting cytosolic phospholipase A2 α in colorectal cancer cells inhibits constitutively activated protein kinase B (AKT) and cell proliferation

A constitutive activation of protein kinase B (AKT) in a hyper-phosphorylated status at Ser473 is one of the hallmarks of anti-EGFR therapy-resistant colorectal cancer (CRC). The aim of this study was to examine the role of cytosolic phospholipase A2a (cPLA2a) on AKT phosphorylation at Ser473 and cell proliferation in CRC cells with mutation in phosphoinositide 3-kinase (PI3K). AKT phosphorylation at Ser473 was resistant to EGF stimulation in CRC cell lines of DLD-1 (PIK3CAE545K mutation) and HT-29 (PIK3CAP499T mutation). Over-expression of cPLA2a by stable transfection increased basal and EGF-stimulated AKT phosphorylation and proliferation in DLD- 1 cells. In contrast, silencing of cPLA2a with siRNA or inhibition with Efipladib decreased basal and EGF-stimulated AKT phosphorylation and proliferation in HT-29. Treating animals transplanted with DLD-1 with Efipladib (10 mg/kg, i.p. daily) over 14 days reduced xenograft growth by > 90% with a concomitant decrease in AKT phosphorylation. In human CRC tissue, cPLA2a expression and phosphorylation were increased in 63% (77/120) compared with adjacent normal mucosa determined by immunohistochemistry. We conclude that cPLA2a is required for sustaining AKT phosphorylation at Ser473 and cell proliferation in CRC cells with PI3K mutation, and may serve as a potential therapeutic target for treatment of CRC resistant to anti-EGFR therapy

Targeting of cytosolic phospholipase A2α impedes cell cycle re-entry of quiescent prostate cancer cells

Cell cycle re-entry of quiescent cancer cells has been proposed to be involved in cancer progression and recurrence. Cytosolic phospholipase A₂α (cPLA₂α) is an enzyme that hydrolyzes membrane glycerophospholipids to release arachidonic acid and lysophospholipids that are implicated in cancer cell proliferation. The aim of this study was to determine the role of cPLA₂α in cell cycle re-entry of quiescent prostate cancer cells. When PC-3 and LNCaP cells were rendered to a quiescent state, the active form of cPLA₂α with a phosphorylation at Ser⁵⁰⁵ was lower compared to their proliferating state. Conversely, the phosphor-cPLA₂α with Efipladib upon induction of cell cycle re-entry inhibited the re-entry process, as manifested by refrained DNA synthesis, persistent high proportion of cells in G₀/G₁ and low percentage of cells in S and G₂/M phases, together with a stagnant recovery of Ki-67 expression. Simultaneously, Efipladib prohibited the emergence of Skp2 while maintained p27 at a high level in the nuclear compartment during cell cycle re-entry. Inhibition of cPLA₂α also prevented an accumulation of cyclin D1/CDK4, cyclin E/CDK2, phosphor-pRb, pre-replicative complex proteins CDC6, MCM7, ORC6 and DNA synthesis-related protein PCNA during induction of cell cycle re-entry. Moreover, a pre-treatment of the prostate cancer cells with Efipladib during induction of cell cycle re-entry subsequently compromised their tumorigenic capacity in vivo. Hence, cPLA₂α plays an important role in cell cycle re-entry by quiescent prostate cancer cells