FASN negatively regulates p65 expression by reducing its stability via Thr254 phosphorylation and isomerization by Pin1.

FASN, the sole cytosolic enzyme responsible for de novo palmitate synthesis in mammalian cells, has been associated with poor prognosis in cancer and shown to cause drug and radiation resistance by upregulating DNA damage repair via suppression of p65 expression. Targeting FASN by repurposing proton pump inhibitors has generated impressive outcomes in triple-negative breast cancer patients. While p65 regulation of DNA damage repair was thought to be due to its suppression of poly(ADP-ribose) polymerase 1 gene transcription, the mechanism of FASN regulation of p65 expression was unknown. In this study, we show that FASN regulates p65 stability by controlling its phosphorylation at Thr254, which recruits the peptidyl-prolyl cis/trans isomerase Pin1 that is known to stabilize many proteins in the nucleus. This regulation is mediated by palmitate, the FASN catalytic product, not by FASN protein per se. This finding of FASN regulation of p65 stability via phosphorylation of Thr254 and isomerization by Pin1 implicates that FASN and its catalytic product palmitate may play an important role in regulating protein stability in general and p65 more specifically.

Translation initiation factor eIF3a regulates glucose metabolism and cell proliferation via promoting small GTPase Rheb synthesis and AMPK activation.

Eukaryotic translation initiation factor 3 subunit A (eIF3a), the largest subunit of the eIF3 complex, has been shown to be overexpressed in malignant cancer cells, potentially making it a proto-oncogene. eIF3a overexpression can drive cancer cell proliferation but contributes to better prognosis. While its contribution to prognosis was previously shown to be due to its function in suppressing synthesis of DNA damage repair proteins, it remains unclear how eIF3a regulates cancer cell proliferation. In this study, we show using genetic approaches that eIF3a controls cell proliferation by regulating glucose metabolism via the phosphorylation and activation of AMP-activated protein kinase alpha (AMPKα) at Thr172 in its kinase activation loop. We demonstrate that eIF3a regulates AMPK activation mainly by controlling synthesis of the small GTPase Rheb, largely independent of the well-known AMPK upstream liver kinase B1 and Ca2+/calmodulin-dependent protein kinase kinase 2, and also independent of mammalian target of rapamycin signaling and glucose levels. Our findings suggest that glucose metabolism in and proliferation of cancer cells may be translationally regulated via a novel eIF3a-Rheb-AMPK signaling axis.

A novel survivin dimerization inhibitor without a labile hydrazone linker induces spontaneous apoptosis and synergizes with docetaxel in prostate cancer cells.

Survivin, a member of the inhibitor of apoptosis protein family, exists as a homodimer and is aberrantly upregulated in a wide spectrum of cancers. It was thought to be an ideal target due to its lack of expression in most adult normal tissues and importance in cancer cell survival. However, it has been challenging to target survivin due to its "undruggable" nature. We previously attempted to target its dimerization domain with a hypothesis that inhibiting survivin dimerization would promote its degradation in proteasome, which led to identification of a lead small-molecule inhibitor, LQZ-7F. LQZ-7F consists of a flat tetracyclic aromatic core with labile hydrazone linking a 1,2,5-oxadiazole moiety. In this study, we tested the hypothesis that LQZ-7F could be developed as a prodrug because the labile hydrazone linker could be hydrolyzed, releasing the tetracyclic aromatic core. To this end, we synthesized the tetracyclic aromatic core (LQZ-7F1) using reported procedure and tested LQZ-7F1 for its biological activities. Here we show that LQZ-7F1 has a significantly improved potency with submicromolar IC50's and induces spontaneous apoptosis in prostate cancer cells. It also more effectively inhibits survivin dimerization and induces survivin degradation in a proteasome-dependent manner than LQZ-7F. We also show that the combination of LQZ-7F1 and docetaxel have strong synergism in inhibiting prostate cancer cell survival. Together, we conclude that the hydrazone linker with the oxadiazole tail is dispensable for survivin inhibition and the survivin dimerization inhibitor, LQZ-7F, may be developed as a prodrug for prostate cancer treatment and to overcome docetaxel resistance.

Translational regulation of Chk1 expression by eIF3a via interaction with the RNA-binding protein HuR.

eIF3a is a putative subunit of the eukaryotic translation initiation factor 3 complex. Accumulating evidence suggests that eIF3a may have a translational regulatory function by suppressing translation of a subset of mRNAs while accelerating that of other mRNAs. Albeit the suppression of mRNA translation may derive from eIF3a binding to the 5'-UTRs of target mRNAs, how eIF3a may accelerate mRNA translation remains unknown. In this study, we show that eIF3a up-regulates translation of Chk1 but not Chk2 mRNA by interacting with HuR, which binds directly to the 3'-UTR of Chk1 mRNA. The interaction between eIF3a and HuR occurs at the 10-amino-acid repeat domain of eIF3a and the RNA recognition motif domain of HuR. This interaction may effectively circularize Chk1 mRNA to form an end-to-end complex that has recently been suggested to accelerate mRNA translation. Together with previous findings, we conclude that eIF3a may regulate mRNA translation by directly binding to the 5'-UTR to suppress or by interacting with RNA-binding proteins at 3'-UTRs to accelerate mRNA translation.

Single-nucleotide polymorphisms in a short basic motif in the ABC transporter ABCG2 disable its trafficking out of endoplasmic reticulum and reduce cell resistance to anticancer drugs.

ATP-binding cassette (ABC) subfamily G member 2 (ABCG2) belongs to the ABC transporter superfamily and has been implicated in multidrug resistance of cancers. Although the structure and function of ABCG2 have been extensively studied, little is known about its biogenesis and the regulation thereof. In this study, using mutagenesis and several biochemical analyses, we show that the positive charges in the vicinity of the RKR motif downstream of the ABC signature drive trafficking of nascent ABCG2 out of the endoplasmic reticulum (ER) onto plasma membranes. Substitutions of and naturally occurring single-nucleotide polymorphisms within these positively charged residues disabled the trafficking of ABCG2 out of the ER. A representative ABCG2 variant in which the RKR motif had been altered underwent increased ER stress-associated degradation. We also found that unlike WT ABCG2, genetic ABCG2 RKR variants have disrupted normal maturation and do not reduce accumulation of the anticancer drug mitoxantrone and no longer confer resistance to the drug. We conclude that the positive charges downstream of the ABC signature motif critically regulate ABCG2 trafficking and maturation. We propose that single-nucleotide polymorphisms of these residues reduce ABCG2 expression via ER stress-associated degradation pathway and may contribute to reduced cancer drug resistance, improving the success of cancer chemotherapy.

FASN regulates cellular response to genotoxic treatments by increasing PARP-1 expression and DNA repair activity via NF-κB and SP1.

Fatty acid synthase (FASN), the sole cytosolic mammalian enzyme for de novo lipid synthesis, is crucial for cancer cell survival and associates with poor prognosis. FASN overexpression has been found to cause resistance to genotoxic insults. Here we tested the hypothesis that FASN regulates DNA repair to facilitate survival against genotoxic insults and found that FASN suppresses NF-κB but increases specificity protein 1 (SP1) expression. NF-κB and SP1 bind to a composite element in the poly(ADP-ribose) polymerase 1 (PARP-1) promoter in a mutually exclusive manner and regulate PARP-1 expression. Up-regulation of PARP-1 by FASN in turn increases Ku protein recruitment and DNA repair. Furthermore, lipid deprivation suppresses SP1 expression, which is able to be rescued by palmitate supplementation. However, lipid deprivation or palmitate supplementation has no effect on NF-κB expression. Thus, FASN may regulate NF-κB and SP1 expression using different mechanisms. Altogether, we conclude that FASN regulates cellular response against genotoxic insults by up-regulating PARP-1 and DNA repair via NF-κB and SP1.

Reversible epigenetic regulation of 14-3-3σ expression in acquired gemcitabine resistance by uhrf1 and DNA methyltransferase 1.

Although gemcitabine is the most commonly used drug for treating pancreatic cancers, acquired gemcitabine resistance in a substantial number of patients appears to hinder its effectiveness in successful treatment of this dreadful disease. To understand acquired gemcitabine resistance, we generated a gemcitabine-resistant pancreatic cancer cell line using stepwise selection and found that, in addition to the known mechanisms of upregulated expression of ribonucleotide reductase, 14-3-3σ expression is dramatically upregulated, and that 14-3-3σ overexpression contributes to the acquired resistance to gemcitabine and cross-resistance to cytarabine. We also found that the increased 14-3-3σ expression in the gemcitabine-resistant cells is due to demethylation of the 14-3-3σ gene during gemcitabine selection, which could be partially reversed with removal of the gemcitabine selection pressure. Most importantly, the reversible methylation/demethylation of the 14-3-3σ gene appears to be carried out by DNA methyltransferase 1 under regulation by Uhrf1. These findings suggest that the epigenetic regulation of gene expression may play an important role in gemcitabine resistance, and that epigenetic modification is reversible in response to gemcitabine treatment.

Spectrin domain of eukaryotic initiation factor 3a is the docking site for formation of the a:b:i:g subcomplex.

eIF3a (eukaryotic translation initiation factor 3a), one of the core subunits of the eIF3 complex, has been implicated in regulating translation of different mRNAs and in tumorigenesis. A subcomplex consisting of eIF3a, eIF3b, eIF3g, and eIF3i (eIF3(a:b:i:g)) has also been identified. However, how eIF3a participates in translational regulation and in formation of the eIF3(a:b:i:g) subcomplex remain to be solved. In this study, we used the tandem affinity purification approach in combination with tandem MS/MS and identified the spectrin domain of eIF3a as the docking site for the formation of eIF3(a:b:i:g) subcomplex. Although eIF3b and eIF3i bind concurrently to the spectrin domain of eIF3a within ∼10-15 amino acids apart, eIF3g binds to eIF3a indirectly via binding to the carboxyl-terminal domain of eIF3b. The binding of eIF3b to the spectrin domain of eIF3a occurs in its RNA recognition motif domain where eIF3j also binds in a mutually exclusive manner. Together, we conclude that the spectrin domain of eIF3a is responsible for the formation of eIF3(a:b:i:g) subcomplex and, because of mutually exclusive nature of bindings of eIF3a and eIF3j to eIF3b, different subcomplexes of eIF3 likely exist and may perform noncanonical functions in translational regulation.

Fatty acid synthase causes drug resistance by inhibiting TNF-α and ceramide production.

Fatty acid synthase (FASN) is a key enzyme in the synthesis of palmitate, the precursor of major nutritional, energetic, and signaling lipids. FASN expression is upregulated in many human cancers and appears to be important for cancer cell survival. Overexpression of FASN has also been found to associate with poor prognosis and higher risk of recurrence of human cancers. Indeed, elevated FASN expression has been shown to contribute to drug resistance. However, the mechanism of FASN-mediated drug resistance is currently unknown. In this study, we show that FASN overexpression causes resistance to multiple anticancer drugs via inhibiting drug-induced ceramide production, caspase 8 activation, and apoptosis. We also show that FASN overexpression suppresses tumor necrosis factor-α production and nuclear factor-κB activation as well as drug-induced activation of neutral sphingomyelinase. Thus, TNF-α may play an important role in mediating FASN function in drug resistance.

Translational regulation of RPA2 via internal ribosomal entry site and by eIF3a.

RPA2 is a subunit of a trimeric replication protein A (RPA) complex important for DNA repair and replication. Although it is known that RPA activity is regulated by post-translational modification, whether RPA expression is regulated and the mechanism therein is currently unknown. eIF3a, the largest subunit of eIF3, is an important player in translational control and has been suggested to regulate translation of a subset of messenger RNAs important for tumorigenesis, metastasis, cell cycle progression, drug response and DNA repair. In the present study, we show that RPA2 expression is regulated at translational level via internal ribosome entry site (IRES)-mediated initiation in response to DNA damage. We also found that eIF3a suppresses RPA2 synthesis and inhibits its cellular IRES activity by directly binding to the IRES element of RPA2 located at -50 to -150 bases upstream of the translation start site. Taken together, we conclude that RPA2 expression is translationally regulated via IRES and by eIF3a and that this regulation is partly accountable for cellular response to DNA damage and survival.

Profibrotic effect of interleukin-18 in HK-2 cells is dependent on stimulation of the Toll-like receptor 4 (TLR4) promoter and increased TLR4 expression.

BACKGROUND: IL-18 induces profibrotic changes in TECs independent of TGF-ß1 activity. RESULTS: IL-18 stimulates the TLR4 promoter via AP-1 activation to increase TLR4 expression in TECs and stimulates profibrotic changes in TECs through increased TLR4 expression/signaling. CONCLUSION: The profibrotic effect of IL-18 in TECs is mediated through stimulation of TLR4 expression via activation of AP-1. SIGNIFICANCE: This represents a novel fibrotic signaling pathway in TECs independent of TGF-ß1. IL-18 is an important mediator of obstruction-induced renal fibrosis and tubular epithelial cell injury independent of TGF-ß1 activity. We sought to determine whether the profibrotic effect of IL-18 is mediated through Toll-like receptor 4 (TLR4). Male C57BL6 wild type and mice transgenic for human IL-18-binding protein were subjected to left unilateral ureteral obstruction versus sham operation. The kidneys were harvested 1 week postoperatively and analyzed for IL-18 production and TLR4 expression. In a separate arm, renal tubular epithelial cells (HK-2) were directly stimulated with IL-18 in the presence or absence of a TLR4 agonist, TLR4 antagonist, or TLR4 siRNA knockdown. Cell lysates were analyzed for TLR4, α-smooth muscle actin, and E-cadherin expression. TLR4 promotor activity, as well as AP-1 activation and the effect of AP-1 knockdown on TLR4 expression, was evaluated in HK-2 cells in response to IL-18 stimulation. The results demonstrate that IL-18 induces TLR4 expression during unilateral ureteral obstruction and induces TLR4 expression in HK-2 cells via AP-1 activation. Inhibition of TLR4 or knockdown of TLR4 gene expression in turn prevents IL-18-induced profibrotic changes in HK-2 cells. These results suggest that IL-18 induces profibrotic changes in tubular epithelial cells via increased TLR4 expression/signaling.

Reversible promoter demethylation of PDGFD confers gemcitabine resistance through STAT3 activation and RRM1 upregulation.

Drug resistance is a major problem in cancer treatment with traditional or targeted therapeutics. Gemcitabine is approved for several human cancers and the first line treatment for locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). However, gemcitabine resistance frequently occurs and is a major problem in successful treatments of these cancers and the mechanism of gemcitabine resistance remains largely unknown. In this study, we identified 65 genes that had reversible methylation changes in their promoters in gemcitabine resistant PDAC cells using whole genome Reduced Representation Bisulfite Sequencing analyses. One of these genes, PDGFD, was further studied in detail for its reversible epigenetic regulation in expression and shown to contribute to gemcitabine resistance in vitro and in vivo via stimulating STAT3 signaling in both autocrine and paracrine manners to upregulate RRM1 expression. Analyses of TCGA datasets showed that PDGFD positively associates with poor outcome of PDAC patients. Together, we conclude that the reversible epigenetic upregulation plays an important role in gemcitabine resistance development and targeting PDGFD signaling alleviates gemcitabine resistance for PDAC treatment.

eIF3a regulation of mTOR signaling and translational control via HuR in cellular response to DNA damage.

eIF3a (eukaryotic translation initiation factor 3a), a subunit of the eIF3 complex, has been suggested to play a regulatory role in protein synthesis and in cellular response to DNA-damaging treatments. S6K1 is an effector and a mediator of mTOR complex 1 (mTORC1) in regulating protein synthesis and integrating diverse signals into control of cell growth and response to stress. Here, we show that eIF3a regulates S6K1 activity by inhibiting mTORC1 kinase via regulating Raptor synthesis. The regulation of Raptor synthesis is via eIF3a interaction with HuR (human antigen R) and binding of the eIF3a-HuR complex to the 5'-UTR of Raptor mRNA. Furthermore, mTORC1 may mediate eIF3a function in cellular response to cisplatin by regulating synthesis of NER proteins and NER activity. Taken together, we conclude that the mTOR signaling pathway may also be regulated by translational control and mediate eIF3a regulation of cancer cell response to cisplatin by regulating NER protein synthesis.

Therapeutic Activity of the Lansoprazole Metabolite 5-Hydroxy Lansoprazole Sulfide in Triple-Negative Breast Cancer by Inhibiting the Enoyl Reductase of Fatty Acid Synthase.

Fatty acid synthase (FASN), a sole cytosolic enzyme responsible for de-novo lipid synthesis, is overexpressed in cancer but not in normal non-lipogenic tissues. FASN has been targeted, albeit no such inhibitor has been approved. Proton pump inhibitors (PPIs), approved for digestive disorders, were found to inhibit FASN with anticancer activities in attempting to repurpose Food and Drug Administration-approved drugs. Indeed, PPI usage benefited breast cancer patients and increased their response rate. Due to structural similarity, we thought that their metabolites might extend anticancer effects of PPIs by inhibiting FASN. Here, we tested this hypothesis and found that 5-hydroxy lansoprazole sulfide (5HLS), the end lansoprazole metabolite, was more active than lansoprazole in inhibiting FASN function and regulation of NHEJ repair of oxidative DNA damage via PARP1. Surprisingly, 5HLS inhibits the enoyl reductase, whereas lansoprazole inhibits the thioesterase of FASN. Thus, PPI metabolites may contribute to the lasting anticancer effects of PPIs by inhibiting FASN.

eIF3i regulation of protein synthesis, cell proliferation, cell cycle progression, and tumorigenesis.

eIF3i, a 36-kDa protein, is a putative subunit of the eIF3 complex important for translation initiation of mRNAs. It is a WD40 domain-containing protein with seven WD40 repeats that forms a ß-propeller structure with an important function in pre-initiation complex formation and mRNA translation initiation. In addition to participating in the eIF3 complex formation for global translational control, eIF3i may bind to specific mRNAs and regulate their translation individually. Furthermore, eIF3i has been shown to bind to TGF-ß type II receptor and participate in TGF-ß signaling. It may also participate in and regulate other signaling pathways including Wnt/ß-catenin pathway via translational regulation of COX-2 synthesis. These multiple canonical and noncanonical functions of eIF3i in translational control and in regulating signal transduction pathways may be responsible for its role in cell differentiation, cell cycle regulation, proliferation, and tumorigenesis. In this review, we will critically evaluate recent progresses and assess future prospects in studying eIF3i.

Proton pump inhibitors suppress DNA damage repair and sensitize treatment resistance in breast cancer by targeting fatty acid synthase.

Human fatty acid synthase (FASN) is the sole cytosolic enzyme responsible for de novo lipid synthesis. FASN is essential for cancer cell survival and contributes to drug and radiation resistance by up-regulating DNA damage repair but not required for most non-lipogenic tissues. Thus, FASN is an attractive target for drug discovery. However, despite decades of effort in targeting FASN, no FASN inhibitors have been approved due to poor pharmacokinetics or toxicities. Here, we show that the FDA-approved proton pump inhibitors (PPIs) effectively inhibit FASN and suppress breast cancer cell survival. PPI inhibition of FASN leads to suppression of non-homologous end joining repair of DNA damages by reducing FASN-mediated PARP1 expression, resulting in apoptosis from oxidative DNA damages and sensitization of cellular resistance to doxorubicin and ionizing radiation. Mining electronic medical records of 6754 breast cancer patients showed that PPI usage significantly increased overall survival and reduced disease recurrence of these patients. Hence, PPIs may be repurposed as anticancer drugs for breast cancer treatments by targeting FASN to overcome drug and radiation resistance.

The effect of eIF3a on anthracycline-based chemotherapy resistance by regulating DSB DNA repair.

BACKGROUND: Anthracycline are inhibitors of topoisomerase II leading to DNA double strand breaks, and it is widely used for treatment of breast cancer. eIF3a is the largest subunit of eukaryotic translation initiation factor 3 (eIF3) and highly expressed in breast cancer. In this study, we investigated the role of eIF3a in DSB DNA repair and the response of breast cancer patients to anthracycline-based chemotherapy. METHODS: MTT assay was used to detect anthracycline sensitivity in cell lines. Real-time reverse transcriptase PCR, western blotting and immunofluorescence were performed to assess changes in gene expression levels. Cometassay and end-joining activity assay were conducted to explore the effect of eIF3a in NHEJ repair. Luciferase reporter assay was performed to detect LIG4 5'UTR activity. Immunohistochemistry was used to detect eIF3a, LIG4 and DNA-PKcs expression levels in breast cancer tissues. RESULTS: The results showed that eIF3a increased cellular response to anthracyclines by regulating DSB repair activity via influencing the expression of LIG4 and DNA-PKcs at translational level. Breast cancer patients with high level of eIF3a or low level of LIG4 or low level of DNA-PKcs had better anthracycline-based chemotherapy prognosis compared. Moreover, Combined expressions of eIF3a, LIG4 and DNA-PKcs could be better to predict PFS in breast cancer patients with anthracycline-based chemotherapy. CONCLUSION: Our findings suggest that eIF3a effects anthracycline-based chemotherapy response by regulating DSB DNA repair.

Inhibiting Fatty Acid Synthase with Omeprazole to Improve Efficacy of Neoadjuvant Chemotherapy in Patients with Operable TNBC.

PURPOSE: Fatty acid synthase (FASN) is overexpressed in 70% of operable triple-negative breast cancer (TNBC) and is associated with poor prognosis. Proton pump inhibitors selectively inhibit FASN activity and induce apoptosis in TNBC cell lines. PATIENTS AND METHODS: Patients with operable TNBC were enrolled in this single-arm phase II study. Patients began omeprazole 80 mg orally twice daily for 4-7 days prior to neoadjuvant anthracycline-taxane-based chemotherapy (AC-T) and continued until surgery. The primary endpoint was pathologic complete response (pCR) in patients with baseline FASN overexpression (FASN+). Secondary endpoints included pCR in all surgery patients, change in FASN expression, enzyme activity, and downstream protein expression after omeprazole monotherapy, safety, and limited omeprazole pharmacokinetics. RESULTS: Forty-two patients were recruited with a median age of 51 years (28-72). Most patients had ≥cT2 (33, 79%) and ≥N1 (22, 52%) disease. FASN overexpression prior to AC-T was identified in 29 of 34 (85%) evaluable samples. The pCR rate was 72.4% [95% confidence interval (CI), 52.8-87.3] in FASN+ patients and 74.4% (95% CI, 57.9-87.0) in all surgery patients. Peak omeprazole concentration was significantly higher than the IC50 for FASN inhibition observed in preclinical testing; FASN expression significantly decreased with omeprazole monotherapy [mean change 0.12 (SD, 0.25); P = 0.02]. Omeprazole was well tolerated with no grade ≥ 3 toxicities. CONCLUSIONS: FASN is commonly expressed in early TNBC. Omeprazole can be safely administered in doses that inhibit FASN. The addition of omeprazole to neoadjuvant AC-T yields a promising pCR rate that needs further confirmation in randomized studies.

Role of 14-3-3σ in poor prognosis and in radiation and drug resistance of human pancreatic cancers.

BACKGROUND: Pancreatic cancer is the fourth leading cause of death in the US. Unlike other solid tumors such as testicular cancer which are now curable, more than 90% of pancreatic cancer patients die due to lack of response to therapy. Recently, the level of 14-3-3σ mRNA was found to be increased in pancreatic cancers and this increased expression may contribute to the failure in treatment of pancreatic cancers. In the present study, we tested this hypothesis. METHODS: Western blot analysis was used to determine 14-3-3σ protein level in fresh frozen tissues and was correlated to clinical outcome. A stable cell line expressing 14-3-3σ was established and the effect of 14-3-3σ over-expression on cellular response to radiation and anticancer drugs were tested using SRB assay and clonogenic assays. Cell cycle distribution and apoptosis analyses were performed using propidium iodide staining and PARP cleavage assays. RESULTS: We found that 14-3-3σ protein level was increased significantly in about 71% (17 of 24) of human pancreatic cancer tissues and that the 14-3-3σ protein level in cancers correlated with lymph node metastasis and poor prognosis. Furthermore, we demonstrated that over-expression of 14-3-3σ in a pancreatic cancer cell line caused resistance to γ-irradiation as well as anticancer drugs by causing resistance to treatment-induced apoptosis and G2/M arrest. CONCLUSION: The increased level of 14-3-3σ protein likely contributes to the poor clinical outcome of human pancreatic cancers by causing resistance to radiation and anticancer drugs. Thus, 14-3-3σ may serve as a prognosis marker predicting survival of pancreatic cancer patients and guide the clinical treatment of these patients.