CIDE family proteins control lipid homeostasis and the development of metabolic diseases.

Dysregulation of lipid homeostasis leads to the development of metabolic disorders including obesity, diabetes, cardiovascular disease and cancer. Lipid droplets (LDs) are subcellular organelles vital in the maintenance of lipid homeostasis by coordinating lipid synthesis, lipid storage, lipid secretion and lipolysis. Under fed condition, free fatty acids (FFAs) are remodeled and esterified into neutral lipids by lipogenesis and stored in the LDs. The lipid storage capacity of LDs is controlled by its growth via local lipid synthesis or by LD fusion. During fasting, neutral lipids are hydrolyzed by lipolysis, released as FFAs and secreted to meet energy demand. Cell death-inducing DNA fragmentation factor alpha (DFFA)-like effector (CIDE) family proteins composed of Cidea, Cideb and Cidec/Fsp27 are ER- and LD-associated proteins and have emerged as important regulators of lipid homeostasis. Notably, when localized on the LDs, CIDE proteins enrich at the LD-LD contact sites (LDCSs) and control LD fusion and growth. Here, we summarize these recent advances made on the role of CIDE proteins in the regulation of lipid metabolism with a particular focus on the molecular mechanisms underlying CIDE-mediated LD fusion and growth.

Polybasic RKKR motif in the linker region of lipid droplet (LD)-associated protein CIDEC inhibits LD fusion activity by interacting with acidic phospholipids.

Lipid droplets (LDs) are intracellular organelles and a central site for lipid synthesis, storage, and mobilization. The size of LDs reflects the dynamic regulation of lipid metabolism in cells. Previously, we found that cell death-inducing DFFA-like effector C (CIDEC) mediates LD fusion and growth by lipid transfer through LD-LD contact sites in adipocytes and hepatocytes. The CIDE-N domains of CIDEC molecules form homodimers, whereas the CIDE-C domain plays an important role in LD targeting and enrichment. Here, using targeted protein deletions and GFP expression coupled with fluorescence microscopy, we identified a polybasic RKKR motif in the linker region that connects the CIDE-N and CIDE-C domains of CIDEC and functions as a regulatory motif for LD fusion. We found that deletion of the linker region or mutation of the RKKR motif increases the formation of supersized LDs compared with LD formation in cells with WT CIDEC. This enhanced LD fusion activity required the interaction between CIDE-N domains. Mechanistically, we found that the RKKR motif interacts with acidic phospholipids via electrostatic attraction. Loss of this motif disrupted the protein-lipid interaction, resulting in enhanced lipid droplet fusion activity and thus formation of larger LDs. In summary, we have uncovered a CIDEC domain that regulates LD fusion activity, a finding that provides insights into the inhibitory regulation of LD fusion through CIDEC-lipid interactions.

Primary angle closure glaucoma (PACG) susceptibility gene PLEKHA7 encodes a novel Rac1/Cdc42 GAP that modulates cell migration and blood-aqueous barrier function.

PLEKHA7, a gene recently associated with primary angle closure glaucoma (PACG), encodes an apical junctional protein expressed in components of the blood aqueous barrier (BAB). We found that PLEKHA7 is down-regulated in lens epithelial cells and in iris tissue of PACG patients. PLEKHA7 expression also correlated with the C risk allele of the sentinel SNP rs11024102 with the risk allele carrier groups having significantly reduced PLEKHA7 levels compared to non-risk allele carriers. Silencing of PLEKHA7 in human immortalized non-pigmented ciliary epithelium (h-iNPCE) and primary trabecular meshwork cells, which are intimately linked to BAB and aqueous humor outflow respectively, affected actin cytoskeleton organization. PLEKHA7 specifically interacts with GTP-bound Rac1 and Cdc42, but not RhoA, and the activation status of the two small GTPases is linked to PLEKHA7 expression levels. PLEKHA7 stimulates Rac1 and Cdc42 GTP hydrolysis, without affecting nucleotide exchange, identifying PLEKHA7 as a novel Rac1/Cdc42 GAP. Consistent with the regulatory role of Rac1 and Cdc42 in maintaining the tight junction permeability, silencing of PLEKHA7 compromises the paracellular barrier between h-iNPCE cells. Thus, downregulation of PLEKHA7 in PACG may affect BAB integrity and aqueous humor outflow via its Rac1/Cdc42 GAP activity, thereby contributing to disease etiology.

DFCP1 associates with lipid droplets.

Double FYVE-containing protein 1 (DFCP1) is ubiquitously expressed, participates in intracellular membrane trafficking and labels omegasomes through specific interactions with phosphatidylinositol-3-phosphate (PI3P). Previous studies showed that subcellular DFCP1 proteins display multi-organelle localization, including in the endoplasmic reticulum (ER), Golgi apparatus and mitochondria. However, its localization and function on lipid droplets (LDs) remain unclear. Here, we demonstrate that DFCP1 localizes to the LD upon oleic acid incubation. The ER-targeted domain of DFCP1 is indispensable for its LD localization, which is further enhanced by double FYVE domains. Inhibition of PI3P binding at the FYVE domain through wortmannin treatment or double mutation at C654S and C770S have no effect on DFCP1's LD localization. These show that the mechanisms for DFCP1 targeting the omegasome and LDs are different. DFCP1 deficiency in MEF cells causes an increase in LD number and reduces LD size. Interestingly, DFCP1 interacts with GTP-bound Rab18, an LD-associated protein. Taken together, our work demonstrates the dynamic localization of DFCP1 is regulated by nutritional status in response to cellular metabolism.

Bosutinib inhibits migration and invasion via ACK1 in KRAS mutant non-small cell lung cancer.

The advent of effective targeted therapeutics has led to increasing emphasis on precise biomarkers for accurate patient stratification. Here, we describe the role of ACK1, a non-receptor tyrosine kinase in abrogating migration and invasion in KRAS mutant lung adenocarcinoma. Bosutinib, which inhibits ACK1 at 2.7 nM IC50, was found to inhibit cell migration and invasion but not viability in a panel of non-small cell lung cancer (NSCLC) cell lines. Knockdown of ACK1 abrogated bosutinib-induced inhibition of cell migration and invasion specifically in KRAS mutant cells. This finding was further confirmed in an in vivo zebrafish metastatic model. Tissue microarray data on 210 Singaporean lung adenocarcinomas indicate that cytoplasmic ACK1 was significantly over-expressed relative to paired adjacent non-tumor tissue. Interestingly, ACK1 expression in "normal" tissue adjacent to tumour, but not tumour, was independently associated with poor overall and relapse-free survival. In conclusion, inhibition of ACK1 with bosutinib attenuates migration and invasion in the context of KRAS mutant NSCLC and may fulfil a therapeutic niche through combinatorial treatment approaches.

Klotho-beta overexpression as a novel target for suppressing proliferation and fibroblast growth factor receptor-4 signaling in hepatocellular carcinoma.

BACKGROUND: We had previously demonstrated overexpression of fibroblast growth factor receptor-4 (FGFR4) in hepatocellular carcinoma (HCC). However, additional molecular mechanisms resulting in amplified FGFR4 signaling in HCC remain under-studied. Here, we studied the mechanistic role of its co-receptor klotho-beta (KLB) in driving elevated FGFR4 activity in HCC progression. RESULTS: Quantitative real-time PCR analysis identified frequent elevation of KLB gene expression in HCC tumors relative to matched non-tumor tissue, with a more than two-fold increase correlating with development of multiple tumors in patients. KLB-silencing in Huh7 cells decreased cell proliferation and suppressed FGFR4 downstream signaling. While transient repression of KLB-FGFR4 signaling decreased protein expression of alpha-fetoprotein (AFP), a HCC diagnostic marker, prolonged inhibition enriched for resistant HCC cells exhibiting increased liver stemness. CONCLUSIONS: Elevated KLB expression in HCC tissues provides further credence to the oncogenic role of increased FGFR4 signaling in HCC progression and represents a novel biomarker to identify additional patients amenable to anti-FGFR4 therapy. The restricted tissue expression profile of KLB, together with the anti-proliferative effect observed with KLB-silencing, also qualifies it as a specific and potent therapeutic target for HCC patients. The enrichment of a liver stem cell-like population in response to extended KLB-FGFR4 repression necessitates further investigation to target the development of drug resistance.

Mitochondrial translocation of cofilin is an early step in apoptosis induction.

Increasing evidence suggests that movement of key proteins in or out of mitochondria during apoptosis is essential for the regulation of apoptosis. Here, we report identification of the actin-binding protein cofilin by a proteomic approach, as such a factor translocated from cytosol into mitochondria after induction of apoptosis. We found that after induction of apoptosis, cofilin was translocated to mitochondria before release of cytochrome c. Reduction of cofilin protein levels with small-interfering RNA (siRNA) resulted in inhibition of both cytochrome c release and apoptosis. Only dephosphorylated cofilin was translocated to mitochondria, and the cofilin S3D mutant, which mimicks the phosphorylated form, suppressed mitochondrial translocation and apoptosis. Translocation was achieved through exposure of an amino-terminal mitochondrial targeting signal in combination with carboxy-terminal sequences. When correctly targeted to mitochondria, cofilin induced massive apoptosis. The apoptosis-inducing ability of cofilin, but not its mitochondrial localization, was dependent on the functional actin-binding domain. Thus, domains involved in mitochondrial targeting and actin binding are indispensable for its pro-apoptotic function. Our data suggest that cofilin has an important function during the initiation phase of apoptosis.

A gel-like condensation of Cidec generates lipid-permeable plates for lipid droplet fusion.

Membrane contact between intracellular organelles is important in mediating organelle communication. However, the assembly of molecular machinery at membrane contact site and its internal organization correlating with its functional activity remain unclear. Here, we demonstrate that a gel-like condensation of Cidec, a crucial protein for obesity development by facilitating lipid droplet (LD) fusion, occurs at the LD-LD contact site (LDCS) through phase separation. The homomeric interaction between the multivalent N terminus of Cidec is sufficient to promote its phase separation both in vivo and in vitro. Interestingly, Cidec condensation at LDCSs generates highly plastic and lipid-permeable fusion plates that are geometrically constrained by donor LDs. In addition, Cidec condensates are distributed unevenly in the fusion plate generating stochastic sub-compartments that may represent unique lipid passageways during LD fusion. We have thus uncovered the organization and functional significance of geometry-constrained Cidec phase separation in mediating LD fusion and lipid homeostasis.

Regulation of Akt(ser473) phosphorylation by choline kinase in breast carcinoma cells.

BACKGROUND: The serine/threonine kinase PKB/Akt plays essential role in various cellular processes including cell growth and proliferation, metabolism and cell survival. The importance of the Akt pathway is highlighted by the mutation of various components of the pathway such as the PTEN and PI3-kinase (P110alpha) in human cancers. In this paper, we employed an RNA interference library targeting all human kinases to screen for kinases involved in the regulation of Akt activation, in particular serine 473 phosphorylation. Here, we transfected the MDA-MB 468 breast cell line with the human kinome siRNA library and measured Akt activation using an antibody specific for phosphoserine 473 of Akt. RESULTS: The screen revealed that phosphorylation of Akt(ser473) can be regulated by more than 90 kinases. Interestingly, phosphorylation of Akt(ser473), but not thr308, can be severely reduced by inhibition of Choline kinase activity via siRNA or small molecule inhibitors. We show here that the regulation of Akt phosphorylation by Choline kinase is PI3K-independent. In addition, xenograft tumors treated with Choline kinase inhibitors demonstrated a statistically significant decrease in Akt(ser473) phosphorylation. Importantly, the reduction in phosphorylation correlates with regression of these xenograft tumors in the mouse model. CONCLUSION: High Choline kinase expression and activity has previously been implicated in tumor development and metastasis. The mechanism by which Choline kinase is involved in tumor formation is still not fully resolved. From our data, we proposed that Choline kinase plays a key role in regulating Akt(ser473) phosphorylation, thereby promoting cell survival and proliferation.

Lipid-exchange Rate Assay for Lipid Droplet Fusion in Live Cells.

Lipid droplets (LDs) are central organelles in maintaining lipid homeostasis. Defective LD growth often results in the development of metabolic disorders. LD fusion and growth mediated by cell death-inducing DNA fragmentation factor alpha (DFFA)-like effector (CIDE) family proteins are crucial for various biological processes including unilocular LD formation in the adipocytes, lipid storage in the liver, milk lipid secretion in the mammary epithelia cells, and lipid secretion in the skin sebocytes. Previous methodology by Gong et al. (2011) first reported a lipid-exchange rate assay to evaluate the fusion ability of each LD pair in the cells mediated by CIDE family proteins and their regulators, but photobleaching issue remains a problem and a detailed procedure was not provided. Here, we provide an improved and detailed protocol for the lipid-exchange rate measurement. The three key steps for this assay are cell preparation, image acquisition, and data analysis. The images of the fluorescence recovery are acquired after photobleaching followed by the measurement of the intensity changes in the LD pair. The difference in fluorescent intensity is used to obtain the lipid exchange rate between the LDs. The accuracy and repetitiveness of the calculated exchange rates are assured with three-cycle of photobleaching process and the linear criteria in data fitting. With this quantitative assay, we are able to identify the functional roles of the key proteins and the effects of their mutants on LD fusion.

Identification of gene products that control lipid droplet size in yeast using a high-throughput quantitative image analysis.

Lipid droplets (LDs) are important organelles involved in energy storage and expenditure. LD dynamics has been investigated using genome-wide image screening methods in yeast and other model organisms. For most studies, genes were identified using two-dimensional images with LD enlargement as readout. Due to imaging limitation, reduction of LD size is seldom explored. Here, we aim to set up a screen that specifically utilizes reduced LD size as the readout. To achieve this, a novel yeast screen is set up to quantitatively and systematically identify genes that regulate LD size through a three-dimensional imaging-based approach. Cidea which promotes LD fusion and growth in mammalian cells was overexpressed in a yeast knockout library to induce large LD formation. Next, an automated, high-throughput image analysis method that monitors LD size was utilized. With this screen, we identified twelve genes that reduced LD size when deleted. The effects of eight of these genes on LD size were further validated in fld1 null strain background. In addition, six genes were previously identified as LD-regulating genes. To conclude, this methodology represents a promising strategy to screen for players in LD size control in both yeast and mammalian cells to aid in the investigation of LD-associated metabolic diseases.

Long noncoding RNA EGFR-AS1 mediates epidermal growth factor receptor addiction and modulates treatment response in squamous cell carcinoma.

Targeting EGFR is a validated approach in the treatment of squamous-cell cancers (SCCs), although there are no established biomarkers for predicting response. We have identified a synonymous mutation in EGFR, c.2361G>A (encoding p.Gln787Gln), in two patients with head and neck SCC (HNSCC) who were exceptional responders to gefitinib, and we showed in patient-derived cultures that the A/A genotype was associated with greater sensitivity to tyrosine kinase inhibitors (TKIs) as compared to the G/A and G/G genotypes. Remarkably, single-copy G>A nucleotide editing in isogenic models conferred a 70-fold increase in sensitivity due to decreased stability of the EGFR-AS1 long noncoding RNA (lncRNA). In the appropriate context, sensitivity could be recapitulated through EGFR-AS1 knockdown in vitro and in vivo, whereas overexpression was sufficient to induce resistance to TKIs. Reduced EGFR-AS1 levels shifted splicing toward EGFR isoform D, leading to ligand-mediated pathway activation. In co-clinical trials involving patients and patient-derived xenograft (PDX) models, tumor shrinkage was most pronounced in the context of the A/A genotype for EGFR-Q787Q, low expression of EGFR-AS1 and high expression of EGFR isoform D. Our study reveals how a 'silent' mutation influences the levels of a lncRNA, resulting in noncanonical EGFR addiction, and delineates a new predictive biomarker suite for response to EGFR TKIs.

Overexpression of Tyro3 and its implications on hepatocellular carcinoma progression.

While various tyrosine kinases have been associated with the pathogenesis of hepatocellular carcinoma (HCC), the identification of a dominant therapeutic target among them remains a challenge. Here, we investigated the role of Tyro3, a relatively uncharacterized member of the TAM (Tyro3, Axl and Mer) receptor family. The present study aimed to profile and identify potential association between Tyro3 expression in HCC and cancer phenotypes. RNAs obtained from 55 HCC patients were quantified for Tyro3 expression in both cancerous tissue and the adjacent normal tissue. Expression profile was correlated with clinical data. These observations were further substantiated with in vitro HCC cell culture investigations.Tyro3 was strongly upregulated (>2-fold elevation) in the tumor tissue of ~42% of the patients. It was shown that higher expression level of Tyro3 was associated with the key tumor marker AFP, and the tumor diameter and liver injury marker ALT. Subsequent cell culture models indicated high expression in various HCC cell lines, in particular Hep3B. Gene silencing of Tyro3 in Hep3B effectively reduced cell proliferation, ERK phosphorylation and cyclin D1 expression, indicating a key in maintaining the proliferative state of these cells. Notably, silencing also suppressed the transcriptional and translational expression of HCC tumor marker AFP. Overall, these data suggest that Tyro3 contributes significantly to tumor growth, aggressiveness and liver dysfunction. Inhibition of Tyro3 and its aberrant signaling in tumors with high expression could present new opportunities for HCC treatment.

Current strategies for inhibiting FGFR activities in clinical applications: opportunities, challenges and toxicological considerations.

Aberrations in fibroblast growth factor receptor (FGFR) signaling are instrumental to the pathophysiology of several malignancies and disorders. Hence, FGFR inhibitors are explored in therapeutics with early candidates developed as competitors for the ATP-binding pocket in the kinase domain. More recent programs yielded compounds of diverse scaffolds with alternative binding modes. Concurrently, monoclonal antibodies and peptide-based agents provide independent options for clinical development. Notwithstanding this rapid progress, we contemplate the toxicological impact of FGFR inhibition based on the defined role of FGFR family members in physiology and homeostasis. The high homology among FGFR1-4 and also with other kinase subfamilies creates an additional challenge in developing selective inhibitors. It orchestrates an ongoing conundrum of moderating a balance between synergism through multitargeting kinase inhibition and minimizing off-target toxicities.

Benzylidene-indolinones are effective as multi-targeted kinase inhibitor therapeutics against hepatocellular carcinoma.

Effective pharmacological intervention of advanced hepatocellular carcinoma (HCC) is currently lacking. Despite the use of tyrosine kinase inhibitors (TKIs) for the targeted therapy of several malignancies, no agent has been developed to specifically interfere with the oncogenic tyrosine kinase signaling aberrations found in HCC. Therefore, we adopted an orthogonal biological phenotypic screening approach to uncover candidate compounds: based on a potent cytotoxicity toward HCC-derived cell lines, and minimal toxicity toward normal liver cells. Given the success of indolinone as a chemical scaffold in deriving potent multi-kinase inhibitors (e.g. sunitinib), we screened a group of newly synthesized benzylidene-indolinones. Among the candidates, E/Z 6-Chloro-3-(3-trifluoromethyl-benzyliden)-1,3-dihydroindol-2-one (compound 47) exhibited potent anti-proliferative, anti-migratory, pro-apoptotic properties and good safety profile as compared to known multi-targeted tyrosine kinase inhibitors sunitinib and sorafenib. Additionally, an accompanying suppression of alpha-fetoprotein (AFP) transcription, an HCC tumor marker, implies a favorable selectivity and efficacy on HCC. The in vivo efficacy was demonstrated in an HCC xenograft where 47 was administered once weekly (60 mg/kg) and suppressed tumor burden to the same extent as sorafenib (30 mg/kg daily). A receptor tyrosine kinase (RTK) array study revealed promising inhibition of multiple tyrosine kinases such as IGF-1R, Tyro3 and EphA2 phosphorylation. Gene silencing of these targets ameliorated the cytotoxic potential of 47 on the HuH7 cell line, thereby implicating their contribution to the tumorigenicity of HCC. Hence, 47 exhibits potent anti-cancer effects on HCC cell lines, and is a suitable lead for developing multi-targeted kinase inhibitors of relevance to HCC.

Somatic mutation in the ACK1 ubiquitin association domain enhances oncogenic signaling through EGFR regulation in renal cancer derived cells.

Activated Cdc42-associated Kinase, ACK1, is a non-receptor tyrosine kinase with numerous interacting partners, including Cdc42 and EGFR. Gene amplification and overexpression of ACK1 were found in many cancer types such as those of the lung and prostate. Previously, we identified both somatic- and germ line missense mutations in the ACK1 coding sequence, by surveying 261 cancer cell lines and 15 control tissues. Here, we verified and characterized the non-synonymous mutation, ACK-S985 N, located in the ubiquitin association domain of the protein. Both overexpression and silencing experiments in MCF7 and A498 cells, respectively, demonstrated a role of the ACK1 S985 N mutation in enhancing cell proliferation, migration and anchorage-independent growth as well as the epithelial-mesenchymal transition. Further, we showed that the ACK1 S985 N mutant is unable to bind ubiquitin, unlike the wild type kinase. This contributed to ACK1 protein stability and stabilized EGFR after EGF stimulation, thereby prolonging mitogenic signaling in cancer cells. In addition, the ACK1 S985 N-EGFR interaction is enhanced, but not the ubiquitination of the receptor. Intriguingly, silencing of ACK1 in A498 cells sensitized the renal carcinoma cells to gefitinib, against which they are otherwise resistant. The work demonstrates that other than gene amplification, a single somatic mutation in ACK1 can result in extended protein stability enabling the oncoprotein to exert its oncogenic function in tumor progression. It also provides a rationale to target ACK1 in combination with other chemotherapeutic drugs, such as EGFR inhibitors, to potentiate therapeutic action against resistant tumors.

The critical role of calpain versus caspase activation in excitotoxic injury induced by nitric oxide.

The pathogenesis of various acute and chronic neurodegenerative disorders has been linked to excitotoxic processes and excess generation of nitric oxide. We investigated the deleterious effects of calpain activation in nitric oxide-elicited neuronal apoptosis. In this model, nitric oxide triggers apoptosis of murine cerebellar granule cells by an excitotoxic mechanism requiring glutamate exocytosis and receptor-mediated intracellular calcium overload. Here, we found that calcium-dependent cysteine proteases, calpains, were activated early in apoptosis of cerebellar granule cells exposed to nitric oxide. Release of the proapoptogenic factors cytochrome c and apoptosis-inducing factor from mitochondria preceded neuronal death. However, caspases-3 was not activated. We observed that procaspase-9 was cleaved by calpains to proteolytically inactive fragments. Inhibition of calpains by different synthetic calpain inhibitors or by adenovirally mediated expression of the calpastatin inhibitory domain prevented mitochondrial release of cytochrome c and apoptosis-inducing factor, calpain-specific proteolysis and neuronal apoptosis. We conclude that (i) signal transduction pathways exist that prevent the entry of neurons into a caspase-dependent death after mitochondrial release of cytochrome c and (ii) that calpain activation links nitric oxide-triggered excitotoxic events with the execution of caspase-independent apoptosis in neurons.