The thermogenic activity of brown adipose tissue (BAT), important for adaptive thermogenesis and energy expenditure, is mediated by the mitochondrial uncoupling protein1 (Ucp1) that uncouples ATP generation and dissipates the energy as heat. We show here that Cidea, a protein of unknown function sharing sequence similarity with the N-terminal region of DNA fragmentation factors Dffb and Dffa, is expressed at high levels in BAT. Cidea-null mice had higher metabolic rate, lipolysis in BAT and core body temperature when subjected to cold treatment. Notably, Cidea-null mice are lean and resistant to diet-induced obesity and diabetes. Furthermore, we provide evidence that the role of Cidea in regulating thermogenesis, lipolysis and obesity may be mediated in part through its direct suppression of Ucp1 activity. Our data thus indicate a role for Cidea in regulating energy balance and adiposity.

Fsp27, a member of the Cide family proteins, was shown to localize to lipid droplet and promote lipid storage in adipocytes. We aimed to understand the biological role of Fsp27 in regulating adipose tissue differentiation, insulin sensitivity and energy balance. Fsp27 −/− mice and Fsp27/lep double deficient mice were generated and we examined the adiposity, whole body metabolism, BAT and WAT morphology, insulin sensitivity, mitochondrial activity, and gene expression changes in these mouse strains. Furthermore, we isolated mouse embryonic fibroblasts (MEFs) from wildtype and Fsp27 −/− mice, followed by their differentiation into adipocytes in vitro. We found that Fsp27 is expressed in both brown adipose tissue (BAT) and white adipose tissue (WAT) and its levels were significantly elevated in the WAT and liver of leptin-deficient ob/ob mice. Fsp27 −/− mice had increased energy expenditure, lower levels of plasma triglycerides and free fatty acids. Furthermore, Fsp27 −/− and Fsp27/lep double-deficient mice are resistant to diet-induced obesity and display increased insulin sensitivity. Moreover, white adipocytes in Fsp27 −/− mice have reduced triglycerides accumulation and smaller lipid droplets, while levels of mitochondrial proteins, mitochondrial size and activity are dramatically increased. We further demonstrated that BAT-specific genes and key metabolic controlling factors such as FoxC2, PPAR and PGC1α were all markedly upregulated. In contrast, factors inhibiting BAT differentiation such as Rb, p107 and RIP140 were down-regulated in the WAT of Fsp27 −/− mice. Remarkably, Fsp27 −/− MEFs differentiated in vitro show many brown adipocyte characteristics in the presence of the thyroid hormone triiodothyronine (T3). Our data thus suggest that Fsp27 acts as a novel regulator in vivo to control WAT identity, mitochondrial activity and insulin sensitivity.

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.

Genomics-driven cancer therapeutics has gained prominence in personalized cancer treatment. However, its utility in indications lacking biomarker-driven treatment strategies remains limited. Here we present a “phenotype-driven precision-oncology” approach, based on the notion that biological response to perturbations, chemical or genetic, in ex vivo patient-individualized models can serve as predictive biomarkers for therapeutic response in the clinic. We generated a library of “screenable” patient-derived primary cultures (PDCs) for head and neck squamous cell carcinomas that reproducibly predicted treatment response in matched patient-derived-xenograft models. Importantly, PDCs could guide clinical practice and predict tumour progression in two n = 1 co-clinical trials. Comprehensive “-omics” interrogation of PDCs derived from one of these models revealed YAP1 as a putative biomarker for treatment response and survival in ~24% of oral squamous cell carcinoma. We envision that scaling of the proposed PDC approach could uncover biomarkers for therapeutic stratification and guide real-time therapeutic decisions in the future.

Cell death-inducing DFF45-like effector (CIDE)-B is a member of the novel family of apoptosis-inducing factors that share homology with the N-terminal region of DFF, the DNA fragmentation factor. The molecular mechanism of CIDE-B-induced apoptosis is unclear. We have shown here that CIDE-B protein is localized in mitochondria and forms homodimers and heterodimers with other family members. Serial deletion analyses suggest that the mitochondria localization signal and dimerization interface are overlapped and localized to the 30 amino acid residues at the C-terminal region of CIDE-B. Mitochondria localization and dimerization are both required for CIDE-B-induced apoptosis. Our study has thus revealed a mechanism for CIDE-B-induced apoptosis by localization to mitochondria and the formation of a high affinity homo-or heterodimeric complex.Mitochondria are major organelles that respond to death stimuli by releasing factors such as cytochrome c and apoptosisinducing factor and altering the cellular reduction-oxidation (redox) potential and oxidative phosphorylation (1-3). A number of pro-and anti-apoptotic proteins reside in mitochondria including various caspases (4), the ced-4 and ced-9 (5) Bcl-2 family proteins (6), and the Nix family proteins (7,8). Mitochondria localization is important for the anti-apoptotic activity of Bcl-2 (6) or the pro-apoptotic activity of Nix (7,8), as deletion of the C-terminal mitochondria localization signal abrogates their activity. Many of the Bcl-2 family members can form either homodimers or heterodimers with other family members (6, 9, 10). Activation of pro-apoptotic Bax appears to induce subcellular translocation from cytosol to mitochondria as well as homodimerization (11). Mutational analyses have revealed that the conserved BH3 domain of the pro-apoptotic Bcl-2 family members such as Bax plays an important role in mediating homo-or heterodimerization (6).The DNA fragmentation factor (DFF) 1 (12, 13) consists of two subunits, a nuclease (CAD/DFF40) and its inhibitor (DFF45/ ICAD). The N-terminal domain of DFF45 is required for its chaperone function by associating with the N-terminal region of DFF40 (14,15). A novel family of cell death-inducing DFF45-like effectors (CIDEs) was identified by its high homology with both of the N-terminal domains of DFF40 and DFF45 (16,17). CIDE proteins can be divided into the N-terminal CIDE-N domain, which shares homology with DFF40/45, and the Cterminal CIDE-C domain, which shares homology within CIDE proteins only (16). Although this class of proteins shares homology with DFF45/40 at the N-terminal region, their functions differ significantly. Unlike DFF45, over-expression of CIDEs in mammalian cells shows strong cell death-inducing activity with the C-terminal domain (CIDE-C) being sufficient for its cell death activity (16). We have recently participated in solving the structure of the N-terminal domain of CIDE-B (or CIDE-N) (18). Our structural analyses suggested that CIDE-N domains interact with each other with low affinity and that t...

The fat-specific protein 27 (Fsp27), a protein localized to lipid droplets (LDs), plays an important role in controlling lipid storage and mitochondrial activity in adipocytes. Fsp27-null mice display increased energy expenditure and are resistant to high fat diet-induced obesity and diabetes. However, little is known about how the Fsp27 protein is regulated. Here, we show that Fsp27 stability is controlled by the ubiquitin-dependent proteasomal degradation pathway in adipocytes. The ubiquitination of Fsp27 is regulated by three lysine residues located in the C-terminal region. Substitution of these lysine residues with alanines greatly increased Fsp27 stability and enhanced lipid storage in adipocytes. Furthermore, Fsp27 was stabilized and rapidly accumulated following treatment with ␤-agonists that induce lipolysis and fatty acid re-esterification in adipocytes. More importantly, Fsp27 stabilization was dependent on triacylglycerol synthesis and LD formation, because knockdown of diacylglycerol acyltransferase in adipocytes significantly reduced Fsp27 accumulation in adipocytes. Finally, we observed that increased Fsp27 during ␤-agonist treatment preferentially associated with LDs. Taken together, our data revealed that Fsp27 can be stabilized by free fatty acid availability, triacylglycerol synthesis, and LD formation. The stabilization of Fsp27 when free fatty acids are abundant further enhances lipid storage, providing positive feedback to regulate lipid storage in adipocytes.Cell death-inducing DNA fragmentation factor-45-like effector (Cide) proteins, including Cidea, Cideb, and fat-specific protein 27 (Fsp27, 3 also known as Cidec in humans), are a family of proteins shown to play critical roles in controlling metabolism homeostasis (1). Our previous work demonstrated that mice with a deficiency in Cidea or Cideb have higher energy expenditure and enhanced insulin sensitivity and are resistant to high fat diet-induced obesity and diabetes (2, 3). Fsp27 is enriched in adipocytes, in both white adipose tissue and brown adipose tissue (2, 4). The Fsp27 protein is detected in the lipid droplet (LD)-enriched fraction (5), and its overexpression can promote triacylglycerol (TAG) storage (6, 7). Interestingly, Fsp27 and Cidea mRNAs have also been detected in fatty livers, where an excess amount of lipids accumulate and large LDs form (8 -10). More recently, Fsp27 was demonstrated to be a direct mediator of peroxisome proliferator-activated receptor ␥-dependent hepatic steatosis (10). In accordance with a role for Fsp27 in LD formation, Fsp27 deficiency results in dramatically reduced white adipose tissue deposits and the acquisition of a brown fat-like morphology in these white adipose tissues, which is characterized by the appearance of smaller LDs and increased mitochondrial size and activity (11, 12). Furthermore, both Fsp27-deficient and Fsp27/leptin double-deficient mice display improved insulin sensitivity and lean phenotype (12). Except for one study showing that Cidea is degraded through the ubiquitin-m...

High-risk human papillomaviruses are causative agents of cervical cancer. Viral protein E7 is required to establish and maintain the pro-oncogenic phenotype in infected cells, but the molecular mechanisms by which E7 promotes carcinogenesis are only partially understood. Our transcriptome analyses in primary human fibroblasts transduced with the viral protein revealed that E7 activates a group of mitotic genes via the activator B-Myb-MuvB complex. We show that E7 interacts with the B-Myb, FoxM1 and LIN9 components of this activator complex, leading to cooperative transcriptional activation of mitotic genes in primary cells and E7 recruitment to the corresponding promoters. E7 interaction with LIN9 and FoxM1 depended on the LXCXE motif, which is also required for pocket protein interaction and degradation. Using E7 mutants for the degradation of pocket proteins but intact for the LXCXE motif, we demonstrate that E7 functional interaction with the B-Myb-MuvB complex and pocket protein degradation are two discrete functions of the viral protein that cooperate to promote acute transcriptional activation of mitotic genes. Transcriptional level of E7 in patient's cervical lesions at different stages of progression was shown to correlate with those of B-Myb and FoxM1 as well as other mitotic gene transcripts, thereby linking E7 with cellular proliferation and progression in cervical cancer in vivo. E7 thus can directly activate the transcriptional levels of cell cycle genes independently of pocket protein stability.

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