CIP2A Research Articles

SIRT1 ISGylation accelerates tumor progression by unleashing SIRT1 from the inactive state to promote its deacetylase activity

ISG15 is an interferon-stimulated ubiquitin-like protein (UBL) with multifaceted roles as a posttranslational modifier in ISG15 conjugation (ISGylation). However, the mechanistic consequences of ISGylation in cancer have not been fully elucidated, largely due to a lack of knowledge on the ISG15 target repertoire. Here, we identified SIRT1, a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase, as a new target for ISGylation. SIRT1 ISGylation impairs the association of SIRT1 with its negative regulator, deleted in breast cancer 1 (DBC1), which unleashes SIRT1 from its inactive state and leads to an increase in its deacetylase activity. Importantly, SIRT1 ISGylation promoted lung cancer progression and limited lung cancer cell sensitivity to DNA damage-based therapeutics in vivo and in vitro models. The levels of ISG15 mRNA and protein were significantly higher in lung cancer tissues than in adjacent normal tissues. Accordingly, elevated expression of SIRT1 and ISG15 was associated with poor prognosis in lung cancer patients, a finding that could be translated for lung cancer patient stratification and disease outcome evaluation. Taken together, our findings provide a mechanistic understanding of the regulatory effect of SIRT1 ISGylation on tumor progression and therapeutic efficacy in lung cancer.

Molecular mechanism of NAD+ and NMN binding to the Nudix homology domains of DBC1

Deleted in breast cancer 1 (DBC1) is a human nuclear protein that modulates the activities of various proteins involved in cell survival and cancer progression. Oxidized form of nicotinamide adenine dinucleotide (NAD+) is suggested to bind to the Nudix homology domains (NHDs) of DBC1, thereby regulating DBC1-Poly (ADP-ribose) polymerase 1 (PARP1) interactions, resulting in the restoration of DNA repair. Using Nuclear Magnetic Resonance (NMR) and Isothermal Titration Calorimetry (ITC), we confirmed NAD+ and its precursor nicotinamide mononucleotide (NMN) both bind the NHD domain of DBC1 (DBC1354–396). NAD+ likely interacts with DBC1354–396 through hydrogen bonding, with a binding affinity (8.99 μM) nearly twice that of NMN (17.0 μM), and the key binding sites are primarily residues E363 and D372, in the agreement with Molecular Docking experiments. Molecular Dynamics (MD) simulation further demonstrated E363 and D372's anchoring role in the binding process. Additional mutagenesis experiments of E363 and D372 confirmed their critical involvement of ligand-protein interactions. These findings lead to a better understanding of how NAD+ and NMN regulate DBC1, thereby offering insights for the development of targeted therapies and drug research focused on DBC1-associated tumors.

DBC1maintains skeletal muscle integrity by enhancing myogenesis and preventing myofibre wasting.

Skeletal muscle atrophy, particularly ageing-related muscular atrophy such as sarcopenia, is a significant health concern. Despite its prevalence, the underlying mechanisms remain poorly understood, and specific approved medications are currently unavailable. Deleted in breast cancer 1 (DBC1) is a well-known regulator of senescence, metabolism or apoptosis. Recent reports suggest that DBC1 may also potentially regulate muscle function, as mice lacking DBC1 exhibit weakness and limpness. However, the function of DBC1 in skeletal muscle and its associated molecular mechanisms remain unknown, thus prompting the focus of this study. Tibialis anterior (TA) muscle-specific DBC1 knockdown C57BL/6J male mice were generated through a single injection of 2.00 E+11vg of adeno-associated virus 9 delivering single-guide RNA for DBC1. Grip strength and endurance were assessed 2months later, followed by skeletal muscle harvest. Muscle atrophy model was generated by cast immobilization of the mouse hindlimb for 2weeks. Molecular markers of atrophy were probed in muscles upon termination. Cardiotoxin (CTX) was injected in TA muscles of DBC1 knockdown mice, and muscle regeneration was assessed by immunohistochemistry, quantitative PCR and western blotting. DBC1 knockdown C2C12 cells and myotubes were investigated using immunofluorescence staining, Seahorse, immunohistology, fluorescence-activated cell sorting and RNA-sequencing analyses. DBC1 knockdown in skeletal muscle of young mice led to signatures of muscle atrophy, including a 28% reduction in muscle grip force (P=0.023), a 54.4% reduction in running distance (P=0.002), a 14.3% reduction in muscle mass (P=0.007) and significantly smaller myofibre cross-sectional areas (P<0.0001). DBC1 levels decrease in age-related or limb immobilization-induced atrophic mouse muscles and overexpress DBC1-attenuated atrophic phenotypes in these mice. Muscle regeneration was hampered in mice with CTX-induced muscle injury by DBC1 knockdown, as evidenced by reductions in myofibre cross-sectional areas of regenerating myofibres with centralized nuclei (P<0.0001), percentages of MyoG+ nuclei (P<0.0001) and fusion index (P<0.0001). DBC1 transcriptionally regulated mouse double minute 2 (MDM2), which mediated ubiquitination and degradation of forkhead box O3 (FOXO3). Increased FOXO3 proteins hampered myogenesis in DBC1 knockdown satellite cells by compromising around 50% of mitochondrial functions (P<0.001) and exacerbated atrophy in DBC1 knockdown myofibres by activating the ubiquitin-proteasome and autophagy-lysosome pathways. DBC1 is essential in maintaining skeletal muscle integrity by protecting against myofibres wasting and enhancing muscle regeneration via FOXO3. This research highlights the significance of DBC1 for healthy skeletal muscle function and its connection to muscular atrophy.

SUMO1-regulated DBC1 promotes p53-dependent stress-induced apoptosis of lens epithelial cells.

Deleted in breast cancer 1 (DBC1) was initially identified from a homozygously deleted region in human chromosome 8p21. It has been well established that DBC1 plays a dual role during cancer development. Depending on the physiological context, it can promote or inhibit tumorigenesis. Whether it plays a role in lens pathogenesis remains elusive. In the present study, we demonstrated that DBC1 is highly expressed in lens epithelial cells from different vertebrates and in retina pigment epithelial cells as well. Moreover, DBC1 is SUMOylated through SUMO1 conjugation at K591 residue in human and mouse lens epithelial cells. The SUMOylated DBC1 is localized in the nucleus and plays an essential role in promoting stress-induced apoptosis. Silence of DBC1 attenuates oxidative stress-induced apoptosis. In contrast, overexpression of DBC1 enhances oxidative stress-induced apoptosis, and this process depends on p53. Mechanistically, DBC1 interacts with p53 to regulate its phosphorylation status at multiple sites and the SUMOylation of DBC1 enhances its interaction with p53. Together, our results identify that DBC1 is an important regulator mediating stress-induced apoptosis in lens, and thus participates in control of lens cataractogenesis.

DBC1 is a key positive regulator of enhancer epigenomic writers KMT2D and p300

Histone modification is a key epigenetic mechanism for regulation of chromatin dynamics and gene expression. Deleted in breast cancer 1 (DBC1) has been shown to act as a negative regulator of epigenetic modifiers and as a co-activator for nuclear receptors and other transcription factors. However, little is known about the role of DBC1 in the regulation of histone modifications and chromatin landscapes. Here, we analyzed genome-wide profiles of active enhancer and promoter marks in colorectal cancer cells and report DBC1 as a critical positive regulator of histone epigenetic writers KMT2D (H3K4 methyltransferase) and p300 (histone acetyltransferase). DBC1 is required for establishing the landscape of active enhancers, for genome-wide chromatin binding and enhancer recruitment of KMT2D and p300, and for gene activation involved in colorectal cancer progression. DBC1 interacts directly with KMT2D and p300, and enhances KMT2D-mediated histone H3K4 methylation (H3K4me1/2/3) and p300-mediated H3 acetylation. Importantly, DBC1 contributes to super-enhancer formation and function by facilitating the recruitment of KMT2D and p300 and by enhancing their functional interaction and cooperative cross-talk. Our results highlight the critical role of DBC1 as a key positive regulator of KMT2D and p300, and provide insights into regulatory mechanisms underlying the interplay between the enhancer epigenomic writers in enhancer activation.

Voluntary exercise training improves body weight of leptin-deficient ob/ob mice by altering hepatic stearoyl-CoA desaturase 1 and deleted in breast cancer 1 protein levels.

[Purpose]Deleted in breast cancer 1 (DBC1) ablation causes obesity, and stearoyl-CoA desaturase 1 (SCD1) induces the biosynthesis of monounsaturated fatty acids. This study examined whether voluntary wheel running (VWR) alters SCD-1 and DBC1 protein levels in the liver of leptin-deficient ob/ob mice.[Methods]Twenty-five Ob/Ob mice were divided into two groups (ob/ob-Sed and ob/ob-Ex). The expression of DBC1 and SCD1 in the mouse liver was determined using western blotting.[Results]After 10 weeks, VWR significantly reduced body weight without affecting the fatty acid synthase and CD36 protein levels. The average daily running distance was 4.0±1.0 km/day. This improvement was associated with changes in the hepatic SCD1 and DBC1 levels. Hepatic SCD-1 protein levels increased significantly, and DBC1 protein levels decreased in ob/ob-Sed animals. On the other hand, VWR inhibited the obesity-induced increase in SCD1 expression and impaired the obesity-induced decrease in DBC1 expression in the liver of leptin-deficient ob/ob mice.[Conclusion]This is the first study showing that VWR has strong effects on hepatic SCD1 and DBC1 in ob/ob mice, and provides key insights into the effects of exercise on obesity.

Interaction of DBC1 with polyoma small T antigen promotes its degradation and negatively regulates tumorigenesis

Deleted in Breast Cancer 1 (DBC1) is an important metabolic sensor. Previous studies have implicated DBC1 in various cellular functions, notably cell proliferation, apoptosis, histone modification, and adipogenesis. However, current reports about the role of DBC1 in tumorigenesis are controversial and designate DBC1 alternatively as a tumor suppressor or a tumor promoter. In the present study, we report that polyoma small T antigen (PyST) associates with DBC1 in mammalian cells, and this interaction leads to the posttranslational downregulation of DBC1 protein levels. When coexpressed, DBC1 overcomes PyST-induced mitotic arrest and promotes the exit of cells from mitosis. Using both transient and stable modes of PyST expression, we also show that cellular DBC1 is subjected to degradation by LKB1, a tumor suppressor and cellular energy sensor kinase, in an AMP kinase-independent manner. Moreover, LKB1 negatively regulates the phosphorylation as well as activity of the prosurvival kinase AKT1 through DBC1 and its downstream pseudokinase substrate, Tribbles 3 (TRB3). Using both transient transfection and stable cell line approaches as well as soft agar assay, we demonstrate that DBC1 has oncogenic potential. In conclusion, our study provides insight into a novel signaling axis that connects LKB1, DBC1, TRB3, and AKT1. We propose that the LKB1–DBC1–AKT1 signaling paradigm may have an important role in the regulation of cell cycle and apoptosis and consequently tumorigenesis.

A KDM4-DBC1-SIRT1 Axis Contributes to TGF-b Induced Mesenchymal Transition of Intestinal Epithelial Cells.

Intestinal fibrosis is one of the common pathophysiological processes in inflammatory bowel diseases (IBDs). Previously it has been demonstrated that epithelial-mesenchymal transition (EMT) can contribute to the development of intestinal fibrosis. Here we report that conditional ablation of SIRT1, a class III lysine deacetylase, in intestinal epithelial cells exacerbated 2, 4, 6-trinitro-benzene sulfonic acid (TNBS) induced intestinal fibrosis in mice. SIRT1 activity, but not SIRT1 expression, was down-regulated during EMT likely due to up-regulation of its inhibitor deleted in breast cancer 1 (DBC1). TGF-β augmented the recruitment of KDM4A, a histone H3K9 demethylase, to the DBC1 promoter in cultured intestinal epithelial cells (IEC-6) leading to DBC1 trans-activation. KDM4A depletion or inhibition abrogated DBC1 induction by TGF-β and normalized SIRT1 activity. In addition, KDM4A deficiency attenuated TGF-β induced EMT in IEC-6 cells. In conclusion, our data identify a KDM4-DBC1-SIRT1 pathway that regulates EMT to contribute to intestinal fibrosis.

Resveratrol-induced Sirt1 phosphorylation by LKB1 mediates mitochondrial metabolism

The NAD+-dependent deacetylase Sirt1 has been implicated in the prevention of many age-related diseases, including cancer, type 2 diabetes, and cardiovascular disease. Resveratrol, a plant polyphenol, exhibits antiaging, antitumor, and vascular protection effects by activating Sirt1. However, the molecular mechanism of Sirt1 activation as induced by resveratrol remains unclear. By knockdown/rescue experiments, fluorometric Sirt1 activity assay, immunoprecipitation, and pull-down assays, we identify here that the tumor suppressor LKB1 (liver kinase B1) as a direct activator of Sirt1 elicited by resveratrol. Resveratrol promotes the binding between LKB1 and Sirt1, which we first reported, and this binding leads to LKB1-mediated phosphorylation of Sirt1 at three different serine residues in the C terminus of Sirt1. Mechanistically, LKB1-mediated phosphorylation increases intramolecular interactions in Sirt1, such as the binding of the C terminus to the deacetylase core domain, thereby eliminating DBC1 (Deleted in Breast Cancer 1, Sirt1 endogenous inhibitor) inhibition and promoting Sirt1–substrate interaction. Functionally, LKB1-dependent Sirt1 activation increases mitochondrial biogenesis and respiration through deacetylation and activation of the transcriptional coactivator PGC-1α. These results identify Sirt1 as a context-dependent target of LKB1 and suggest that a resveratrol-stimulated LKB1-Sirt1 pathway plays a vital role in mitochondrial metabolism, a key physiological process that contributes to numerous age-related diseases.

Material basis, effect, and mechanism of ethanol extract of Pinellia ternata tubers on oxidative stress-induced cell senescence

BackgroundThe tuber of Pinellia ternata has been used for a thousand years in China. P. ternata possessed the activities of anti-emetic, sedative-hypnotic, anti-cancer, anti-asthmatic, anti-tussive, and anti-inflammatory. It is the representative of expectorant medicines in Traditional Chinese Medicine (TCM). Phlegm is the pathological product and a new pathogenic factor of the metabolite, which is analogous to the damage of oxidative stress. PurposeThe objectives of the study were to investigate the protective activity and mechanism of ethanol extract of P. ternata tubers (PTE) and its main constituents on oxidative stress-induced cell senescence. MethodsH2O2 and AAPH were used to establish cellular senescence models. The anti-aging effects of PTE and its components were evaluated by SA-β-gal staining, flow cytometry, scanning electron microscope (SEM), and multiple microplate reader, the molecular mechanisms of them were investigated by qRT-PCR and Western Blot. ResultsWe found PTE exhibited the apparent effect on cell senescence, evidenced by inhibiting senescence β-Galactosidase (SA-β-gal) expression, lipofuscin accumulation, cell cycle arrest at the G2/M phase, oxidative damage and apoptosis, and increasing telomerase activity. Their mechanisms were related to increase expressions of SIRT1, forkhead box 3a (Foxo3a), Bcl-2, active regulator of SIRT1, RPS19BP1 (AROS), and Hu antigen R (HuR), but decrease Bax, p53 and deleted in breast cancer 1 (DBC1) levels. Furthermore, adenosine, and succinic acid, as the critical substances in PTE, could also inhibit SA-β-gal expression and cell cycle arrest, down-regulate the expression of Bax, and up-regulate Bcl-2, SirT1, and Foxo3a. ConclusionsWe have demonstrated that PTE slows down oxidative stress-induced cell senescence, and adenosine and succinic acid are the key active components.

The protein Deleted in Breast Cancer-1 (DBC1) regulates vascular response and formation of aortic dissection during Angiotensin II infusion

Cardiovascular diseases are among the main causes of morbimortality in the adult population. Among them, hypertension is a leading cause for stroke, heart disease and kidney failure. Also, as a result of arterial wall weakness, hypertension can lead to the development of dissecting aortic aneurysms, a rare but often fatal condition if not readily treated. In this work, we investigated the role of DBC1 in the regulation of vascular function in an ANGII-induced hypertension mouse model. We found that WT and DBC1 KO mice developed hypertension in response to ANGII infusion. However, DBC1 KO mice showed increased susceptibility to develop aortic dissections. The effect was accompanied by upregulation of vascular remodeling factors, including MMP9 and also VEGF. Consistent with this, we found decreased collagen deposition and elastic fiber fragmentation, suggesting that increased expression of MMPs in DBC1 KO mice weakens the arterial wall, promoting the formation of aortic dissections during treatment with ANGII. Finally, DBC1 KO mice had reduced cell proliferation in the intima-media layer in response to ANGII, paralleled with an impairment to increase wall thickness in response to hypertension. Furthermore, VSMC purified from DBC1 KO mice showed impaired capacity to leave quiescence, confirming the in vivo results. Altogether, our results show for the first time that DBC1 regulates vascular response and function during hypertension and protects against vascular injury. This work also brings novel insights into the molecular mechanisms of the development of aortic dissections.

A novel form of Deleted in breast cancer 1 (DBC1) lacking the N-terminal domain does not bind SIRT1 and is dynamically regulated in vivo

The protein Deleted in Breast Cancer-1 is a regulator of several transcription factors and epigenetic regulators, including HDAC3, Rev-erb-alpha, PARP1 and SIRT1. It is well known that DBC1 regulates its targets, including SIRT1, by protein-protein interaction. However, little is known about how DBC1 biological activity is regulated. In this work, we show that in quiescent cells DBC1 is proteolytically cleaved, producing a protein (DN-DBC1) that misses the S1-like domain and no longer binds to SIRT1. DN-DBC1 is also found in vivo in mouse and human tissues. Interestingly, DN-DBC1 is cleared once quiescent cells re-enter to the cell cycle. Using a model of liver regeneration after partial hepatectomy, we found that DN-DBC1 is down-regulated in vivo during regeneration. In fact, WT mice show a decrease in SIRT1 activity during liver regeneration, coincidentally with DN-DBC1 downregulation and the appearance of full length DBC1. This effect on SIRT1 activity was not observed in DBC1 KO mice. Finally, we found that DBC1 KO mice have altered cell cycle progression and liver regeneration after partial hepatectomy, suggesting that DBC1/DN-DBC1 transitions play a role in normal cell cycle progression in vivo after cells leave quiescence. We propose that quiescent cells express DN-DBC1, which either replaces or coexist with the full-length protein, and that restoring of DBC1 is required for normal cell cycle progression in vitro and in vivo. Our results describe for the first time in vivo a naturally occurring form of DBC1, which does not bind SIRT1 and is dynamically regulated, thus contributing to redefine the knowledge about its function.

DBC1 Regulates p53 Stability via Inhibition of CBP-Dependent p53 Polyubiquitination

SUMMARYThe control of p53 protein stability is critical to its tumor suppressor functions. The CREB binding protein (CBP) transcriptional co-activator co-operates with MDM2 to maintain normally low physiological p53 levels in cells via exclusively cytoplasmic E4 polyubiquitination activity. Using mass spectrometry to identify nuclear and cytoplasmic CBP-interacting proteins that regulate compartmentalized CBP E4 activity, we identified deleted in breast cancer 1 (DBC1) as a stoichiometric CBP-interacting protein that negatively regulates CBP-dependent p53 polyubiquitination, stabilizes p53, and augments p53-dependent apoptosis. TCGA analysis demonstrated that solid tumors often retain wild-type p53 alleles in conjunction with DBC1 loss, supporting the hypothesis that DBC1 is selected for disruption during carcinogenesis as a surrogate for p53 functional loss. Because DBC1 maintains p53 stability in the nucleus, where p53 exerts its tumor-suppressive transcriptional function, replacement of DBC1 functionality in DBC1-deleted tumors might enhance p53 function and chemosensitivity for therapeutic benefit.

Deleted in breast cancer 1 as a potential prognostic biomarker in human cancers: a pooled analysis of 2,254 patients.

BackgroundDeleted in breast cancer 1 (DBC1) is believed to be involved in human cancers. However, it is still uncertain whether DBC1 expression can be regarded as a prognostic factor in patients with various cancers. This meta-analysis aimed to evaluate the relationship between high levels of DBC1 and prognosis in tumor patients.MethodsElectronic databases were searched and 14 studies meeting the selection criteria were included. Overall survival (OS), relapse-free survival (RFS), and 95% CIs were extracted and analyzed. HRs from individual studies were pooled using fixed-or random-effects models, depending on the heterogeneity of the included studies, and publication bias analyses were also performed to increase the reliability of the results.ResultsA total of 2,254 patients with tumors from 14 published studies were included in the meta-analysis. DBC1 overexpression was associated with worse OS (univariate analysis: HR=2.94; 95% CI: [2.38–3.63]; multivariate analysis: HR=1.98, 95% CI: [1.21–3.25]) and RFS (univariate analysis: HR=2.83, 95% CI: [2.30–3.49]; multivariate analysis: HR=2.71, 95% CI: [2.07–3.53]) for various tumors. No publication bias was observed according to test of funnel plot asymmetry and Egger’s test.ConclusionCurrent evidence supports the conclusion that the upregulation of DBC1 is correlated with poor survival among tumor patients, suggesting that DBC1 represents an independent prognostic factor significantly associated with OS and RFS, and could serve as a novel therapeutic target in patients with tumors. Nevertheless, further large-scale prospective trials and well-designed studies are warranted to confirm this finding.

The c-MYC/NAMPT/SIRT1 feedback loop is activated in early classical and serrated route colorectal cancer and represents a therapeutic target.

We have recently identified a positive feedback loop in which c-MYC increases silent information regulator 1 (SIRT1) protein level and activity through transcriptional activation of nicotinamide phosphoribosyltransferase (NAMPT) and NAD+ increase. Here, we determined the relevance of the c-MYC-NAMPT-SIRT1 feedback loop, including the SIRT1 inhibitor deleted in breast cancer 1 (DBC1), for the development of conventional and serrated colorectal adenomas. Immunohistochemical analyses of 104 conventional adenomas with low- and high-grade dysplasia and of 157 serrated lesions revealed that elevated expression of c-MYC, NAMPT, and SIRT1 characterized all conventional and serrated adenomas, whereas DBC1 was not differentially regulated. Analyzing publicly available pharmacogenomic databases from 43 colorectal cancer cell lines demonstrated that responsiveness towards a NAMPT inhibitor was significantly associated with alterations in PTEN and TGFBR2, while features such as BRAF or RNF43 alterations, or microsatellite instability typical for serrated route colorectal cancer, showed increased sensitivities for inhibition of NAMPT and SIRT1. Our findings suggest an activation of the c-MYC-NAMPT-SIRT1 feedback loop that may crucially contribute to initiation and development of both routes to colorectal cancer. Targeting of NAMPT or SIRT1 may represent novel therapeutic strategies with putative higher sensitivity of the serrated route colorectal cancer subtype.

NANOGP8 expression regulates gastric cancer cell progression by transactivating DBC1 in gastric cancer MKN-45 cells.

NANOGP8 is one of the NANOG pseudogenes and is expressed together with NANOG in multiple tumor tissues and cell lines. The biological functions of NANOGP8 in progression of gastric cancer are unclear. In the present study, the role of NANOGP8 was investigated in gastric cancer cells. The gathered data demonstrated that NANOG expression in both mRNA and protein was elevated in gastric cancer cell lines relative to a normal gastric epithelial cell line. Downregulation of NANOGP8 inhibited cell proliferation and increased apoptosis in human gastric carcinoma cell lines. Furthermore, silencing of NANOGP8 suppressed tumor growth in vivo. Interestingly, it was identified that deleted in breast cancer 1 (DBC1) expression was also markedly downregulated following NANOGP8 knockdown. DNA microarray and dual-luciferase assays further indicated that NANOGP8 may bind to the DBC1 promoter region and regulate DBC1 expression. Therefore, the gathered data provided evidence that NANOGP8 contributes to progression of gastric cancer via DBC1 and may have potential translational significance.

Loss of DBC1 (CCAR2) affects TNFα-induced lipolysis and Glut4 gene expression in murine adipocytes.

STAT5A (signal transducer and activator of transcription 5A) is a transcription factor that plays a role in adipocyte development and function. In this study, we report DBC1 (deleted in breast cancer 1; also known as CCAR2) as a novel STAT5A-interacting protein. DBC1 has been primarily studied in tumor cells, but there is evidence that loss of this protein may promote metabolic health in mice. Currently, the functions of DBC1 in mature adipocytes are largely unknown. Using immunoprecipitation and immunoblotting techniques, we confirmed that there is an association between endogenous STAT5A and DBC1 proteins under physiological conditions in the adipocyte nucleus that is not dependent upon STAT5A tyrosine phosphorylation. We used siRNA to knockdown DBC1 in 3T3-L1 adipocytes to determine the impact on STAT5A activity, adipocyte gene expression, and TNFα (tumor necrosis factor α)-regulated lipolysis. The loss of DBC1 did not affect the expression of several STAT5A target genes including Socs3, Cish, Bcl6, Socs2, and Igf1 However, we did observe decreased levels of TNFα-induced glycerol and free fatty acids released from adipocytes with reduced DBC1 expression. In addition, DBC1-knockdown adipocytes had increased Glut4 expression. In summary, DBC1 can associate with STAT5A in adipocyte nucleus, but it does not appear to impact regulation of STAT5A target genes. Loss of adipocyte DBC1 modestly increases Glut4 gene expression and reduces TNFα-induced lipolysis. These observations are consistent with in vivo observations that show loss of DBC1 promotes metabolic health in mice.

ZNF326 promotes a malignant phenotype of breast cancer by interacting with DBC1

The biological role and underlying mechanism of action of zinc-finger protein 326 (ZNF326) in malignant tumors, including breast cancer, are still not clear. In this study, we detected high expression of ZNF326 in breast cancer specimens (60/111, 54.1%) and breast cancer cell lines (7/7); the expression level of ZNF326 was inversely associated with advanced pTNM stage (P = 0.002), positive lymph node metastasis (P = 0.004), poor prognosis in patients with breast cancer (P = 0.0097), and ER/PR/Her2 status (P = 0.013). Meanwhile, the ectopic expression of ZNF326 significantly upregulated MMP7, EMT-related proteins (Snail and Slug), and cell cycle-related proteins (cyclinA2 and cyclinB1); downregulated E-cadherin expression; and promoted the proliferation and invasiveness of breast cancer cells both in vivo and in vitro. Mechanistically, co-immunoprecipitation and immunofluorescence assays both demonstrated that ZNF326 interacted with deleted in breast cancer-1 (DBC1) in breast cancer cells. Additionally, DBC1 knockdown eliminated the up-regulation of MMP7, EMT-related proteins, and cell cycle-related proteins as well as the enhanced proliferation and invasiveness induced by ZNF326. Therefore, we concluded that ZNF326 is highly expressed in breast cancer, is associated with poor prognosis, and plays a vital role in promoting the malignant phenotype of breast cancer cells by interacting with DBC1.

SIRT7 promotes thyroid tumorigenesis through phosphorylation and activation of Akt and p70S6K1 via DBC1/SIRT1 axis.

SIRT7 is an NAD+-dependent histone/non-histone deacetylase, which is highly expressed in different types of cancer including thyroid cancer; however, its biological function in thyroid cancer is still undiscovered. In this study, we found that SIRT7 expression was elevated in papillary thyroid cancers (PTCs), and demonstrated that SIRT7 knockdown dramatically inhibited the proliferation, colony formation, migration and invasion of thyroid cancer cells, and induced thyroid cancer cell cycle arrest and apoptosis. Conversely, SIRT7 re-expression markedly enhanced thyroid cancer cell growth, invasiveness and tumorigenic potential in nude mice. Further studies revealed that SIRT7 exerted an oncogenic function in thyroid tumorigenesis by phosphorylation of Akt and p70S6K1. Mechanistically, SIRT7 binds to the promoter of deleted in breast cancer-1 (DBC1), an endogenous inhibitor of SIRT1, and represses its transcription via deacetylation of H3K18Ac. This results in enhanced interactions between SIRT1 and Akt or p70S6K1, thereby promoting deacetylation and subsequent phosphorylation of Akt and p70S6K1 through a SIRT1-dependent manner. Altogether, our results show that DBC1 is a downstream target of SIRT7, and first uncover that SIRT7 promotes thyroid tumorigenesis through phosphorylation and activation of Akt and p70S6K1 via the modulation of DBC1/SIRT1 axis.

DBC1, Deleted in Breast Cancer 1, Is a Nuclear STAT5A-Interacting Protein in Adipocytes, but Its Primary Effects on GLUT4 Gene Expression and Lipolysis Are STAT5 Independent

STAT5A (signal transducer and activator of transcription 5A) is a transcription factor that plays a role in adipocyte development and function. Although the role of STAT5A in fat cell development is well characterized, its specific roles in mature fat cells are still largely unknown. Moreover, the potential role of STAT5A in metabolic diseases, in particular type 2 diabetes, remains unclear. To further understand the function of STAT5A in adipocytes, we employed a non-biased co-immunoprecipitation and mass-spectrometry-based approach to identify novel STAT5A-interacting proteins. One of the proteins we identified was DBC1 (deleted in breast cancer 1; also known as CCAR2). DBC1 is typically localized in the nucleus and has been primarily studied in tumor cells. However, the functions of DBC1 in adipocytes are relatively unknown. Using mouse fat cells, we confirmed that there is a physical association between endogenous STAT5A and DBC1 proteins under physiological conditions in the nucleus that is not dependent upon STAT5 tyrosine phosphorylation. Knockdown of DBC1 in 3T3-L1 adipocytes using siRNA did not affect the expression of several STAT5A target genes including socs3, cish, bcl-6, socs2, and igf-1. However, we did observe increased levels glycerol and free fatty acids released from adipocytes with reduced DBC1 following TNF (tumor necrosis factor) α stimulation. Although loss of DBC1 did not alter STAT5 transcriptional activity for the genes examined, it appears to modulate TNFα-mediated lipolysis in a STAT5-independent manner. In addition, DBC1 knockdown increased GLUT4 expression in murine fat cells but did not have a profound effect on TNFα-mediated changes in gene expression. On-going studies are being performed to elucidate the function of the DBC1/STAT5A interaction and to clarify the impact of DBC1 on lipolysis in adipocytes. Disclosure A. Able: None. A.J. Richard: None. J.M. Stephens: None.