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Hirotaka Kanzaki,1 Avradip Chatterjee,1 Hanieh Hossein Nejad Ariani,1 Xinfeng Zhang,2 Stacey Chung,2 Nan Deng,3,4 V Krishnan Ramanujan,4 Xiaojiang Cui,1,2,4 Mark I Greene,5 Ramachandran Murali1 1Department of Biomedical Sciences, Research Division of Immunology; 2Department of Surgery; 3Biostatistics and Bioinformatics Research Center; 4Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA; 5Department of Pathology and Laboratory of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USACorrespondence: Ramachandran Murali Email ramachandran.murali@csmc.eduIntroduction: Constitutive activation of NF-κB has been implicated as being contributive to cancer cell growth, drug resistance, and tumor recurrence in many cancers including breast cancer. Activation of NF-κB leads to nuclear translocation of RelA, a critical component of the NF-κB transcription factor complex, which subsequently binds to specific DNA sites and activates a multitude of genes involved in diverse cell functions. Studies show that triple-negative breast cancer (TNBC) cells possess constitutively active NF-κB and concomitantly have higher levels of nuclear localization of RelA than cytoplasmic RelA. This feature is considered to be associated with the response to chemotherapy. However, currently, there is no specific inhibitor to block nuclear translocation of RelA.Methods: A structure-based approach was used to develop a small-molecule inhibitor of RelA nuclear translocation. The interaction between this molecule and RelA was verified biophysically through isothermal titration calorimetry and microscale thermophoresis. TNBC cell lines MDA-MB-231 and MDA-MB-468 and a human TNBC xenograft model were used to verify in vitro and in vivo efficacy of the small molecule, respectively.Results: We found that the small molecule, CRL1101, bound specifically to RelA as indicated by the biophysical assays. Further, CRL1101 blocked RelA nuclear translocation in breast cancer cells in vitro, and markedly reduced breast tumor growth in a triple-negative breast cancer xenograft model.Conclusion: Our study demonstrates that CRL1101 may lead to new NF-κB-targeted therapeutics for TNBC. Further, blocking of nuclear translocation of shuttling transcription factors may be a useful general strategy in cancer drug development.Keywords: transcription factors, breast cancer, computer aided drug design, nuclear transport, drug-target

Background: APOE4 has been hypothesized to increase Alzheimer’s disease risk by increasing neuroinflammation, though the specific neuroinflammatory pathways involved are unclear. Objective: Characterize cerebrospinal fluid (CSF) proteomic changes related to APOE4 copy number. Methods: We analyzed targeted proteomic data from ADNI CSF samples using a linear regression model adjusting for age, sex, and APOE4 copy number, and additional linear models also adjusting for AD clinical status or for CSF Aβ, tau, or p-tau levels. False discovery rate was used to correct for multiple comparisons correction. Results: Increasing APOE4 copy number was associated with a significant decrease in a CRP peptide level across all five models (q

Background: Postoperative cognitive dysfunction (POCD), a syndrome of cognitive deficits occurring 1–12 months after surgery primarily in older patients, is associated with poor postoperative outcomes. POCD is hypothesized to result from neuroinflammation; however, the pathways involved remain unclear. Unbiased proteomic analyses have been used to identify neuroinflammatory pathways in multiple neurologic diseases and syndromes but have not yet been applied to POCD. Objective: To utilize unbiased mass spectrometry-based proteomics to identify potential neuroinflammatory pathways underlying POCD. Methods: Unbiased LC-MS/MS proteomics was performed on immunodepleted cerebrospinal fluid (CSF) samples obtained before, 24 hours after, and 6 weeks after major non-cardiac surgery in older adults who did (n = 8) or did not develop POCD (n = 6). Linear mixed models were used to select peptides and proteins with intensity differences for pathway analysis. Results: Mass spectrometry quantified 8,258 peptides from 1,222 proteins in > 50%of patient samples at all three time points. Twelve peptides from 11 proteins showed differences in expression over time between patients with versus without POCD (q

Objective:To determine the relationship between affective measures and cognition before and after non-cardiac surgery in older adults.Methods:Observational prospective cohort study in 103 surgical patients age ≥ 60 years old. All participants underwent cognitive testing, Center for Epidemiologic Studies-Depression, and State Anxiety Inventory screening before and 6 weeks after surgery. Cognitive test scores were combined by factor analysis into 4 cognitive domains, whose mean was defined as the continuous cognitive index (CCI). Postoperative global cognitive change was defined by CCI change from before to after surgery, with negative CCI change indicating worsened postoperative global cognition and vice versa.Results:Lower global cognition before surgery was associated with greater baseline depression severity (Spearman’s r = −0.30, p = 0.002) and baseline anxiety severity (Spearman’s r = −0.25, p = 0.010), and these associations were similar following surgery (r = −0.36, p < 0.001; r = −0.26, p = 0.008, respectively). Neither baseline depression or anxiety severity, nor postoperative changes in depression or anxiety severity, were associated with pre- to postoperative global cognitive change.Conclusions:Greater depression and anxiety severity were each associated with poorer cognitive performance both before and after surgery in older adults. Yet, neither baseline depression or anxiety symptoms, nor postoperative change in these symptoms, were associated with postoperative cognitive change.

BackgroundAPOE4 has been hypothesized to increase Alzheimer’s disease risk by increasing neuroinflammation, though the specific neuroinflammatory pathways involved are unclear.ObjectivesTo characterize CSF proteomic changes as a function of APOE4 copy number.MethodsWe analyzed targeted proteomic data obtained on ADNI CSF samples using a linear regression model adjusting for age, sex, and APOE4 copy number, and a second linear model also adjusting for AD clinical status. False Discovery Rate (FDR) was used to correct for multiple comparisons.ResultsIn the first model, increasing APOE4 copy number was associated with significant expression decreases in a CRP peptide (q=0.006), and significant expression increases in peptides from ALDOA, CH3L1 (YKL-40), and FABPH (qAPOE4 copy number was associated with significant expression decreases in a CRP peptide (q=0.009). In both models, increased APOE4 copy number was associated with trends towards lower expression of all 24 peptides from all 8 different complement proteins measured here, although none of these differences were statistically significant. The odds of this happening by chance for 24 unrelated peptides would be less than 1 in 16 million.ConclusionsIncreasing APOE4 copy number was associated with decreased CSF CRP levels and increased CSF ALDOA, CH3L1 and FABH levels; the CRP decrease remained significant after controlling for AD clinical status. Increased APOE4 copy number may also be associated with decreased CSF complement pathway protein levels, a hypothesis for investigation in future studies.

BACKGROUND: The global obesity epidemic has sobering consequences to human health. Especially concerning is obesity-associated hepato-steatosis (HS), a common cause of chronic liver disease in the Americas and Western Europe that precedes non-alcoholic steatohepatitis (NASH). Maintenance of normal body weight is the only current means to prevent HS and NASH. We hypothesized that excess liver fat in obesity-associated HS could act as a pathophysiologic chemical depot for fat-soluble vitamins and alter normal physiology. Because clinical trials with Vitamin E (α-T) have shown that NASH partially responds to this supplement, we selected α-T as a model vitamin to test the sequestration hypothesis. INTERVENTIONS: Under an IND and IRB-approved protocol, two deuterium-labeled α-tocopherols (d3-α-T and d6-α-T) were administered orally and intravenously, respectively, to 10 healthy women and 6 women with HS. Serial blood samples obtained over 72 h were analyzed by LC-MS/MS. In parallel, we performed studies in hepatocytes in cell culture and mouse model. RESULTS: In healthy women who received oral d3- and intravenous d6-α-T, 85% of the initial plasma peak d6-α-T disappeared within 20 minute and reappeared in the plasma peaking between 6-8 h. Compared to healthy subjects, subjects with HS had similar d6-α-T entry rates into liver, but reduced release rates into plasma (p CONCLUSION: These findings suggest the unique role of the liver in vitamin E physiology which is dysregulated by excess liver fat (measured by magnetic resonance spectroscopy). Considered together, the findings imply that obesity-associated HS may produce unrecognized hepatic α-T sequestration, which might subsequently drive liver disease. The data here raise the intriguing possibility that timely α-T supplementation might attenuate progression of HS to NASH, perhaps by correcting an unrecognized fat-induced, localized, hepatic vitamin E deficiency prior to onset of inflammation, hepatitis, and fibrosis. Additionally, our findings raise the possibility that HS may similarly alter hepatic physiology of other fat-soluble vitamins.

// Liting Jin 1, 2, * , Ying Qu 2, * , Liliana J. Gomez 2 , Stacey Chung 2 , Bingchen Han 2 , Bowen Gao 2 , Yong Yue 3 , Yiping Gong 1 , Xuefeng Liu 4 , Farin Amersi 2 , Catherine Dang 2 , Armando E. Giuliano 2 and Xiaojiang Cui 2 1 Department of Breast Surgery, Hubei Cancer Hospital, Wuhan, 430079, China 2 Department of Surgery, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA 3 Department of Radiation, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA 4 Department of Pathology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA * These authors contributed equally to the work Correspondence to: Xiaojiang Cui, email: Xiaojiang.Cui@cshs.org Keywords: conditional reprogramming; estrogen receptor; mammary epithelial cells; luminal cells; myoepithelial cells Received: April 07, 2017 Accepted: October 30, 2017 Published: December 22, 2017 ABSTRACT Purpose: Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. Results: We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. Materials and Methods: We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. Conclusions: The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro . We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

The Forkhead box C1 (FOXC1) transcription factor is involved in normal embryonic development and regulates the development and function of many organs. Most recently, a large body of literature has shown that FOXC1 plays a critical role in tumor development and metastasis. Clinical studies have demonstrated that elevated FOXC1 expression is associated with poor prognosis in many cancer subtypes, such as basal-like breast cancer (BLBC). FOXC1 is highly and specifically expressed in BLBC as opposed to other breast cancer subtypes. Its functions in breast cancer have been extensively explored. This review will summarize current knowledge on the function and regulation of FOXC1 in tumor development and progression with a focus on BLBC, as well as the implications of these new findings in cancer diagnosis and treatment.

BACKGROUND: We hypothesized that obesity-associated hepatosteatosis is a pathophysiological chemical depot for fat-soluble vitamins and altered normal physiology. Using α-tocopherol (vitamin E) as a model vitamin, pharmacokinetics and kinetics principles were used to determine whether excess liver fat sequestered α-tocopherol in women with obesity-associated hepatosteatosis versus healthy controls. METHODS: Custom-synthesized deuterated α-tocopherols (d(3)- and d(6)-α-tocopherols) were administered to hospitalized healthy women and women with hepatosteatosis under investigational new drug guidelines. Fluorescently labeled α-tocopherol was custom-synthesized for cell studies. RESULTS: In healthy subjects, 85% of intravenous d(6)-α-tocopherol disappeared from the circulation within 20 minutes but reappeared within minutes and peaked at 3–4 hours; d(3)- and d(6)-α-tocopherols localized to lipoproteins. Lipoprotein redistribution occurred only in vivo within 1 hour, indicating a key role of the liver in uptake and re-release. Compared with healthy subjects who received 2 mg, subjects with hepatosteatosis had similar d(6)-α-tocopherol entry rates into liver but reduced initial release rates (P < 0.001). Similarly, pharmacokinetics parameters were reduced in hepatosteatosis subjects, indicating reduced hepatic d(6)-α-tocopherol output. Reductions in kinetics and pharmacokinetics parameters in hepatosteatosis subjects who received 2 mg were echoed by similar reductions in healthy subjects when comparing 5- and 2-mg doses. In vitro, fluorescent-labeled α-tocopherol localized to lipid in fat-loaded hepatocytes, indicating sequestration. CONCLUSIONS: The unique role of the liver in vitamin E physiology is dysregulated by excess liver fat. Obesity-associated hepatosteatosis may produce unrecognized hepatic vitamin E sequestration, which might subsequently drive liver disease. Our findings raise the possibility that hepatosteatosis may similarly alter hepatic physiology of other fat-soluble vitamins. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00862433. FUNDING: National Institute of Diabetes and Digestive and Kidney Diseases and NIH grants DK053213-13, DK067494, and DK081761.

Ras genes potently drive human cancers, with mutated proto-oncogene GTPase KRAS4B (K-Ras4B) being the most abundant isoform. Targeted inhibition of oncogenic gene products is considered the "holy grail" of present-day cancer therapy, and recent discoveries of small-molecule KRas4B inhibitors were made thanks to a deeper understanding of the structure and dynamics of this GTPase. Because interactions with biological membranes are key for Ras function, Ras-lipid interactions have become a major focus, especially because such interactions evidently involve both the Ras C terminus for lipid anchoring and its G-protein domain. Here, using NMR spectroscopy and molecular dynamics simulations complemented by biophysical- and cell-biology assays, we investigated the interaction between K-Ras4B with the signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2). We discovered that the β2 and β3 strands as well as helices 4 and 5 of the GTPase G-domain bind to PIP2 and identified the specific residues in these structural elements employed in these interactions, likely occurring in two K-Ras4B orientation states relative to the membrane. Importantly, we found that some of these residues known to be oncogenic when mutated (D47K, D92N, K104M, and D126N) are critical for K-Ras-mediated transformation of fibroblast cells, but do not substantially affect basal and assisted nucleotide hydrolysis and exchange. Moreover, the K104M substitution abolished localization of K-Ras to the plasma membrane. The findings suggest that specific G-domain residues can critically regulate Ras function by mediating interactions with membrane-associated PIP2 lipids; these insights that may inform the future design of therapeutic reagents targeting Ras activity.

T-cell lymphoma invasion and metastasis-inducing factor (Tiam)1 is an established proto-oncogene that drives cancer cell migration and metastasis. The correlation between Tiam1 integrity and patient survival, epithelial-mesenchymal transition, and angiogenesis, render it as an important prognostic factor in multiple human malignancies. Tiam1 is a guanine nucleotide exchange factor (GEF) that facilitates the activation of the small GTPase Rac1, thereby controlling cytoskeletal organization, cell polarity, motility, and invasion. In light of the clinical relevance of Tiam1 signaling, we investigated the utility of small molecule inhibitors that target Tiam1 as a therapeutic intervention strategy. We used an in silico computational approach to screen 10 million compounds for candidate drugs that bind to a select patch on Tiam1's surface. We then identified 13 compounds that inhibit the migration of Tiam1-expressing A549 cells without affecting overall viability. Our studies open the door for a new targeted therapy approach in Tiam1-relevant cancers. Citation Format: Claire Fritz, Michaela Stamper, Stephen Valentino, Lynn Ulatowski, Meghana Gupta, Varsha Thakur, Stacey Chung, Nick DeHann, Christian Laggner, Danny Manor. Potential small-molecule inhibitors of the Tiam1 oncogene product [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2271.

α-Tocopherol (vitamin E) is an essential nutrient for all vertebrates. From the eight naturally occurring members of the vitamin E family, α-tocopherol is the most biologically active species and is selectively retained in tissues. The hepatic α-tocopherol transfer protein (TTP) preferentially selects dietary α-tocopherol and facilitates its transport through the hepatocyte and its secretion to the circulation. In doing so, TTP regulates body-wide levels of α-tocopherol. The mechanisms by which TTP facilitates α-tocopherol trafficking in hepatocytes are poorly understood. We found that the intracellular localization of TTP in hepatocytes is dynamic and responds to the presence of α-tocopherol. In the absence of the vitamin, TTP is localized to perinuclear vesicles that harbor CD71, transferrin, and Rab8, markers of the recycling endosomes. Upon treatment with α-tocopherol, TTP- and α-tocopherol-containing vesicles translocate to the plasma membrane, prior to secretion of the vitamin to the exterior of the cells. The change in TTP localization is specific to α-tocopherol and is time- and dose-dependent. The aberrant intracellular localization patterns of lipid binding-defective TTP mutants highlight the importance of protein-lipid interaction in the transport of α-tocopherol. These findings provide the basis for a proposed mechanistic model that describes TTP-facilitated trafficking of α-tocopherol through hepatocytes.

OBJECTIVES: Hepato-steatosis (HS) due to obesity is now the most common cause of chronic liver disease in the Americas and Western Europe. The only means to prevent disease is avoidance of obesity. α-Tocopherol at doses of 800 I.U. daily was reported to have partial treatment effects for NASH. Because alpha tocopherol is a fat-soluble vitamin, we hypothesized that excess fat in liver, as found in HS, could act unintentionally sequester vitamin E, thereby altering its normal physiology and contributing to development of NASH. Using oral and intravenous deuterated tocopherols, evidence showing HS altered a-tocopherol physiology was reported based on pharmacokinetics studies in obese women with HS. Here we further tested the sequestration hypothesis in vitro, and in vivo. METHODS: In vitro, we investigated effects of fat on intracellular vitamin E localization. Control human and mouse hepatocytes and hepatocytes pre-loaded with fat were incubated with fluorescent α-tocopherol (BDP-α-tocopherol). In vivo experiments were performed using mice fed a high fat diet with different vitamin E doses. RESULTS: Compared to controls, fat- loaded cells contained more a-tocopherol, and BDP-a-tocopherol was specifically localized into intracellular fat droplets. In cells incubated with BDP a-tocopherol, we found that fat loading decreased a-tocopherol release. Induced expression of TPP, which mediates vitamin E intracellular disposition under normal conditions, was not observed in fat loaded cells, further confirming vitamin E was trapped in fat. Livers of mice fed high fat diet had more vitamin E compared to controls. By further increasing vitamin E content of the high fat diet, we observed a reduction in liver size and liver fat in the high vitamin E group. Using a mouse metabolic chamber, we observed a slight reduction of oxygen consumption rate in the high vitamin E group compared to controls. CONCLUSIONS: Considered together, these findings imply that fat in the liver may produce unrecognized hepatic vitamin E sequestration, which could drive liver disease. These results are consistent with the possibility that increased vitamin E intake might, if begun at an early stage, restore vitamin E physiology, potentially decreasing or preventing progression of HS to NASH. FUNDING SOURCES: NIH intramural program (DK053213–14).

Ras genes are potent drivers of human cancers, with mutated K-Ras4B being the most abundant isoform. Targeted inhibition of oncogenic gene products is considered the holy grail of present-day cancer therapy, and recent discoveries of small molecule inhibitors for K-Ras4B greatly benefited from a deeper understanding of the protein structure and dynamics of the GTPase. Since interactions with biological membranes are key for Ras function, the details of Ras - lipid interactions have become a major focus of study, especially since it is becoming clear that such interactions not only involve the Ras C-terminus for lipid anchoring, but also the G-protein domain. Here we investigated the interaction between K-Ras4B with the signaling lipid phosphatidyl inositol (4,5) phosphate (PIP2) using NMR spectroscopy and molecular dynamics simulations, complemented by biophysical and cell biology assays. We discovered that the β2 and β3 strands as well as helices 4 and 5 of the GTPase G-domain bind to PIP2, and that these secondary structural elements employ specific residues for these interactions. These likely occur in two orientation states of the protein relative to the membrane. Importantly, we found that some of these residues, which are known to be oncogenic when mutated (D47K, D92N, K104M and D126N), are critical for K-Ras-mediated transformation of fibroblast cells, while not substantially affecting basal and assisted nucleotide hydrolysis and exchange. We further showed that mutation K104M can indeed abolish localization of mutant K-Ras to the plasma membrane. These findings suggest that specific G-domain residues play an important, previously-unknown role in regulating Ras function by mediating interactions with membrane PIP2 lipids. Thus, a detailed description of the novel K-Ras-PIP2 binding surfaces is likely to inform the future design of therapeutic reagents.

Triple-negative breast cancer (TNBC) has high rates of local recurrence and distant metastasis, partially due to its high invasiveness. The Forkhead box C1 (FOXC1) transcription factor has been shown to be specifically overexpressed in TNBC and associated with poor clinical outcome. How TNBC's high invasiveness is driven by FOXC1 and its downstream targets remains poorly understood. In the present study, pathway-specific PCR array assays revealed that WNT5A and matrix metalloproteinase-7 (MMP7) were upregulated by FOXC1 in TNBC cells. Interestingly, WNT5A mediates the upregulation of MMP7 by FOXC1 and the WNT5A-MMP7 axis is essential for FOXC1-induced invasiveness of TNBC cells in vitro. Xenograft models showed that the lung extravasation and metastasis of FOXC1-overexpressing TNBC cells were attenuated by knocking out WNT5A, but could be restored by MMP7 overexpression. Mechanistically, FOXC1 can bind directly to the WNT5A promoter region to activate its expression. Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP), coupled with mass spectrometry, identified FOXC1-interacting proteins including a group of heterogeneous nuclear ribonucleoproteins involved in WNT5A transcription induction. Finally, we found that WNT5A activates NF-κB signaling to induce MMP7 expression. Collectively, these data demonstrate a FOXC1-elicited non-canonical WNT5A signaling mechanism comprising NF-κB and MMP7 that is essential for TNBC cell invasiveness, thereby providing implications toward developing an effective therapy for TNBC.

Ewing Sarcoma (ES) is an aggressive malignancy that arises in children and young adults. Although the survival for patients with localized tumors is relatively high, the metastatic form carries a dismal prognosis, particularly when bone metastases are present. The frequency of metastases and osseous dissemination increases in patients with necrotic ES tumors. In line with this, hypoxia, the major cause of tumor necrosis, has been shown to increase metastatic potential of ES cells. Previous data from our laboratory demonstrated that in an ES orthotopic xenograft model, primary tumor hypoxia specifically promotes bone metastasis, which is associated with accumulation of cells with enlarged nuclei and frequent chromosome gains in bone invasion areas. We have also shown that the progeny of hypoxia-induced polyploid ES cells (ES≈4n cells) preferentially metastasized to bone. Thus, the goal of our study was to determine the mechanisms enabling osseous dissemination of ES≈4n cells. To this end, we used FUCCI Cell Cycle Sensor followed by DNA staining with Hoechst 33342 and cell sorting to isolate tetraploid cells (4n) from hypoxia-exposed SK-ES1 ES cells. Subsequently, we compared the metastatic properties of their progeny (H-SK-ES1≈4n) with a diploid cell population selected from normoxic SK-ES1 cells (N-SK-ES1-2n cells). We have found that H-SK-ES1≈4n cells have increased motility and invasiveness, as compared to the N-SK-ES1-2n cells. H-SK-ES1≈4n cells had also an increased ability to grow in hypoxia in 2D culture and in soft agar. In line with this, H-SK-ES1≈4n cells were highly sensitive to a glycolysis inhibitor, 2D-glucose, but not an inhibitor of oxidative phosphorylation, metformin. Moreover, H-SK-ES1≈4n cells were more sensitive than controls to a growth-inhibitory effect of AICAR, a blocker of anabolic metabolism. In contrary, the tetraploid cell progeny were highly resistant to doxorubicin, particularly under hypoxic conditions, which in contrast sensitized N-SK-ES1-2n and wild type SK-ES1 cells to chemotherapy. Altogether, our data implicate the following mechanisms underlying osseous dissemination of the hypoxia-induced poplyploid cell progeny: 1) increased motility and invasiveness facilitating escape from the primary tumor; 2) ability to survive under low oxygen tension characteristic for bone tissue. Moreover, we have shown that the cells initiating bone dissemination are highly resistant to conventional chemotherapy, while strategies targeting their metabolic dependencies may be more successful in treatment of patients with bone metastases. Citation Format: Shiya Zhu, Akanksha Mahajan, Sung-Hyeok Hong, Susana Galli, Congyi Lu, You-Shin Chen, Sara Misiukiewicz, Stacey Chung, Jason Tilan, Joanna B. Kitlinska. Hypoxia-induced phenotypic and metabolic changes in Ewing sarcoma cells trigger bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3664.

Human adenoviruses (Ads) generally cause mild self-limiting infections but can lead to serious disease and even be fatal in high-risk individuals, underscoring the importance of understanding how the virus counteracts host defense mechanisms. This study had two goals. First, we wished to determine the molecular basis of cholesterol homeostatic responses induced by the early region 3 membrane protein RIDα via its direct interaction with the sterol-binding protein ORP1L, a member of the evolutionarily conserved family of oxysterol-binding protein (OSBP)-related proteins (ORPs). Second, we wished to determine how this interaction regulates innate immunity to adenovirus. ORP1L is known to form highly dynamic contacts with endoplasmic reticulum-resident VAP proteins that regulate late endosome function under regulation of Rab7-GTP. Our studies have demonstrated that ORP1L-VAP complexes also support transport of LDL-derived cholesterol from endosomes to the endoplasmic reticulum, where it was converted to cholesteryl esters stored in lipid droplets when ORP1L was bound to RIDα. The virally induced mechanism counteracted defects in the predominant cholesterol transport pathway regulated by the late endosomal membrane protein Niemann-Pick disease type C protein 1 (NPC1) arising during early stages of viral infection. However, unlike NPC1, RIDα did not reconstitute transport to endoplasmic reticulum pools that regulate SREBP transcription factors. RIDα-induced lipid trafficking also attenuated proinflammatory signaling by Toll-like receptor 4, which has a central role in Ad pathogenesis and is known to be tightly regulated by cholesterol-rich “lipid rafts.” Collectively, these data show that RIDα utilizes ORP1L in a way that is distinct from its normal function in uninfected cells to fine-tune lipid raft cholesterol that regulates innate immunity to adenovirus in endosomes. IMPORTANCE Early region 3 proteins encoded by human adenoviruses that attenuate immune-mediated pathology have been a particularly rich source of information regarding intracellular protein trafficking. Our studies with the early region 3-encoded RIDα protein also provided fundamental new information regarding mechanisms of nonvesicular lipid transport and the flow of molecular information at membrane contacts between different organelles. We describe a new pathway that delivers cholesterol from endosomes to the endoplasmic reticulum, where it is esterified and stored in lipid droplets. Although lipid droplets are attracting renewed interest from the standpoint of normal physiology and human diseases, including those resulting from viral infections, experimental model systems for evaluating how and why they accumulate are still limited. Our studies also revealed an intriguing relationship between lipid droplets and innate immunity that may represent a new paradigm for viruses utilizing these organelles.

Human mammary gland development and differentiation are tightly regulated by hormones, growth factors, and microenvironmental cues. Rodent models have been used to help gain knowledge about mammary gland biology, but there are significant structural and hormonal response differences between the human and rodent mammary glands. Moreover, cultured immortalized human mammary cell lines have been widely used for in vitro experiments to study epithelial cell biology, but it has been questioned whether they faithfully recapitulate normal breast cells. Conditional reprogramming has recently emerged as an efficient method to induce rapid and inexhaustible in vitro proliferation of primary epithelial cells from normal and malignant tissues in two-dimensional (2D) culture conditions. However, studies using this method have not shown whether conditionally reprogrammed mammary epithelial cells can form defined structures in three-dimensional (3D) culture conditions. Therefore, our goal is to develop an appropriate in vitro model using conditional reprogramming to study human mammary cell and tissue function under 2D and 3D culture conditions. We cultured primary human mammary cells from normal prophylactic tissues or breast tumors using this method. Cell type heterogeneity, cellular marker expression, and structural arrangement were examined using immunofluorescence staining. We found that normal breast cells grown under this culture condition exhibited morphologic features of luminal cells (CK18, desmoglein 3, and CK19) and myoepithelial cells (vimentin, p63, and CK14), indicating maintenance of in vivo heterogeneity. CD49f and EpCAM double staining is commonly used to separate luminal, basal, and progenitor populations. Immunofluorescence and FACS analysis further revealed subpopulations with varying CD49f and EpCAM expression profiles in the normal primary cultures, as well as detectable expression of ERα in earlier passages. Treatment with estradiol also stimulated cellular proliferation as detected by positive EdU staining. When grown in Matrigel/Collagen I gel, normal primary cells self-organize into two distinct 3D structures that are composed of densely packed cells or a spherical structure containing a lumen, which express either luminal or myoepithelial cell markers, respectively. CK8-positive luminal cells that form the lumen can differentiate into milk-producing cells in the presence of a prolactogenic growth condition. Tumor cells extracted from breast cancer patients showed expression for either basal (CK18 and FOXC1) or luminal (CK14 and ER-positive) markers in 2D cultures. Our ongoing work entails delineating the long-term culture effect on primary mammary cell fate and function and the tumorigenic property of primary breast tumor cells. The current findings uncover an in vitro model that may be a valuable tool to study mammary cell function and can potentially be used to elucidate mechanisms involved in mammary tumorigenesis. Citation Format: Stacey Chung, Liting Jin, Ying Qu, Liliana J. Gomez, Bingchen Han, Bowen Gao, Xuefeng Liu, Farin Amersi, Catherine Dang, Armando E. Giuliano, Xiaojiang Cui. Assessment of conditional reprogramming to generate 2D and 3D primary human mammary cell culture models [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A59.

Mutations in the PLEKHG4 (puratrophin-1) gene are associated with the heritable neurological disorder autosomal dominant spinocerebellar ataxia. However, the biochemical functions of this gene product have not been described. We report here that expression of Plekhg4 in the murine brain is developmentally regulated, with pronounced expression in the newborn midbrain and brainstem that wanes with age and maximal expression in the cerebellar Purkinje neurons in adulthood. We show that Plekhg4 is subject to ubiquitination and proteasomal degradation, and its steady-state expression levels are regulated by the chaperones Hsc70 and Hsp90 and by the ubiquitin ligase CHIP. On the functional level, we demonstrate that Plekhg4 functions as a bona fide guanine nucleotide exchange factor (GEF) that facilitates activation of the small GTPases Rac1, Cdc42, and RhoA. Overexpression of Plekhg4 in NIH3T3 cells induces rearrangements of the actin cytoskeleton, specifically enhanced formation of lamellopodia and fillopodia. These findings indicate that Plekhg4 is an aggregation-prone member of the Dbl family GEFs and that regulation of GTPase signaling is critical for proper cerebellar function.

Vitamin E, a plant-derived neutral lipid, is an essential nutrient for all vertebrates that scavenges free radicals in biological membranes, thereby preventing oxidative stress. Of the eight natura...

The mammalian embryo relies on maternal circulating retinoids (vitamin A derivatives) for development. β-Carotene is the major human dietary provitamin A. β-Carotene-15,15′-oxygenase (CMOI) has been proposed as the main enzyme generating retinoid from β-carotene in vivo. CMOI is expressed in embryonic tissues, suggesting that β-carotene provides retinoids locally during development. We performed loss of CMOI function studies in mice lacking retinol-binding protein (RBP), an established model of embryonic vitamin A deficiency (VAD). We show that, unexpectedly, lack of CMOI in the developing tissues further exacerbates the severity of VAD and thus the embryonic malformations of RBP−/− mice. Since β-carotene was not present in any of the mouse diets, we unveiled a novel action of CMOI independent from its β-carotene cleavage activity. We also show for the first time that CMOI exerts an additional function on retinoid metabolism by influencing retinyl ester formation via modulation of lecithin:retinol acyltransferase (LRAT) activity, at least in developing tissues. Finally, we demonstrate unequivocally that β-carotene can serve as an alternative vitamin A source for the in situ synthesis of retinoids in developing tissues by the action of CMOI.—Kim, Y.-K., Wassef, L., Chung, S., Jiang, H., Wyss, A., Blaner, W. S., Quadro, L. β-Carotene and its cleavage enzyme β-carotene-15,15′-oxygenase (CMOI) affect retinoid metabolism in developing tissues.