Faculty Bibliography

The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER), and the transcription factor X-box binding protein 1 spliced (XBP1S), a regulator of the UPR, is known to be important for ER stress (ERS)-mediated apoptosis and cell growth, but the molecular mechanism underlying these processes remains unexplored. Here, we report that knockdown of XBP1S by an siRNA silencing approach increased the expression of ERS-associated molecules. The overexpression of XBP1S stimulated, whereas its knockdown inhibited, cell proliferation in chondrocytes and chondrosarcoma cells; in addition, overexpression of XBP1S inhibited, while its repression enhanced, ERS-mediated apoptosis in chondrocytes and chondrosarcoma cells. Furthermore, XBP1S-mediated inhibition of apoptosis in response to ERS is through the Erk1/2 signaling pathway and down-regulation CHOP transcription factor. CHOP is one of the key downstream molecules known to be involved in ERS-mediated apoptosis. Collectively, these findings reveal a novel critical role of XBP1S in ERS-mediated apoptosis and the molecular mechanisms involved.

The pathobiology of vitiligo, characterized by the spread of depigmented skin patches due to localized melanocyte loss, is not fully understood. Oxidative stress is thought to play a role in disease onset with a subsequent autoimmune response underlying progression. We therefore sought to identify mechanisms that linked oxidative stress and autoimmune responses. Melanocytes at the periphery of vitiligo lesions have distended endoplasmic reticuli (ER). We hypothesized that oxidative stress disrupted homeostasis of the ER where oxidation/reduction reactions facilitate disulfide bond formation. As a result, misfolded peptides would accumulate, dilating the ER and activating the unfolded protein response (UPR). The UPR is a stress response pathway, initiated by three regulators (IRE1, PERK and ATF6). It first promotes cell survival, however sustained activation induces apoptosis. In order to identify a potential role for the UPR in vitiligo we dosed melanocytes with 4-tertiary butyl phenol (4- TBP) and monobenzyl ether of hydroquinone (MBEH), phenols known to trigger vitiligo. The phenols caused an increase in expression of IRE1 and PERK. PERK activation leads to enhancement of the antioxidant response by recruitment of the transcription factor NRF2 to the nucleus and increased expression of the antioxidant HMOX1. The IRE1 effector, X-box binding protein-1 (XBP1) was also activated by phenol treatment which led to increased production of interleukin-6 (IL6) and IL8, cytokines expressed at increased levels in perilesional skin in vitiligo. Treatment with XBP1 inhibitors reduced phenol-induced IL6 and IL8 production, while over-expression of active XBP1 increased their expression. Thus, chemicals known to cause vitiligo trigger a UPR-mediated increase in cytokine production. There are a number of potential roles for the UPR in vitiligo. The UPR may (i) induce apoptosis following sustained oxidative stress causing release of melanocyte specific antigens, (ii) be dysregulated resulting in a muted PERK-!

The field of label-free biophysical technologies used to quantitatively characterize macromolecular interactions with each other and with small molecules has grown enormously in the last 10 years. The most widely used analytical technologies for characterizing biomolecular interactions are surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), biolayer interferometry (BLI), and analytical ultracentrifugation (AUC). Measuring interaction parameters accurately and quantitatively is challenging, as it requires specialized expertise, training, and instrumentation. The Molecular Interaction Research Group (MIRG) conducted an online survey designed to capture the current profile of label-free technologies, including ITC, SPR, and other biosensors used in academia and the pharmaceutical industry sector. The main goal of the survey was to take a snapshot of laboratory, instrumentation, applications for measuring various biophysical parameters, confidence in data interpretation, data validation and acceptability, and limitations of using various technologies. Through this survey, we anticipate that the participating laboratories will be able to gauge their own capabilities and gain insights into the relative success of the different technologies that they use for characterizing molecular interactions.

Bone metastasis is a common and serious consequence of breast cancer. Bidirectional interaction between tumor cells and the bone marrow microenvironment drives a so-called 'vicious cycle' that promotes tumor cell malignancy and stimulates osteolysis. Targeting these interactions and pathways in the tumor-bone microenvironment has been an encouraging strategy for bone metastasis therapy. In the present study, we examined the effects of plumbagin on breast cancer bone metastasis. Our data indicated that plumbagin inhibited cancer cell migration and invasion, suppressed the expression of osteoclast-activating factors, altered the cancer cell induced RANKL/OPG ratio in osteoblasts, and blocked both cancer cell- and RANKL-stimulated osteoclastogenesis. In mouse model of bone metastasis, we further demonstrated that plumbagin significantly repressed breast cancer cell metastasis and osteolysis, inhibited cancer cell induced-osteoclastogenesis and the secretion of osteoclast-activating factors in vivo. At the molecular level, we found that plumbagin abrogated RANKL-induced NF-kappaB and MAPK pathways by blocking RANK association with TRAF6 in osteoclastogenesis, and by inhibiting the expression of osteoclast-activating factors through the suppression of NF-kappaB activity in breast cancer cells. Taken together, our data demonstrate that plumbagin inhibits breast tumor bone metastasis and osteolysis by modulating the tumor-bone microenvironment and that plumbagin may serve as a novel agent in the treatment of tumor bone metastasis.

Background: A pancreaticoduodenectomy (PD) offers the only chance of a cure for pancreatic cancer and can be performed with low mortality and morbidity. However, little is known about outcomes of a PD in octogenarians. Methods: Differences in two groups of patients (Group Y, <80 and Group O, >/=80 year-old) who underwent a PD for pancreatic adenocarcinoma were analysed. Study end-points were length of post-operative stay, overall morbidity, 30-day mortality and overall survival. Results: There were 175 patients in Group Y (mean age 64 years) and 25 patients in Group O (mean age 83 years). Octogenarians had worse Eastern Cooperative Oncology Group (ECOG) Performance Status (PS >/=1: 90% vs. 51%) and American Society of Anesthesiology (ASA) score (>2: 71% vs. 47%). The two groups were similar in underlying co-morbidities, operative time, rates of portal vein resection, intra-operative complications, blood loss, pathological stage and status of resection margins. Octogenarians had a longer post-operative stay (20 vs. 14 days) and higher overall morbidity (68% vs. 44%). There was a single death in each group. At a median follow-up of 13 months median survival appeared similar in the two groups (17 vs. 13 months). Conclusions: As 30-day mortality and survival are similar to those observed in younger patients, a PD can be offered to carefully selected octogenarians.

INTRODUCTION: Chronic pancreatitis (CP) and pancreatic cancer (PDAC) are characterized by an intense inflammatory infiltration. Regulation of this process is incompletely understood. We found that TLR7 expression is increased in CP and PDAC. We postulated that CP and PDAC depend on TLR7 signaling. METHODS: CP was induced in C57BL/6 mice using 7X daily caerulein injections (50mug/kg) for three weeks. KrasG12D mice which harbor a Kras mutation in pancreatic progenitor cells were used to model PDAC. TLR7 ligand (ssRNA40; 100mug/kg) was administered thrice-weekly. Blockade of TLR7 was accomplished with olignonucleotide inhibitors of TLR7 (IRS661). RESULTS: In CP and PDAC we observed substantially-increased TLR7-expression in both inflammatory and parenchymal cells, and significantly elevated levels of TLR7 ligands (Table). Ligation of TLR7 exacerbated CP in WT mice and accelerated tumor progression in KrasG12D mice (Table). TLR7 ligation altered epithelial cell expression of numerous oncogenic targets in PDAC including loss of PTEN, p16, and Cyclin D1 and up-regulation of p21, p27, p53, c-myc, SHPTP1, and TGF-beta. Further, TLR7 blockade protected against CP and pancreatic carcinogenesis, with marked reduction of inflammatory infiltration and complete arrest of tumor progression. TLR7 activation affected intra-pancreatic inflammation by interfacing with Notch pathway intermediates via STAT3 activation, and by inducing canonical NF-kB and MAP kinase signaling pathways- with corresponding blockade mitigating the pro-inflammatory and neoplastic effects of TLR7 activation. (Table presented) CONCLUSIONS: TLR7 ligation drives pancreatic inflammation and neoplasia via novel signaling mechanisms. Conversely, TLR7 blockade is powerfully protective. Targeting TLR7 holds promise for the treatment of CP and PDAC

INTRODUCTION: Non-alcoholic steatohepatitis (NASH) is the most common cause of chronic liver dysfunction in theUnited States and can lead to cirrhosis, liver failure, and the need for liver transplantation. Dendritic cells (DC) are antigen presenting cells with an emerging role in hepatic inflammation. However, the role of DC in the progression of NASH is unknown. METHODS: NASH was induced in C57BL/6 mice by feeding a methionine-choline deficient diet. NASH progression was assessed by changes in liver inflammation, steatosis, and fibrosis. DC recruitment and activation was assessed by flow cytometry. DC depletion using diphtheria toxin (4ng/g/day) was achieved in C57BL/6 mice made chimeric with CD11c. DTR bone marrow. RESULTS: DC expand in NASH 4-5 fold (Table) and upregulate their expression of MHCII, CD40, ICAM-1, B7-1, and B7-2. DC also accumulate intracellular lipid in NASH (Table) and are primary in the clearance of necrotic hepatocytes, thereby limiting byproducts of sterile inflammation. Conversely, DC depletion in NASH (NASH-DC) markedly exacerbated intrahepatic inflammation, evidenced by increased liver cytokines, markedly increased CD45+ inflammatory infiltrate (Table), and activation of Kupffer cells (KC) and neutrophils with concomitant decrease in regulatory T cells. In NASH-DC, KC, inflammatory monocytes, and neutrophils underwent lower rates of apoptosis and produced increased TNF-alpha, IL-6 and pro IL-1beta. Further, end-organ fibrosis was significantly higher in NASH-DC mice (Table). (Table presented) CONCLUSIONS: DC become activated in NASH and contribute to steatosis by accumulating lipids. However, DC protect against intrahepatic fibro-inflammation by clearance of necrotic debris, thus limiting KC, monocyte, and neutrophil activation. Targeting DC may hold promise for NASH treatment

Regulation of hepatic very low density lipoprotein (VLDL) assembly and maturation is crucial in controlling lipid homeostasis and in the development of metabolic disorders, including obesity, hepatic steatosis, and insulin resistance. Cideb, a member of cell death-inducing DFF45-like effector (CIDE) protein family, has been previously shown to promote VLDL lipidation and maturation. However, the precise subcellular location of Cideb-mediated VLDL lipidation and the factors modulating its activity remain elusive. In addition to its localization to endoplasmic reticulum (ER) and lipid droplets (LD), we observed that Cideb was also localized to the Golgi apparatus. Mature and lipid-rich VLDL particles did not accumulate in the Golgi apparatus in Cideb(-/-) livers. Interestingly, we observed that hepatic perilipin 2/adipose differentiation-related protein (ADRP) levels were markedly increased in Cideb(-/-) mice. Liver-specific knockdown of perilipin 2 in Cideb(-/-) mice resulted in the reduced accumulation of hepatic triglycerides (TAG), increased VLDL-TAG secretion, and the accumulation of mature TAG-rich VLDL in the Golgi apparatus. These data reveal that Cideb and perilipin 2 play opposing roles in controlling VLDL lipidation and hepatic lipid homeostasis.

Allergic asthma is a chronic disease of the lung characterized by underlying Th2- and IgE-mediated inflammation, structural alterations of the bronchial wall, and airway hyperresponsiveness. Initial allergic sensitization and later development of chronic disease are determined by close interactions between lung structural cells and the resident and migratory immune cells in the lung. Epithelial cells play a crucial role in allergic sensitization by directly influencing dendritic cells induction of tolerant or effector T cells and production of type 2 cytokines by innate immune cells. During chronic disease, the bronchial epithelium, stroma, and smooth muscle become structurally and functionally altered, contributing to the perpetuation of tissue remodeling. Thus, targeting tissue-driven pathology in addition to inflammation may increase the effectiveness of asthma treatment.