Faculty Bibliography

Estrogen receptors (ER) play important roles in the development and progression of breast and ovarian cancers. ERs mediate transcriptional regulation through interaction with cofactors and binding to response elements within the regulatory elements of target genes. Here, we examined the expression and function of TBLR1/TBL1XR1, a core component of NCoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoic acid and thyroid receptor) corepressor complexes, in breast and ovarian cancers. We found that although TBLR1 is present in both the nucleus and cytoplasm of normal and neoplastic breast and ovarian cells, it is expressed at significantly higher levels in the nucleus of malignant breast and ovarian cells compared to benign cells. TBLR1 functions as an ER corepressor to inhibit ER-mediated transcriptional activation in both breast and ovarian cell lines, but it has no effect on androgen receptor (AR) mediated transcriptional activation in these cells. Furthermore, ectopic expression of nuclear TBLR1 in breast and ovarian cancer cells stimulates cell proliferation. The increased cell proliferation by nuclear TBLR1 is through both ER-independent and ER-dependent mechanisms as evidenced by increased growth in hormone-free medium and estrogen medium, as well as reduced growth with ER knockdown by siRNA. Nuclear TBLR1 overexpression also increased migration and invasion in both breast and ovarian cancer cells. Determining the functional relationship between TBLR1 and ER may provide insights to develop novel treatment strategies and improve response to hormonal therapy in breast and ovarian cancers.

Infantile hemangioma (IH) is the most common tumor of infancy. Its cellular origin and biological signals for uncontrolled growth are poorly understood, and specific pharmacological treatment is unavailable. To understand the process of hemangioma-genesis we characterized the progenitor hemangioma-derived stem cell (HemSC) and its lineage and non-lineage derivatives. For this purpose we performed a high-throughput (HT) phenotypic and gene expression analysis of HemSCs, and analyzed HemSC-derived tumorspheres. We found that IH is characterized by high expression of genes involved in vasculogenesis, angiogenesis, tumorigenesis and associated signaling pathways. These results show that IH derives from a dysregulated stem cell that remains in an immature, arrested stage of development. The potential biomarkers we identified can afford the development of diagnostic tools and precision-medicine therapies to "rewire" or redirect cellular transitions at an early stage, such as signaling pathways or immune response modifiers.

BACKGROUND AND AIMS: Human liver chimeric mice are useful models of human hepatitis virus infection, including hepatitis B (HBV) and C virus infections. Independently, immunodeficient mice reconstituted with CD34+ hematopoietic stem cells (HSC) derived from fetal liver reliably develop human T and B lymphocytes. Combining these systems has long been hampered by inefficient liver reconstitution of human fetal hepatoblasts. Our study aimed to enhance hepatoblast engraftment in order to create a mouse model with syngeneic human liver and immune cells. METHODS: The effects of human oncostatin-M administration on fetal hepatoblast engraftment into immunodeficient fah-/- mice was tested. Mice were then transplanted with syngeneic human hepatoblasts and HSC after which human leukocyte chimerism and functionality were analyzed by flow cytometry, and mice were challenged with HBV. RESULTS: Addition of human oncostatin-M enhanced human hepatoblast engraftment in immunodeficient fah-/- mice by 5-100 fold. In contrast to mice singly engrafted with HSC, which predominantly developed human T and B lymphocytes, mice co-transplanted with syngeneic hepatoblasts also contained physiological levels of human monocytes and natural killer cells. Upon infection with HBV, these mice displayed rapid and sustained viremia. CONCLUSIONS: Our study provides a new mouse model with improved human fetal hepatoblast engraftment and an expanded human immune cell repertoire. With further improvements, this model may become useful for studying human immunity against viral hepatitis. LAY ABSTRACT: Important human pathogens such as hepatitis B virus (HBV), hepatitis C virus (HCV) and HIV only infect human cells which complicates the development of mouse models for the study of these pathogens. One way to make mice permissive for human pathogens is the transplantation of human cells into immune-compromised mice. For instance, the transplantation of human liver cells will allow the infection of these so-called "liver chimeric mice" with HBV and HCV. The co-transplantation of human immune cells into liver chimeric mice will further allow the study of human immune responses to HBV or HCV. However, for immunological studies it will be crucial that the transplanted human liver and immune cells are derived from the same human donor. In our study we describe the efficient engraftment of human fetal liver cells and immune cells derived from the same donor into mice. We show that liver co-engraftment resulted in an expanded human immune cell repertoire, including monocytes and natural killer cells in the liver. We further demonstrate that these mice could be infected with HBV which lead to an expansion of natural killer cells. In conclusion we have developed a new mouse model that could be useful to study human immune responses to human liver pathogens.

PURPOSE: Although the existence of cancer stem cells (CSC) in adenoid cystic carcinoma (ACC) has been proposed, lack of assays for their propagation and uncertainty about molecular markers prevented their characterization. Our objective was to isolate CSC from ACC and provide insight into signaling pathways that support their propagation. EXPERIMENTAL DESIGN: To isolate CSC from ACC and characterize them, we used ROCK inhibitor-supplemented cell culture, immunomagnetic cell sorting, andin vitro/in vivoassays for CSC viability and tumorigenicity. RESULTS: We identified in ACC CD133-positive CSC that expressed NOTCH1 and SOX10, formed spheroids, and initiated tumors in nude mice. CD133(+)ACC cells produced activated NOTCH1 (N1ICD) and generated CD133(-)cells that expressed JAG1 as well as neural differentiation factors NR2F1, NR2F2, and p27Kip1. Knockdowns ofNOTCH1, SOX10, and their common effectorFABP7had negative effects on each other, inhibited spheroidogenesis, and induced cell death pointing at their essential roles in CSC maintenance. Downstream effects ofFABP7knockdown included suppression of a broad spectrum of genes involved in proliferation, ribosome biogenesis, and metabolism. Among proliferation-linked NOTCH1/FABP7 targets, we identified SKP2 and its substrate p27Kip1. A gamma-secretase inhibitor, DAPT, selectively depleted CD133(+)cells, suppressed N1ICD and SKP2, induced p27Kip1, inhibited ACC growthin vivo, and sensitized CD133(+)cells to radiation. CONCLUSIONS: These results establish in the majority of ACC the presence of a previously uncharacterized population of CD133(+)cells with neural stem properties, which are driven by SOX10, NOTCH1, and FABP7. Sensitivity of these cells to Notch inhibition and their dependence on SKP2 offer new opportunities for targeted ACC therapies.Clin Cancer Res; 22(8); 2083-95. (c)2016 AACR.

Endothelin-1 (ET-1) is a vasoactive peptide that also plays a role in the tanning response of the skin. Animal and cell culture studies have also implicated ET-1 in melanoma progression, but no association studies have been performed to link ET-1 expression and melanoma in humans. Here, we present the first in-vivo study of ET-1 expression in pigmented lesions in humans: an ET-1 immunohistochemical screen of melanocytic nevi, melanoma in situ lesions, invasive melanomas, metastatic melanomas, and blue nevi was performed. Twenty-six percent of melanocytic nevi and 44% of melanoma in situ lesions demonstrate ET-1 expression in the perilesional microenvironment, whereas expression in nevus or melanoma cells was rare to absent. In striking contrast, 100% of moderately to highly pigmented invasive melanomas contained numerous ET-1-positive cells in the tumor microenvironment, with 79% containing ET-1-positive melanoma cells, confirmed by co-staining with melanoma tumor marker HMB45. Hypopigmented invasive melanomas had reduced ET-1 expression, suggesting a correlation between ET-1 expression and pigmented melanomas. ET-1-positive perilesional cells were CD68-positive, indicating macrophage origin. Sixty-two percent of highly pigmented metastatic melanomas demonstrated ET-1 expression in melanoma cells, in contrast to 28.2% of hypopigmented specimens. Eighty-nine percent of benign nevi, known as blue nevi, which have a dermal localization, were associated with numerous ET-1-positive macrophages in the perilesional microenvironment, but no ET-1 expression was detected in the melanocytes. We conclude that ET-1 expression in the microenvironment increases with advancing stages of melanocyte transformation, implicating a critical role for ET-1 in melanoma progression, and the importance of the tumor microenvironment in the melanoma phenotype.

BACKGROUND: Long considered to be inert organelles for lipid storage, lipid droplets (LDs) have recently attracted great interest as dynamic structures central to cellular lipid and energy metabolism. hypoxia-inducible gene (HIG2) and perilipin 2 (PLIN2, adipophilin) are LD-associated proteins known to be upregulated by hypoxia (Bensaad, 2014) and/or following von Hippel-Lindau (VHL) gene inactivation (Togashi, 2005; Yao, 2005). We sought to determine whether overexpression of HIG2 and PLIN2 in response to hypoxia or pseudohypoxia may be involved in these histopathologic features of glioma and hemangioblastoma. METHODS: Tumor specimens from 12 patients with glioma grade II-IV (age 3-59 y) and 23 patients with CNS hemangioblastoma (age 15-63 y) were analyzed by immunohistochemistry (IHC) to delineate their expression of HIG2 and PLIN2. To evaluate the role of hypoxia, glioblastoma (GBM) tissues (n = 2) were double-label immunostained for HIF-1alpha and PLIN2 (Zagzag, 2008). Additionally, cultures of tumor spheres isolated from GBM patients (n = 2) (Bayin, 2014) were exposed to hypoxic (1% O2) conditions for 24-72 h, and the cell proteins analyzed by Western blots. RESULTS: HIG2 and PLIN2 were consistently expressed on LDs in hypoxic glioma tumor cells, including pseudopalisading cells in GBMs, but not in adjacent hyperplastic vessels, inflammatory cells or normal brain tissue, independently of tumor grade or the presence of IDH1 (n = 3) and/or TP53 (n = 7) mutations. Likewise, LDs in stromal tumor cells in hemangioblastoma were intensely immunopositive for HIG2 and PLIN2. Double-label IHC showed tight co-expression of HIF-1a and PLIN2 in glioma tumor cells, consistent with the hypoxic regulation of PLIN2. Similarly, expression of HIF-1a and HIG2 proteins was upregulated in GBM tumor spheres under hypoxic conditions. CONCLUSIONS: Our results suggest that HIG2 and PLIN2 are involved in the hypoxic adaptation of lipid metabolism during tumorigenesis, and may serve as specific biomarkers of glioma tumor cells and stromal cells in CNS hemangioblastoma

At least 170 million people are chronically infected with hepatitis C virus (HCV). Owing to the narrow host range of HCV and restricted use of chimpanzees, there is currently no suitable animal model for HCV pathogenesis studies or the development of a HCV vaccine. To identify cellular determinants of interspecies transmission and establish a novel immunocompetent model system, we examined the ability of HCV to infect hepatocytes from a small nonhuman primate, the rhesus macaque (Macaca mulatta). We show that the rhesus orthologs of critical HCV entry factors support viral glycoprotein-dependent virion uptake. Primary hepatocytes from rhesus macaques are also permissive for HCV-RNA replication and particle production, which is enhanced when antiviral signaling is suppressed. We demonstrate that this may be owing to the diminished capacity of HCV to antagonize mitochondrial antiviral-signaling protein-dependent innate cellular defenses. To test the ability of HCV to establish persistent replication in vivo, we engrafted primary rhesus macaque hepatocytes into immunocompromised xenorecipients. Inoculation of resulting simian liver chimeric mice with either HCV genotype 1a or 2a resulted in HCV serum viremia for up to 10 weeks. CONCLUSION: Together, these data indicate that rhesus macaques may be a viable model for HCV and implicate host immunity as a potential species-specific barrier to HCV infection. We conclude that suppression of host immunity or further viral adaptation may allow robust HCV infection in rhesus macaques and creation of a new animal model for studies of HCV pathogenesis, lentivirus coinfection, and vaccine development. (Hepatology 2015;62:57-67).

The TLR7/8 agonist, Resiquimod has been used as an immune adjuvant in cancer vaccines. We evaluated the safety and immunogenicity of the cancer testis antigen NY-ESO-1 given in combination with Montanide with or without Resiquimod in high risk melanoma patients. In Part I of the study, patients received 100ug full length NY-ESO-1 protein emulsified in 1.25mL Montanide (day 1) followed by topical application of 1000mg of 0.2% Resiquimod gel on days 1 and 3 (Cohort 1) versus days 1, 3, and 5 (Cohort 2) of a 21 day cycle. In Part II, patients were randomized to receive 100ug NY-ESO-1 protein plus Montanide (day 1) followed by topical application of placebo gel (Arm-A; N=8) or 1000mg of 0.2% Resiquimod gel (Arm-B; N=12) using the dosing regimen established in Part I. The vaccine regimens were generally well-tolerated. NY-ESO-1 specific humoral responses were induced or boosted in all patients, many with high titers. In Part II, 16 of 20 patients in both arms had NY-ESO-1-specific CD4+ T cell responses. CD8+ T cell responses were only seen in 3 of 12 patients in Arm B. Patients with TLR7 SNP rs179008 had a greater likelihood of developing NY-ESO-1-specific CD8+ responses. In conclusion, NY-ESO-1 protein in combination with Montanide with or without topical Resiquimod is safe and induces both antibody and CD4+ cell responses in the majority of patients; the small proportion of CD8+ cell responses suggests that the addition of topical Resiquimod to Montanide is not sufficient to induce consistent NY-ESO-1 specific CD8+ cell responses.