Benign Epithelial Cells Research Articles

Dissipative particle dynamics simulation of cell entry into a micro-channel

Cell deformability is an important biomarker which can be used to distinguish and sort between healthy and cancer cells. In this paper, we presented a dissipative particle dynamics (DPD) model for investigating cell entry into micro-channels. The cell membrane is represented by a network of DPD particles (beads) connected by worm-like chain (WLC) springs, which is able to mimic the viscoelastic effect of the membrane. The entry process of benign breast epithelial cells (MCF-10A) and non-metastatic tumor breast cells (MCF-7) through a constricted micro-channel are comparatively investigated using this DPD model. It is shown that both the time histories of the cell displacement and the dynamic behaviors of cell entry agree with experimental observations. The entry time of MCF-10A cell is approximately four times of that of MCF-7 cell since MCF-10A cells are stiffer than MCF-7 cells. It is demonstrated that the presented DPD method is effective in modeling cell deformability, and the obtained results can be helpful in understanding how cells with different mechanical properties respond to physical loads.

E‐cadherin is downregulated in benign prostatic hyperplasia and required for tight junction formation and permeability barrier in the prostatic epithelial cell monolayer

We previously reported the presence of prostate-specific antigen (PSA) in the stromal compartment of benign prostatic hyperplasia (BPH). Since PSA is expressed exclusively by prostatic luminal epithelial cells, PSA in the BPH stroma suggests increased tissue permeability and the compromise of epithelial barrier integrity. E-cadherin, an important adherens junction component and tight junction regulator, is known to exhibit downregulation in BPH. These observations suggest that the prostate epithelial barrier is disrupted in BPH and E-cadherin downregulation may increase epithelial barrier permeability. The ultra-structure of cellular junctions in BPH specimens was observed using transmission electron microscopy (TEM) and E-cadherin immunostaining analysis was performed on BPH and normal adjacent specimens from BPH patients. In vitro cell line studies using benign prostatic epithelial cell lines were performed to determine the impact of small interfering RNA knockdown of E-cadherin on transepithelial electrical resistance and diffusion of fluorescein isothiocyanate (FITC)-dextran in transwell assays. The number of kiss points in tight junctions was reduced in BPH epithelial cells as compared with the normal adjacent prostate. Immunostaining confirmed E-cadherin downregulation and revealed a discontinuous E-cadherin staining pattern in BPH specimens. E-cadherin knockdown increased monolayer permeability and disrupted tight junction formation without affecting cell density. Our results indicate that tight junctions are compromised in BPH and loss of E-cadherin is potentially an important underlying mechanism, suggesting targeting E-cadherin loss could be a potential approach to prevent or treat BPH.

UV Light-inactivated HSV-1 Stimulates Natural Killer Cell-induced Killing of Prostate Cancer Cells.

Herein we demonstrate that ultraviolet light-inactivated Herpes Simplex Virus-1 (UV-HSV-1) stimulates peripheral blood mononuclear cells (PBMCs) to lyse both androgen-sensitive and androgen-independent prostate cancer (PrCA) cell lines, but not the benign prostatic hyperplastic epithelial cell line, BPH-1, and is 1000-10,000-fold more potent at stimulating this killing than ultraviolet light-inactivated Vesicular Stomatitis Virus, adenovirus, reovirus or cytomegalovirus. Among PBMCs, natural killer (NK) cells appear to be a major cell type involved in this killing and UV-HSV-1 appears to directly and potently stimulate NK cell expression of CD69, degranulation, cytokine production, and migration to IL-8 in PC3 conditioned medium. We also found that UV-HSV-1 stimulates glycolysis in PBMCs and NK cells, and that 2-deoxyglucose and the protein kinase C inhibitor, Go6976, and the NFκB inhibitor, Bay 11-7082, all abrogate UV-HSV-1 activated killing of PC3 cells by PBMCs and NK cells. Using neutralizing anti-Toll-like receptor 2 (TLR2) we found that UV-HSV-1, like HSV-1, activates NK cells via TLR2. Taken together, these results are consistent with Toll-like receptor 2 ligands on UV-HSV-1 stimulating TLR2 on NK cells to activate protein kinase C, leading to enhanced glycolysis and NFκB activation, both of which play a critical role in this anti-PrCA innate immune response. Importantly, UV-HSV-1 synergizes with IL-15 to increase the cytolytic activity of PBMCs against PC3 cells and there was considerable donor-to-donor variation in killing ability. These results support the preclinical development of UV-HSV-1 as an adjuvant, in combination with IL-15, for cell infusions of healthy, preselected NK cells to treat PrCA.

Downregulation of RalGTPase-activating protein promotes invasion of prostatic epithelial cells and progression from intraepithelial neoplasia to cancer during prostate carcinogenesis.

RalGTPase-activating protein (RalGAP) is an important negative regulator of small GTPases RalA/B that mediates various oncogenic signaling pathways in various cancers. Although the Ral pathway has been implicated in prostate cancer (PCa) development and progression, the significance of RalGAP in PCa has been largely unknown. We examined RalGAPα2 expression using immunohistochemistry on two independent tissue microarray sets. Both datasets demonstrated that the expression of RalGAPα2 was significantly downregulated in PCa tissues compared to adjacent benign prostatic epithelia. Silencing of RalGAPα2 by short hairpin RNA enhanced migration and invasion abilities of benign and malignant prostate epithelial cell lines without affecting cell proliferation. Exogenous expression of wild-type RalGAP, but not the GTPase-activating protein activity-deficient mutant of RalGAP, suppressed migration and invasion of multiple PCa cell lines and was phenocopied by pharmacological inhibition of RalA/B. Loss of Ralgapa2 promoted local microscopic invasion of prostatic intraepithelial neoplasia without affecting tumor growth in a Pten-deficient mouse model for prostate tumorigenesis. Our findings demonstrate the functional significance of RalGAP downregulation to promote invasion ability, which is a property necessary for prostate carcinogenesis. Thus, loss of RalGAP function has a distinct role in promoting progression from prostatic intraepithelial neoplasia to invasive adenocarcinoma.

Expression of tetraspanins NET-6 and CD151 in breast cancer as a potential tumor biomarker.

Tetraspanins have been implicated in multiple biological functions including protein networking and cell signaling. NET-6 (TSPAN 13) has been demonstrated to be a tumor suppressor gene in breast cancer, while CD151 is more likely to act as an oncogene. However, the biological function of both proteins is still inconclusive. Immunohistochemistry was used to analyze the expression of NET-6 and CD151 proteins in breast tumors and benign epithelial cells. The cellular expression of both markers was correlated with HER2, ER, and PR status as well as tumor grade, Ki-67 scores, invasion, and metastasis. Expression of NET-6 and CD151 was variable both in tumors and in benign epithelial cells. Expression of NET-6 and CD151 was stronger in tumors than in benign epithelial cells. The expression of NET-6 was also stronger in HER2-negative, low-grade, lymphovascular invasion-negative, and non-metastatic breast tumors. There was no correlation between NET-6 expression and ER, or PR, or triple-negative status. There was no correlation between CD151 expression and HER2, ER, PR, or triple-negative status, tumor grade, or Ki-67 scores, invasion, and metastasis. The expression of tetraspanins NET-6 and CD151 may indicate an alteration of their biological function during neoplastic transformation. NET-6 expression in tumors might be a potential marker indicating the outcome of breast cancer.

SUN-010 BRCA1 Mutation Influences Progesterone Response in Human Benign Mammospheres

Women, who carry a germline mutation in the breast cancer-associated gene 1 (BRCA1) have an increased risk (60-85%) of developing breast cancer during their lifetime. While BRCA1 mutated patients usually develop triple-negative, basal like, aggressive breast tumors, hormone signaling is important in the genesis of BRCA1 mutant breast cancers. Estrogen and progesterone promote carcinogenesis in the breast. Progesterone promotes proliferation of both benign epithelial cells and cancer cells and can expand the mammary stem cell pool. It is unclear how PR functions in the background of BRCA1 mutation in normal non-cancerous mammary epithelial cells before breast cancer develops. In order to determine whether BRCA1 mutation affects hormone response in benign mammary cells, human mammospheres from patients with wild type (WT; n=6) or BRCA1 mutation (n=6) were treated in vitro with a stepwise menstrual cycle hormone regimen of estrogen (E2) and progesterone (P4) over the course of 28 days. During the last 14 days of treatment, the SPRM, telapristal acetate (TPA) was added to the mammospheres. The mammospheres grown from benign breast tissues retained the expression of hormone receptors (ER and PR) even after the 28 day hormone treatment in vitro. Human mammary epithelial cells expressed aldehyde dehydrogenese-1 ALDH-1 with increasing levels observed in the BRCA1 mutated mammospheres compared to the WT mammospheres. RNA sequencing (RNA-seq) analysis revealed distinct differentially expressed genes between the WT and BRCA1 groups. Specifically, genes associated with extracellular matrix (ECM) organization were differentially regulated by TPA in the BRCA1 mutation group but not in the WT group. In fact, these ECM genes were differentially expressed in response to E2+P4 in WT versus BRCA1 mammospheres. In addition, cell cycle genes were responsive to TPA but in the WT group only. Taken together, these data show that BRCA1 mutation influences the hormone response and in particular to TPA compared to WT cells. Our physiologically relevant model systems have provided important insights into the role of progesterone in BRCA1 mutated breast cells. Further analysis of the molecular mechanism of BRCA1 and PR crosstalk is warranted using this model system.

OR34-3 The Androgen Receptor Malignancy Shift Can Be Caused by Mutations Commonly Found in Primary Prostate Cancer

Prostate cancer (PC) is the second most diagnosed cancer and sixth leading cause of cancer mortality in men in the world. The androgen receptor (AR) plays key roles in the development of prostate tissue as well as in the development and progression of PC. Following male development, AR guides cytodifferentiation and homeostasis in benign luminal epithelial cells. However, in PC, AR instead drives the uncontrolled proliferation of these cells; this "AR malignancy shift" is a central event in tumorigenesis, as the AR becomes a primary driver of growth in malignant neoplastic cells. Despite this change in AR behavior having been recognized for decades, the details of the AR malignancy shift remain unclear. Using a ChIP-seq approach in primary human tissues, cell lines, and mouse models, we demonstrate that the AR malignancy shift occurs in every sample analyzed, suggesting that it is necessary for PC development. Using molecular and genetic techniques, we demonstrate that FOXA1, HOXB13, GATA2, JUN, and CEBPA are involved in the regulation of the AR malignancy shift. AR binding sites (ARBS) are enriched for FOX, HOX, and GATA motifs in PC cells but for JUN and CEBP motifs in benign cells. We show that mutations commonly found in localized PCs, including ERG, SPOP and FOXA1 alterations, can CAUSE the AR malignancy shift. Furthermore, these alterations are not transformative on their own but must be coupled to other mutations to transform cells, suggesting that the AR malignancy shift is not sufficient for tumorigenesis. We show that the AR malignancy shift occurs in mouse models of PC as well and that chronic low T, which is associated with increased PC risk and aggressiveness in humans, also causes the AR malignancy shift in mouse prostate tissue without the development of frank carcinoma. In conclusion, we have discovered a previously-unrecognized, fundamental tenet of PC, one which explains how and why AR signaling is different in cancer and benign cells. Our work has the potential to create genomic classifiers that accurately predict clinical outcomes and could be used to stratify patients with localized PC for specific treatments. Furthermore, our work suggests that the AR malignancy shift is a novel target for the treatment and/or prevention of PC.

Extracellular Vesicle Integrins Distinguish Unique Cancers.

The proteomic profile of extracellular vesicles (EVs) has been of increasing interest, particularly in understanding cancer growth, drug resistance, and metastatic behavior. Emerging data suggest that cancer-derived EVs carry an array of oncogenic cargo, including certain integrin proteins that may, in turn, promote cell detachment, migration, and selection of future metastatic sites. We previously reported a large comparison of secreted vesicle protein cargo across sixty diverse human cancer cell lines. Here, we analyze the distinct integrin profiles of these cancer EVs. We further demonstrate the enrichment of integrin receptors in cancer EVs compared to vesicles secreted from benign epithelial cells. The total EV integrin levels, including the quantity of integrins α6, αv, and β1 correlate with tumor stage across a variety of epithelial cancer cells. In particular, integrin α6 also largely reflects breast and ovarian progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of certain primary tumors. This study provides preliminary evidence of the value of vesicle-associated integrin proteins in detecting the presence of cancer cells and prediction of tumor stage. Differential expression of integrins across cancer cells and selective packaging of integrins into EVs may contribute to further understanding the development and progression of tumor growth and metastasis across a variety of cancer types.

The role of NLRP3 inflammasome in regulating the progression of prostate cancer

Chronic inflammation is a hallmark of cancers and plays a critical role in modulating tumor progression and metastasis. Inflammasomes are a group of cytosolic protein complexes, composed of an NOD‐like protein (NLR), the adaptor apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), and caspase‐1, which are responsible for the cleavage of pro‐IL‐1β and pro‐IL‐18 proteins into mature and biologically active IL‐1β and IL‐18, respectively. IL‐1β levels in tumor tissues were previously connected to poor prognosis for cancer patients. However, it remains unclear the function of inflammasomes in tumor growth and metastasis. The aim of this study is to examine whether the activation of NLRP3 inflammasome regulates the progression of prostate cancer. First, we screened the ability of NLRP3 inflammasome activation in prostate cells due to cancer heterogeneity. Prostate cell lines were primed with LPS, followed by treated with ATP or nigericin to induce NLRP3 inflammasome activation. Our results showed that the secretion of IL‐1β was significantly increased in PC3 (human prostate cancer cell line), BPH‐1 (human benign prostate hyperplasia epithelial cell line) and RWPE‐1 (human prostate epithelial cell line) cells. Second, we tested the effect of hypoxic condition in regulating the activation of NLRP3 inflammasome. Our results showed that hypoxia also induced the secretion of IL‐1β in PC3, BPH‐1 and RWPE‐1 cells. Third, we found that NLRP3 inflammasome activation involved in regulating tumor growth and metastasis. Our results indicated that the activation of NLRP3 inflammation plays an important role in modulating the progression of prostate cancer. Therefore, targeting NLRP3 inflammasome could be a strategy for prostate cancer therapy.Support or Funding InformationThis study was supported by the Ministry of Science and Technology, Taiwan, R.O.C. (Grant No. MOST 107‐2320‐B‐037‐019).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abi1 loss drives prostate tumorigenesis through activation of EMT and noncanonical WNT signaling.

280 Background: Prostate cancer is characterized by heterogeneity of mechanisms which are poorly understood but pointing to epithelial plasticity as the key mechanism in progression to metastatic disease. ABI1, a member of WAVE complex and actin cytoskeleton regulator and adaptor protein, is proposed to act as tumor suppressor in prostate cancer, but the mechanism of tumor progression due to Abi1 loss is not clear. Methods: To address Abi1’s role in prostate cancer we used CRISPR-based gene editing and retroviral expression to manipulate Abi1 levels in prostate cancer cell lines. Levels of Abi1 expression in prostate organoid tumor cell lines were evaluated by Western blotting and/or RNA sequencing. Association of Abi1 loss with tumor grade was evaluated by immunohistochemistry. Results: Abi1 expression is downregulated in tumor organoid cell lines from metastatic bone and lymph node biopsies. Moreover, low Abi1 expression is associated with high-grade prostate tumors (GG3 or higher, p < 0.001). Disruption of Abi1 gene in a benign prostate epithelial cell line RWPE-1 resulted in a gain of invasive phenotype, which is characterized by loss of cell-cell adhesion markers and increased migratory ability of RWPE-1 Abi1 KO spheroids. Through RNA sequencing and protein expression analysis we discovered that Abi1 loss leads to activation of non-canonical WNT signaling and EMT pathways, which are rescued by re-expression of Abi1. Furthermore, increase in STAT3 phosphorylation upon Abi1 inactivation and evidence for high affinity interaction of FYN-SH2 domain with Abi1 pY421 support the model that Abi1 acts as a gatekeeper of the non-canonical WNT-EMT pathway activation downstream from FZD2 receptor. The gene expression profile of Abi1-EMT-WNT pathway overlaps with the reported gene signature of high-risk prostate tumors. Conclusions: Abi1 contributes to prostate cancer progression and epithelial plasticity through regulation of EMT-WNT pathways. Understanding of Abi1’s role may provide more mechanistic understanding of prostate cancer tumor progression.

Involvement of Actin Cytoskeletal Components in Breast Cancer Cell Fusion with Human Mesenchymal Stroma/Stem-Like Cells.

Cell fusion as a rare event was observed following the co-culture of human MDA-MB-231cherry breast cancer cells or benign neoplastic MCF10Acherry breast epithelial cells together with different mesenchymal stroma/stem-like cells (MSCGFP) cultures, respectively, resulting in the generation of double-fluorescing hybrid cells. Analysis of potential molecular mechanisms for the formation of cancer hybrid cells revealed cytoskeletal components, including F-actin. Thus, a sub-lethal concentration of cytochalasin D, which blocks elongation of actin filaments, was able to significantly reduce cancer hybrid cell formation. Simultaneously, cell cycle progression of the different co-cultures remained unaffected following treatment with cytochalasin D, indicating continued proliferation. Moreover, exposure to 50 nM cytochalasin D revealed little if any effect on the expression of various integrins and cell adhesion molecules in the different co-cultures. However, LC-MS proteome analysis of the different control co-cultures compared to corresponding cytochalasin-treated co-cultures demonstrated predominant differences in the expression of actin-associated cytoskeletal proteins. In addition, the requirement of structured actin to provide an appropriate cytoskeletal network for enabling subsequent fusion processes was also substantiated by the actin filament disrupting latrunculin B, which inhibits the fusion process between the breast cancer populations and mesenchymal stroma/stem-like cells (MSC). Together, these findings suggest an important role of distinct actin structures and associated cytoskeletal components during cell fusion and the formation of breast cancer hybrid cells.

Cynomorium songaricum Rupr demonstrates phytoestrogenic or phytoandrogenic like activities that attenuates benign prostatic hyperplasia via regulating steroid 5-α-reductase

Ethnopharmacological relevanceCynomorium songaricum Rupr. (CS) belongs to the genus of parasitic perennial flowering plants, mostly used in Chinese traditional medicine for benign prostatic hyperplasia (BPH) treatment. BPH is a chronic disease in men that both androgen and estrogen play a crucial role in promoting its development via their receptors. Previously we have showed that compounds from CS have the phytoestrogenic and/or phytoandrogenic activities that may have the potential suppressive effects on BPH, while the mechanism remains unclear. Aim of the studyIn this study, we aim to investigate the effect of CS and its derived compounds: luteolin (LUT), gallic acid (GA), protocatechuic acid (PA) and protocatechualdehyde (Pra) on inhibition of rat BPH and proliferation of BPH-1 cell line respectively, and further uncover whether it is related with the phytoestrogenic and / or phytoandrogenic activities. Materials and methodsEstradiol/testosterone (1:100) was subcutaneous injected to induce BPH in a castrated rat model, and CS was orally administrated for 45 days. Then the weights of the body and prostate were recorded, the pathogenesis changes of prostate were analyzed by Hematoxylin and eosin (H&E) and immunohistochemical (IHC). The levels of 17β-estradiol (E2), testosterone, and dihydrotestosterone (DHT) from rats’ serum were measured by enzyme-linked immunosorbent assay (ELISA). In vitro, human benign prostatic epithelial cell BPH-1 was cultured and treated with or without different CS compounds and DHT or E2. MTT and CCK-8 assays were performed to detect the regulatory effects on cell proliferation. The expressions of PCNA, AR, ERα, ERβ, and steroid 5-α-reductases (SRD5A1 and SRD5A2) were further analyzed by western blotting upon treatment. ResultsTreatment with CS significantly inhibited rat prostate enlargement, improved the pathological feature and reduced the thickness of smooth muscle layer. The up-regulated AR and ERα expressions and down-regulated ERβ in BPH rat prostate were significantly blocked after CS administration. Moreover, the enhanced values of E2/testosterone and the level of DHT in serum were also strongly inhibited in CS group compared with those in BPH groups. In cellular level, LUT, GA, PA, or Pra significantly inhibited DHT- or E2- induced BPH-1 cell proliferation and PCNA expressions. Consistently with the data in vivo, compounds from CS interfered the DHT or E2-regulated AR, ERα and ERβ expressions in BPH-1 cells as well. Importantly, the dramatic increased SRD5A1 and SRD5A2 expressions were observed in BPH rat prostates and DHT or E2-stimulated BPH-1 cells. However, treatment with CS in rat or with compounds isolated from CS in BPH-1 cells significantly blocked the induction of SRD5A1 and SRD5A2. ConclusionsCS suppressed BPH development through interfering with prostatic AR, ERα/β, and SRD5A1/2 expressions, which provided evidence of CS for BPH treatment.

Performance of A Convolutional Neural Network in Screening Liquid Based Cervical Cytology Smears.

Context:Cervical cancer is the second most common cancer in women. The liquid based cervical cytology (LBCC) is a useful tool of choice for screening cervical cancer.Aims:To train a convolutional neural network (CNN) to identify abnormal foci from LBCC smears.Settings and Design:We have chosen retrospective study design from archived smears of patients undergoing screening from cervical cancer by LBCC smears.Materials and Methods:2816 images, each of 256 × 256 pixels, were prepared from microphotographs of these LBCC smears, which included 816 “abnormal” foci (low grade or high grade squamous intraepithelial lesion) and 2000 ‘normal’ foci (benign epithelial cells and reactive changes). The images were split into three sets, Training, Testing, and Evaluation. A convolutional neural network (CNN) was developed with the python programming language. The CNN was trained with the Training dataset; performance was assayed concurrently with the Testing dataset. Two CNN models were developed, after 20 and 10 epochs of training, respectively. The models were then run on the Evaluation dataset.Statistical Analysis Used:A contingency table was prepared from the original image labels and the labels predicted by the CNN.Results:Combined assessment of both models yielded a sensitivity of 95.63% in detecting abnormal foci, with 79.85% specificity. The negative predictive value was high (99.19%), suggesting potential utility in screening. False positives due to overlapping cells, neutrophils, and debris was the principal difficulty met during evaluation.Conclusions:The CNN shows promise as a screening tool; however, for its use in confirmatory diagnosis, further training with a more diverse dataset will be required.

Amylase crystalloids in a cystic lesion of the parotid gland diagnosed by and #64257;ne needle aspiration cytology

Several types of crystalloids, including tyrosine, collagenous, intraluminal, and amylase crystalloids, may be encountered in fine-needle aspiration biopsy (FNAB) of salivary glands. They have been reported in benign and malignant lesions of the salivary glands. Amylase crystalloids mainly found in benign salivary gland lesions. Microscopic identification of the crystalloids are made according to differences in structural appearance. As it can helpful in differential diagnosis of benign and malignant salivary gland lesions, it is important to identify types of crystalloid. In this report, a 73-year-old man with painless mass in the left parotid gland is presented. Ultrasonographic examination demonstrated a well-defined, hypoechoic lesion in the tail portion of left parotid. Microscopic examination of smears revealed many non-birefringent, rectangular, rhomboid and rod-shaped with parallel sides crystalloids varying in size. Crystalloids stained pink with Hematoxylin & Eosin (H&E), bright orange with Papanicaloau (PAP), and deep blue with May Grunwald Giemsa (MGG). Inflammatory cells and rare benign ductal epithelial cells were observed in a mucoid background. The cytopathological diagnosis was reported as benign non-specific cystic lesion with amylase crystalloids. Since amylase crystalloids are only seen in benign lesions of the salivary glands, it is important to distinguish between amylase and the other crystalloids.

Suppressive effects of iron chelation in clear cell renal cell carcinoma and their dependency on VHL inactivation

Increasing data implicate iron accumulation in tumorigenesis of the kidney, particularly the clear cell renal cell carcinoma (ccRCC) subtype. The von Hippel Lindau (VHL)/hypoxia inducible factor-α (HIF-α) axis is uniquely dysregulated in ccRCC and is a major regulator and regulatory target of iron metabolism, yet the role of iron in ccRCC tumorigenesis and its potential interplay with VHL inactivation remains unclear. We investigated whether ccRCC iron accumulation occurs due to increased cell dependency on iron for growth and survival as a result of VHL inactivation. Free iron levels were compared between four VHL-mutant ccRCC cell lines (786-0, A704, 769-P, RCC4) and two benign renal tubule epithelial cell lines (RPTEC, HRCEp) using the Phen Green SK fluorescent iron stain. Intracellular iron deprivation was achieved using two clinical iron chelator drugs, deferasirox (DFX) and deferoxamine (DFO), and chelator effects were measured on cell line growth, cell cycle phase, apoptosis, HIF-1α and HIF-2α protein levels and HIF-α transcriptional activity based on expression of target genes CA9, OCT4/POU5F1 and PDGFβ/PDGFB. Similar assays were performed in VHL-mutant ccRCC cells with and without ectopic wild-type VHL expression. Baseline free iron levels were significantly higher in ccRCC cell lines than benign renal cell lines. DFX depleted cellular free iron more rapidly than DFO and led to greater growth suppression of ccRCC cell lines (>90% at ~30–150 µM) than benign renal cell lines (~10–50% at up to 250 µM). Similar growth responses were observed using DFO, with the exception that a prolonged treatment duration was necessary to deplete cellular iron adequately for differential growth suppression of the less susceptible A704 ccRCC cell line relative to benign renal cell lines. Apoptosis and G1-phase cell cycle arrest were identified as potential mechanisms of chelator growth suppression based on their induction in ccRCC cell lines but not benign renal cell lines. Iron chelation in ccRCC cells but not benign renal cells suppressed HIF-1α and HIF-2α protein levels and transcriptional activity, and the degree and timing of HIF-2α suppression correlated with the onset of apoptosis. Restoration of wild-type VHL function in ccRCC cells was sufficient to prevent chelator-induced apoptosis and G1 cell cycle arrest, indicating that ccRCC susceptibility to iron deprivation is VHL inactivation-dependent. In conclusion, ccRCC cells are characterized by high free iron levels and a cancer-specific dependency on iron for HIF-α overexpression, cell cycle progression and apoptotic escape. This iron dependency is introduced by VHL inactivation, revealing a novel interplay between VHL/HIF-α dysregulation and ccRCC iron metabolism. Future study is warranted to determine if iron deprivation using chelator drugs provides an effective therapeutic strategy for targeting HIF-2α and suppressing tumor progression in ccRCC patients.

Treatment Effects Can Mimic Recurrent Extramammary Paget Disease in Perianal Skin.

The histologic differential diagnosis of perianal Paget disease includes malignant melanoma, pagetoid spread of squamous cell carcinoma, and secondary involvement by colorectal carcinoma. While consideration of these entities is useful when establishing a diagnosis, it does not apply when patients with Paget disease undergo surveillance for recurrent disease. Treatment of perianal Paget disease consists of a combination of surgical excision with skin grafts and topical chemotherapeutic agents that induce cytologic alterations in benign cells and simulate recurrent malignancy. To evaluate the therapy-related changes and possible diagnostic pitfalls in patients with Paget disease, we reviewed 412 posttreatment tissue samples from 3 women with primary perianal Paget disease who underwent wide excision, skin grafting, and topical 5-fluorouracil therapy. Biopsy samples from engrafted skin often displayed single and clustered cells with hyperchromatic nuclei dispersed in the deep epidermis. Similar cells were scattered throughout all levels of the epidermis in biopsy samples following topical chemotherapy. The abnormal cells were negative for cytokeratin 7 (CK7) and mucicarmine in both situations. Disease ultimately recurred in all patients; some Paget cells showed classic features with eosinophilic or mucinous cytoplasm and eccentric nuclei, whereas others were smaller with less conspicuous atypia. All Paget cells showed strong, membranous CK7 staining. In short, treatment of perianal Paget disease can elicit cytologic abnormalities in benign epithelial cells that simulate the cytologic features of recurrent disease, and can diminish the atypia of Paget cells. Immunohistochemical stains for CK7 can be helpful when evaluating surveillance samples from these patients.

Physical characterization and uptake of iron oxide nanoparticles of different prostate cancer cells

Abstract Among nanoparticles, magnetic nanoparticles are the most appealing candidate for diagnosis and cancer therapy. The researchers are tempting to improve the particles properties, including the size, shape, coating, and magnetic behavior or heating characteristics. Core shell type of magnetic nanoparticle is an important property that modulates their internalization via normal and cancer cells. In this study, magnetite nanoparticles (MNP) covered by N-(2-aminoethyl)-3-aminopropyltriethoxysilane (aminosilane – APTES) were synthesized by co-precipitation of aqueous solution of ferric chloride and ferrous sulfate iron salts with ammonium hydroxide as a base and functionalized by APTES to increase the viability and affinity of the particles to the cancer cells. The structural and morphological properties of these particles were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier transformed infrared spectroscopy (FTIR). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT assay) was carried out, to check the viability of the cells treatment with MNP and APTES-MNP. To study the cellular uptake in vitro, two prostate cell lines were investigated: PC3 as a cancerous cell line and BPH1 as a benign epithelial cell line (normal cells). Both cell lines were incubated for 24 h with different concentrations of MNP and APTES-MNP (100 and 500 μg/ml and one untreated sample as control). TEM and flow cytometry (FC) analyses were subsequently carried out to monitor the cellular uptake of MNP and APTES-MNP. FC data revealed an increase in cell granularity following the treatment with high concentration of the particles. Data showed that PC3 cancer cells take up more APTES-MNP with respect to control cells than BPH1 benign cells and in contrast BPH1 cell uptake MNP correlated to control cells more efficient than PC3 cells. The results from FC and TEM analyses demonstrate increasing of affinity of particles to cancer cell line (PC3). In this project we investigated the effect of surface functionalization of NP to affinity of the MNP and APTES-MNP on PC3 cells as a malignancy prostate cell and BPH1 benign cells as a normal cells. This approach may help to optimize the efficiency of hyperthermia for prostate cancer through internalization of particles to the cells or attaching to the membrane.

Paraesophageal Bronchogenic Cyst

Bronchogenic cysts are congenital abnormalities along the bronchial tree and are usually found in the mediastinum. Upper endoscopy and endoscopic ultrasound may be used to establish the diagnosis. A 52-year-old female with history of hypertension, presented with chest pain. As part of cardiac work up, a calcium scoring CT scan was done which showed an incidental mediastinal mass. Patient denied any dysphagia or odynophagia symptoms. A dedicated CT chest with contrast demonstrated a lobulated soft tissue mass within the azygoesophageal recess measuring 34 x 18 mm with the lateral aspect of the mass appearing confluent with the distal esophagus. The lateral aspect of the mass extended into the medial right lung base. The remainder of the lungs were clear with patent central airways (Figure 1). Given concern for malignancy, a PET CT was obtained and demonstrated a mediastinal soft tissue mass inseparable from the distal esophagus without significant FDG update. Further evaluation was donw by EGD and EUS. Endoscopically a mild extrinsic impression was noted in the lower third esophagus which was easily transversed. EUS revealed a 28 x 20 mm hypoechoic mass with well-defined borders in the mediastinum, at about 34cm from the incisors (Firgure 2). It was soft and compressible. FNA with a 22 G needle was performed. Histological examination showed clusters of benign bronchial epithelial cells and squamous epithelial cells in a background of abundant mucin, suggestive of a bronchogenic cyst (Figure 3). Patient underwent robotic resection of the mass, which appeared as a bluish brown cyst in the lower posterior mediastinum and was distinct from the esophagus and mediastinum, but intimately attached to the lower lobe of the right lung. Surgical pathology confirmed a benign bronchogenic cyst. Patient remained asymptomatic since resection. Bronchogenic cyst is a rare cause of a paraesophageal mass. Cross sectional imaging may be helpful in evaluation but tissue acquisition is essential for further evaluation of any malignancy and to determine appropriate surgical planning. EUS is useful modality to further characterize a cyst and FNA provided adequate tissue diagnosis in case of a paraesophageal bronchial cyst.2114_A Figure 1. A, B and C. Coronal, sagittal and cross sectional CT images of the paraesophageal bronchogenic cyst (yellow arrow)2114_C Figure 3. A and B: Fine needle aspiration showed clusters of benign bronchial epithelial cells and squamous epithelial cells in a background of abundant mucin, suggestive of a bronchogenic cyst, HE stain, 100X. C: Surgical resection showed a cyst lined by benign bland ciliated columnar epithelium with cartilage and submucosal glands in the wall, consistent with a bronchogenic cyst.2114_B Figure 2. Endosonography demonstrating a hypoechoic mass with well-defined borders arising from the mediastinum.v

Abstract B068: Methionine aminopeptidase II (MetAP2) activated in situ self-assembly of small-molecule probes for imaging prostate cancer

Abstract Prostate cancer is the most common noncutaneous malignancy among men in the United States and a leading cause of cancer death worldwide. The major clinical challenges in prostate cancer diagnosis include accurate staging, localizing tumors within the prostate, and developing imaging prognostic biomarkers. PET is routinely used in the clinical management of many cancers dues to its high sensitivity (10-15 M), spatial resolution (2-4 mm3), rapid whole-body scan times (<20 min), noninvasiveness, and its ability to provide both anatomic and molecular information when combined with computed tomography (PET/CT). Several PET tracers (e.g., 18F-FDG, 18F-NaF, 11C-choline, 11C-acetate, 18F-fluciclovine, etc.) have been studied for early stage prostate cancer, bone metastases, and recurrent disease; nevertheless, all of these tracers rely on cell uptake and their applications are often limited by the low metabolic activity and a low growth rate of prostate cancer. In this project, we aim to develop a novel enzymatic activity-based PET tracer for in vivo imaging of prostate cancer by targeting methionine aminopeptidase II (MetAP2), a cytosolic metalloprotease that catalyzes the cotranslational removal of the N-terminus initiator methionine residue from nascent proteins. We hypothesize that MetAP2 can serve as a novel diagnostic biomarker of prostate cancer. A MetAP2 activatable molecular imaging probe that self-aggregates in situ can be utilized to image and define this disease. To test our hypothesis, we first measured the expression level of MetAP2 in primary benign human prostate epithelial cells (BS403) and prostate tumor cell lines by Western blot. BS403 cells express significantly lower levels of MetAP2 than PC3 (by 4-fold), DU145 (3-fold), and 22Rv1 (3-fold). The expression of MetAP2 in DU145 tumor xenografts is 12-fold higher and for 22Rv1 14-fold higher, compared to BS403 cells. Immunohistologic analysis of tissues derived from DU145 and 22Rv1 xenografts confirms strong staining of MetAP2. We performed immunohistochemistry on a prostate cancer tissue microarray containing 225 independent patient samples represented in quadruplicate. Positive staining for MetAP2 was found in nearly all of the cancer samples. Encouraged by these data, we designed and synthesized a nonradioactive MetAP2 sensitive nano-aggregation probe called M-SNAP. Incubation of M-SNAP with recombinant MetAP2 enzyme confirmed the formation of the cyclized and aggregated product M-SNAP-cycl by HPLC, TEM (transmission electron microscopy), and DLS (dynamic light scattering). Treatment of PC3 and DU145 cells with or without M-SNAP and the MetAP2 specific inhibitor TNP470 demonstrated MetAP2 dependent intracellular probe aggregation/signal retention using post fluorescent labeling of the cyclized and aggregated products. In future work, we will prepare and study an MetAP2 activatable radioisotope-labeled probe in vitro for sensing and imaging and will evaluate the PET probe in different human prostate xenograft models. MetAP2 sensing probes could provide a highly sensitive and noninvasive PET imaging approach for prostate cancer detection that could be useful in prostate cancer detection, staging, and monitoring of therapeutic response. Citation Format: Jinghang Xie, Meghan Rice, Yunfeng Cheng, Guosheng Song, Christian Kunder, James D. Brooks, Tanya Stoyanova, Jianghong Rao. Methionine aminopeptidase II (MetAP2) activated in situ self-assembly of small-molecule probes for imaging prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B068.

PO-245 Fructose supports tumour growth and aggressiveness through a metabolic reprogramming in prostate cancer

IntroductionPET-scanning can detect primary tumours relaying on their high glucose uptake ability through overexpression of the glucose transporter Glut-1. However, this method has shown limited clinical applicability for prostate cancer (PCa) diagnosis, suggesting that PCa cells do not use glucose as its primary source of energy. Preliminary data from our laboratory has shown overexpression of the fructose transporter Glut-5 in PCa cell lines and in clinical specimens of PCa, which suggest that fructose could play an important role in PCa biology.Material and methodsWe analysed the effect of fructose on aggressiveness and metabolic reprogramming in benign prostate epithelial and PCa cell lines. Cells were incubated with glucose or fructose in the media for 24, 48, and 72 hour and then we evaluated: 1) the proliferative rate of benign and PCa cells, 2) the invasion and migration capacity of PCa cells, and 3) the mRNA levels of the enzymes involved in glycolysis, pentose phosphate and de novo lipogenesis pathways using real-time PCR in PCa cells. The effect of fructose on tumour growth was analysed by a PC3 cell line xenograft in immunosuppressed NSG mice. 15% fructose was added to the drinking water for 8 weeks. Tumour weight and the expression of glycolytic enzymes by qPCR was evaluated with respect to the control (water without additives).Results and discussionsPCa cells incubated with fructose or glucose, showed similar proliferative rate, invasion and migration capacities. However, fructose evokes a different expression profile of the enzymes involve in glycolysis, pentose phosphate and de novo lipogenesis pathways. Fructose promotes tumour growth of PC3 cells in the NSG mice.ConclusionOur data suggest that fructose could play an important role in PCa pathophysiology, promoting proliferation and migration of PCa cells in vitro, and increasing tumour growth in vivo thanks to a reprogram in their metabolism that allows PCa cells to use fructose as effectively as glucose.