CUL4high Lung Adenocarcinomas Are Dependent on the CUL4-p21 Ubiquitin Signaling for Proliferation and Survival

Cullin (CUL) 4A and 4B ubiquitin ligases are often highly accumulated in human malignant neoplasms and are believed to possess oncogenic properties. However, the underlying mechanisms by which CUL4A and CUL4B promote pulmonary tumorigenesis remain largely elusive. This study reports that CUL4A and CUL4B are highly expressed in patients with non–small cell lung cancer (NSCLC), and their high expression is associated with disease progression, chemotherapy resistance, and poor survival in adenocarcinomas. Depletion of CUL4A (CUL4A^k/d) or CUL4B (CUL4B^k/d) leads to cell cycle arrest at G₁ and loss of proliferation and viability of NSCLC cells in culture and in a lung cancer xenograft model, suggesting that CUL4A and 4B are oncoproteins required for tumor maintenance of certain NSCLCs. Mechanistically, increased accumulation of the cell cycle–dependent kinase inhibitor p21/Cip1/WAF1 was observed in lung cancer cells on CUL4 silencing. Knockdown of p21 rescued the G₁ arrest of CUL4A^k/d or CUL4B^k/d NSCLC cells, and allowed proliferation to resume. These findings reveal that p21 is the primary downstream effector of lung adenocarcinoma dependence on CUL4, highlight the notion that not all substrates respond equally to abrogation of the CUL4 ubiquitin ligase in NSCLCs, and imply that CUL4A^high/CUL4B^high may serve as a prognostic marker and therapeutic target for patients with NSCLC.

Lung cancer is the most common cause of cancer mortality worldwide,¹ accounting for 19.4% of all cancer-related deaths and representing a significant clinical burden.² Among the subtypes of lung cancer, non–small cell lung cancer (NSCLC) accounts for 80% to 85% of cases.3, 4, 5 Although multimodality treatments, including targeted therapies and immunotherapies, have been applied to NSCLCs, with high rates of local and distant failure, the overall cure and survival rates for NSCLC remain low.^6,7 Thus, understanding the molecular mechanisms underlying NSCLC development and progression is of fundamental importance for the development of new therapeutic strategies for patients with NSCLC.Cullin (CUL) 4, a molecular scaffold of the CUL4-RING ubiquitin ligase (CRL4), plays an important role in regulating key cellular processes through modulating the ubiquitylation and degradation of various protein substrates.⁸ Two CUL4 proteins, CUL4A and CUL4B, share an 82% sequence homology, with similar but distinct functions.⁹ CUL4 has been extensively studied in the process of nucleotide excision repair (NER) after UV irradiation.10, 11, 12, 13 Loss of CUL4A, but not CUL4B, elevates global genomic NER activity and confers increased protection against UV-induced skin carcinogenesis.¹¹ In addition to DNA repair, CUL4 also plays a significant role in a wide spectrum of physiologic processes, such as the cell cycle, cell signaling, and histone methylation, which have direct relevance to the development of human cancers.14, 15, 16 Accumulating studies have found that CUL4A is amplified or expressed at abnormally high levels in multiple cancers, including breast cancer, squamous cell carcinoma, hepatocellular carcinomas, and lung cancer.^9,17, 18, 19 More importantly, CUL4A and 4B overexpression is implicated in tumor progression, metastasis, and a poorer survival rate for patients with cancer.^9,20,21 CUL4A, but not CUL4B, is inversely correlated with the NER protein xeroderma pigmentosum, complementation group C and the G₁/S DNA damage checkpoint protein p21 in patients with lung squamous cell carcinoma, highlighting a reduced DNA damage response⁹ as well as promoting cell growth and tumorigenesis.^22,23 Increased expression of CUL4A caused hyperplasia as well as lung adenocarcinomas in mice.²⁴ However, the mechanistic basis and clinical significance of CUL4A dysregulation in NSCLC remain unclear.The CUL4A paralog CUL4B shares extensive sequence homology and redundant functions with CUL4A.⁹ To date, research on CUL4B has been focused mainly on its genetic association with human X-linked mental retardation.25, 26, 27, 28 Recently, CUL4B was found to be overexpressed in colon cancer and correlated with tumor stage, histologic differentiation, vascular invasion, and distant metastasis.²⁹ Patients with lung and colon cancer with high levels of CUL4B had lower overall survival (OS) and disease-free survival (DFS) rates than those with low CUL4B expression.^9,29 CUL4B is also overexpressed in cervical, esophageal, and breast cancers and associated with tumor invasion and lymph node metastasis.^16,30,31 Furthermore, CUL4B overexpression promotes the development of spontaneous liver tumors at a high rate and enhances diethylnitrosamine-induced hepatocarcinogenesis in transgenic mice.³²The molecular mechanisms underlying the capacity of CUL4 to promote pulmonary tumorigenesis remain largely elusive. CUL4A promotes NSCLC cell growth.²² CUL4 targets a panel of cell cycle regulators for ubiquitination and degradation, including Cdc6, Cdt1, p21, cyclin E, minichromosome maintenance 10 replication initiation factor, and forkhead box M1.³³ However, which of the cell cycle substrates of CUL4 play a key role in tumor dependence on dysregulated CUL4A or CUL4B remains to be defined. This study found that attenuation of CUL4, especially CUL4B, inhibited NSCLC cell proliferation and tumorigenesis through increased accumulation of p21 and cell cycle arrest in G₁.     

Endometrial Cancer Side-Population Cells Show Prominent Migration and Have a Potential to Differentiate into the Mesenchymal Cell Lineage

Cancer stem-like cell subpopulations, referred to as “side-population” (SP) cells, have been identified in several tumors based on their ability to efflux the fluorescent dye Hoechst 33342. Although SP cells have been identified in the normal human endometrium and endometrial cancer, little is known about their characteristics. In this study, we isolated and characterized the SP cells in human endometrial cancer cells and in rat endometrial cells expressing oncogenic human K-Ras protein. These SP cells showed i) reduction in the expression levels of differentiation markers; ii) long-term proliferative capacity of the cell cultures; iii) self-renewal capacity in vitro; iv) enhancement of migration, lamellipodia, and, uropodia formation; and v) enhanced tumorigenicity. In nude mice, SP cells formed large, invasive tumors, which were composed of both tumor cells and stromal-like cells with enriched extracellular matrix. The expression levels of vimentin, α-smooth muscle actin, and collagen III were enhanced in SP tumors compared with the levels in non-SP tumors. In addition, analysis of microdissected samples and fluorescence in situ hybridization of Hec1-SP-tumors showed that the stromal-like cells with enriched extracellular matrix contained human DNA, confirming that the stromal-like cells were derived from the inoculated cells. Moreober, in a Matrigel assay, SP cells differentiated into α-smooth muscle actin-expressing cells. These findings demonstrate that SP cells have cancer stem-like cell features, including the potential to differentiate into the mesenchymal cell lineage.Recently, adult stem cells have been identified in several mature tissues, such as the adult intestine,¹ skin,² muscle,³ blood,⁴ and the nervous system^5–7 A stem cell is an undifferentiated cell that is defined by its ability to both self-renew and to produce mature progeny cells.⁸ Stem cells are classified based on their developmental potential as totipotent, pluripotent, oligopotent, and unipotent. Adult somatic stem cells were originally thought to be tissue specific and only able to give rise to progeny cells corresponding to their tissue of origin. Recent studies, however, have shown that adult mammalian stem cells are able to differentiate across tissue lineage boundaries,^9,10 although this “plasticity” of adult somatic stem cells remains controversial.Stem cell subpopulations (“side-population” (SP) cells) have been identified in many mammals, including humans, based on the ability of these cells to efflux the fluorescent dye Hoechst 33342.¹¹ Recent evidence suggests that the SP phenotype is associated with a high expression level of the ATP-binding cassette transporter protein ABCG2/Bcrp1.¹² Most recently, established malignant cell lines, which have been maintained for many years in culture, have also been shown to contain SP cells as a minor subpopulation.¹³The human endometrium is a highly dynamic tissue undergoing cycles of growth, differentiation, shedding, and regeneration throughout the reproductive life of women. Endometrial adult stem/progenitor cells are likely responsible for endometrial regeneration.¹⁴ Rare populations of human endometrial epithelial and stromal colony-forming cells¹⁵ and SP cells^16,17 have been identified. Although coexpression of CD146 and PDGFRβ isolates a population of mesenchymal stem like cells from human endometrium,¹⁸ specific stem cell markers of endometrium remain unclear. Recently, Gotte et al¹⁹ demonstrated that the adult stem cell marker Musashi-1 was coexpressed with Notch-1 in a subpopulation of endometrial cells. Furthermore, they showed that telomerase and Musashi-1-expressing cells were significantly increased in proliferative endometrium, endometriosis, and endometrial carcinoma tissue, compared with secretary endometrium, suggesting the concept of a stem cell origin of endometriosis and endometrial carcinoma.Recent evidence suggests that cancer stem-like cells exist in several malignant tumors, such as leukemia^20,21 breast cancer,²² and brain tumors,²³ and that these stem cells express surface markers similar to those expressed by normal stem cells in each tissue.^20,24Development of endometrial carcinoma is associated with a variety of genetic alterations. For example, increased expression and activity of telomerase^25,26 and frequent dysregulation of signaling pathways have been observed in endometrial carcinoma. Some of these pathways are important determinants of stem cell activity (Wnt-β-catenin and PTEN).^27–29 These suggest a stem cell contribution to endometrial carcinoma development.Recently, we isolated SP cells from the human endometrium. These SP cells showed long-term proliferating capacity in cultures and produced both gland and stromal-like cells. Additionally, they were able to function as progenitor cells.¹⁶ In this study, we isolated and characterized SP cells from human endometrial cancer cells and from rat endometrial cells expressing oncogenic [¹²Val] human K-Ras protein and demonstrated their cancer stem-like cell phenotypes.     

Cell Fusion Connects Oncogenesis with Tumor Evolution

Cell fusion likely drives tumor evolution by undermining chromosomal and DNA stability and/or by generating phenotypic diversity; however, whether a cell fusion event can initiate malignancy and direct tumor evolution is unknown. We report that a fusion event involving normal, nontransformed, cytogenetically stable epithelial cells can initiate chromosomal instability, DNA damage, cell transformation, and malignancy. Clonal analysis of fused cells reveals that the karyotypic and phenotypic potential of tumors formed by cell fusion is established immediately or within a few cell divisions after the fusion event, without further ongoing genetic and phenotypic plasticity, and that subsequent evolution of such tumors reflects selection from the initial diverse population rather than ongoing plasticity of the progeny. Thus, one cell fusion event can both initiate malignancy and fuel evolution of the tumor that ensues.The multiple genetic changes that convert a normal cell to a malignant cell likely occur in one of the following two pathways: the pathway involving the accretion of point mutations with or without ensuing chromosomal damage over time^1–4 or the pathway involving a catastrophic event causing manifold genetic changes, including those underlying malignant transformation.^5–7 Inherited defects in DNA repair, exposure to ionizing radiation, and infection with oncogenic viruses accelerate the accumulation of multiple discrete mutations or DNA damage and hence the development of cancer.⁴ However, inheritance, infection, or instantaneous exposure to an environmental carcinogen cannot explain the inception of most cancers. Hence, identification of discrete events that cause normal cells to undergo oncogenesis remains a compelling challenge.For many years, cell fusion has been considered in theory an appealing explanation for oncogenesis. Cell fusion can be detected in existing cancers.^8–10 Cell fusion can generate aneuploidy, chromosomal instability, and DNA damage, all of which might cause multiple genetic changes and cancer.^11–19 Cell fusion might explain how terminally differentiated, nonproliferating cells initiate tumors.^11,13,20However, cell fusion by itself has never been proven to initiate malignancy. Lack of such proof reflects the exigencies of experimental systems used for analysis of karyotype and malignant transformation (ie, proliferation of parent and fused cells over multiple generations). Formation of tumors has never been found to occur as a consequence of spontaneous fusion of cells in whole animal systems.^{14,15,21–25} Therefore, the question of whether cell fusion can initiate cancer remains a matter of speculation.We tested whether cell fusion can initiate tumors using IE-6 cells. Originally isolated as outgrowths from fragments of rat intestine,²⁶ IEC-6 cells are considered the archetype of normal intestinal crypt epithelial cells.^26–28 As in normal crypt epithelium, the proliferation and differentiation of IEC-6 cells are likely governed by the caudal type homeobox genes Cdx1 and Cdx2,^29–31 and the cells have a stable karyotype, nontransformed phenotype and are unable to form tumors at repeated passage in cultures.^26–28 To generate sufficient numbers of fused and unfused cells, we used polyethylene glycol (PEG), which does not promote and in fact might inhibit oncogenesis,³² to facilitate fusion and isolated and cloned the ensuing hybrids. Analysis of these clones reveals that cell fusion engenders aneuploidy, DNA damage, phenotypic heterogeneity, transformation, and the capacity to form tumors and that these properties are established immediately or within a few cell divisions after the fusion event.     

Neuropilin 1 Receptor Is Up-Regulated in Dysplastic Epithelium and Oral Squamous Cell Carcinoma

Neuropilins are receptors for disparate ligands, including proangiogenic factors such as vascular endothelial growth factor and inhibitory class 3 semaphorin (SEMA3) family members. Differentiated cells in skin epithelium and cutaneous squamous cell carcinoma highly express the neuropilin-1 (NRP1) receptor. We examined the expression of NRP1 in human and mouse oral mucosa. NRP1 was significantly up-regulated in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC). NRP1 receptor localized to the outer suprabasal epithelial layers in normal tongue, an expression pattern similar to the normal skin epidermis. However, dysplastic tongue epithelium and OSCC up-regulated NRP1 in basal and proliferating epithelial layers, a profile unseen in cutaneous squamous cell carcinoma. NRP1 up-regulation is observed in a mouse carcinogen-induced OSCC model and in human tongue OSCC biopsies. Human OSCC cell lines express NRP1 protein in vitro and in mouse tongue xenografts. Sites of capillary infiltration into orthotopic OSCC tumors correlate with high NRP1 expression. HSC3 xenografts, which express the highest NRP1 levels of the cell lines examined, showed massive intratumoral lymphangiogenesis. SEMA3A inhibited OSCC cell migration, suggesting that the NRP1 receptor was bioactive in OSCC. In conclusion, NRP1 is regulated in the oral epithelium and is selectively up-regulated during epithelial dysplasia. NRP1 may function as a reservoir to sequester proangiogenic ligands within the neoplastic compartment, thereby recruiting neovessels toward tumor cells.Oral squamous cell carcinoma (OSCC) is the most malignant tumor of the oral cavity. OSCC is more aggressive than cutaneous squamous cell carcinoma (CSCC). Although the incidence of OSCC is 20 times lower than CSCC with 30,000 (0.01%) and 700,000 (0.2%) new cases each year in the United States, respectively^{1, 2}; two-thirds of OSCC patients have evidence of disseminated disease at diagnosis,¹ yet CSCC is rarely malignant with only 4% of cases developing nodal metastases.² All tumor growth beyond the volume of 1 to 2 mm³ is angiogenesis dependent.³ The extent of tumor angiogenesis and lymphangiogenesis are two of the most important prognostic factors in OSCC.⁴ Understanding the mechanisms that control tumor neovascularization may lead to new therapeutic options for cancer patients.Neuropilin 1 (NRP1) has been studied extensively in the vascular system where it acts as a coreceptor for angiogenic proteins such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) and in the neuronal system where it serves as a receptor for guidance molecules called class 3 semaphorins (SEMA3s).^{5, 6} Our laboratory previously demonstrated that epithelial cells in the skin and CSCC tumor cells express high levels of NRP1.^{7, 8, 9} However, the function of NRP1 in epithelial cells and carcinoma cells is not as well understood as its role in endothelial cells.^{10, 11}Several studies have reported that overexpressing the NRP1 receptor via transfection into tumor cells results in enhanced tumor size in vivo, although NRP1 does not directly increase proliferation of tumor cells in vitro.^{12, 13} In addition, tumors overexpressing NRP1 formed hypervascular xenografts, suggesting that NRP1 expression in the tumor compartment influences neovascularization from the stromal compartment.^{14, 15} This effect is apparently caused by the binding (and release) of angiogenic factors [VEGF, placenta growth factor (PlGF), HGF] to the NRP1 receptor on tumor cells, resulting in an increased chemogradient of these ligands within the local tumor microenvironment that attracts and induces sprouting of neighboring endothelial cells.To test our hypothesis that NRP1 may increase the vascularity and invasiveness of OSCC, we investigated the expression and function of NRP1 in oral (tongue) epithelial cells and OSCC cells. Our results found an up-regulation in NRP1 receptor expression in oral dysplastic epithelium that persists throughout the stages of oral carcinogenesis and progression. In addition, the expression of NRP1 protein within the OSCC tumor compartment correlates with the pattern of blood vessel infiltration within the tumor microenvironment.     

Inhibition of Hepatic Stellate Cell Activation Suppresses Tumorigenicity of Hepatocellular Carcinoma in Mice

Transdifferentiation (or activation) of hepatic stellate cells (HSCs) to myofibroblasts is a key event in liver fibrosis. Activated HSCs in the tumor microenvironment reportedly promote tumor progression. This study analyzed the effect of an inhibitor of HSC activation, retinol-binding protein–albumin domain III fusion protein (R-III), on protumorigenic functions of HSCs. Although conditioned medium collected from activated HSCs enhanced the migration, invasion, and proliferation of the hepatocellular carcinoma cell line Hepa-1c1c7, this effect was not observed in Hepa-1c1c7 cells treated with conditioned medium from R-III–exposed HSCs. In a subcutaneous tumor model, larger tumors with increased vascular density were formed in mice transplanted with Hepa-1c1c7+HSC than in mice transplanted with Hepa-1c1c7 cells alone. Intriguingly, when Hepa-1c1c7+HSC–transplanted mice were injected intravenously with R-III, a reduction in vascular density and extended tumor necrosis were observed. In an orthotopic tumor model, co-transplantation of HSCs enhanced tumor growth, angiogenesis, and regional metastasis accompanied by increased peritumoral lymphatic vessel density, which was abolished by R-III. In vitro study showed that R-III treatment affected the synthesis of pro-angiogenic and anti-angiogenic factors in activated HSCs, which might be the potential mechanism underlying the R-III effect. These findings suggest that the inhibition of HSC activation abrogates HSC-induced tumor angiogenesis and growth, which represents an attractive therapeutic strategy.

Cancers develop in complex tissue environments, which is essential for sustained growth, invasion, and metastasis.¹ Tumor angiogenesis is an essential process for tumor progression, and lymphatic vessels provide an alternate route for tumor cell dissemination.² The tumor microenvironment (TME) comprises a mass of heterogeneous cell types, among which the two most prominent types of protumorigenic cells are cancer-associated fibroblasts (CAFs) and tumor-associated macrophages.³ CAFs, also called myofibroblasts, are reportedly associated with the progression of several types of cancers, and can originate as a result of the activation of resident fibroblasts, bone marrow–derived fibrocytes, epithelial cells, endothelial cells, or from certain specialized cells such as stellate cells (SCs) in the pancreas and liver.⁴ The presence of activated SCs has been shown in the stroma surrounding cancer cells, and bidirectional interactions between SCs and cancer cells, by which tumor-derived factors activate SCs, and, in turn, activated SCs promote metastatic growth.^5,6 Conditioned media from activated SCs promotes the proliferation, migration, and invasion of tumor cells in vitro, and co-transplantation of these activated SCs and tumor cells into mice resulted in an enlarged tumor mass that correlated with enhanced angiogenesis.^7,8Hepatic stellate cells (HSCs) are pericytes residing in the space of Disse between the sinusoidal endothelial cells and the parenchymal cells, and constitute 5% to 10% of the total number of cells in the liver.⁹ In normal liver, HSCs maintain a nonproliferative, quiescent phenotype and store approximately 80% of vitamin A (retinol) in the whole body as retinyl esters in lipid droplets in the cytoplasm. In response to fibrogenic stimuli, HSCs become activated, transdifferentiating from vitamin A–storing cells to myofibroblast-like cells.¹⁰ Upon activation, HSCs lose cytoplasmic vitamin A–containing lipid droplets, proliferate vigorously, and produce a large amount of extracellular matrix proteins. When cultured on plastic, HSCs undergo spontaneous activation in vitro. Activation of HSCs is a widely accepted key event in liver fibrosis, which is characterized by excessive accumulation of extracellular matrix proteins.¹¹ Cirrhosis is the advanced stage of liver fibrosis and is the leading risk factor for the development of hepatocellular carcinoma (HCC).¹² Cells resembling HSCs were isolated from the pancreas in the late 1990s,¹³ and these pancreatic stellate cells also play a central role in pancreatic fibrogenesis in a manner similar to HSCs.¹⁴Albumin is the most abundant plasma protein with a mol. wt. of approximately 66 kDa produced in the liver.¹⁵ It is composed of three homologous domains (I to III) and performs a variety of functions. A previous study showed that albumin was expressed in quiescent SCs, but not in activated SCs, and that its forced expression in activated SCs induced the phenotypic reversion to fat-storing, early activated cells.¹⁶ Building on these results, a recombinant fusion protein (designated R-III; mol. wt., approximately 45 kDa) was developed as an antifibrotic agent, in which the domain III of albumin was fused to the C-terminus of retinol-binding protein.¹⁷ Retinol-binding protein was adopted for targeted delivery to SCs because the protein and its membrane receptor STRA6 coordinate the cellular uptake of retinol into HSCs.¹⁸ A follow-up study showed that R-III inhibited SC activation in vitro and reduced liver and kidney fibrosis in vivo.19, 20, 21 In this study, the effect of R-III on the protumorigenic functions of activated HSCs was examined, and R-III was found to suppress HSC-induced HCC angiogenesis and growth.     

Generation of a Dual-Functioning Antitumor Immune Response in the Peritoneal Cavity

Tumor cell metastasis to the peritoneal cavity is observed in patients with tumors of peritoneal organs, particularly colon and ovarian tumors. Following release into the peritoneal cavity, tumor cells rapidly attach to the omentum, a tissue consisting of immune aggregates embedded in adipose tissue. Despite their proximity to potential immune effector cells, tumor cells grow aggressively on these immune aggregates. We hypothesized that activation of the immune aggregates would generate a productive antitumor immune response in the peritoneal cavity. We immunized mice i.p. with lethally irradiated cells of the colon adenocarcinoma line Colon38. Immunization resulted in temporary enlargement of immune aggregates, and after challenge with viable Colon38 cells, we did not detect tumor growth on the omentum. When Colon38-immunized mice were challenged with cells from the unrelated breast adenocarcinoma line E0771 or the melanoma line B16, these tumors also did not grow. The nonspecific response was long-lived and not present systemically, highlighting the uniqueness of the peritoneal cavity. Cellular depletions of immune subsets revealed that NK1.1⁺ cells were essential in preventing growth of unrelated tumors, whereas NK1.1⁺ cells and T cells were essential in preventing Colon38 tumor growth. Collectively, these data demonstrate that the peritoneal cavity has a unique environment capable of eliciting potent specific and nonspecific antitumor immune responses.The peritoneal cavity is a unique immunologic environment that includes immune aggregates present in the peritoneal wall, mesentery, and omentum as well as free cells present in the peritoneal fluid.^1,2 This fluid, which mechanically acts to lubricate organ movement, also distributes a variety of immune subsets throughout the peritoneal cavity. The immune cells present in the peritoneal fluid are primarily macrophages and B cells but also include other lymphocyte and dendritic cell populations.³ These free-floating immune cells have a dynamic relationship with the organized immune aggregates also present in the peritoneal cavity.^4,5 These structures contain immune cell subsets similar to those in the peritoneal fluid but in a highly organized manner, similar to many other tertiary immune structures.^3,6,7 One site of these immune aggregates, the omentum, is of particular interest because of the high density of aggregates found there.The omentum is a thin adipose tissue located in the peritoneal cavity that is appreciated as a guardian of the peritoneal cavity, especially for its immunologic role in controlling infections. For example, peritoneal dialysis, which can introduce bacteria into the cavity, leads to increases in the number and size of omental immune aggregates, which further increase on complications of peritonitis.^8,9 In addition, omental immune aggregates are the primary site of leukocyte extravasation in models of peritonitis.^10,11 Furthermore, bacteria are rapidly sequestered in the omentum shortly after introduction to the peritoneal cavity,¹² a process that slows bacterial dissemination throughout the peritoneal cavity.⁸ Collectively, these data suggest that omental immune aggregates are capable of responding against foreign pathogens.Similar to bacterial localization to the omentum, following tumor cell metastasis to the peritoneal cavity, the initial and most common site of tumor formation is the omentum.⁷ Tumor cell metastasis to the peritoneal cavity is generally a poor prognostic indicator, and limited effective therapies are available to diagnosed individuals.^13,14 Omental metastasis is a common occurrence in individuals diagnosed as having cancers of the ovary and colon as well as other peritoneal organs.^15,16 It is specifically immune aggregates to which metastasizing cells originally bind and subsequently divide.⁷ Tumor growth on the omentum is suggested to be a result of preferential binding to this site and the presence of factors that promote tumor growth.^7,17,18 After tumor formation on the omentum, tumor cells often further disseminate to other sites in the peritoneal cavity, as well as systemically, further propagating disease.Despite data demonstrating the immune capabilities of the omentum,^4,6 the omental immune response to tumor metastasis is relatively understudied. Limited work shows that after cells adhere to the omentum, the vasculature of omental immune aggregates is well-suited to supporting rapid tumor growth. Under normal conditions, the vasculature of omental immune aggregates exhibits a phenotype that may be capable of rapid expansion after an immunologic stimulus, which is exploited by metastasizing tumor cells.⁷ Despite the abundance of immune cells present at the site of tumor growth, a productive immune response does not occur naturally, and tumors grow progressively.^3,19In an attempt to determine whether the omental immune microenvironment is capable of promoting antitumor responses, we immunized mice with lethally irradiated tumor cells. Because the omentum is the initial site of tumor cell binding, i.p. immunization with these lethally irradiated tumor cells allows us to target the omentum to potentially generate an antitumor immune response. Herein, we found that i.p. immunization with lethally irradiated tumor cells led to the production of an antitumor immune response that was effective in controlling the growth of both specific and unrelated tumors after a secondary challenge with viable tumor cells. The nonspecific antitumor response was unique to the peritoneal cavity and was sustained for ≥60 days after immunization. In addition, depletion of NK1.1⁺ cells reversed the protective effects elicited by immunization only when challenged with an unrelated tumor challenge. In contrast, depletion of NK1.1⁺ cells and conventional T-cell populations was required to reverse the protective effects against specific tumor challenge. Thus, activation of peritoneal NK1.1⁺ cells in addition to conventional T-cell populations may have potent antitumor capabilities that could be exploited to benefit patients therapeutically.     

Fatty Acid Ethyl Esters Are Less Toxic Than Their Parent Fatty Acids Generated during Acute Pancreatitis

Although ethanol causes acute pancreatitis (AP) and lipolytic fatty acid (FA) generation worsens AP, the contribution of ethanol metabolites of FAs, ie, FA ethyl esters (FAEEs), to AP outcomes is unclear. Previously, pancreata of dying alcoholics and pancreatic necrosis in severe AP, respectively, showed high FAEEs and FAs, with oleic acid (OA) and its ethyl esters being the most abundant. We thus compared the toxicities of FAEEs and their parent FAs in severe AP. Pancreatic acini and peripheral blood mononuclear cells were exposed to FAs or FAEEs in vitro. The triglyceride of OA (i.e., glyceryl tri-oleate) or OAEE was injected into the pancreatic ducts of rats, and local and systemic severities were studied. Unsaturated FAs at equimolar concentrations to FAEEs induced a larger increase in cytosolic calcium, mitochondrial depolarization, and necro-apoptotic cell death. Glyceryl tri-oleate but not OAEE resulted in 70% mortality with increased serum OA, a severe inflammatory response, worse pancreatic necrosis, and multisystem organ failure. Our data show that FAs are more likely to worsen AP than FAEEs. Our observations correlate well with the high pancreatic FAEE concentrations in alcoholics without pancreatitis and high FA concentrations in pancreatic necrosis. Thus, conversion of FAs to FAEE may ameliorate AP in alcoholics.Although fat necrosis has been associated with severe cases of pancreatitis for more than a century,^{1, 2} and alcohol consumption is a well-known risk factor for acute pancreatitis (AP),³ only recently have we started understanding the mechanistic basis of these observations.^{4, 5, 6, 7} High amounts of unsaturated fatty acids (UFAs) have been noted in the pancreatic necrosis and sera of severe AP (SAP) patients by multiple groups.^{8, 9, 10, 11, 12} These high UFAs seem pathogenically relevant because several studies show UFAs can cause pancreatic acinar injury or can worsen AP.^{11, 12, 13, 14} Ethanol may play a role in AP by distinct mechanisms,³ including a worse inflammatory response to cholecystokinin,⁴ increased zymogen activation,¹⁵ basolateral enzyme release,¹⁶ sensitization to stress,⁷ FA ethyl esters (FAEEs),¹⁷ cytosolic calcium,¹⁸ and cell death.¹⁹Because the nonoxidative ethanol metabolite of fatty acids (FAs), FAEEs, were first noted to be elevated in the pancreata of dying alcoholics, they have been thought to play a role in AP.^{17, 19, 20, 21, 22} Conclusive proof of the role of FAEEs in AP in comparison with their parent UFAs is lacking. Uncontrolled release of lipases into fat, whether in the pancreas or in the peritoneal cavity, may result in fat necrosis, UFA generation, which has been associated with SAP.^{11, 12} Pancreatic homogenates were also noted to have an ability to synthesize FAEEs from FAs and ethanol,^{20, 23} and the putative enzyme for this was thought to be a lipase.^{24, 25} It has been shown that the FAEE synthase activity of the putative enzyme exceeds its lipolytic capacity by several fold.²⁵Triglyceride (TG) forms >80% of the adipocyte mass,^{26, 27, 28} oleic acid (OA) being the most enriched FA.^{9, 29} We recently showed that lipolysis of intrapancreatic TG worsens pancreatitis.^{11, 12} Therefore, after noting the ability of the pancreas to cause lipolysis of TG into FAs and also to have high FAEE synthase activity and FAEE concentrations, we decided to compare the relative ability of FAEEs and their parent FAs to initiate deleterious signaling in pancreatitis and to investigate their impact on the severity of AP.     

Synergistic Actions of Blocking Angiopoietin-2 and Tumor Necrosis Factor-α in Suppressing Remodeling of Blood Vessels and Lymphatics in Airway Inflammation

Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response.Remodeling of blood vessels and lymphatics contributes to the pathophysiology of many chronic inflammatory diseases, including asthma, chronic bronchitis, chronic obstructive pulmonary disease, inflammatory bowel disease, and psoriasis.^{1, 2, 3} When inflammation is sustained, capillaries acquire venule-like properties that expand the sites of plasma leakage and leukocyte influx. Consistent with this transformation, the remodeled blood vessels express P-selectin, intercellular adhesion molecule 1 (ICAM-1), EphB4, and other venular markers.^{4, 5, 6} The changes are accompanied by remodeling of pericytes and disruption of pericyte-endothelial crosstalk involved in blood vessel quiescence.⁷ Remodeling of blood vessels is accompanied by plasma leakage, inflammatory cell influx, and sprouting lymphangiogenesis.^{6, 8, 9}Mycoplasma pulmonis infection causes sustained inflammation of the respiratory tract of rodents.¹⁰ This infection has proved useful for dissecting the features and mechanisms of vascular remodeling and lymphangiogenesis.^{6, 9, 10} At 7 days after infection, there is widespread conversion of capillaries into venules, pericyte remodeling, inflammatory cell influx, and lymphatic vessel sprouting in the airways and lung.^{4, 5, 6, 7, 8, 9} Many features of chronic M. pulmonis infection in mice are similar to Mycoplasma pneumoniae infection in humans.¹¹Angiopoietin-2 (Ang2) is a context-dependent antagonist of Tie2 receptors^{12, 13} that is important for prenatal and postnatal remodeling of blood vessels and lymphatic vessels.^{13, 14, 15} Ang2 promotes vascular remodeling,^{4, 5} lymphangiogenesis,^{15, 16, 17} and pericyte loss¹⁸ in disease models in mice. Mice genetically lacking Ang2 have less angiogenesis, lymphangiogenesis, and neutrophil recruitment in inflammatory bowel disease.³ Ang2 has proved useful as a plasma biomarker of endothelial cell activation in acute lung injury, sepsis, hypoxia, and cancer.¹⁹Like Ang2, tumor necrosis factor (TNF)-α is a mediator of remodeling of blood vessels and lymphatics.^{8, 9, 20, 21} TNF triggers many components of the inflammatory response, including up-regulation of expression of vascular cell adhesion molecule-1, ICAM-1, and other endothelial cell adhesion molecules.²² TNF inhibitors reduce inflammation in mouse models of inflammatory disease^{23, 24} and are used clinically in the treatment of rheumatoid arthritis, ankylosing spondylitis, Crohn''s disease, psoriatic arthritis, and some other inflammatory conditions.^{24, 25} Indicative of the complex role of TNF in disease, inhibition or deletion of TNF can increase the risk of serious infection by bacterial, mycobacterial, fungal, viral, and other opportunistic pathogens.²⁶TNF and Ang2 interact in inflammatory responses. TNF increases Ang2 expression in endothelial cells in a time- and dose-dependent manner, both in blood vessels²⁷ and lymphatics.¹⁶ Administration of TNF with Ang2 increases cell adhesion molecule expression more than TNF alone.^{16, 28} Similarly, Ang2 can promote corneal angiogenesis in the presence of TNF, but not alone.²⁹ In mice that lack Ang2, TNF induces leukocyte rolling but not adherence to the endothelium.²⁸ Ang2 also augments TNF production by macrophages.^{30, 31} Inhibition of Ang2 and TNF together with a bispecific antibody can ameliorate rheumatoid arthritis in a mouse model.³²With this background, we sought to determine whether Ang2 and TNF act together to drive the remodeling of blood vessels and lymphatics in the initial inflammatory response to M. pulmonis infection. In particular, we asked whether Ang2 and TNF have synergistic actions in this setting. The approach was to compare the effects of selective inhibition of Ang2 or TNF, individually or together, and then assess the severity of vascular remodeling, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic sprouting. Functional consequences of genome-wide changes in gene expression were analyzed by Ingenuity Pathway Analysis (IPA)^{33, 34} and the Database for Annotation, Visualization and Integrated Discovery (DAVID).³⁵ The studies revealed that inhibition of Ang2 and TNF together, but not individually, completely prevented the development of vascular remodeling and lymphatic sprouting and had synergistic effects in suppressing gene expression and cellular pathways activated during the initial stage of the inflammatory response.     

Survivin Blockade Sensitizes Rhabdomyosarcoma Cells for Lysis by Fetal Acetylcholine Receptor–Redirected T Cells

Cellular immunotherapy may provide a strategy to overcome the poor prognosis of metastatic and recurrent rhabdomyosarcoma (RMS) under the current regimen of polychemotherapy. Because little is known about resistance mechanisms of RMS to cytotoxic T cells, we investigated RMS cell lines and biopsy specimens for expression and function of immune costimulatory receptors and anti-apoptotic molecules by RT-PCR, Western blot analysis, IHC, and cytotoxicity assays using siRNA or transfection-modified RMS cell lines, together with engineered RMS-directed cytotoxic T cells specific for the fetal acetylcholine receptor. We found that costimulatory CD80 and CD86 were consistently absent from all RMSs tested, whereas inducible T-cell co-stimulator ligand (ICOS-L; alias B7H2) was expressed by a subset of RMSs and was inducible by tumor necrosis factor α in two of five RMS cell lines. Anti-apoptotic survivin, along with other inhibitor of apoptosis (IAP) family members (cIAP1, cIAP2, and X-linked inhibitor of apoptosis protein), was overexpressed by RMS cell lines and biopsy specimens. Down-regulation of survivin by siRNA or pharmacologically in RMS cells increased their susceptibility toward a T-cell attack, whereas induction of ICOS-L did not. Treatment of RMS-bearing Rag^−/− mice with fetal acetylcholine receptor–specific chimeric T cells delayed xenograft growth; however, this happened without definitive tumor eradication. Combined blockade of survivin and application of chimeric T cells in vivo suppressed tumor proliferation during survivin inhibition. In conclusion, survivin blockade provides a strategy to sensitize RMS cells for T-cell–based therapy.Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue malignancy. Although the cure rates increased from 25% in 1970 to 70% in 1990, based on multimodal approaches with chemotherapy, surgery, and irradiation, no further improvement has been achieved during the past 20 years. In addition, patients with primary metastatic and recurrent disease, particularly those with alveolar RMS, have an extremely poor prognosis (<20% cure rate).^{1, 2} Therefore, new therapeutic approaches are urgently needed. Immunotherapies provide alternative approaches, the most promising of which are vaccination toward tumor antigens^{3, 4} and adoptive transfer of redirected cytotoxic T lymphocytes with engineered specificity provided by a chimeric antigen receptor (CAR).⁵Vaccination against RMS is tested in clinical trials using RMS-specific neopeptide or peptides from broadly expressed tumor antigens, such as WT1.^{3, 4} Complex vaccination protocols are required to achieve efficacy, including the use of autologous T cells, peptide-pulsed dendritic cells, and cytokines to maintain survival of RMS-specific T cells in vivo.³ A variety of factors, however, affect the vaccination efficiency, including the expression levels of major histocompatibility complex classes I and II and costimulatory and co-inhibitory molecules on tumor cells.^{6, 7, 8} The frequency of natural precursors of tumor-reactive T cells, moreover, varies widely in different patients and may be too low to achieve therapeutic efficacy.⁶An alternative strategy is the adoptive transfer of chimeric T cells that are genetically engineered with a CAR with predefined specificity. Chimeric T cells are redirected in an antibody-based, major histocompatibility complex–independent manner toward predefined cell surface targets. Because CD80 costimulation is indispensable for full T-cell activation, amplification, and long-term survival, the CD28 signaling domain was added to the CD3ζ signal in the CAR to sustain the CAR-redirected T-cell response in the long-term.^{9, 10, 11} CAR-redirected T cells are explored in clinical trials, with significant success.¹² The CAR used in the current study is directed against the fetal acetylcholine receptor (fAChR)^{13, 14, 15, 16} that is suitable for targeting RMS because it is expressed on various RMS subtypes but not on postnatal muscle or other relevant healthy tissue cells.^{15, 17}The cytolytic efficacy of adoptively transferred cytotoxic T cells can be affected by anti-apoptotic protection of the target cell; granzyme-mediated mitochondrial release of pro-apoptotic smac is blocked in some tumor cells, protecting from cell death by cytotoxic T lymphocytes.¹⁸ Similarly, overexpression of X-linked inhibitor of apoptosis protein (XIAP) can block execution of T-cell–mediated cytolysis, which can be overcome by phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1)-mediated enhancement of mitochondrial second mitochondria-derived activator of caspase release.¹⁹ Survivin, another IAP family member, stabilizes high XIAP levels in malignant cells, thereby contributing to apoptosis resistance.^{20, 21} Consistently, we observed that lysis of RMS cells by RMS-directed cytotoxic T cells was unexpectedly poor,²² suggesting that some resistance mechanisms may protect RMS cells.Herein, we report that RMS resistance to a cytolytic T-cell attack is the result of at least two mechanisms: lack of costimulatory molecules required for sustained T-cell activation and overexpression of anti-apoptotic molecules. Among these, survivin became crucial because survivin repression by siRNA or blockade by pharmacological interference substantially increased the susceptibility of RMS cells to a cytolytic T-cell attack, implying survivin as a key target to improve RMS sensitivity for adoptive immunotherapy.     

Role of Collagen Matrix in Tumor Angiogenesis and Glioblastoma Multiforme Progression

Glioblastoma is a highly vascularized brain tumor, and antiangiogenic therapy improves its progression-free survival. However, current antiangiogenic therapy induces serious adverse effects including neuronal cytotoxicity and tumor invasiveness and resistance to therapy. Although it has been suggested that the physical microenvironment has a key role in tumor angiogenesis and progression, the mechanism by which physical properties of extracellular matrix control tumor angiogenesis and glioblastoma progression is not completely understood. Herein we show that physical compaction (the process in which cells gather and pack together and cause associated changes in cell shape and size) of human glioblastoma cell lines U87MG, U251, and LN229 induces expression of collagen types IV and VI and the collagen crosslinking enzyme lysyl oxidase and up-regulates in vitro expression of the angiogenic factor vascular endothelial growth factor. The lysyl oxidase inhibitor β-aminopropionitrile disrupts collagen structure in the tumor and inhibits tumor angiogenesis and glioblastoma multiforme growth in a mouse orthotopic brain tumor model. Similarly, d-penicillamine, which inhibits lysyl oxidase enzymatic activity by depleting intracerebral copper, also exhibits antiangiogenic effects on brain tumor growth in mice. These findings suggest that tumor microenvironment controlled by collagen structure is important in tumor angiogenesis and brain tumor progression.Glioblastoma multiforme (GBM), a highly malignant glioma that exhibits various glial lineages such as a mixed oligodendroglial–astrocytic phenotype,¹ is the most common primary brain tumor. Every year in the United States, GBM is diagnosed in about 8000 persons. Despite a great deal of effort to develop effective therapies targeting GBM, current approaches including surgery, radiotherapy, and chemotherapy have not been successful, with median survival of only 1 to 2 years.² Angiogenesis, the formation and development of new blood vessels, is an important step in tumor growth and metastasis.^3,4 Since GBM is a highly vascularized tumor and the presence of neovascularization is a key diagnostic criteria for GBM, antiangiogenic therapy is thought to represent a promising strategy for treatment of GBM.⁴ Indeed, the antivascular endothelial growth factor (VEGF) agent bevacizumab improves progression-free survival of GBM.⁵ However, antiangiogenic therapy for brain tumors potentially induces neuronal cytotoxicity, invasiveness of the tumor cells,^6,7 and resistance to therapy,^3–5,8,9 which diminish the benefits of current antiangiogenic therapy for GBM. Thus, to establish safe and efficient therapies that have long-term anti–tumor activity and fewer adverse effects, we need to understand the comprehensive mechanisms that govern brain tumor angiogenesis.Most of the work in tumor angiogenesis has been focused on identifying the genetic and chemical signals that control neovascularization.^3,10 Thus, most US Food and Drug Administration (FDA)–approved angiogenesis inhibitors target soluble angiogenic factors such as VEGF.^5,8,9,11 These chemical signaling cascades are clearly necessary for tumor growth and vascular development; however, the insoluble extracellular matrix (ECM) and mechanical forces have equally important roles in tumor angiogenesis and progression.^12–19 Cell shape controls the growth of capillary endothelial cells,²⁰ and mechanical forces such as matrix stiffness control cell migration, angiogenesis, tumor progression, and metastasis, both in vitro and in vivo.^15,21–23Normal brain ECM is composed of hyaluronan, proteoglycans, and tenascin-C and is devoid of rigid ECM structures formed by fibrillar collagens,²⁴ whereas ECM in GBM is associated with a large increase in components such as collagens, laminin, and fibronectin,^25,26 primarily in the basement membrane of the blood vessels, which suggests that modifying these tumor-specific ECM components could be a potentially promising strategy for treatment of brain tumors.Physical compaction is the process in which cells gather and pack together, causing associated changes in cell shape and size. Mesenchymal condensation, in which dispersed mesenchymal cells are tightly packed together, results in differentiation of these cells into specific tissue types that occur during early development of various organs (eg, tooth, cartilage, bone, muscle, tendon, kidney, and lung) in mice.^27,28 During the course of mesenchymal condensation, cell shape is changed, cell size is decreased, and cell density is increased, which results in changes in mechanical and chemical signaling in the cells and dictates various cellular behaviors such as proliferation, migration, and cell fate determination that are critical for organ-specific morphogenesis. Physical compaction of the mesenchyme during early organ development increases collagen expression and governs subsequent organogenesis.²⁹ In addition, changes in cell shape control cell growth through the Rho signaling pathway in capillary endothelial cells,²⁰ and mechanical forces elicited by ECM stiffness, which also change cell shape and size, control angiogenesis and vascular function.^22,30 Proliferating cancer cells in a confined space undergo compressive stress,^16,31 and physically compacted cancer cells exhibit aggressive phenotypes.^32,33 For example, rapidly growing mammary tumor cells in confined spaces become smaller and packed (ie, physically compacted),³² which affects tumor cell behavior and collagen VI expression.³⁴ Therefore, in addition to soluble factors, mechanical forces such as physical compaction of the cells and subsequent changes in expression and structure of collagen may contribute to tumor angiogenesis and GBM progression. However, the role of physical properties of the tumor microenvironment on brain tumor angiogenesis and progression has not been well elucidated.The present study was initiated to examine whether tumor cell compaction and associated changes in cell shape and density control collagen and angiogenic factor expression using in vitro experimental methods (ie, microcontact printing system and mechanical compressor) (Supplemental Figure S1). We have previously used these methods to examine the effects of physical compaction in normal development,²⁹ in which we could precisely change cell size, shape, and density to mimic physical compaction in vitro. We also altered collagen expression and structure by changing the crosslinking ability of collagens and examined whether it controls brain tumor angiogenesis and tumor growth in vivo. Compaction of GBM cells changes collagen expression and structure, resulting in increased VEGF expression in vitro. Inhibition of collagen crosslinking attenuated the effects of physical compaction of GBM cells on tumor angiogenesis and progression of GBM in a mouse orthotopic brain tumor model. Because this novel approach targets tumor-specific ECM structures, it may lead to development of more stable and efficient antiangiogenic therapy for GBM and other types of hypervascular brain tumors.     

Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas

Cholangiocarcinomas (CCAs) comprise a mucin-secreting form, intrahepatic or perihilar, and a mixed form located peripherally. We characterized cancer stem cells (CSCs) in CCA subtypes and evaluated their cancerogenic potential. CSC markers were investigated in 25 human CCAs in primary cultures and established cell lines. Tumorigenic potential was evaluated in vitro or in xenografted mice after s.c. or intrahepatic injection in normal and cirrhotic (carbon tetrachloride-induced) mice. CSCs comprised more than 30% of the tumor mass. Although the CSC profile was similar between mucin-intrahepatic and mucin-perihilar subtypes, CD13⁺ CSCs characterized mixed-intrahepatic, whereas LGR5⁺ characterized mucin-CCA subtypes. Many neoplastic cells expressed epithelial-mesenchymal transition markers and coexpressed mesenchymal and epithelial markers. In primary cultures, epithelial-mesenchymal transition markers, mesenchymal markers (vimentin, CD90), and CD13 largely predominated over epithelial markers (CD133, EpCAM, and LGR5). In vitro, CSCs expressing epithelial markers formed a higher number of spheroids than CD13⁺ or CD90⁺ CSCs. In s.c. tumor xenografts, tumors dominated by stromal markers were formed primarily by CD90⁺ and CD13⁺ cells. By contrast, in intrahepatic xenografts in cirrhotic livers, tumors were dominated by epithelial traits reproducing the original human CCAs. In conclusion, CSCs were rich in human CCAs, implicating CCAs as stem cell–based diseases. CSC subpopulations generate different types of cancers depending on the microenvironment. Remarkably, CSCs reproduce the original human CCAs when injected into cirrhotic livers.Cholangiocarcinoma (CCA) is the second most common primary hepatic malignancy and arises from the neoplastic transformation of cells in the cholangiocytic lineage.¹ CCA is associated with a very bad prognosis with virtually no response to current chemotherapeutics or radiation therapies.¹ CCA is classified as intrahepatic (IHCCA), perihilar (pCCA), or distal, characterized by significant differences in terms of epidemiology, pathobiology, and molecular biology.¹ Recent studies reveal that IHCCA comprises two different forms: mucin-IHCCA constituted by pure mucin-secreting cells and displaying similarities with pCCA, and mixed-IHCCA comprising areas of hepatocytic differentiation and neoplastic ductular reaction.²The cancer stem cell (CSC) hypothesis has been validated recently by the identification of a subpopulation of self-renewing stem cells that give rise to maturational lineages with a hierarchical organization and are able to divide symmetrically and asymmetrically to generate the tumor mass.^3,4 CSCs, also referred to as tumor-initiating cells or tumor-propagating cells, are tumorigenic, metastatic, resistant to chemoradio therapies, and responsible for tumor recurrence.^3,4 For all these reasons, CSCs represent a primary therapeutic target.^3,4Recently, several CSC markers have been reported in human CCA, including CD133,⁵ epithelial cell adhesion molecule (EpCAM),⁶ CD44,⁷ CD13,⁸ and CD90.⁹ In addition, most cells in human CCAs have been demonstrated to coexpress cytokeratin (K)19 and albumin, a feature characterizing hepatobiliary stem/progenitor cells.¹⁰ Recent reports support further investigations on the role of CSCs in CCA. Unfortunately, very little information exists with respect to CSCs in CCA and its subtypes.Our aim was to analyze CSCs in different human CCA subtypes, primary cultures obtained from human CCA, and established CCA cell lines.     

p120-Catenin Expressed in Alveolar Type II Cells Is Essential for the Regulation of Lung Innate Immune Response

The integrity of the lung alveolar epithelial barrier is required for the gas exchange and is important for immune regulation. Alveolar epithelial barrier is composed of flat type I cells, which make up approximately 95% of the gas-exchange surface, and cuboidal type II cells, which secrete surfactants and modulate lung immunity. p120-catenin (p120; gene symbol CTNND1) is an important component of adherens junctions of epithelial cells; however, its function in lung alveolar epithelial barrier has not been addressed in genetic models. Here, we created an inducible type II cell–specific p120-knockout mouse (p120EKO). The mutant lungs showed chronic inflammation, and the alveolar epithelial barrier was leaky to ¹²⁵I-albumin tracer compared to wild type. The mutant lungs also demonstrated marked infiltration of inflammatory cells and activation of NF-κB. Intracellular adhesion molecule 1, Toll-like receptor 4, and macrophage inflammatory protein 2 were all up-regulated. p120EKO lungs showed increased expression of the surfactant proteins Sp-B, Sp-C, and Sp-D, and displayed severe inflammation after pneumonia caused by Pseudomonas aeruginosa compared with wild type. In p120-deficient type II cell monolayers, we observed reduced transepithelial resistance compared to control, consistent with formation of defective adherens junctions. Thus, although type II cells constitute only 5% of the alveolar surface area, p120 expressed in these cells plays a critical role in regulating the innate immunity of the entire lung.Lungs are constantly exposed to pathogens; therefore, a highly restrictive alveolar epithelial barrier and finely tuned host defense mechanisms are indispensable for their protection.^1,2 Unchecked inflammation is linked to various acute and chronic diseases, including edema, acute respiratory distress syndrome, and fibrosis.^3,4 Although it is abundantly clear that the alveolar epithelial barrier regulates the transport of gases, liquid, and ions,^5,6 the role of the barrier in the regulation of the innate immune function of lungs remains poorly understood.The restrictiveness of the alveolar epithelial barrier is dependent on a series of interacting proteins comprising the adherens junctions (AJs) and tight junctions (TJs).^7,8 The core of the epithelial AJs is composed of E-cadherin, which links cells to one another in the monolayer.⁹ The cytoplasmic domain of E-cadherin associates with α-catenin, β-catenin, and p120-catenin (p120, official name catenin delta 1; CTNND1).⁹ The α- and β-catenins can recruit proteins that link E-cadherin to the actin cytoskeleton,⁹ and together, these interactions maintain the tension landscape in the epithelial monolayer.¹⁰ β-Catenin also plays an essential role in the Wnt signaling pathway and thereby contributes to cell proliferation and differentiation.¹¹ However, p120 has received comparatively less attention, although recent studies have shown that p120 has important functions in regulating cadherin stability and turnover¹² and innate immunity.¹³Here, we focused on the role of p120 expressed in alveolar epithelial type II cells in regulating the innate immune function of lungs. Although alveolar type II cells cover only 5% of the alveolar surface area, these cells are metabolically active.¹⁴ They produce surfactants, serve as facultative progenitor cells to repair alveolar injury, and regulate innate immune function of the lung.¹⁴ These cells express Toll-like receptors (TLRs) and tumor necrosis factor receptors.¹⁵ Interactions with pathogens or endotoxins activate these receptors to initiate NF-κB signaling to produce tumor necrosis factor,¹⁶ IL-1 and IL-6,¹⁶ regulated on activation normal T cell expressed and secreted,¹⁷ and chemokine C-X-C motif ligand 1.¹⁸ These factors play key roles in recruiting inflammatory cells.^19–21 Alveolar type II cells also secrete the surfactant proteins (Sp)-A, -B, -C, and -D,²² which regulate innate and adaptive immunity by binding to antigen through interactions with surface receptors on inflammatory cell membranes.²³ Here, we studied the function of p120 through disrupting the p120 gene in alveolar type II cells in mice using the rtTA/TetO system coupled with a type II cell–specific SPC promoter. In these mice, we observed unchecked chronic lung inflammation associated with increased NF-κB activity and a persistently leaky alveolar epithelial barrier. These results provide the first genetic evidence that p120 in type II cells is a central regulator of innate immunity of lungs.     

Dipeptidyl Peptidase 4 Distribution in the Human Respiratory Tract: Implications for the Middle East Respiratory Syndrome

Dipeptidyl peptidase 4 (DPP4, CD26), a type II transmembrane ectopeptidase, is the receptor for the Middle Eastern respiratory syndrome coronavirus (MERS-CoV). MERS emerged in 2012 and has a high mortality associated with severe lung disease. A lack of autopsy studies from MERS fatalities has hindered understanding of MERS-CoV pathogenesis. We investigated the spatial and cellular localization of DPP4 to evaluate an association MERS clinical disease. DPP4 was rarely detected in the surface epithelium from nasal cavity to conducting airways with a slightly increased incidence in distal airways. DPP4 was also found in a subset of mononuclear leukocytes and in serous cells of submucosal glands. In the parenchyma, DPP4 was found principally in type I and II cells and alveolar macrophages and was also detected in vascular endothelium (eg, lymphatics) and pleural mesothelia. Patients with chronic lung disease, such as chronic obstructive pulmonary disease and cystic fibrosis, exhibited increased DPP4 immunostaining in alveolar epithelia (type I and II cells) and alveolar macrophages with similar trends in reactive mesothelia. This finding suggests that preexisting pulmonary disease could increase MERS-CoV receptor abundance and predispose individuals to MERS morbidity and mortality, which is consistent with current clinical observations. We speculate that the preferential spatial localization of DPP4 in alveolar regions may explain why MERS is characterized by lower respiratory tract disease.Middle East respiratory syndrome (MERS) was recognized as a significant illness on the Saudi Arabian peninsula in mid-2012, and the causative agent was rapidly identified as a novel coronavirus (CoV)—MERS-CoV.¹ Since its emergence, the World Health Organization has been notified of 1542 laboratory-confirmed cases of MERS-CoV infection in >2 dozen countries, resulting in at least 544 related deaths (http://www.who.int/emergencies/mers-cov/en; last accessed September 12, 2015). Available data indicate that men are more commonly infected than women, with a median age of 47 years.^{2, 3, 4} Although human-to-human or zoonotic spread of MERS has not reached epidemic or pandemic levels, its potential to spread among individuals was found in health care settings in the Middle East⁵ and by the recent outbreak in South Korea caused by a single infected individual.⁶Most fatal MERS cases have occurred in individuals 60 years or older, frequently associated with significant comorbidities, such as obesity, renal or cardiac disease, diabetes, lung disease, or immunocompromise.⁷ Severely affected individuals have manifested significant respiratory symptoms, including cough, fever, dyspnea, and chest pain.^{2, 3, 4} Many seriously ill patients have progressed to respiratory failure and required ventilatory support. These patients exhibited dense airspace and interstitial lesions on chest radiography and computed tomography.^{1, 3, 8} In addition to the pulmonary manifestations, other reported problems in seriously ill patients include hyperkalemia, disseminated intravascular coagulopathy, pericardial effusion, central nervous system manifestations,⁹ and multiorgan failure.^{2, 3, 4} To date, a lack of autopsy pathology data from patients who have died of MERS has hindered understanding of disease pathogenesis.Epidemiologic studies have established that MERS is zoonotic in origin, with evidence of a closely related virus in dromedary camels on the Arabian peninsula and throughout Africa.^{10, 11, 12} Spread from camels to humans is documented,¹³ as well as person-to-person spread among health care workers in hospital settings.⁵ Unlike the ‘super spreader’ cases described with SARS-CoV,^{14, 15} the spread of MERS-CoV from person-to-person is inefficient, but this could change with virus evolution.^{16, 17} MERS-CoV has also been detected in individuals with mild, influenza-like illnesses, those with a dengue-like illness, and those without obvious disease signs or symptoms,^{18, 19, 20, 21} suggesting that there may be a larger disease burden than currently recognized.Shortly after MERS-CoV was discovered, its cellular receptor, dipeptidyl peptidase 4 (DPP4, CD26), was identified.²² The structural residues comprising the receptor-binding domain have been defined by co-crystallization of the MERS-CoV spike glycoprotein and DPP4.²³ DPP4 is a single-pass type II transmembrane glycoprotein with a short N-terminal cytoplasmic tail. The native protein is a homodimer. DPP4 cleaves X-proline dipeptides from N-terminus of polypeptides and in doing so may functionally modify many substrates, including growth factors, neuropeptides, cytokines, chemokines, and vasoactive peptides.²⁴DPP4 is expressed in many tissues and cell types, including kidney, intestine, liver, thymocytes, and several cells of hematopoietic lineage.²⁴ DPP4 expression is increased on activation of T, B, and natural killer cells and is considered a marker of functional activation.²⁴ DPP4 is also shed from the surface of many cell types and is present in soluble forms in plasma.²⁵ Although there are limited reports describing aspects of DPP4 expression in animal and human tissues and cell types,^{25, 26, 27} there has been no comprehensive survey of its cellular expression in the human respiratory tract. We localize DPP4 expression in normal and diseased human respiratory tissues to identify the pulmonary cell types that may be susceptible to MERS-CoV infection and thereby obtain insight into MERS pathogenesis.