AQP1 in the Gastrointestinal Tract of Mice: Expression Pattern and Impact of AQP1 Knockout on Colonic Function.

Aquaporin 1 (AQP1) is one of thirteen known mammalian aquaporins. Its main function is the transport of water across cell membranes. Lately, a role of AQP has been attributed to other physiological and pathological functions including cell migration and peripheral pain perception. AQP1 has been found in several parts of the enteric nervous system, e.g., in the rat ileum and in the ovine duodenum. Its function in the intestine appears to be multifaceted and is still not completely understood. The aim of the study was to analyze the distribution and localization of AQP1 in the entire intestinal tract of mice. AQP1 expression was correlated with the hypoxic expression profile of the various intestinal segments, intestinal wall thickness and edema, as well as other aspects of colon function including the ability of mice to concentrate stools and their microbiome composition. AQP1 was found in a specific pattern in the serosa, the mucosa, and the enteric nervous system throughout the gastrointestinal tract. The highest amount of AQP1 in the gastrointestinal tract was found in the small intestine. AQP1 expression correlated with the expression profiles of hypoxia-dependent proteins such as HIF-1α and PGK1. Loss of AQP1 through knockout of AQP1 in these mice led to a reduced amount of bacteroidetes and firmicutes but an increased amount of the rest of the phyla, especially deferribacteres, proteobacteria, and verrucomicrobia. Although AQP-KO mice retained gastrointestinal function, distinct changes regarding the anatomy of the intestinal wall including intestinal wall thickness and edema were observed. Loss of AQP1 might interfere with the ability of the mice to concentrate their stool and it is associated with a significantly different composition of the of the bacterial stool microbiome.

Perfusion-Based Bioreactor Culture and Isothermal Microcalorimetry for Preclinical Drug Testing with the Carbonic Anhydrase Inhibitor SLC-0111 in Patient-Derived Neuroblastoma.

Neuroblastoma is a rare disease. Rare are also the possibilities to test new therapeutic options for neuroblastoma in clinical trials. Despite the constant need to improve therapy and outcomes for patients with advanced neuroblastoma, clinical trials currently only allow for testing few substances in even fewer patients. This increases the need to improve and advance preclinical models for neuroblastoma to preselect favorable candidates for novel therapeutics. Here we propose the use of a new patient-derived 3D slice-culture perfusion-based 3D model in combination with rapid treatment evaluation using isothermal microcalorimetry exemplified with treatment with the novel carbonic anhydrase IX and XII (CAIX/CAXII) inhibitor SLC-0111. Patient samples showed a CAIX expression of 18% and a CAXII expression of 30%. Corresponding with their respective CAIX expression patterns, the viability of SH-EP cells was significantly reduced upon treatment with SLC-0111, while LAN1 cells were not affected. The inhibitory effect on SH-SY5Y cells was dependent on the induction of CAIX expression under hypoxia. These findings corresponded to thermogenesis of the cells. Patient-derived organotypic slice cultures were treated with SLC-0111, which was highly effective despite heterogeneity of CAIX/CAXII expression. Thermogenesis, in congruence with the findings of the histological observations, was significantly reduced in SLC-0111-treated samples. In order to extend the evaluation time, we established a perfusion-based approach for neuroblastoma tissue in a 3D perfusion-based bioreactor system. Using this system, excellent tissue quality with intact tumor cells and stromal structure in neuroblastoma tumors can be maintained for 7 days. The system was successfully used for consecutive drug response monitoring with isothermal microcalorimetry. The described approach for drug testing, relying on an advanced 3D culture system combined with a rapid and highly sensitive metabolic assessment, can facilitate development of personalized treatment strategies for neuroblastoma.

Increased Proliferation of Neuroblastoma Cells under Fructose Metabolism Can Be Measured by Isothermal Microcalorimetry.

Neuroblastoma, like other cancer types, has an increased need for energy. This results in an increased thermogenic profile of the cells. How tumor cells optimize their energy efficiency has been discussed since Warburg described the fact that tumor cells prefer an anaerobic to an aerobic metabolism in the 1920s. An important question is how far the energy efficiency is influenced by the substrate. The aim of this study was to investigate how the metabolic activity of neuroblastoma cells is stimulated by addition of glucose or fructose to the medium and if this can be measured accurately by using isothermal microcalorimetry. Proliferation of Kelly and SH-EP Tet-21/N cells was determined in normal medium, in fructose-enriched, in glucose-enriched and in a fructose/glucose-enriched environment. Heat development of cells was measured by isothermal microcalorimetry. The addition of fructose, glucose or both to the medium led to increases in the metabolic activity of the cells, resulting in increased proliferation under the influence of fructose. These changes were reflected in an enhanced thermogenic profile, mirroring the results of the proliferation assay. The tested neuroblastoma cells prefer fructose metabolism over glucose metabolism, a quality that provides them with a survival benefit under unfavorable low oxygen and low nutrient supply when fructose is available. This can be quantified by measuring thermogenesis.

Metastatic Esophageal Carcinoma Cells Exhibit Reduced Adhesion Strength and Enhanced Thermogenesis.

Despite continuous improvements in multimodal therapeutic strategies, esophageal carcinoma maintains a high mortality rate. Metastases are a major life-limiting component; however, very little is known about why some tumors have high metastatic potential and others not. In this study, we investigated thermogenic activity and adhesion strength of primary tumor cells and corresponding metastatic cell lines derived from two patients with metastatic adenocarcinoma of the esophagus. We hypothesized that the increased metastatic potential of the metastatic cell lines correlates with higher thermogenic activity and decreased adhesion strength. Our data show that patient-derived metastatic esophageal tumor cells have a higher thermogenic profile as well as a decreased adhesion strength compared to their corresponding primary tumor cells. Using two paired esophageal carcinoma cell lines of primary tumor and lymph nodes makes the data unique. Both higher specific thermogenesis profile and decreased adhesion strength are associated with a higher metastatic potential. They are in congruence with the clinical patient presentation. Understanding these functional, biophysical properties of patient derived esophageal carcinoma cell lines will enable us to gain further insight into the mechanisms of metastatic potential of primary tumors and metastases. Microcalorimetric evaluation will furthermore allow for rapid assessment of new treatment options for primary tumor and metastases aimed at decreasing the metastatic potential.

Perspective on Similarities and Possible Overlaps of Congenital Disease Formation-Exemplified on a Case of Congenital Diaphragmatic Hernia and Neuroblastoma in a Neonate.

The coincidence of two rare diseases such as congenital diaphragmatic hernia (CDH) and neuroblastoma is exceptional. With an incidence of around 2-3:10,000 and 1:8000 for either disease occurring on its own, the chance of simultaneous presentation of both pathologies at birth is extremely low. Unfortunately, the underlying processes leading to congenital malformation and neonatal tumors are not yet thoroughly understood. There are several hypotheses revolving around the formation of CDH and neuroblastoma. The aim of our study was to put the respective hypotheses of disease formation as well as known factors in this process into perspective regarding their similarities and possible overlaps of congenital disease formation. We present the joint occurrence of these two rare diseases based on a patient presentation and immunochemical prognostic marker evaluation. The aim of this manuscript is to elucidate possible similarities in the pathogeneses of both disease entities. Discussed are the role of toxins, cell differentiation, the influence of retinoic acid and NMYC as well as of hypoxia. The detailed discussion reveals that some of the proposed pathophysiological mechanisms of both malformations have common aspects. Especially disturbances of the retinoic acid pathway and NMYC expression can influence and disrupt cell differentiation in either disease. Due to the rarity of both diseases, interdisciplinary efforts and multi-center studies are needed to investigate the reasons for congenital malformations and their interlinkage with neonatal tumor disease.

AQP1 Is Up-Regulated by Hypoxia and Leads to Increased Cell Water Permeability, Motility, and Migration in Neuroblastoma.

The water channel aquaporin 1 (AQP1) has been implicated in tumor progression and metastasis. It is hypothesized that AQP1 expression can facilitate the transmembrane water transport leading to changes in cell structure that promote migration. Its impact in neuroblastoma has not been addressed so far. The objectives of this study have been to determine whether AQP1 expression in neuroblastoma is dependent on hypoxia, to demonstrate whether AQP1 is functionally relevant for migration, and to further define AQP1-dependent properties of the migrating cells. This was determined by investigating the reaction of neuroblastoma cell lines, particularly SH-SY5Y, Kelly, SH-EP Tet-21/N and SK-N-BE(2)-M17 to hypoxia, quantitating the AQP1-related water permeability by stopped-flow spectroscopy, and studying the migration-related properties of the cells in a modified transwell assay. We find that AQP1 expression in neuroblastoma cells is up-regulated by hypoxic conditions, and that increased AQP1 expression enabled the cells to form a phenotype which is associated with migratory properties and increased cell agility. This suggests that the hypoxic tumor microenvironment is the trigger for some tumor cells to transition to a migratory phenotype. We demonstrate that migrating tumor cell express elevated AQP1 levels and a hypoxic biochemical phenotype. Our experiments strongly suggest that elevated AQP1 might be a key driver in transitioning stable tumor cells to migrating tumor cells in a hypoxic microenvironment.

AQP1-Driven Migration Is Independent of Other Known Adverse Factors but Requires a Hypoxic Undifferentiated Cell Profile in Neuroblastoma.

Neuroblastoma is a biologically very heterogeneous tumor with its clinical manifestation ranging from spontaneous regression to highly aggressive metastatic disease. Several adverse factors have been linked to oncogenesis, tumor progression and metastases of neuroblastoma including NMYC amplification, the neural adhesion molecule NCAM, as well as CXCR4 as a promoter of metastases. In this study, we investigate to what extent the expression of AQP1 in neuroblastoma correlates with changing cellular factors such as the hypoxic status, differentiation, expression of known adverse factors such as NMYC and NCAM, and CXCR4-related metastatic spread. Our results show that while AQP1 expression leads to an increased migratory behavior of neuroblastoma cells under hypoxic conditions, we find that hypoxia is associated with a reduction of NMYC in the same cells. A similar effect can be observed when using the tetracycline driven mechanism of SH-EP/Tet cells. When NMYC is not expressed, the expression of AQP1 is increased together with an increased expression of HIF-1α and HIF-2α. We furthermore show that when growing cells in different cell densities, they express AQP1, HIF-1α, HIF-2α, NMYC and NCAM to different degrees. AQP1 expression correlates with a hypoxic profile of these cells with increased HIF-1α and HIF-2α expression, as well as with NMYC and NCAM expression in two out of three neuroblastoma cell lines. When investigating cell properties of the cells that actually migrate, we find that the increased APQ1 expression in the migrated cells correlates with an increased NMYC and NCAM expression again in two out of three cell lines. Expression of the tumor cell homing marker CXCR4 varies between different tumor areas and between cell lines. While some migrated tumor cells highly express CXCR4, cells of other origin do not. In the initial phase of migration, we determined a dominant role of AQP1 expression of migrating cells in the scratch assay.

Correction: The lipogenic LXR-SREBF1 signaling pathway controls cancer cell DNA repair and apoptosis and is a vulnerable point of malignant tumors for cancer therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The lipogenic LXR-SREBF1 signaling pathway controls cancer cell DNA repair and apoptosis and is a vulnerable point of malignant tumors for cancer therapy.

Cancer cells are defective in DNA repair, so they experience increased DNA strand breaks, genome instability, gene mutagenesis, and tumorigenicity; however, multiple classic DNA repair genes and pathways are strongly activated in malignant tumor cells to compensate for the DNA repair deficiency and gain an apoptosis resistance. The mechanisms underlying this phenomenon in cancer are unclear. We speculate that a key DNA repair gene or signaling pathway in cancer has not yet been recognized. Here, we show that the lipogenic liver X receptor (LXR)-sterol response element binding factor-1 (SREBF1) axis controls the transcription of a key DNA repair gene polynucleotide kinase/phosphatase (PNKP), thereby governing cancer cell DNA repair and apoptosis. Notably, the PNKP levels were significantly reduced in 95% of human pancreatic cancer (PC) patients, particularly deep reduction for sixfold in all of the advanced-stage PC cases. PNKP is also deficient in three other types of cancer that we examined. In addition, the expression of LXRs and SREBF1 was significantly reduced in the tumor tissues from human PC patients compared with the adjacent normal tissues. The newly identified LXR-SREBF1-PNKP signaling pathway is deficient in PC, and the defect in the pathway contributes to the DNA repair deficiency in the cancer. Strikingly, further diminution of the vulnerable LXR-SREBF1-PNKP signaling pathway using a small molecule triptonide, a new LXR antagonist identified in this investigation, at a concentration of 8 nM robustly activated tumor-suppressor p53 and readily elevated cancer cell DNA strand breaks over an apoptotic threshold, and selectively induced PC cell apoptosis, resulting in almost complete elimination of tumors in xenograft mice without obvious complications. Our findings provide new insight into DNA repair and apoptosis in cancer, and offer a new platform for developing novel anticancer therapeutics.

Atorvastatin enhances endothelial adherens junctions through promoting VE-PTP gene transcription and reducing VE-cadherin-Y731 phosphorylation.

Vascular endothelial protein tyrosine phosphatase (VE-PTP) is essential for endothelial cells (ECs) adherens junction and vascular homeostasis; however, the regulatory mechanism of VE-PTP transcription is unknown, and a drug able to promote VE-PTP expression in ECs has not yet been reported in the literature. In this study, we used human ECs as a model to explore small molecule compounds able to promote VE-PTP expression, and found that atorvastatin, a HMG-CoA reductase inhibitor widely used in the clinic to treat hypercholesterolemia-related cardiovascular diseases, strongly promoted VE-PTP transcription in ECs through activating the VE-PTP promoter and upregulating the expression of the transcription factor, specificity protein 1 (SP1). Additionally, atorvastatin markedly reduced VE-cadherin-Y731 phosphorylation induced by cigarette smoke extract and significantly enhanced stability of endothelial adherens junctions. Together, our findings reveal that atorvastatin up-regulates VE-PTP expression, increases VE-cadherin protein levels, and decreases VE-cadherin-Y731 phosphorylation to strengthen EC adherens junctions and maintain vascular cell monolayer integrity, offering a new mechanism of atorvastatin against CSE-induced disruption of vascular integrity and relevant cardio-cerebrovascular disease.

Cigarette smoke extracts induce overexpression of the proto-oncogenic gene interleukin-13 receptor α2 through activation of the PKA-CREB signaling pathway to trigger malignant transformation of lung vascular endothelial cells and angiogenesis.

Cigarette smoking is a major cause of lung cancer. Tumor-associated endothelial cells (TAECs) play important roles in tumor angiogenesis and metastasis. However, whether cigarette smoking can trigger genesis of lung TAECs has not been reported yet. In the current study, we used lung endothelial cell (EC) lines as a model to study the pathological effect of cigarette smoke extracts (CSEs) on human lung ECs, and found that a lower dose of 4% CSEs obviously caused abnormal morphological changes in ECs, increased the permeability of endothelial monolayer, while a higher concentration of 8% CSEs caused EC apoptosis. Strikingly, CSEs induced a 117-fold overexpression of a pro-tumorigenic interleukin-13 receptor α2 gene (IL-13Rα2, also named as CT-19) through activation of the protein kinase A (PKA) and cAMP response element-binding protein (CREB) signaling pathway. A PKA specific inhibitor H89 completely abolished CSEs-induced IL-13Rα2 overexpression. The overexpression of IL-13Rα2 in lung ECs significantly increased the tumorigenic, migratory, and angiogenic capabilities of the cells, suggesting that IL-13Rα2 promotes genesis of lung TAECs. Together, our data show that CSEs activate the PKA, CREB, and IL-13Rα2 axis in lung ECs, and IL-13Rα2 promotes the malignant transformation of lung ECs and genesis of TAECs with robust angiogenic and oncogenic capabilities. Our study provides new insight into the mechanism of CSEs-triggered lung cancer angiogenesis and tumorigenesis, suggesting that the PKA-CREB-IL-13Rα2 axis is a potential target for novel anti-lung tumor angiogenesis and anti-lung cancer drug discovery.

Thrombomodulin reduces tumorigenic and metastatic potential of lung cancer cells by up-regulation of E-cadherin and down-regulation of N-cadherin expression.

Thrombomodulin (TM) is an endothelial cell membrane protein and plays critical roles in anti-thrombosis, anti-inflammation, vascular endothelial protection, and is traditionally regarded as a "vascular protection god". In recent years, although TM has been reported to be down-regulated in a variety of malignant tumors including lung cancer, the role and mechanism of TM in lung cancer are enigmatic. In this study, we found that induction of TM overexpression by cholesterol-reducing drug atorvastatin significantly diminished the tumorigenic capability of the lung cancer cells. Moreover, we demonstrated that TM overexpression caused G0/G1 phase arrest and markedly reduced the colony forming capability of the cells. Furthermore, overexpression of TM inhibited cell migration and invasion. Consistently, depletion of TM promoted cell growth, reduced the cell population at the G0/G1 phase, and enhanced cell migratory ability. Mechanistic study revealed that TM up-regulated E-cadherin but down-regulated N-cadherin expression, resulting in reversal of epithelial-mesenchymal transition (EMT) in the lung cancer cells. Moreover, silencing TM expression led to decreased E-cadherin and increased N-cadherin. Taken together, our study suggests that TM functions as a tumor suppressive protein, providing a conceptual framework for inducing TM overexpression as a sensible strategy and approach for novel anti-lung cancer drug discovery.

Oroxin B selectively induces tumor-suppressive ER stress and concurrently inhibits tumor-adaptive ER stress in B-lymphoma cells for effective anti-lymphoma therapy.

Cancer cells have both tumor-adaptive and -suppressive endoplasmic reticulum (ER) stress machineries that determine cell fate. In malignant tumors including lymphoma, constant activation of tumor-adaptive ER stress and concurrent reduction of tumor-suppressive ER stress favors cancer cell proliferation and tumor growth. Current ER stress-based anti-tumor drugs typically activate both tumor-adaptive and -suppressive ER stresses, resulting in low anti-cancer efficacy; hence, selective induction of tumor-suppressive ER stress and inhibition of tumor-adaptive ER stress are new strategies for novel anti-cancer drug discovery. Thus far, specific tumor-suppressive ER stress therapeutics have remained absent in clinical settings. In this study, we explored unique tumor-suppressive ER stress agents from the traditional Chinese medicinal herb Oroxylum indicum, and found that a small molecule oroxin B selectively induced tumor-suppressive ER stress in malignant lymphoma cells, but not in normal cells, effectively inhibited lymphoma growth in vivo, and significantly prolonged overall survival of lymphoma-xenografted mice without obvious toxicity. Mechanistic studies have revealed that the expression of key tumor-adaptive ER-stress gene GRP78 was notably suppressed by oroxin B via down-regulation of up-stream key signaling protein ATF6, while tumor-suppressive ER stress master gene DDIT3 was strikingly activated through activating the MKK3-p38 signaling pathway, correcting the imbalance between tumor-suppressive DDIT3 and tumor-adaptive GRP78 in lymphoma. Together, selective induction of unique tumor-suppressive ER stress and concurrent inhibition of tumor-adaptive ER stress in malignant lymphoma are new and feasible approaches for novel anti-lymphoma drug discovery and anti-lymphoma therapy.

Cancer/testis antigens trigger epithelial-mesenchymal transition and genesis of cancer stem-like cells.

Malignant tumors aberrantly overexpress various embryonic genes and proto-oncogenes, including a variety of cancer-testis antigens (CTAs). CTAs belong to a class of testis-derived proteins which are only expressed in germ cells in the male testis, and the expression of CTA genes is entirely silenced in the adult somatic tissues. They are, however, aberrantly overexpressed in a variety of malignant tumor tissues. Emerging evidence shows that a number of CTAs promote epithelialmesenchymal transition (EMT) and genesis of cancer stem like cells, escalating tumorigenesis, invasion, and metastasis. The can cer-testis antigens, such as SSX, MAGE-D4B, CAGE, piwil2, and CT45A1, upregulate EMT and metastatic genes, promoting EMT and tumor dissemination. In addition, certain members of CTAs, including Piwil2, DNAJB8, CT45A1, MAGE-A, GAGE, and SPANX, are implicated in the initiation or maintenance, of cancer stem-like cells, promoting tumorigenesis and malignant progression. Clinically CTAs are closely associated with poor prognosis in cancer patients. Intriguely, CTAs are strongly immunogenic and normally restricted to the male testis after birth, however, these proteins are aberrantly overexpressed in cancer stem-like cells and in a variety of cancers, suggesting their target potential for cancer immunotherapy, as diagnostic biomarkers, and as targets for novel anticancer drug discovery. Thus, the targeting of tumorigenic CTAs is a promising strategy to eradicate cancer stem-like cells and inhibit tumorigenesis for effective cancer treatment.