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Targeting immune checkpoints such as programmed cell death protein 1 (PD-1) and programmed death ligand-1 (PD-L1) have been approved for treating melanoma, gastric cancer (GC) and bladder cancer with clinical benefit. Nevertheless, many patients failed to respond to anti-PD-1/PD-L1 treatment, so it is necessary to seek an alternative strategy for traditional PD-1/PD-L1 targeting immunotherapy. Here with the data from The Cancer Genome Atlas (TCGA) and our in-house tissue library, PD-L1 expression was found to be positively correlated with the expression of ubiquitin-specific processing protease 7 (USP7) in GC. Furthermore, USP7 directly interacted with PD-L1 in order to stabilize it, while abrogation of USP7 attenuated PD-L1/PD-1 interaction and sensitized cancer cells to T cell killing in vitro and in vivo. Besides, USP7 inhibitor suppressed GC cells proliferation by stabilizing P53 in vitro and in vivo. Collectively, our findings indicate that in addition to inhibiting cancer cells proliferation, USP7 inhibitor can also downregulate PD-L1 expression to enhance anti-tumor immune response simultaneously. Hence, these data posit USP7 inhibitor as an anti-proliferation agent as well as a novel therapeutic agent in PD-L1/PD-1 blockade strategy that can promote the immune response of the tumor., Graphical abstract This study supports that small molecule USP7 inhibitors may be used as novel promoter of the tumor immune response.Image 1

Human mutT homolog 1(MTH1), the oxidized dNTP pool sanitizer enzyme, has been reported to be highly expressed in various malignant tumors. However, the oncogenic role of MTH1 in gastric cancer remains to be determined. In the current study, we found that MTH1 was overexpressed in human gastric cancer tissues and cells. Using an in vitro MTH1 inhibitor screening system, the compounds available in our laboratory were screened and the small molecules containing 5-cyano-6-phenylpyrimidine structure were firstly found to show potently and specifically inhibitory effect on MTH1, especially compound MI-743 with IC

Background One of the remarkable metabolic characteristics of cancer cells is that they prefer glycolysis rather than oxidative phosphorylation (OXPHOS). Pyruvate dehydrogenase E1 alpha subunit (PDHA1) is an important prerequisite for OXPHOS. Our previous studies have shown that low level of PDHA1 protein expression in esophageal squamous cell cancer (ESCC) was correlated with poor prognosis. However, the effect of PDHA1 inhibition on metabolism and biological behavior of esophageal cancer cells remains unclear. Methods and results In this study, a KYSE450 PDHA1 knockout (KO) cell line of esophageal cancer was established by CRISPR/Cas9 technology. Then, the glycose metabolism, cell proliferation and migration abilities, chemotherapeutic tolerance and angiogenesis of the PDHA1 KO cells were investigated in vitro and in vivo. In the PDHA1 KO cells, the glycolysis and the consumption of glucose and glutamine were significantly enhanced, while the OXPHOS was significantly suppressed, implying Warburg effect in the PDHA1 KO cells. Furthermore, it was also proved in vitro experiments that the PDHA1 KO cell obtained proliferation advantage, as well as significantly greater chemotherapy tolerance and migration ability. Xenograft experiments discovered not only larger tumors but also increased angiogenesis in the PDHA1 KO cell group. Conclusion Inhibition of PDHA1 gene expression in human ESCC leads to metabolic reprogramming of Warburg effect and increased malignancies. Targeting ESCC metabolic reprogramming may become a potential therapeutic target.

Pentraxin 3 (PTX3) is an inflammatory molecule that is closely related to the proliferation, invasion, and metastasis of cancer. In order to explore the role of PTX3 in the occurrence and development of esophageal carcinoma (ESCA), we modified the PTX3 gene in ESCA cell lines to obtain the model of gene knockout and overexpression and studied cell proliferation, cycle, apoptosis, migration ability, energy metabolism, and sensitivity to chemotherapy and radiotherapy. We observed the increase in cell proliferation, cycle, apoptosis, migration ability, and sensitivity to chemotherapy and radiotherapy in the PTX3 knockout model, while in the PTX3 overexpression model, these phenomena were significantly reduced. Knockout of the PTX3 also resulted in decreased cell glycolysis and increased oxidative phosphorylation, which is consistent with other findings that PTX3 affects the tumorigenic ability of cells and their sensitivity to docetaxel. In ESCA, SOX9 directly regulates the expression of PTX3, while human leukocyte antigen (HLA)-system-related genes are significantly up-regulated when lacking PTX3. These results indicate that SOX9 may play a crucial role in regulating PTX3 and affecting the HLA system in ESCA.

Meng Zhang,1 Xiaoli Li,1,2 Zhirui Fan,1 Jing Zhao,1 Shuzheng Liu,3 Mingzhi Zhang,1 Huixiang Li,4 Mariusz Adam Goscinski,5 Huijie Fan,1 Zhenhe Suo1,2,6 1Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou, Henan Province, China; 2Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, University of Oslo, Oslo, Norway; 3Henan Office for Cancer Research and Control, Henan Cancer Hospital, Zhengzhou, Henan Province, China; 4Department of Pathology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China; 5Department of Surgery, The Norwegian Radium Hospital, Oslo University Hospital, University of Oslo, Oslo, Norway; 6Department of Pathology, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Background: Sushi repeat-containing protein X-linked 2 (SRPX2) is overexpressed in a variety of different tumor tissues and correlated with poor prognosis in patients. Little research focuses on the role of SRPX2 expression in prostate cancer (PCa), and the clinicopathological significance of the protein expression in this tumor is relatively unknown. However, our previous transcriptome data from those cancer stem-like cells indicated the role of SRPX2 in PCa. Materials and methods: In this study, RT-PCR and Western blotting were firstly used to examine the SRPX2 expression in three PCa cell lines including LNCaP, DU145, and PC3, and then SRPX2 protein expression was immunohistochemically investigated and statistically analyzed in a series of 106 paraffin-embedded PCa tissue specimens. Results: Significantly lower levels of SRPX2 expression were verified in the LNCaP cells, compared with the expression in the aggressive DU145 and PC3 cells, in both mRNA and protein levels. Immunohistochemically, there were variable SRPX2 protein expressions in the clinical samples. Moreover, high levels of SRPX2 expression in the PCa tissues were significantly associated with Gleason score (P=0.008), lymph node metastasis (P=0.009), and distant metastasis (P=0.021). Furthermore, higher levels of SRPX2 expression in the PCa tissues were significantly associated with shorter overall survival (OS) (P

Background/Aims: Retinoic acid receptor beta (RAR beta) is a retinoic acid receptor gene that has been shown to play key roles during multiple cancer processes, including cell proliferation, apoptosis, migration and invasion. Numerous studies have found that methylation of the RAR beta promoter contributed to the occurrence and development of malignant tumors. However, the connection between RAR beta promoter methylation and prostate cancer (PCa) remains unknown. This meta-analysis evaluated the clinical significance of RAR beta promoter methylation in PCa. Materials and Methods: We searched all published records relevant to RAR beta and PCa in a series of databases, including PubMed, Embase, Cochrane Library, ISI Web of Science and CNKI. The rates of RAR beta promoter methylation in the PCa and control groups (including benign prostatic hyperplasia and normal prostate tissues) were summarized. In addition, we evaluated the source region of available samples and the methods used to detect methylation. To compare the incidence and variation in RAR beta promoter methylation in PCa and non-PCa tissues, the odds ratio (OR) and 95% confidence interval (CI) were calculated accordingly. All the data were analyzed with the statistical software STATA 12.0. Results: Based on the inclusion and exclusion criteria, 15 articles assessing 1,339 samples were further analyzed. These data showed that the RAR beta promoter methylation rates in PCa tissues were significantly higher than the rates in the non-PCa group (OR=21.65, 95% CI: 9.27-50.57). Subgroup analysis according to the source region of samples showed that heterogeneity in Asia was small (I2=0.0%, P=0.430). Additional subgroup analysis based on the method used to detect RAR beta promoter methylation showed that the heterogeneity detected by MSP (methylation-specific PCR) was relatively small (I2=11.3%, P=0.343). Conclusion: Although studies reported different rates for RAR beta promoter methylation in PCa tissues, the total analysis demonstrated that RAR beta promoter methylation may be correlated with PCa carcinogenesis and that the RAR beta gene is particularly susceptible. Additional studies with sufficient data are essential to further evaluate the clinical features and prognostic utility of RAR beta promoter methylation in PCa.

// Xiaoli Li 1, 2, * , Gaoyang Han 3, * , Xiaoran Li 2 , Quancheng Kan 4 , Zhirui Fan 1 , Yaqing Li 1, 2 , Yasai Ji 1 , Jing Zhao 1 , Mingzhi Zhang 1 , Mantas Grigalavicius 2 , Viktor Berge 5 , Mariusz Adam Goscinski 6 , Jahn M. Nesland 2 and Zhenhe Suo 1, 2 1 Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, 450000, China 2 Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, 0379, Norway 3 Department of Thoracic Surgery, The First Affiliated Hospital of Xinxiang Medical University, Weihui City, Henan Province, 453000, China 4 Department of Clinical Pharmacology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, 450000, China 5 Department of Urology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, 0379, Norway 6 Department of Surgery, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, 0379, Norway * These authors have contributed equally to this work Correspondence to: Zhenhe Suo, email: zhenhes@medisin.uio.no , zhenhesuo@aliyun.com Keywords: MPC, OXPHOS, TCA, glycolysis, stem cell Received: November 09, 2016 Accepted: May 05, 2017 Published: May 25, 2017 ABSTRACT One of the remarkable features of cancer cells is aerobic glycolysis, a phenomenon known as the “Warburg Effect”, in which cells rely preferentially on glycolysis instead of oxidative phosphorylation (OXPHOS) as the main energy source even in the presence of high oxygen tension. Cells with dysfunctional mitochondria are unable to generate sufficient ATP from mitochondrial OXPHOS, and then are forced to rely on glycolysis for ATP generation. Here we report our results in a prostate cancer cell line in which the mitochondrial pyruvate carrier 1 (MPC1) gene was knockout. It was discovered that the MPC1 gene knockout cells revealed a metabolism reprogramming to aerobic glycolysis with reduced ATP production, and the cells became more migratory and resistant to both chemotherapy and radiotherapy. In addition, the MPC1 knockout cells expressed significantly higher levels of the stemness markers Nanog, Hif1α, Notch1, CD44 and ALDH. To further verify the correlation of MPC gene function and cell stemness/metabolic reprogramming, MPC inhibitor UK5099 was applied in two ovarian cancer cell lines and similar results were obtained. Taken together, our results reveal that functional MPC may determine the fate of metabolic program and the stemness status of cancer cells in vitro .

This Article contains an error in Figure 3. In panel g, images representing miR-222-TuD and miR-424-TuD were both taken from the miR-222-TuD image. The correct version of Figure 3 is shown in the accompanying Author Correction.

Human mutT homolog 1(MTH1), the oxidized dNTP pool sanitizer enzyme, has been reported to be highly expressed in various malignant tumors. However, the oncogenic role of MTH1 in gastric cancer remains to be determined. In the current study, we found that MTH1 was overexpressed in human gastric cancer tissues and cells. Using an in vitro MTH1 inhibitor screening system, the compounds available in our laboratory were screened and the small molecules containing 5-cyano-6-phenylpyrimidine structure were firstly found to show potently and specifically inhibitory effect on MTH1, especially compound MI-743 with IC50 = 91.44 ± 1.45 nM. Both molecular docking and target engagement experiments proved that MI-743 can directly bind to MTH1. Moreover, MI-743 could not only inhibit cell proliferation in up to 16 cancer cell lines, especially gastric cancer cells HGC-27 and MGC-803, but also significantly induce MTH1-related 8-oxo-dG accumulation and DNA damage. Furthermore, the growth of xenograft tumours derived by injection of MGC-803 cells in nude mice was also significantly inhibited by MI-743 treatment. Importantly, MTH1 knockdown by siRNA in those two gastric cancer cells exhibited the similar findings. Our findings indicate that MTH1 is highly expressed in human gastric cancer tissues and cell lines. Small molecule MI-743 with 5-cyano-6-phenylpyrimidine structure may serve as a novel lead compound targeting the overexpressed MTH1 for gastric cancer treatment.

Neddylation of the Cullin-RING E3 ligases (CRLs) regulates the homeostasis of approximately 20% of cellular proteins. Defective in cullin neddylation 1 (DCN1), as a co-E3 ligase, interacts with UBE2M to enhance the activation of CRLs, and this interaction is emerging as a therapeutic target for human diseases. Here, we present a series of pyrimidin-based small molecular inhibitors targeting DCN1-UBE2M interaction. After finding a novel inhibitor DC-1 with IC50 = 1.2 μM, we performed a series of chemical optimizations, which finally led to the discovery of a potent thiazole containing 5-cyano-6-phenylpyrimidin-based inhibitor DC-2 (IC50 = 15 nM). Next, using protein and cellular thermal shift assays, coimmunoprecipitation, molecular docking, and site-specific mutation experiments, we further proved that DC-2 specifically inhibited the interaction of UBE2M and DCN1 at molecule and cellular levels, resulting in the decrease of cullin3 neddylation and accumulation of its substrate, NRF2. Our findings indicate that DC-2 may serve as a novel lead compound for specific derivatives targeting DCN1-UBE2M interaction.

Abnormalities in tricarboxylic acid (TCA) cycle function were related to a variety of pathological processes. Fumarate hydratase (FH) is a required enzyme in the TCA cycle. To explore the general influence of FH knockout, we isolated FH+/- rat and normal rat lung fibroblasts and cultured these cells in vitro. The isolated fibroblasts with the current method were rather homogeneous and were confirmed spindle in morphology, positive for vimentin and negative for α-SMA (α-smooth muscle actin). Sequencing of the PCR (polymerase chain reaction) products flanking the FH gene mutation verified the FH+/- status, and the FH gene and protein expression were confirmed to be reduced in the FH+/- cells. No sign of ageing for the FH+/- cells after 61 passages was observed, but the controls died out at this stage. Flow cytometry revealed increased S-phase and decreased G1/G0 proportions with significantly less early apoptosis in FH+/- cells compared to that in control cells. At the same time, increased glucose consumption, intracellular fumarate production and extracellular lactate secretion were verified in the FH+/- cells. Correspondingly, FH+/- cells showed a lower basal oxygen consumption rate (OCR) but a higher level of reactive oxygen species (ROS) production. Single cell cloning and cell line establishment were successfully performed with the FH+/- cells at the 84th passage. All the above results indicate an important role for FH+/- in the longevity or immortality of the FH+/- cells, in which increased p53 and TERT (telomerase reverse transcriptase) protein expression, decreased p21 and p16 protein expression and negative SA-β-Gal (senescence-associated beta-galactosidase) were verified along with metabolic reprogramming.

// Xiaoli Li 1, 2 , Yaqing Li 1, 2 , Gaoyang Han 3 , Xiaoran Li 2 , Yasai Ji 1 , Zhirui Fan 1 , Yali Zhong 1 , Jing Cao 1, 4 , Jing Zhao 1 , Goscinski Mariusz 5 , Mingzhi Zhang 1 , Jianguo Wen 6 , Jahn M. Nesland 2 , Zhenhe Suo 1, 2 1 Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China 2 Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Institute of Clinical Medicine, University of Oslo, Montebello, Oslo, Norway 3 Department of Thoracic Surgery, The First Affiliated Hospital of Xinxiang Medical University, Weihui City, Henan Province, China 4 Department of Pathology, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China 5 Department of Surgery, The Norwegian Radium Hospital, Oslo University Hospital, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Norway 6 The Institute of Clinical Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China Correspondence to: Zhenhe Suo, email: zhenhes@medisin.uio.no, zhenhesuo@aliyun.com Keywords: MPC1, CRISPR/Cas9, diabetes, gene knockout, reproductive capability Received: June 13, 2016 Accepted: October 19, 2016 Published: November 08, 2016 ABSTRACT Pyruvate plays a critical role in the mitochondrial tricarboxylic acid (TCA) cycle, and it is the center product for the synthesis of amino acids, carbohydrates and fatty acids. Pyruvate transported across the inner mitochondrial membrane appears to be essential in anabolic and catabolic intermediary metabolism. The mitochondrial pyruvate carrier (MPC) mounted in the inner membrane of mitochondria serves as the channel to facilitate pyruvate permeating. In mammals, the MPC is formed by two paralogous subunits, MPC1 and MPC2. It is known that complete ablation of MPC2 in mice causes death on the 11th or 12th day of the embryonic period. However, MPC1 deletion and the knowledge of gene function in vivo are lacking. Using the new technology of gene manipulation known as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 (CRISPR/Cas9) systems, we gained stable MPC1 gene heterozygous mutation mice models, and the heterozygous mutations could be stably maintained in their offsprings. Only one line with homozygous 27 bases deletion in the first exon was established, but no offsprings could be obtained after four months of mating experiments, indicating infertility of the mice with such homozygous deletion. The other line of MPC1 knockout (KO) mice was only heterozygous, which mutated in the first exon with a terminator shortly afterwards. These two lines of MPC1 KO mice showed lower fertility and significantly higher bodyweight in the females. We concluded that heterozygous MPC1 KO weakens fertility and influences the metabolism of glucose and fatty acid and bodyweight in mice.

// Yaqing Li 1, 2, 3 , Xiaoran Li 2, 3 , Xiaoli Li 1, 2 , Yali Zhong 1 , Yasai Ji 1 , Dandan Yu 1, 2 , Mingzhi Zhang 1 , Jian-Guo Wen 4 , Hongquan Zhang 5 , Mariusz Adam Goscinski 6 , Jahn M. Nesland 2, 3 , Zhenhe Suo 1, 2, 3 1 Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China 2 Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, University of Oslo, Montebello, Oslo, Norway 3 Department of Pathology, The Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Montebello, Oslo, Norway 4 Institute of Clinical Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China 5 Department of Anatomy, Histology and Embryology, Peking University Health Science Center, Peking University, Beijing, China 6 Department of Surgery, the Norwegian Radium Hospital, Oslo University Hospital, University of Oslo, Oslo, Norway Correspondence to: Zhenhe Suo, email: zhenhes@medisin.uio.no Keywords: PDHA1 gene knockout, metabolic reprogramming, glutamine dependence, prognosis, GC-MS analysis Received: April 06, 2016 Accepted: July 10, 2016 Published: July 22, 2016 ABSTRACT Alternative pathways of metabolism endowed cancer cells with metabolic stress. Inhibiting the related compensatory pathways might achieve synergistic anticancer results. This study demonstrated that pyruvate dehydrogenase E1α gene knockout (PDHA1 KO) resulted in alterations in tumor cell metabolism by rendering the cells with increased expression of glutaminase1 (GLS1) and glutamate dehydrogenase1 (GLUD1), leading to an increase in glutamine-dependent cell survival. Deprivation of glutamine induced cell growth inhibition, increased reactive oxygen species and decreased ATP production. Pharmacological blockade of the glutaminolysis pathway resulted in massive tumor cells apoptosis and dysfunction of ROS scavenge in the LNCaP PDHA1 KO cells. Further examination of the key glutaminolysis enzymes in human prostate cancer samples also revealed that higher levels of GLS1 and GLUD1 expression were significantly associated with aggressive clinicopathological features and poor clinical outcome. These insights supply evidence that glutaminolysis plays a compensatory role for cell survival upon alternative energy metabolism and targeting the glutamine anaplerosis of energy metabolism via GLS1 and GLUD1 in cancer cells may offer a potential novel therapeutic strategy.

Hypoxia promotes an aggressive tumor phenotype with increased genomic instability, partially due to downregulation of DNA repair pathways. However, genome stability is also surveilled by cell cycle checkpoints. An important issue is therefore whether hypoxia also can influence the DNA damage‐induced cell cycle checkpoints. Here, we show that hypoxia (24 h 0.2% O2) alters the expression of several G2 checkpoint regulators, as examined by microarray gene expression analysis and immunoblotting of U2OS cells. While some of the changes reflected hypoxia‐induced inhibition of cell cycle progression, the levels of several G2 checkpoint regulators, in particular Cyclin B, were reduced in G2 phase cells after hypoxic exposure, as shown by flow cytometric barcoding analysis of individual cells. These effects were accompanied by decreased phosphorylation of a Cyclin dependent kinase (CDK) target in G2 phase cells after hypoxia, suggesting decreased CDK activity. Furthermore, cells pre‐exposed to hypoxia showed increased G2 checkpoint arrest upon treatment with ionizing radiation. Similar results were found following other hypoxic conditions (∼0.03% O2 20 h and 0.2% O2 72 h). These results demonstrate that the DNA damage‐induced G2 checkpoint can be altered as a consequence of hypoxia, and we propose that such alterations may influence the genome stability of hypoxic tumors.

Although abnormal metabolism in metabolic syndrome and tumours has been well described, the relationship between oxoglutarate dehydrogenase (OGDH) and obesity-related diseases is still largely unknown. This study aimed to investigate whether it was possible to use transcription activator-like effector nuclease (TALEN) technology to establish OGDH−/− rats and then study the effect of a high-fat diet (HFD) on these rats. However, after OGDH+/−rats were generated, we were unable to identify any OGDH−/− rats by performing mating experiments with the OGDH+/− rats for almost one year. During the past three years, only OGDH+/− rats were stably established, and correspondingly reduced OGDH expression in the tissues of the OGDH+/− rats was verified. No significant abnormal behaviour was observed in the OGDH+/− rats compared to the wild-type (WT) control rats. However, the OGDH+/− rats were revealed to have higher body weight, and the difference was even significantly greater under the HFD condition. Furthermore, blood biochemical and tissue histological examinations uncovered no abnormalities with normal diets, but a HFD resulted in liver dysfunction with pathological alterations in the OGDH+/− rats. Our results strongly indicate that OGDH homologous knockout is lethal in rats but heterologous OGDH knockout results in vulnerable liver lesions with a HFD. Therefore, the current study may provide a useful OGDH+/− rat model for further investigations of metabolic syndrome and obesity-related hepatic carcinogenesis.

The high prevalence of urinary tract infection in aging adults is a challenging aspect of geriatric care. Incontinence and cognitive/functional impairment make collection of urine samples difficult and often require either catheterization for sample collection, which is a risk factor for infections, or more lenient criteria for initiating antibiotic treatment. We report the development of a diaper inlay with absorbent materials, superabsorbent polymer-based valve and chemical reaction pads for rapid screening of urinary tract infection of incontinent diaper-wearing elderly receivers of home care services. The developed diaper inlay was capable of collecting, isolating, analyzing samples and retaining results > 8 h. The diaper inlay can therefore be compatible with the diaper changing routines of nurses in home care services, without requiring much time or effort. A nurse can insert a diaper inlay in a diaper and the results can be recorded during a later diaper change. Although the research focuses on tools for home care services, the nursing home sector has similar problems and may benefit from technological development for rapid screening to avoid unnecessary catheterization and overuse of antibiotics.

Zinc finger E-box-binding homeobox 1 (ZEB1) is a transcriptional factor known to repress E-cadherin promoter and thus induce EMT. Expression of ZEB-1 has in numerous cancers been associated with aggressive disease and poor clinical outcome. Our aim was to investigate the expression of ZEB1 in esophageal squamous- and small-cell carcinomas. Immunohistochemical staining was performed on tissue sections obtained from 151 patients with esophageal squamous cell carcinoma (ESCC) and 25 patients with primary small-cell carcinoma of the esophagus (PSCCE). Semi-quantitative analysis, and thus statistical analysis, has been accomplished on the samples. Immunohistochemistry revealed ZEB1 expression in the cytoplasm (64.9% of cases), in nuclei (11.3% of cases) and in tumor stroma (80.1% of cases) of ESCC. In PSCCE only nuclear staining (88.0% of cases) was observed. Weak cytoplasmic expression of ZEB1 in ESCC was associated with longer survival. Immunohistochemical evaluation of ZEB1 cytoplasmic expression in ESCC may have clinical prognostic value, but further studies are needed to fully understand the function as well as potential clinical and therapeutic implications of ZEB1 expression in cancers.

A growing body of evidence indicates that aberrant activation of epithelial-to-mesenchymal transition (EMT) plays a key role in tumor cell invasion and metastasis. Zinc finger E-box-binding homeobox factor 1 (ZEB1), as a crucial mediator of EMT, contributes to the malignant progression of various epithelial tumors. To determine whether ZEB1 is involved in the progression of ovarian cancer, we immunohistochemically evaluated the expression of ZEB1 in 238 cases of epithelial ovarian cancer (EOC) and analyzed its associations with clinicopathological parameters. Positive expression of ZEB1 was observed in 32.8% (78/238) of EOCs and it was found to be significantly associated with advanced tumor stage (P=0.001). The survival analysis indicated that the expression of ZEB1 was associated with a poor 5-year progression-free survival (PFS) (P=0.021). A similar tendency was also observed between the expression of ZEB1 and 5-year overall survival, although it did not reach statistical significance (P=0.118). Moreover, the multivariate analysis demonstrated that ZEB1 expression was an independent risk factor for 5-year PFS in ovarian cancer. Taken together, our data provide evidence that ZEB1 may play a crucial role in promoting aggressive ovarian carcinoma progression. Therefore, ZEB1 may serve as an effectively predictive marker and a potential target for therapeutic intervention in EOC.

Aerobic glycolysis is one of the important hallmarks of cancer cells and eukaryotic cells. In this study, we have investigated the relationship between blocking mitochondrial pyruvate carrier (MPC) with UK5099 and the metabolic alteration as well as stemness phenotype of prostatic cancer cells. It was found that blocking pyruvate transportation into mitochondrial attenuated mitochondrial oxidative phosphorylation (OXPHOS) and increased glycolysis. The UK5099 treated cells showed significantly higher proportion of side population (SP) fraction and expressed higher levels of stemness markers Oct3/4 and Nanog. Chemosensitivity examinations revealed that the UK5099 treated cells became more resistant to chemotherapy compared to the non-treated cells. These results demonstrate probably an intimate connection between metabolic reprogram and stem-like phenotype of LnCap cells in vitro. We propose that MPC blocker (UK5099) application may be an ideal model for Warburg effect studies, since it attenuates mitochondrial OXPHOS and increases aerobic glycolysis, a phenomenon typically reflected in the Warburg effect. We conclude that impaired mitochondrial OXPHOS and upregulated glycolysis are related with stem-like phenotype shift in prostatic cancer cells.

The aim of this study was to investigate the effect of hypoxic conditions on the expression of enamel genes and on the secretion of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), cytokines, and interleukins by an ameloblast-derived cell line. Murine ameloblast-derived cells (LS-8 cells) were exposed to 1% oxygen for 24 and 48 h and harvested after 1, 2, 3, and 7 d. The effect of culture in hypoxic conditions on the expression of structural enamel matrix genes and on the secretion of cytokines and interleukins, as well as ALP and LDH, into the cell-culture medium was calculated relative to the expression and secretion of these factors by untreated cells (controls) at each time point. Hypoxia increased expression of the structural enamel matrix genes amelogenin (Amelx), ameloblastin (Ambn), and enamelin (Enam), and the enamel protease matrix metalloproteinase-20 (Mmp20). Expression of hypoxia-inducible factor 1-alpha (Hif1α), and secretion of several vascularization factors and pro-inflammatory factors, were increased after 24 and 48 h of hypoxia. The ALP activity was reduced after 24 and 48 h of hypoxia, whereas the LDH level in the cell-culture medium was higher after 24 h of hypoxic conditions compared with 48 h. In conclusion, hypoxic exposure may disrupt the controlled fine-tuned expression and processing of enamel genes, and promote the secretion of pro-inflammatory factors.

// Benling Xu 1,2,* , Long Yuan 3,* , Quanli Gao 2 , Peng Yuan 2 , Peng Zhao 4 , Huijuan Yuan 5 , Huijie Fan 1 , Tiepeng Li 2 , Peng Qin 2 , Lu Han 2 , Weijia Fang 4 and Zhenhe Suo 1,6 1 Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, P. R. China 2 Department of Cancer Biotherapy, The Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou, Henan, P. R. China 3 Department of Surgery, The Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou, Henan, P. R. China 4 Department of Oncology, The First Affiliated Hospital of Zhejiang University, Hangzhou, Zhejiang, P. R. China 5 Department of Endocrinology, Henan Provincial People’s Hospital, Zhengzhou, Henan, P. R. China 6 Department of Pathology, Oslo University Hospital and Clinical Institute, Faculty of Medicine, University of Oslo, Oslo, Norway * These authors have contributed equally to the work Correspondence to: Zhenhe Suo, email: // Keywords : colorectal cancer; T cell exhaustion; PD-1; Tim-3 Received : March 12, 2015 Accepted : April 22, 2015 Published : May 12, 2015 Abstract T-cell exhaustion represents a progressive loss of T-cell function. The inhibitory receptor PD-1 is known to negatively regulate CD8 + T cell responses directed against tumor antigen, but the blockades of PD-1 pathway didn’t show the objective responses in patients with colorectal cancer (CRC). Thus, further exploring the molecular mechanism responsible for inducing T-cell dysfunction in CRC patients may reveal effective strategies for immune therapy. This study aims to characterize co-inhibitory receptors on T cells in CRC patients to identify novel targets for immunotherapy. In this study, peripheral blood samples from 20 healthy controls and 54 consented CRC patients, and tumor and matched paraneoplastic tissues from 7 patients with advanced CRC, subjected to multicolor flow cytometric analysis of the expression of PD-1 and Tim-3 receptors on CD8 + T cells. It was found that CRC patients presented with significantly higher levels of circulating Tim-3 + PD-1 + CD8 + T cells compared to the healthy controls (medians of 3.12% and 1.99%, respectively, p = 0.0403). A similar increase of Tim-3 + PD-1 + CD8 + T cells was also observed in the tumor tissues compared to paraneoplastic tussues. Tim-3 + PD-1 + CD8 + T cells in tumor tissues produced even less cytokine than that in paraneoplastic tissues. Functional ex vivo experiments showed that Tim-3 + PD-1 + CD8 + T cells produced significantly less IFN-γ than Tim-3 - PD-1 - CD8 + T cells, followed by Tim-3 + PD-1 - CD8 + T cells, and Tim-3 - PD-1 + CD8 + T cells, indicating a stronger inhibition of IFN-γ production of Tim-3 + CD8 + T cells . It is also found in this study that Tim-3 + PD-1 + CD8 + T cell increase in circulation was correlated with clinical cancer stage but not histologic grade and serum concentrations of cancer biomarker CEA. Our results indicate that upregulation of the inhibitory receptor Tim-3 may restrict T cell responses in CRC patients, and therefore blockage of Tim-3 and thus restoring T cell responses may be a potential therapeutic approach for CRC patients.

Sex hormone binding globulin (SHBG) is known as a carrier protein, classically thought to be mainly synthesized in the liver and then secreted into the circulating system, where it binds to sex steroids with a high affinity and modulates the bioavailability of these hormones. In humans, the organs other than the liver known to produce SHBG include the brain, uterus, testis, prostate, breast and ovary, and the locally expressed SHBG is considered to play an important role in various physiological and pathological processes. A few studies of SHBG in rats were reported, but systemic SHBG studies in consideration of different organs and aging are currently missing. So we examined the SHBG expression in the brain, liver, prostate, and serum in 40 Sprague-Dawley male rats in four different groups (newborn, 2, 6, and 12 months old, respectively) with 10 in each group by immunohistochemistry, immunofluorescience microscopy, qRT-PCR, ELISA, western blotting, and laser confocal microscopy. We discovered that SHBG was increasingly expressed in all the three tissues along with age, and the SHBG protein expression was observed in the cytoplasm and membrane of neurons, hepatocyte, and prostate epithelial cells. The ELISA assay of the sera also supported an increasing SHBG level along with age. It is concluded that the locally synthesized SHBG in the liver, brain, and prostate and the circulating SHBG of male SD rats are positively associated with age, further indicating a potential role of SHBG in aging.

Flavopiridol (FP) is a pan-cyclin dependent kinase inhibitor, which shows strong efficacy in inducing cancer cell apoptosis. Although FP is potent against most cancer cells in vitro, unfortunately it proved less efficacious in clinical trials in various aggressive cancers. To date, the molecular mechanisms of the FP resistance are mostly unknown. Here, we report that a small fraction human prostate cancer DU145 cells can survive long-term FP treatment and emerge as FP-resistant cells (DU145FP). These DU145FP cells show accumulated mitochondrial lesions with stronger glycolytic features, and they proliferate in slow-cycling and behave highly migratory with strong anti-apoptotic potential. In addition, the cells are less sensitive to cisplatin and docetaxel-induced apoptotic pressure, and over-express multiple stem cell associated biomarkers. Our studies collectively uncover for the first time that FP-resistant prostate cancer cells show metabolic remodeling, and the metabolic plasticity might be required for the FP resistance-associated cancer cell stemness up-regulation.

Amyloid-β deposition in senile plaques is one of the main pathological changes in Alzheimer's disease (AD). We previously reported that aquaporin-4 (AQP4) is redistributed within the astrocytes in cerebral amyloid angiopathy in the tg-ArcSwe mouse model of AD, suggesting that AQP4 may participate in amyloid-β deposition. However, the role of AQP4 in plaque formation is not currently clear. The objective of the current study was to explore the AQP4 distribution within plaques in the tg-ArcSwe mice in more depth by the combined application of immunofluorescence cytochemistry and immunogold electron microscopy. In addition, the astrocyte marker, glial fibrillary acidic protein (GFAP), was studied in association with AQP4. We demonstrated a robust upregulation of AQP4 expression in areas of plaques. Compared to GFAP, AQP4 appeared predominantly at later stages of plaque formation, in older mice, and within the processes of astrocytes. In combination with GFAP, AQP4 differentiated plaques into three progression stages under light microscopy. This suggests that AQP4 expression was associated with amyloid deposition and astrocyte pathology in the Tg-ArcSwe mouse model of AD. This provides novel proof for the involvement of AQP4 in the process of amyloid deposition in AD.

Photodynamic therapy (PDT) with a light-activated drug is an approved modality for cancer treatment. Hexaminolevulinate (HAL), a hexylester of 5-aminolevulinic acid as the photosensitising protoporphyrin IX (PpIX) precursor, is clinically used for both PDT and photodetection. Our previous studies have shown that HAL–PDT can effectively induce apoptosis in several human blood malignant cell lines. However, the mechanisms involved in the apoptotic induction are still not fully elucidated. In this study we have focused on the role of cellular lamin A/C in the apoptotic induction. HAL–PDT-mediated apoptosis was confirmed by various techniques including fluorescence microscopy and electron microscopy in both human B-cell lymphoma Ramos and Daudi cell lines. The lamin A/C, together with caspases-6 and -3, was cleaved during the apoptosis. Western blots, immunocytochemistry, fluorescence microscopy and electron microscopy demonstrated that the specific caspase-6inhibitor abrogated the HAL-PDT-mediated cleavages of both caspase-6 and lamin A/C and subsequent apoptosis in these two cell lines, suggesting that the cleavage of lamin A/C by the caspase-6 activation is crucial for such apoptotic induction.

Aims To investigate the aberrant expression of CD117 in oesophageal squamous cell carcinoma (SCC) and its prognostic significance. Methods and results Immunohistochemical staining for CD117 was performed on tissue microarray and routine tissue sections from 157 oesophageal SCC patients and 10 normal oesophageal epithelia adjacent to tumour. The positive rate of CD117 expression was 29.9% in oesophageal SCC tissues, whereas no CD117 expression was detected in the 10 normal oesophageal epithelia. CD117 expression was significantly associated with T stage (P

// Yali Zhong 1, 2, 3, 4 , Xiaoli Li 1 , Yasai Ji 1 , Xiaoran Li 3, 4 , Yaqing Li 1 , Dandan Yu 1 , Yuan Yuan 5 , Jian Liu 6 , Huixiang Li 7 , Mingzhi Zhang 1 , Zhenyu Ji 8 , Dandan Fan 8 , Jianguo Wen 9 , Mariusz Adam Goscinski 10 , Long Yuan 11 , Bin Hao 12 , Jahn M Nesland 3, 4 , Zhenhe Suo 1, 3, 4 1 Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China 2 Department of Gastroenterology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China 3 Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, University of Oslo, Montebello, Oslo, Norway 4 Department of Pathology, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway 5 Department of Pathology, Capital Medical University, Beijing, China 6 Institute of Health Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, China 7 Department of Pathology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China 8 Henan Academy of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 9 Institute of Clinical Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Henan, China 10 Department of Surgery, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway 11 Department of Surgery, The Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou, Henan, China 12 Department of Urology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China Correspondence to: Zhenhe Suo, email: zhenhes@medisin.uio.no Keywords: PDHA1, glycolysis, stemness, prostate cancer Received: September 30, 2016 Accepted: December 28, 2016 Published: January 05, 2017 ABSTRACT Cells generate adenosine-5′-triphosphate (ATP), the major currency for energy-consuming reactions, through mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis. One of the remarkable features of cancer cells is aerobic glycolysis, also known as the “Warburg Effect”, in which cancer cells rely preferentially on glycolysis instead of mitochondrial OXPHOS as the main energy source even in the presence of high oxygen tension. One of the main players in controlling OXPHOS is the mitochondrial gatekeeperpyruvate dehydrogenase complex (PDHc) and its major subunit is E1α (PDHA1). To further analyze the function of PDHA1 in cancer cells, it was knock out (KO) in the human prostate cancer cell line LnCap and a stable KO cell line was established. We demonstrated that PDHA1 gene KO significantly decreased mitochondrial OXPHOS and promoted anaerobic glycolysis, accompanied with higher stemness phenotype including resistance to chemotherapy, enhanced migration ability and increased expression of cancer stem cell markers. We also examined PDHA1 protein expression in prostate cancer tissues by immunohistochemistry and observed that reduced PDHA1 protein expression in clinical prostate carcinomas was significantly correlated with poor prognosis. Collectively, our results show that negative PDHA1 gene expressionis associated with significantly higher cell stemness in prostate cancer cells and reduced protein expression of this gene is associated with shorter clinical outcome in prostate cancers.

Aerobic glycolysis is one of the emerging hallmarks of cancer cells. In this study, we investigated the relationship between blocking mitochondrial pyruvate carrier (MPC) with MPC blocker UK5099 and the metabolic alteration as well as aggressive features of esophageal squamous carcinoma. It was found that blocking pyruvate transportation into mitochondria attenuated mitochondrial oxidative phosphorylation (OXPHOS) and triggered aerobic glycolysis, a feature of Warburg effect. In addition, the HIF-1α expression and ROS production were also activated upon UK5099 application. It was further revealed that the UK5099-treated cells became significantly more resistant to chemotherapy and radiotherapy, and the UK5099-treated tumor cells also exhibited stronger invasive capacity compared to the parental cells. In contrast to esophageal squamous epithelium cells, decreased MPC protein expression was observed in a series of 157 human squamous cell carcinomas, and low/negative MPC1 expression predicted an unfavorable clinical outcome. All these results together revealed the potential connection of altered MPC expression/activity with the Warburg metabolic reprogramming and tumor aggressiveness in cell lines and clinical samples. Collectively, our findings highlighted a therapeutic strategy targeting Warburg reprogramming of human esophageal squamous cell carcinomas.

Ovarian cancer is the most lethal gynecologic malignancy, in which cancer stem cells (CSC) have been reported to be the driving force of relapse and therapy-resistance. It is therefore important to explore CSC markers in ovarian cancer. This project aimed to explore the correlation between the expression of potential CSC maker Cacna2d1 and clinicopathological parameters in 238 epithelial ovarian cancer (EOC) samples. Immunohistochemically, positive Cacna2d1 expression was observed in 83.6% (199/238) of the EOC tumors, among which 107 tumors (44.9%) were highly positive and 92 (38.7%) tumors were weakly positive for the Cacna2d1 protein expression. Among the 158 serous carcinomas, the Cacna2d1 positivity was 148 (93.7%), in which 88 (55.7%) were highly positive, and 60 (38.0%) were weakly positive for the Cacna2d1 protein expression. Most strikingly, the Cacna2d1 was specifically expressed in the infiltration front areas of the EOC tumors. Statistical analyses showed that positive expression of Cacna2d1 was significantly associated with advanced FIGO stage (P

Aldehyde dehydrogenase-1 (ALDH1) has been shown to be a potential cancer stem cell marker in different types of cancer. However, the role of its expression in tumor cells and the microenvironment in different types of cancer is still controversial.ALDH1 protein was immunohistochemically investigated and analyzed in 157 samples of surgically dissected esophageal squamous cell carcinoma (ESCC) tissues.ALDH1 protein expression in ESCC tumor cells was significantly associated with poor differentiation (p0.05) and strongly positive ALDH1 expression in tumor cells was related with shorter overall survival (p0.05), while the expression of ALDH1 in ESCC stromal cells had no significant relationship with clinicopathological features (p0.05).High expression of cancer stem cell marker ALDH1 in ESCC cells may thus portend a poor prognosis. However, its expression in the tumor microenvironment did not appear to have a role in ESCC behavior. More studies are warranted to find out the mechanisms to explain this.