Apc Mice Research Articles

Hypoxia-induced hsa_circ_0000826 is linked to liver metastasis of colorectal cancer.

Backgroundhsa_circ_0000826 has been previously linked to CRC through the competing endogenous RNA network; however, the upstream driver of hsa_circ_0000826 elevation remains unknown. In this study, we aim to elucidate the effect of hypoxia‐induced hsa_circ_0000826 on CRC tumorigenesis and metastasis.MethodsRNA scope assay was used to evaluate the expression of hsa_circ_0000826 in CRC cells under hypoxia condition. The effects of hsa_circ_0000826 on phenotypes of CRC cells were evaluated through cell migration and invasion assay. The nude, AOM‐DSS model mice and APCMin /+ mice were used to investigate the relationship between circ_0000826, hypoxia, and CRC in mice. A total of 100 CRC tissue samples, as well as the paired adjacent tissues, were collected, and qRT‐PCR assay was used to detect the expression of hsa_circ_0000826 in these samples.ResultsHypoxia‐induced hsa_circ_0000826 overexpression can increase the malignant phenotypes, tumor formation, and metastasis capability of CRC cells in vitro. mmu_circ_0000826 levels were significantly increased in the CRC tissues from AOM‐DSS and APC mice model under hypoxia conditions. Further, the hypoxia‐induced upregulation of mmu_circ_0000826 can also promote CRC tumorigenesis and liver metastasis in vivo. The expression of hsa_circ_0000826 in serum was significantly increased in CRC tissues in 100‐pair of CRC and according to the adjacent normal tissues by qRT‐PCR assays. Moreover, the expression levels of hsa_circ_0000826 in serum of patient with liver metastasis were significantly increased than those without metastasis.ConclusionOur results suggested that hsa_circ_0000826 was induced by the hypoxia in CRC, which can be a potential biomarker of CRC liver metastasis.

IKKα as a potential novel target for treatment of colorectal cancer.

174 Background: Colorectal cancer (CRC) is a heterogeneous disease leading to different survival outcomes for patients with the same stage of disease. The non-canonical NF-κB pathway has been shown to have a key role in tumorigenesis, and the aim of this study was to investigate the role of IKKα, the main catalytic component of this pathway in CRC. Methods: A tissue microarray was retrospectively constructed from a patient cohort (1033) with stage I-III CRC who underwent surgery. IHC was utilised to examine cytoplasmic and punctate IKKα expression and determine any association with clincopathological features and cancer specific survival (CSS). To assess IKKα inhibition, organoids were prepared from wild type (WT) mouse colon, mouse models of CRC (Apc and Apc.KRAS.pT53.TGFbR2 (AKPT)) and patient derived human organoids. These were treated with an IKKα inhibitor, SU1433 and organoid size and cell viability assessed. Results: High cytoplasmic expression of IKKα was associated with increasing T stage (p = 0.012), poor tumour differentiation (p = 0.010), tumour necrosis (p = 0.013) and low proliferation status (p = 0.013) but was not associated with CSS. High punctate IKKα expression associated with tumour differentiation (p = 0.001), necrosis (p = 0.004), proliferation (p = 0.044) and MMR competence (p < 0.001) and was also significantly associated with reduced CSS (HR1.20 95%CI 1.02-1.42, p < 0.001). SU1433 did not significantly impact on WT (C57/B16) organoid viability up to a concentration of 1 uM, however organoid size and cell viability was significantly reduced in a dose dependent manner in organoids from both Apc and AKPT mouse models. A similar reduction was observed in patient derived human organoids. Conclusions: Punctate IKKα expression was associated with poor cancer specific survival in CRC patients, and inhibition with SU1433, impacted on CRC mouse and patient derived human organoid size and cell viability. These results suggest that, following further investigation and confirmation, IKKα may be employed as a novel therapeutic target in CRC.

CRISPR gene-engineered CYBBko THP-1 cell lines highlight the crucial role of NADPH-induced reactive oxygen species for regulating inflammasome activation

Summary CRISPR-Cas9 engineered CYBBko THP-1 cell lines display an inflammatory profile with increased IL-1β, IL-6 and TNF-α secretion as consequence of NADPH-induced ROS deficiency. This phenotype is reminiscent of the one observed in phagocytes from CGD patients.

HASPIN kinase inhibitor CHR-6494 suppresses intestinal polyp development, cachexia, and hypogonadism in Apcmin/+ mice.

HASPIN has been identified as a nuclear Ser/Thr kinase specifically expressed in haploid germ cells. HASPIN kinase inhibitors were recently isolated, and their antitumor activity reported. Colorectal cancer occurs with high incidence worldwide. In this study, we examined whether HASPIN inhibitor CHR-6494 suppresses cancer progression in ApcMin/+ mice, a familial colon tumor disease model. Mice were treated by intraperitoneal injection of CHR-6494 for 50 days. Following the treatment period, intestinal polyps were counted and testosterone and spermatogenesis levels were observed. Intraperitoneal administration of CHR-6494 significantly inhibited intestinal polyp development and recovered body weight in ApcMin/+ mice. Although spermatogenesis was inhibited with increasing age in ApcMin/+ mice, CHR-6494 significantly improved blood testosterone levels and spermatogenesis. Our results suggest that HASPIN inhibitors may be useful as anti-cancer agents and for the treatment of hypogonadism in colorectal cancer patients.

Cachexia Disrupts Diurnal Regulation of Activity, Feeding, and Muscle Mechanistic Target of Rapamycin Complex 1 in Mice.

Cancer cachexia is characterized by severe skeletal muscle mass loss, which is driven by decreased muscle protein synthesis and increased protein degradation. Daily physical activity and feeding behaviors exhibit diurnal fluctuations in mice that can impact the systemic environment and skeletal muscle signaling. We investigated the effect of cancer cachexia on the diurnal regulation of feeding, physical activity, and skeletal muscle mechanistic target of rapamycin complex 1 (mTORC1) signaling in tumor-bearing mice. We also examined the impact of increased physical activity on diurnal behaviors and skeletal muscle mTROC1 signaling in the cancer environment. Physical activity and feeding behaviors were measured for four consecutive days before sacrifice in male C57BL/6 (B6; n = 24) and Apc (MIN; n = 22) mice at 7:00 AM and 7:00 PM under ad libitum condition. A subset of B6 (n = 16) and MIN (n = 19) mice were given wheel access for 2 wk before diurnal behavior measurements. Gastrocnemius muscle protein expression was examined. The MIN mice demonstrated altered diurnal fluctuations in feeding and activity compared with the B6. Interestingly, cachexia did not alter MIN total food intake, but dramatically reduced cage physical activity. As a measurement of mTORC1 activity, 4E-BP1 phosphorylation increased after the dark cycle in B6 and precachectic MIN mice, whereas rpS6 phosphorylation was only increased after the dark cycle in MIN mice. MIN 4E-BP1 phosphorylation at the end of the light cycle was significantly correlated with cachexia progression and reduced physical activity. Voluntary wheel running increased light cycle MIN 4E-BP1 phosphorylation and attenuated muscle mass loss. The cancer environment can alter diurnal feeding and physical activity behaviors in tumor-bearing mice, which are linked to the progression of cachexia and muscle wasting. Furthermore, suppressed physical activity during cachexia is associated with decreased skeletal muscle mTORC1 signaling.

The guanine nucleotide exchange factor, Spata13, influences social behaviour and nocturnal activity

Spermatogenesis-associated protein 13 (Spata13) is a guanine nucleotide exchange factor (GEF) enriched in discrete brain regions in the adult, with pronounced expression in the extended central amygdala (CeA). Loss of Spata13, also known as the adenomatous polyposis coli exchange factor Asef2, has no identifiable phenotype although it has been shown to reduce the number and size of intestinal tumours in Apc (Min/+) mice. Nevertheless, its brain-related functions have not been investigated. To pursue this, we have generated a Spata13 knockout mouse line using CRISPR-mediated deletion of an exon containing the GTPase domain that is common to multiple isoforms. Homozygous mutants were viable and appeared normal. We subjected both male and female cohorts to a comprehensive battery of behavioural tests designed to investigate particular CeA-related functions. Here, we show that Spata13 modulates social behaviour with homozygous mutants being subordinate to wildtype controls. Furthermore, female homozygotes show increased activity in home cages during the dark phase of the light–dark cycle. In summary, Spata13 modulates social hierarchy in both male and female mice in addition to affecting voluntary activity in females.

Gankyrin Contributes to Tumorigenesis and Chemoresistance in Sporadic Colorectal Cancer

Background: Although Gankyrin is overexpressed in many malignancies, the role of Gankyrin for tumorigenesis and chemoresistance remains to be elucidated in sporadic colorectal cancer (CRC). Aims: We investigate whether Gankyrin affects Adenomatous polyposis coli (Apc) inactivation-induced tumorigenesis and therapeutic response to anti-angiogenic agents. Methods: Epithelial cell-specific APC and/or Gankyrin-deficient mice were used. The patients with metastatic CRC (n = 53) who were enrolled in this study underwent resection of primary cancer followed by systemic chemotherapy containing bevacizumab. We determined whether gene expression in CRC tissues before chemotherapy is associated with radiological responses. Results: Deletion of Gankyrin in epithelial cell reduced the expression of c-Myc, a critical mediator of the APC signaling pathway, and interleukin-6. Gankyrin deficiency decreased the expression of Bmi1, a downstream molecule of c-Myc, and the activity of V-Akt murine thymoma viral oncogene homolog and extracellular signal-regulated protein kinase, leading to reduced Apc inactivation-induced tumorigenesis. Of 53 patients, 38 (72%) had increased Gankyrin expression in tumor cells. The enhanced Gankyrin expression in tumor cells was associated with unfavorable progression-free survival (log-rank test p = 0.026). Conclusion: Gankyrin in epithelial cell contributes to the development of sporadic CRC and the expression could serve as a biomarker to predict therapeutic response in patients with metastatic CRC.

Abstract 4074: Pten haploinsufficiency promotes tumor invasion and carcinogenesis in mouse colon epithelium with Apc deficiency

Abstract Background and Aim: The loss of PTEN (phosphatase and tensin homologue) expression in human colorectal cancer (CRC) is found in approximately 40% of cases, and its functional contribution is not fully understood. To address this question, the colon tumors from the Pten-deficient mice with a disruption of the Adenomatous polyposis coli (Apc) were compared with those from the mice with Apc deficiency alone. Method: To recapitulate human CRC, mouse models carrying transgenes regulated by a 9.5-kb fragment containing sequences from the human CDX2 promoter (CDX2P9.5), which were previously shown to have tightly restricted transgene expression in the colon epithelium, were prepared. CDX2P9.5-NLSCre;Apcflox/+;Ptenflox/+ (referred to as CPC;Apc+Pten mouse) and CDX2P9.5-NLSCre;Apcflox/+(;Pten+/+) mice (referred to as CPC;Apc mouse) were generated, and the overall survival and tumor phenotypes (number, size and severity) at 4, 6, 9, 12 and 15 weeks of age were compared between the two models. In addition, the loss of heterozygosity status of the Apc and Pten wild-type alleles in tumor tissue and normal mucosa in the two mouse models were compared with multiplex polymerase chain reaction (PCR), and Pten transcripts and protein expression were evaluated by quantitative reverse transcription PCR and immunohistochemistry, respectively. Results: CPC;Apc+Pten mice had a significantly shorter life span than CPC;Apc mice (median survival time of CPC;Apc+Pten mice and CPC;Apc mice: 14.4 weeks [n=20] vs. 21.6 weeks [n=20], respectively). The CPC;Apc+Pten mice had more tumors than CPC;Apc mice at all time points measured, but the tumor size in CPC;Apc+Pten mice was significantly larger than that in CPC;Apc mice only at 9 weeks of age. Invasion into the submucosa was more frequently observed in CPC;Apc+Pten mice than in CPC;Apc mice (CPC;Apc+Pten mice and CPC;Apc mice: 36.3%/58.3% and 4.9%/10% at 12/15 weeks of age, respectively). Regarding the findings on multiplex PCR, while the tumors from both models showed biallelic Apc inactivation, the tumors from CPC;Apc+Pten mice showed no loss of the wild-type allele of Pten. Regarding the Pten transcript levels, the levels in the tumors and mucosa from CPC;Apc+Pten mice were almost half of those in CPC;Apc mice. The Pten protein expression level in tumors from CPC;Apc+Pten mice was lower than that in tumors from CPC;Apc mice but not completely suppressed. Conclusion: Pten promotes tumor invasion and carcinogenesis without two hits on the gene. Consistent with the previous finding that two hits on the PTEN gene was a rare event in human CRC with PTEN loss, our data strongly suggest the haploinsufficient tumor-suppressive activity of Pten. These findings show that this CPC;Apc+Pten mouse model recapitulates human CRC with PTEN loss. Citation Format: Haruki Sada, Takao Hinoi, Hiroaki Niitsu, Masatoshi Kochi, Naoya Sakamoto, Kazuhiro Sentani, Naohide Oue, Wataru Yasui, Hideki Ohdan. Pten haploinsufficiency promotes tumor invasion and carcinogenesis in mouse colon epithelium with Apc deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4074.

Abstract 1266: Downregulation of PD-L1 by NSAID administration augments the effects of a multi-antigen vaccine for the prevention of adenomatous polyps in APC(Min/+) mice

Abstract Introduction: We have shown that treatment of APC(Min/+) mice with NSAIDs will inhibit development of adenomatous polyps and induce significant levels of polyp-infiltrating CD8 T-cells. We sought to identify the mechanism of NSAID induced immune stimulation and questioned whether administration of NSAIDs concurrent with vaccination could further reduce polyp formation. Methods: PD-L1 expression and T-cell infiltrates were assessed by IHC and flow cytometry. MC38 and RKO (murine/human colorectal carcinoma cell lines) were treated with naproxen (200uM-1000 uM) and harvested at 24, 48 and 72h. At 4-6 weeks, APC(Min/+) mice were given a multi-antigen peptide vaccine (COX2, CDC25B, EGFR) with CFA/IFA or adjuvant alone. Two groups received 400ppm naproxen orally 7d/wk for 18 weeks. Polyps were quantified at ≤24 weeks. Spleens and polyps were collected for IFN-gamma ELISPOT, flow cytometry, and IHC. Results: Polyps from the APC(Min/+) mouse and both cancer cell lines highly express PD-L1. PD-L1 expression was significantly decreased in MC38 (p<0.01) and RKO (p<0.01) as compared to control after incubation with naproxen at all doses. The inhibitory effect of NSAIDs on PD-L1 expression was both time and dose dependent. We evaluated the in vivo effect of combination immunoprevention in the APC(Min/+). Animals receiving vaccine alone showed a 33% inhibition of polyp formation while naproxen alone showed 54% inhibition (p<0.0001) compared to adjuvant alone. Combination treatment demonstrated significantly greater inhibition of polyps than either modality (p<0.001), 81% inhibition vs. adjuvant. Antigen specific T-cells could be detected at higher levels than control antigen in both the vaccine alone (p=0.0001) and combination-treated animals (p<0.0001). The antigen specific responses seen in combination treated animals were nearly two-fold that of vaccine alone (p=0.008). No antigen specific immunity was detected in naproxen and adjuvant only treated mice. The magnitude of the immune response was significantly correlated with lower polyp counts, with a Pearson correlation coefficient of -0.55 (p=0.0014). Detailed immunologic analysis of tumors will be presented. Conclusions: NSAIDs, via modulation of PD-L1, synergize with vaccines to increase immunogenicity and enhance influx of polyp infiltrating lymphocytes. This synergy results in superior prevention of polyp formation compared to treatment with NSAID or vaccine alone and has high potential for clinical application. Citation Format: Mary L. Disis, Lauren R. Corulli, Clinton Grubbs, Ronald A. Lubet, Paul Cowan, Ekram Gad. Downregulation of PD-L1 by NSAID administration augments the effects of a multi-antigen vaccine for the prevention of adenomatous polyps in APC(Min/+) mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1266.

PO-346 Defining microRNA mediated regulation of metabolic pathways involved in colon cancer progression (BST1-microRNA interactions)

IntroductionColorectal cancer (CRC) is the third most common cause of cancer related death in the world. Progressive accumulation of mutations in oncogenic and tumour suppressor pathways (e.g., KRAS and p53) and also microRNA (miR) deregulation are associated with CRC development. Very little has been done to dissect specific CRC pathways involved in miR regulation in response to stress and metabolic changes. Therefore, we previously characterised gene (mRNA) and miR expression as well as metabolic changes in different CRC genotypes. Here, we investigated the interaction between miR-mediated regulation of bone marrow stromal cell antigen 1 (BST1) or CD157, a metabolic gene involved in the conversion of nicotinamide adenine dinuclease (NAD) to paracrine factor cyclic ADP ribose (cADPR), following the acquisition of KRAS mutation in metastatic CRC.Material and methodsA combination of array analysis data, in-silico prediction tools and nuclear magnetic resonance (NMR) system were used to analyse gene and miR expression changes associated with different rounds of knock in/out mutations in Apc, Kras, and p53 CRC mouse models and their tumour-derived organoids (TDOs). Subsequently, we genetically modified human CRC cell lines and patients KRAS mutant (mut) TDOs from CRC metastases to modulate the candidate gene and miR. Organoids formation, growth rate and viability were measured with IncuCyte® Live-Cell Analysis System and Celigo® imaging cytometer.Results and discussionsWe identified mRNA/miR networks involved in cancer metabolic pathways and assessed miRs of potential interest among the candidates. MiR-203 appeared as one of the most significant candidates. Furthermore target validation and analysis of human tissues microarrays showed an association between KRAS mutations, miR-203 down-regulation and over-expression of BST1, which was identified as a direct target of miR-203 regulation in our studies. Repressing BST1 and over-expressing miR-203 had a significant effect on the proliferation and migration abilities of organoids in 3D culture in normal and calorie-deprived conditions.ConclusionThis project aimed at characterising the functional consequences of miR-203 loss and BST1 up regulation in normal and nutrient-deprived conditions in order to find a promising candidate as a therapeutic target in KRAS mut CRC. Although we showed KRAS mut CRC cells lost a growth advantage with miR-203 and BST1 deregulation ex vivo, work is on going to confirm the consequence of their interaction in vivo.

Insulin Action on Endothelial Cells Limits Leukocyte-Endothelial Interaction through CXCR4 and Counteracts Intestinal Tumor Formation in Obesity

An activated, pro-inflammatory endothelium is a key feature of obesity and type 2 diabetes but little is known about its contribution to the increased cancer risk in these conditions. We recently published that insulin resistance in endothelial cells increases VCAM-1 expression, increases recruitment of tumor-associated neutrophils and promotes intestinal tumor formation. Here, we report that tumor-prone Apc(Min/+) mice have 2.1-fold more intestinal tumors during high-fat feeding. However, gain of insulin signaling specifically in endothelial cells by overexpression of IRS1 prevented 44% of tumors in diet-induced obesity. RNA sequencing in primary human endothelial cells identified 17 genes changed by insulin treatment with a log(2) change of at least ±0.5, among them C-X-C motif chemokine receptor 4 (CXCR4), which was downregulated by 54%. Downregulation of CXCR4 after insulin treatment was completely prevented by pretreatment with wortmannin but not by U0126, an inhibitor of MAPK signaling. Using FACS of enzymatically dissociated tissue, CXCR4 mRNA in CD31+ cells was 77% higher in mice with diet-induced obesity compared to lean controls and 37% higher in db/db mice compared to db/+ controls, consistent with upregulation of CXCR4 in endothelial cell insulin resistance. CXCL12, the ligand for CXCR4, increased leukocyte adhesion to cultured endothelial cells and this was completely prevented by plerixafor, a clinically approved CXCR4 antagonist. In vivo microscopy of mesenteric venules showed an increase in leukocyte rolling and adhesion after intravenous injection of CXCL12 and this change was abrogated in transgenic mice with endothelial overexpression of IRS1. We conclude that endothelial cell insulin signaling limit leukocyte-endothelial cell interaction through downregulation of CXCR4 and that improving insulin signaling in endothelial cells may protect against tumor development in obesity. Disclosure T. Rathjen: Employee; Self; Novo Nordisk A/S, Bayer AG. Q. Li: None. K. Park: None. G. Povlsen: Employee; Self; Novo Nordisk A/S. G.S. Olsen: Employee; Self; Novo Nordisk A/S. G.L. King: Research Support; Self; Sanofi-Aventis. C. Rask-Madsen: Research Support; Self; Novo Nordisk A/S.

Phenotypic Characterization of Adherent Cells Population CD34+ CD90+ CD105+ Derived from Wharton's Jelly.

BackgroundMesenchymal stromal cells, MSCs, show expression of specific antigens on their surface. The aim of the study is to assess the phenotype of stem cells like isolated from the umbilical cord with respect to the presence of surface antigens CD34, CD90, and CD105 and differences in the expression of surface antigens in cells isolated from freshly sampled material in comparison with the phenotype of cells from in vitro culture.Material/MethodsStem cells collected from the umbilical cord from healthy patients and then cultured in vitro. To assess the phenotype of stem cells, cytometric analysis was carried out. To assess the phenotype of cells we used fluorescently labelled monoclonal antibodies: APC Mouse anti-human CD34, PC5 Mouse anti-human CD90 and PE Mouse anti-human CD105.ResultsIn the case of cells from the umbilical cord and then cultured in vitro for the period of 10–14 days CD34 expression is lower (69,5%) in comparison with the group of cells not cultured. Not cultured cells were demonstrated 37% of cells co-expression of antigens CD34 and CD105, over 21% of CD34/CD90 cells and over 24% of CD105/CD90. Cultured cells group was showed higher percentage of CD90, CD105, CD34/CD105, CD34/CD90, CD105/CD90 in comparison with not cultured cells.ConclusionsOur reults suggested that adherent cells population from umbilical cord, demonstrate CD34 expression In vivo. Moreover, the phenotype of MSCs, mainly in the context of CD34 expression, may vary depending on the place of collection of cells and the length of growing the cell culture.

Immunogenicity of murine mPEG-red blood cells and the risk of anti-PEG antibodies in human blood donors.

The immunocamouflage of non-ABO blood group antigens by membrane-grafted methoxypoly(ethylene glycol) (mPEG) may attenuate the risk of red blood cell (RBC) alloimmunization. However, concerns have been raised over the immunogenic risk of PEG and PEG-RBCs. To assess this risk, murine and human studies were performed. Mice were exposed to soluble PEG prior to, or between, multiple transfusions (∼60-day intervals) of control or mPEG-RBCs, and cell survival was determined by flow cytometry. In some studies, the control and mPEG-RBC groups were reversed after one or more transfusions. Furthermore, human blood donors and commercial intravenous immunoglobulin products were examined to detect anti-PEG antibodies and to assess the risk for false positives. Naïve mice receiving chronic mPEG-RBC transfusions had normal RBC survival curves with no evidence of anti-PEG antibodies. Similarly, challenge with soluble PEG did not elicit anti-PEG antibodies in mice. Studies in humans revealed no evidence of a high prevalence of anti-PEG antibodies in either blood donors or commercial intravenous immunoglobulin. However, by use of the methods employed by studies identifying high levels of anti-PEG antibodies, a significant level (∼15%) of "false positives" were detected in commercial antibodies of known (non-PEG) specificities. These findings suggest that methodologic problems yielded a high rate of false positives in these earlier studies. These data continue to support the clinical utility of cellular PEGylation and the low immunogenic risk of grafted mPEG.

Wnt signalling modulates transcribed-ultraconserved regions in hepatobiliary cancers

ObjectiveTranscribed-ultraconserved regions (T-UCR) are long non-coding RNAs which are conserved across species and are involved in carcinogenesis. We studied T-UCRs downstream of the Wnt/β-catenin pathway in liver cancer.DesignHypomorphic Apc mice...

Abstract 3675: Loss of Pkhd1 promotes intestinal tumorigenesis in Apc mice

Abstract Objectives: PKHD1 (Polycystic Kidney and Hepatic Disease 1), a causal gene for ARPKD (Autosomal Recessive Polycystic Kidney Disease), has been proposed to play a role in the colorectal carcinogenesis. This study is to explore the role of PKHD1 in colorectal carcinogenesis. Methods: Mouse model with Pkhd1 mutation (Pkhd1-/-) was used to mate with ApcMin/+ mouse in the same genetic background. The resulting ApcMin/+ mice with and without Pkhd1-/- alleles were randomly sorted into two experimental groups (i.e. Pkhd1-/-;ApcMin/+ n = 36; ApcMin/+ n = 23). Tumorigenic susceptibility of Pkhd1-/-;ApcMin/+ and ApcMin/+ mice was compared. The number, size, pathology of intestinal tumors in the mice were analyzed at the age of 1-, 3-, and 6-month. The survival rates were investigated in the two groups of mice. Results: There was no statistical difference in tumor number and size between 1-month-old ApcMin/+ and Pkhd1-/-;ApcMin/+ mice. However, the number and size of the intestinal tumors in 3- and 6-month-old Pkhd1-/-;ApcMin/+ mice were significantly increased compared with those of ApcMin/+ mice alone (P = 0.017,P = 0.022). In addition, the pathology of tumors was also analyzed between the two groups. There was no pathological difference between 1-month-old ApcMin/+ and Pkhd1-/-;ApcMin/+ mice. However, at 3-month-old, Pkhd1-/-;ApcMin/+ mice exhibited severer atypical hyperplasia in the intestinal tumor than those of ApcMin/+ mice; while at 6-month-old, focal necrosis were often seen in intestinal tumors of Pkhd1-/-;ApcMin/+ mice, but none from ApcMin/+ mice. Strikingly, tumor infiltration into the muscular mucosa was also seen in a 6-month-old Pkhd1-/-;ApcMin/+ mice. There was no statistically survival difference between ApcMin/+ and Pkhd1-/-;ApcMin/+ mice. Conclution: Loss of Pkhd1 promotes the intestinal tumorigenesis and induces tumor malignant transformation in ApcMin/+ mice. Citation Format: Bo Hu, Wei Li, Qingchao Qiu, Zhengxi He, Xiusheng He, Guanqing Wu. Loss of Pkhd1 promotes intestinal tumorigenesis in Apc mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3675.

Abstract 952: Genome-wide enhancer analysis identifies PVT1 as an oncogenic enhancer of MYC

Abstract Introduction: An enhancer is a short stretch of DNA that can be targeted by specific proteins to activate gene transcription, and recent evidence suggests that enhancers can act as key regulators of oncogenes in cancer. Recently, the FANTOM5 project comprehensively characterized enhancer activities in various cell types. Interestingly, several enhancers coding for lncRNAs were commonly up-regulated in multiple cancer types, suggesting that these enhancers may play an important oncogenic role through enhancer-promoter-lncRNA interaction, and may help mediate chemoresistance. However, specific lncRNAs with oncogenic enhancer function and their clinical significance in colorectal cancer (CRC) remains unexplored. Aims: We analyzed genome-wide cancer-specific enhancer activation in multiple cancers to identify potential oncogenic lncRNAs, and assessed their clinical significance in two independent patient cohorts with CRC. We thereafter evaluated the functional role of candidate enhancer lncRNAs in CRC. Methods: Using the FANTOM5 enhancer database, cancer-specific enhancer activity was analyzed in cancers of the colon, stomach, lung, prostate and melanoma. LncRNA candidates that encode these cancer-specific enhancers were identified. We then screened these lncRNAs in two independent CRC cohorts, as well as the APC(Min/+) mouse model. The functional role of target lncRNAs was assessed in parental and chemoresistant CRC cell lines. The oncogenic enhancer activity was confirmed using the chromatin conformation capture (3C) assay. Results: We identified ten lncRNA candidates with consistent enhancer activities in all five cancer types. In particular, PVT1 appeared to have one of the highest enhancer activities. Using two independent CRC cohorts, we confirmed upregulation of PVT1 in cancer tissues. Patients with high-PVT1 expression had shorter overall survival in Stage II and III CRCs, suggesting that the PVT1 expression could discriminate high-risk patients, and identify those who may benefit from adjuvant chemotherapy. Intriguingly, we discovered that PVT1 was also upregulated in human colorectal adenomas as well as adenomas isolated from APC(Min/+) mice, indicating that PVT1 may be involved in the early stages of the adenoma-carcinoma sequence. The expression level of PVT1 significantly correlated with c-MYC expression in CRC specimens and the 3C assay showed that the enhancer region in PVT1 can target c-MYC. Finally, we demonstrated that PVT1 and c-MYC were co-overexpressed in several chemoresistant CRC cell lines, and that suppression of PVT1 led to a significant reduction in proliferation of chemoresistant cells, further suggesting that PVT1 may play a key role in chemoresistance. Conclusion: PVT1 acts as an oncogenic enhancer of c-MYC in CRC. High expression of PVT1 served as a predictive marker for identifying high-risk Stage II and III CRC patients. PVT1 could serve as a potential therapeutic target in chemoresistant CRCs. Citation Format: Kunitoshi Shigeyasu, Shusuke Toden, Takeshi Nagasaka, Toshiyoshi Fujiwara, C. Richard Boland, Ajay Goel. Genome-wide enhancer analysis identifies PVT1 as an oncogenic enhancer of MYC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 952.

Loss of TGFβ signaling promotes colon cancer progression and tumor-associated inflammation.

TGFβ has both tumor suppressive and tumor promoting effects in colon cancer. Also, TGFβ can affect the extent and composition of inflammatory cells present in tumors, contextually promoting and inhibiting inflammation. While colon tumors display intratumoral inflammation, the contributions of TGFβ to this process are poorly understood. In human patients, we found that epithelial loss of TGFβ signaling was associated with increased inflammatory burden; yet overexpression of TGFβ was also associated with increased inflammation. These findings were recapitulated in mutant APC models of murine tumorigenesis, where epithelial truncation of TGFBR2 led to lethal inflammatory disease and invasive colon cancer, mediated by IL8 and TGFβ1. Interestingly, mutant APC mice with global suppression of TGFβ signals displayed an intermediate phenotype, presenting with an overall increase in IL8-mediated inflammation and accelerated tumor formation, yet with a longer latency to the onset of disease observed in mice with epithelial TGFBR-deficiency. These results suggest that the loss of TGFβ signaling, particularly in colon epithelial cells, elicits a strong inflammatory response and promotes tumor progression. This implies that treating colon cancer patients with TGFβ inhibitors may result in a worse outcome by enhancing inflammatory responses.

Inhibition of ER stress and unfolding protein response pathways causes skeletal muscle wasting during cancer cachexia.

Cachexia is a devastating syndrome that causes morbidity and mortality in a large number of patients with cancer. However, the mechanisms of cancer cachexia remain poorly understood. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes stress. The ER responds to this stress through activating certain pathways commonly known as the unfolding protein response (UPR). The main function of UPR is to restore homeostasis, but excessive or prolonged activation of UPR can lead to pathologic conditions. In this study, we examined the role of ER stress and UPR in regulation of skeletal muscle mass in naïve conditions and during cancer cachexia. Our results demonstrate that multiple markers of ER stress are highly activated in skeletal muscle of Lewis lung carcinoma (LLC) and Apc(Min/+) mouse models of cancer cachexia. Treatment of mice with 4-phenylbutyrate (4-PBA), a chemical chaperon and a potent inhibitor of ER stress, significantly reduced skeletal muscle strength and mass in both control and LLC-bearing mice. Blocking the UPR also increased the proportion of fast-type fibers in soleus muscle of both control and LLC-bearing mice. Inhibition of UPR reduced the activity of Akt/mTOR pathway and increased the expression of the components of the ubiquitin-proteasome system and autophagy in LLC-bearing mice. Moreover, we found that the inhibition of UPR causes severe atrophy in cultured myotubes. Our study provides initial evidence that ER stress and UPR pathways are essential for maintaining skeletal muscle mass and strength and for protection against cancer cachexia.-Bohnert, K. R., Gallot, Y. S., Sato, S., Xiong, G., Hindi, S. M., Kumar, A. Inhibition of ER stress and unfolding protein response pathways causes skeletal muscle wasting during cancer cachexia.

Olfactomedin 4 expression and functions in innate immunity, inflammation, and cancer.

Olfactomedin 4 (OLFM4) is an olfactomedin domain-containing glycoprotein. Multiple signaling pathways and factors, including NF-κB, Wnt, Notch, PU.1, retinoic acids, estrogen receptor, and miR-486, regulate its expression. OLFM4 interacts with several other proteins, such as gene associated with retinoic-interferon-induced mortality 19 (GRIM-19), cadherins, lectins, nucleotide oligomerization domain-1 (NOD1) and nucleotide oligomerization domain-2 (NOD2), and cathepsins C and D, known to regulate important cellular functions. Recent investigations using Olfm4-deficient mouse models have provided important clues about its in vivo biological functions. Olfm4 inhibited Helicobacter pylori-induced NF-κB pathway activity and inflammation and facilitated H. pylori colonization in the mouse stomach. Olfm4-deficient mice exhibited enhanced immunity against Escherichia coli and Staphylococcus aureus infection. Olfm4 deletion in a chronic granulomatous disease mouse model rescued them from S. aureus infection. Olfm4 deletion in mice treated with azoxymethane/dextran sodium sulfate led to robust intestinal inflammation and intestinal crypt hyperplasia. Olfm4 deletion in Apc (Min/+) mice promoted intestinal polyp formation as well as adenocarcinoma development in the distal colon. Further, Olfm4-deficient mice spontaneously developed prostatic epithelial lesions as they age. OLFM4 expression is correlated with cancer differentiation, stage, metastasis, and prognosis in a variety of cancers, suggesting its potential clinical value as an early-stage cancer marker or a therapeutic target. Collectively, these data suggest that OLFM4 plays important roles in innate immunity against bacterial infection, gastrointestinal inflammation, and cancer. In this review, we have summarized OLFM4's initial characterization, expression, regulation, protein interactions, and biological functions.

Hsp70 exerts oncogenic activity in the Apc mutant Min mouse model.

Colorectal cancer (CRC) develops from colonic epithelial cells that lose expression of key tumor suppressor genes and/or gain expression of proproliferative and antiapoptotic genes like heat shock protein 70 (Hsp70). Heat shock protein 70 is overexpressed in CRC, but it is not known whether this is in response to the proteotoxic stress induced by transformation, or if it contributes to the process of transformation itself. Here, using the Apc (Min/+) mouse model of CRC, we show that Hsp70 regulates mitogenic signaling in intestinal epithelial cells through stabilization of proteins involved in the receptor tyrosine kinase (RTK) and WNT signaling pathways. Loss of Hsp70 reduced tumor size with decreased proliferation and increased tumor cell death. Hsp70 loss also led to decreased expression of ErbB2, Akt, ERK and β-catenin along with decreased β-catenin transcriptional activity as measured by c-myc and axin2 expression. Upregulation of RTK or WNT signals are frequent oncogenic events in CRC and many other cancers. Thus, in addition to the role of Hsp70 in cell-survival after transformation, Hsp70 stabilization of β-catenin, Akt, ERK and ErbB2 are predicted to contribute to transformation. This has important implications not only for understanding the pathophysiology of these cancers, but also for treatment since anti-EGFR antibodies are in clinical use for CRC and EGFR is a major ErbB2 heterodimeric partner. Targeting Hsp70, therefore, might provide an alternative or complementary strategy for achieving better outcomes for CRC and other related cancer types.