Therapeutic effect of c‐Jun N‐terminal kinase inhibition on pancreatic cancer

c‐Jun N‐terminal kinase (JNK) is a member of the mitogen‐activated protein kinase (MAPK) family, and it is reportedly involved in the development of several cancers. However, the role of JNK in pancreatic cancer has not been elucidated. We assessed t he involvement of JNK in the development of pancreatic cancer and investigated the therapeutic effect of JNK inhibitors on this deadly cancer. Small interfering RNAs against JNK or the JNK inhibitor SP600125 were used to examine the role of JNK in cellular proliferation and the cell cycles of pancreatic cancer cell lines. Ptf1a^cre/+;LSL‐Kras^G12D/+;Tgfbr2^flox/flox mice were treated with the JNK inhibitor to examine pancreatic histology and survival. The effect of JNK inhibition on tumor angiogenesis was also assessed using cell lines and murine pancreatic cancer specimens. JNK was frequently activated in human and murine pancreatic cancer in vitro and in vivo. Growth of human pancreatic cancer cell lines was suppressed by JNK inhibition through G1 arrest in the cell cycle with decreased cyclin D1 expression. In addition, oncogenic K‐ras expression led to activation of JNK in pancreatic cancer cell lines. Treatment of Ptf1a^cre/+;LSL‐Kras^G12D/+;Tgfbr2^flox/flox mice with the JNK inhibitor decreased growth of murine pancreatic cancer and prolonged survival of the mice significantly. Angiogenesis was also decreased by JNK inhibition in vitro and in vivo. In conclusion, activation of JNK promotes development of pancreatic cancer, and JNK may be a potential therapeutic target for pancreatic cancer.     

Vasohibin‐2 plays an essential role in metastasis of pancreatic ductal adenocarcinoma

Vasohibin‐2 (VASH2) is expressed in various cancers and promotes their progression. We recently reported that pancreatic cancer patients with higher VASH2 expression show poorer prognosis. Herein, we sought to characterize the role of VASH2 in pancreatic cancer. We used LSL‐Kras^G12D; LSL‐Trp53^R172H; Pdx‐1‐Cre (KPC) mice, a mouse model of pancreatic ductal adenocarcinoma (PDAC), and cells isolated from them (KPC cells). Knockdown of Vash2 from PDAC cells did not affect their proliferation, but decreased their migration. When Vash2‐knockdown PDAC cells were orthotopically inoculated, liver metastasis and peritoneal dissemination were reduced, and the survival period was significantly prolonged. When KPC mice were crossed with Vash2‐deficient mice, metastasis was significantly decreased in Vash2‐deficient KPC mice. VASH2 was recently identified to have tubulin carboxypeptidase activity. VASH2 knockdown decreased, whereas VASH2 overexpression increased tubulin detyrosination of PDAC cells, and tubulin carboxypeptidase (TCP) inhibitor parthenolide inhibited VASH2‐induced cell migration. We next clarified its role in the tumor microenvironment. Tumor angiogenesis was significantly abrogated in vivo when VASH2 was knocked down or deleted. We further examined genes downregulated by Vash2 knockdown in KPC cells, and found chemokines and cytokines that were responsible for the recruitment of myeloid derived suppressor cells (MDSC). Indeed, MDSC were accumulated in PDAC of KPC mice, and they were significantly decreased in Vash2‐deficient KPC mice. These findings suggest that VASH2 plays an essential role in the metastasis of PDAC with multiple effects on both cancer cells and the tumor microenvironment, including tubulin detyrosination, tumor angiogenesis and evasion of tumor immunity.     

Reduced IL‐6 levels and tumor‐associated phospho‐STAT3 are associated with reduced tumor development in a mouse model of lung cancer chemoprevention with myo‐inositol

Several promising chemopreventive agents have for lung cancer emerged in preclinical models and in retrospective trials. These agents have been shown to modulate pathways altered in carcinogenesis and reduce markers of carcinogenesis in animal and cell culture models. Cancer‐prone transgenic mice with oncogenic Kras expressed in the airway epithelium (Ccsp^Cre/+; Kras^LSL‐G12D/+) were raised on diets compounded with myo‐inositol. These animals form lung premalignant lesions in a stereotypical fashion over the ten weeks following weaning. Mice raised on myo‐inositol containing diets showed potent reduction in the number, size, and stage of lesions as compared to those raised on control diets. myo‐inositol has previously been reported to inhibit phosphoinositide 3‐kinase (PI3K) signaling. However, in mice raised on myo‐inositol, total PI3K signaling was largely unaffected. Proteomic and cytokine analyses revealed large reduction in IL‐6 related pathways, including STAT3 phosphorylation. This effect was not due to direct inhibition of IL‐6 production and autocrine signaling within the tumor cell, but rather through alteration in macrophage recruitment and in phenotype switching, with an increase in antitumoral M1 macrophages.     

Epistatic interactions govern chemically‐induced lung tumor susceptibility and Kras mutation site in murine C57BL/6J‐ChrA/J chromosome substitution strains

Cancer susceptibility results from interactions between sensitivity and resistance alleles. We employed murine chromosome substitution strains to study how resistance alleles affected sensitive alleles during chemically‐induced lung carcinogenesis. The C57BL/6J‐Chr#^A/J strains, constructed by selectively breeding sensitive A/J and resistant C57BL/6J (B6) mice, each contain one pair of A/J chromosomes within an otherwise B6 genome. Pas1, the major locus responsible for this differential strain response to urethane carcinogenesis, resides on Chr 6, but C57BL/6J‐Chr6^A/J mice (hereafter CSS‐6) developed few tumors following a single urethane injection, which demonstrates epistatic interactions with other B6 alleles. CSS6 mice developed dozens of lung tumors after chronic urethane exposure, however, indicating that these epistatic interactions could be overcome by repeated carcinogen administration. Unlike A/J, but similar to B6 mice, CSS6 mice were resistant to lung carcinogenesis induced by 3‐methylcholanthrene (MCA). Tumor multiplicity increased if BHT administration followed urethane exposure, showing that a Chr 6 gene(s) regulates sensitivity to chemically‐induced tumor promotion. Unlike A/J tumors (predominantly codon 61 A→T transversions), Kras mutations in tumors induced by urethane in CSS‐6 mice were similar to B6 tumors (codon 61 A→G transitions). DNA repair genes not located on Chr 6 may determine the nature of Kras mutations. CSS‐6 mice are a valuable resource for testing the ability of candidate genes to modulate lung carcinogenesis.     

Development of Kras mutant lung adenocarcinoma in mice with knockout of the airway lineage‐specific gene Gprc5a

Despite the urgency for prevention and treatment of lung adenocarcinoma (LUAD), we still do not know drivers in pathogenesis of the disease. Earlier work revealed that mice with knockout of the G‐protein coupled receptor Gprc5a develop late onset lung tumors including LUADs. Here, we sought to further probe the impact of Gprc5a expression on LUAD pathogenesis. We first surveyed GPRC5A expression in human tissues and found that GPRC5A was markedly elevated in human normal lung relative to other normal tissues and was consistently downregulated in LUADs. In sharp contrast to wild‐type littermates, Gprc5a^–/– mice treated chronically with the nicotine‐specific carcinogen NNK developed LUADs by 6 months following NNK exposure. Immunofluorescence analysis revealed that the LUADs exhibited abundant expression of surfactant protein C and lacked the clara cell marker Ccsp, suggesting that these LUADs originated from alveolar type II cells. Next, we sought to survey genome‐wide alterations in the pathogenesis of Gprc5a^–/– LUADs. Using whole exome sequencing, we found that carcinogen‐induced LUADs exhibited markedly higher somatic mutation burdens relative to spontaneous tumors. All LUADs were found to harbor somatic mutations in the Kras oncogene (p. G12D or p. Q61R). In contrast to spontaneous lesions, carcinogen‐induced Gprc5a^–/– LUADs exhibited mutations (variants and copy number gains) in additional drivers (Atm, Kmt2d, Nf1, Trp53, Met, Ezh2). Our study underscores genomic alterations that represent early events in the development of Kras mutant LUAD following Gprc5a loss and tobacco carcinogen exposure and that may constitute targets for prevention and early treatment of this disease.     

c‐Jun N‐terminal kinase in pancreatic tumor stroma augments tumor development in mice

Pancreatic ductal adenocarcinoma (PDAC) is a life‐threatening disease and there is an urgent need to develop improved therapeutic approaches. The role of c‐Jun N‐terminal kinase (JNK) in PDAC stroma is not well defined even though dense desmoplastic reactions are characteristic of PDAC histology. We aimed to explore the role of JNK in PDAC stroma in mice. We crossed Ptf1a^Cre/+;Kras^G12D/+ mice with JNK1^?/? mice to generate Ptf1a^Cre/+;Kras^G12D/+;JNK1^?/? (Kras;JNK1^?/?) mice. Tumor weight was significantly lower in Kras;JNK1^?/? mice than in Kras;JNK1^+/? mice, whereas histopathological features were similar. We also transplanted a murine PDAC cell line (mPC) with intact JNK1 s.c. into WT and JNK1^?/? mice. Tumor diameters were significantly smaller in JNK1^?/? mice. Phosphorylated JNK (p‐JNK) was activated in α‐smooth muscle actin (SMA)‐positive cells in tumor stroma, and mPC‐conditioned medium activated p‐JNK in tumor‐associated fibroblasts (TAF) in vitro. Relative expression of Ccl20 was downregulated in stimulated TAF. Ccl20 is an important chemokine that promotes CD8⁺ T‐cell infiltration by recruitment of dendritic cells, and the number of CD8⁺ T cells was decreased in Kras;JNK1^+/? mice compared with Kras;JNK1^?/? mice. These results suggest that the cancer secretome decreases Ccl20 secretion from TAF by activation of JNK, and downregulation of Ccl20 secretion might be correlated with reduction of infiltrating CD8⁺ T cells. Therefore, we concluded that inhibition of activated JNK in pancreatic tumor stroma could be a potential therapeutic target to increase Ccl20 secretion from TAF and induce accumulation of CD8⁺ T cells, which would be expected to enhance antitumor immunity.     

Ikaros is a critical target during simultaneous exposure to X‐rays and N‐ethyl‐N‐nitrosourea in mouse T‐cell lymphomagenesis

Cancer risk associated with radiation exposure is considered the result of concurrent exposure to other natural and manmade carcinogens. Available data on the molecular characteristics of cancer after simultaneous exposure to radiation and chemicals are insufficient. In our study, we used a mouse thymic lymphoma (TL) model that was synergistically induced by simultaneous exposure to X‐rays and N‐ethyl‐N‐nitrosourea (ENU) at subcarcinogenic doses and analyzed the mutation frequency and spectrum of the TL‐associated genes Ikaros, Notch1, p53 and Kras. We found that the point mutation frequency in Ikaros was significantly increased to 47% for simultaneous exposure compared to 13 and 0% for X‐ray and ENU exposure alone, respectively. These mutations were mostly G:C > A:T at non‐CpG sites and T:A > C:G, both of which are characteristic of ENU mutagenesis. About half of the point mutations were accompanied by loss of heterozygosity (LOH), typical of X‐irradiation. The remaining half did not include LOH, which suggests that they were dominant‐negative mutations. In Notch1, the frequency of abnormalities was high (>58%) regardless of the treatment, suggesting that Notch1 aberration may be important for T‐cell lymphomagenesis. The p53 and Kras mutation frequencies were low for all treatments (<23%). Importantly, the frequency of TLs containing mutations in multiple genes, especially both Ikaros and Notch1, increased after simultaneous exposure. Thus, after simultaneous exposure, Ikaros is a critical target and is inactivated by ENU‐induced point mutations and/or X‐ray‐induced LOH in T‐cell lymphomagenesis. Furthermore, concomitant alterations of multiple tumor‐associated genes may contribute to enhanced lymphomagenesis after simultaneous exposure.     

Peripubertal high‐fat diet promotes c‐Myc stabilization in mammary gland epithelium

Dietary fat consumption during accelerated stages of mammary gland development, such as peripubertal maturation or pregnancy, is known to increase the risk for breast cancer. However, the underlying molecular mechanisms are not fully understood. Here we examined the gene expression profile of mouse mammary epithelial cells (MMECs) on exposure to a high‐fat diet (HFD) or control diet (CD). Trp53^?/? female mice were fed with the experimental diets for 5 weeks during the peripubertal period (3‐8 weeks of age). The treatment showed no significant difference in body weight between the HFD‐fed mice and CD‐fed mice. However, gene set enrichment analysis predicted a significant enrichment of c‐Myc target genes in animals fed HFD. Furthermore, we detected enhanced activity and stabilization of c‐Myc protein in MMECs exposed to a HFD. This was accompanied by augmented c‐Myc phosphorylation at S62 with a concomitant increase in ERK phosphorylation. Moreover, MMECs derived from HFD‐fed Trp53^?/? mouse showed increased colony‐ and sphere‐forming potential that was dependent on c‐Myc. Further, oleic acid, a major fatty acid constituent of the HFD, and TAK‐875, an agonist to G protein‐coupled receptor 40 (a receptor for oleic acid), enhanced c‐Myc stabilization and MMEC proliferation. Overall, our data indicate that HFD influences MMECs by stabilizing an oncoprotein, pointing to a novel mechanism underlying dietary fat‐mediated mammary carcinogenesis.     

A newly synthesized compound, 4′‐geranyloxyferulic acid–N(omega)‐nitro‐l‐arginine methyl ester suppresses inflammation‐associated colorectal carcinogenesis in male mice

We previously reported the cancer chemopreventive activity of 4′‐geranyloxyferulic acid (GOFA, Miyamoto et al., Nutr Cancer 2008; 60:675‐84) and a β‐cyclodextrin inclusion compound of GOFA (Tanaka et al., Int J Cancer 2010; 126:830‐40) in colitis‐related colorectal carcinogenesis. In our study, the chemopreventive effects of a newly synthesized GOFA‐containing compound, GOFA–N(omega)‐nitro‐l ‐arginine methyl ester (L‐NAME), which inhibits inducible nitric oxide (iNOS) and cyclooxygenase‐2 (COX) enzymes, were investigated using a colitis‐associated mouse colorectal carcinogenesis model with azoxymethane (AOM) and dextran sodium sulfate (DSS). The dietary administration of GOFA–L‐NAME after the AOM and DSS treatments significantly reduced the multiplicity of adenocarcinomas (inhibition rates: 100 ppm, 84%, p < 0.001; 500 ppm, 94%, p < 0.001) compared with the AOM + DSS group. Dietary GOFA–L‐NAME significantly decreased the proliferation (p < 0.001) and increased the apoptosis (p < 0.001) of colonic adenocarcinoma cells. A subsequent short‐term experiment revealed that dietary GOFA–L‐NAME decreased the mRNA expression of inflammatory enzymes, such as iNOS and COX‐2, and proinflammatory cytokines, such as tumor necrosis factor‐α, interleukin (IL)?1β, IL‐6 and macrophage inflammatory protein (MIP)?2 in the colonic mucosa of mice that received 1.5% DSS in their drinking water for 7 days. Our findings indicate that GOFA–L‐NAME is able to inhibit colitis‐associated colon carcinogenesis by modulating inflammation, proliferation, apoptosis and the expression of proinflammatory cytokines in mice.     

Mebendazole disrupts stromal desmoplasia and tumorigenesis in two models of pancreatic cancer

The five-year survival rate for metastatic pancreatic cancer is currently only 3%, which increases to 13% with local invasion only and to 39% with localized disease at diagnosis. Here we evaluated repurposed mebendazole, an approved anthelminthic drug, to determine how mebendazole might work at the different stages of pancreatic cancer formation and progression. We asked if mebendazole could prevent initiation of pancreatic intraepithelial neoplasia precursor lesions, interfere with stromal desmoplasia, or suppress tumor growth and liver metastasis. In both the Kras^LSL.G12D/+; Pdx1-Cre (KC) mouse model of caerulein-induced inflammatory pancreatitis and the Kras^LSL.G12D/+; Tp53^R172H/+; Pdx1-Cre (KPC) mouse model of advanced pancreatic cancer, mebendazole significantly reduced pancreas weight, dysplasia and intraepithelial neoplasia formation, compared to controls. Mebendazole significantly reduced trichrome-positive fibrotic connective tissue and α-SMA-positive activated pancreatic stellate cells that heralds fibrogenesis. In the aggressive KPC model, mebendazole significantly suppressed pancreatic tumor growth, both as an early and late intervention. Mebendazole reduced the overall incidence of pancreatic cancer and severity of liver metastasis in KPC mice. Using early models of pancreatic cancer, treatment with mebendazole resulted in less inflammation, decreased dysplasia, with the later stage model additionally showing a decreased tumor burden, less advanced tumors, and a reduction of metastasis. We conclude that mebendazole should be investigated further as a component of adjuvant therapy to slow progression and prevent metastasis, and well as for primary prevention in the highest risk patients.     

Inhibition of mutant Kras and p53‐driven pancreatic carcinogenesis by atorvastatin: Mainly via targeting of the farnesylated DNAJA1 in chaperoning mutant p53

Recent studies have indicated that using statins to inhibit the mevalonate pathway induces mutant p53 degradation by impairing the interaction of mutant p53 with DnaJ subfamily A member 1 (DNAJA1). However, the role of the C‐terminus of DNAJA1 with a CAAX box for farnesylation in the binding, folding, and translocation of client proteins such as mutant p53 is not known. In the present study, we used a genetically engineered mouse model of pancreatic carcinoma and showed that atorvastatin significantly increased animal survival and inhibited pancreatic carcinogenesis. There was a dramatic decrease in mutant p53 protein accumulation in the pancreatic acini, pancreas intraepithelial neoplasia lesions, and adenocarcinoma. Supplementation with farnesyl pyrophosphate, a substrate for protein farnesylation, rescued atorvastatin‐induced mutant p53 degradation in pancreatic cancer cells. Tipifarnib, a farnesyltransferase inhibitor, mirrored atorvastatin's effects on mutant p53, degraded mutant p53 in a dose‐dependent manner, and converted farnesylated DNAJA1 into unfarnesylated DNAJA1. Farnesyltransferase gene knockdown also significantly promoted mutant p53 degradation. Coimmunoprecipitation either by an anti‐DNAJA1 or p53 antibody confirmed the direct interaction of mutant p53 and DNAJA1 and higher doses of atorvastatin treatments converted more farnesylated DNAJA1 into unfarnesylated DNAJA1 with much less mutant p53 pulled down by DNAJA1. Strikingly, C394S mutant DNAJA1, in which the cysteine of the CAAX box was mutated to serine, was no longer able to be farnesylated and lost the ability to maintain mutant p53 stabilization. Our results show that farnesylated DNAJA1 is a crucial chaperone in maintaining mutant p53 stabilization and targeting farnesylated DNAJA1 by atorvastatin will be critical for inhibiting p53 mutant cancer.     

Mass profiling of serum to distinguish mice with pancreatic cancer induced by a transgenic Kras mutation

Mass spectrometry (MS) has the unique ability to profile, in an easily accessible body tissue (peripheral blood/serum,) the sizes and relative amounts of a wide variety of biomolecules in a single platform setting. Using electrospray ionization (ESI)‐MS, we distinguished individual serum from wild‐type control mice from serum of mice containing an oncogenic Kras mutation, which leads to development of pancreatic ductal adenocarcinoma (PDAC) similar to that observed in humans. Identification of differences in significant ESI‐MS sera mass peaks between Kras‐activated mice and control mice was performed using t tests and a “nested leave one out” cross‐validation procedure. Peak distributions in serum of control mice from mice with Kras‐mutant‐dependent PDAC were distinguished from those of pancreatic intraepithelial neoplasia (PanIN) lesions (p = 0.00024). In addition, Kras mutant mice with PDAC were distinguished from Kras mutant mice with PanIN alone (p = 0.0057). Test specificity, a measure of the false positives, was greater for the control vs. Kras mutated mice, and the test sensitivity, a measure of false negatives, was greater for the PDAC vs. PanIN containing mice. Receiver‐operating characteristic (ROC) curve discriminatory values were 0.85 for both comparisons. These studies indicate ESI‐MS serum mass profiling can detect physiological changes associated with pancreatic cancer initiation and development in a GEM (genetic engineered mouse) model that mimics pancreatic cancer development in humans. Such technology has the potential to aid in early detection of pancreatic cancer and in developing therapeutic drug interventions.     

Catechol‐O‐methyltransferase,a new target for pancreatic cancer therapy

Catechol‐O‐methyltransferase (COMT) is an important molecule in different types of cancers. Its biological effect and therapeutic significance, however, rarely been investigated fully in pancreatic cancer. Immunohistologically, high COMT expression was significantly correlated with the longer overall survival of patients (P < 0.05), indicating its protective nature. The effects of COMT on cell growth, apoptosis, and invasion were evaluated using overexpression and silencing methods. In detail, we carried out experiments using one stably transduced and two transiently transfected pancreatic cancer cell lines in vitro, and one stably transduced cell line in vivo mice xenograft models. In vitro experiments showed that COMT inhibited cell proliferation, enhanced gemcitabine‐induced apoptosis, and inhibited cell invasion in stably transduced and transiently transfected cell lines by regulating the PI3K/Akt pathway, p53, and E‐cadherin. The COMT overexpressed and silenced cell lines showed significantly inhibited and enhanced growth capacities in in vivo xenograft models, respectively. In conclusion, COMT suppressed pancreatic cancer and its high expression predicted longer survival time. The interaction of COMT with the PI3K/Akt pathway makes it a potential target for therapy.     

Metabolic activation of 2‐amino‐1‐methyl‐6‐phenylimidazo [4,5‐b]pyridine and DNA adduct formation depends on p53: Studies in Trp53(+/+),Trp53(+/−) and Trp53(−/−) mice

The expression of the tumor suppressor p53 can influence the bioactivation of, and DNA damage induced by, the environmental carcinogen benzo[a]pyrene, indicating a role for p53 in its cytochrome P450 (CYP)‐mediated biotransformation. The carcinogen 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), which is formed during the cooking of food, is also metabolically activated by CYP enzymes, particularly CYP1A2. We investigated the potential role of p53 in PhIP metabolism in vivo by treating Trp53(+/+), Trp53(+/?) and Trp53(?/?) mice with a single oral dose of 50 mg/kg body weight PhIP. N‐(Deoxyguanosin‐8‐yl)‐2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP‐C8‐dG) levels in DNA, measured by liquid chromatography‐tandem mass spectrometry, were significantly lower in liver, colon, forestomach and glandular stomach of Trp53(?/?) mice compared to Trp53(+/+) mice. Lower PhIP‐DNA adduct levels in the livers of Trp53(?/?) mice correlated with lower Cyp1a2 enzyme activity (measured by methoxyresorufin‐O‐demethylase activity) in these animals. Interestingly, PhIP‐DNA adduct levels were significantly higher in kidney and bladder of Trp53(?/?) mice compared to Trp53(+/+) mice, which was accompanied by higher sulfotransferase (Sult) 1a1 protein levels and increased Sult1a1 enzyme activity (measured by 2‐naphthylsulfate formation from 2‐naphthol) in kidneys of these animals. Our study demonstrates a role for p53 in the metabolism of PhIP in vivo, extending previous results on a novel role for p53 in xenobiotic metabolism. Our results also indicate that the impact of p53 on PhIP biotransformation is tissue‐dependent and that in addition to Cyp1a enzymes, Sult1a1 can contribute to PhIP‐DNA adduct formation.     

Cooperation of tumor‐derived HBx mutants and p53‐249ser mutant in regulating cell proliferation,anchorage‐independent growth and aneuploidy in a telomerase‐immortalized normal human hepatocyte‐derived cell line

Hepatocellular carcinoma (HCC) is a common cancer, and hepatitis B virus (HBV) is a major etiological agent. Convincing epidemiological and experimental evidence also links HCC to aflatoxin, a naturally occurring mycotoxin that produces a signature p53‐249^ser mutation. Recently, we have reported that tumor‐derived HBx variants encoded by HBV exhibited attenuated transactivation and proapoptotic functions but retained their ability to block p53‐mediated apoptosis. These results indicate that mutations in HBx may contribute to the development of HCC. In this study, we determined whether tumor‐derived HBx mutants along, or in cooperation with p53‐249^ser, could alter cell proliferation and chromosome stability of normal human hepatocytes. To test this hypothesis, we established a telomerase immortalized normal human hepatocycte line HHT4 that exhibited a near diploid karyotype and expressed many hepatocyte‐specific genes. We found that overexpression one of the tumor‐derived HBx mutants, CT, significantly increased colony forming efficiency (CFE) while its corresponding wild‐type allele CNT significantly decreased CFE in HHT4 cells. p53‐249^ser rescued CNT‐mediated inhibition of colony formation. Although HHT4 cells lacked an anchorage independent growth capability as they did not form any colonies in soft agar, the CT‐expressing HHT4 cells could form colonies, which could be significantly enhanced by p53‐249^ser. Induction of aneuploidy could be observed in HHT4 cells expressing CT, but additionally recurring chromosome abnormalities could only be detected in cells coexpressing CT and p53‐249^ser. Our results are consistent with the hypothesis that certain mutations in HBx and p53 at codon 249 may cooperate in contributing to liver carcinogenesis.     

CENP‐50 is required for papilloma development in the two‐stage skin carcinogenesis model

CENP‐50/U is a component of the CENP‐O complex (CENP‐O/P/Q/R/U) and localizes to the centromere throughout the cell cycle. Aberrant expression of CENP‐50/U has been reported in many types of cancers. However, as Cenp‐50/U‐deficient mice die during early embryogenesis, its functions remain poorly understood in vivo. To investigate the role of Cenp‐50/U in skin carcinogenesis, we generated Cenp‐50/U conditional knockout (K14Cre^ER‐Cenp‐50/U^fl/fl) mice and subjected them to the 7,12‐dimethylbenz(a)anthracene (DMBA)/terephthalic acid (TPA) chemical carcinogenesis protocol. As a result, early‐stage papillomas decreased in Cenp‐50/U‐deficient mice. In contrast, Cenp‐50/U‐deficient mice demonstrated almost the same carcinoma incidence as control mice. Furthermore, mRNA expression analysis using DMBA/TPA‐induced papillomas and carcinomas revealed that Cenp‐50/U expression levels in papillomas were significantly higher than in carcinomas. These results suggest that Cenp‐50/U functions mainly in early papilloma development and it has little effect on malignant conversion.