Neil M. Kernohan

The Barrett’s Antigen Anterior Gradient-2 Silences the p53 Transcriptional Response to DNA Damage

The esophageal epithelium is subject to damage from bile acid reflux that promotes normal tissue injury resulting in the development of Barrett’s epithelium. There is a selection pressure for mutating p53 in this preneoplastic epithelium, thus identifying a physiologically relevant model for discovering novel regulators of the p53 pathway. Proteomic technologies were used to identify such p53 regulatory factors by identifying proteins that were overexpressed in Barrett’s epithelium. A very abundant polypeptide selectively expressed in Barrett’s epithelium was identified as anterior gradient-2. Immunochemical methods confirmed that anterior gradient-2 is universally up-regulated in Barrett’s epithelium, relative to normal squamous tissue derived from the same patient. Transfection of the anterior gradient-2 gene into cells enhances colony formation, similar to mutant oncogenic p53 encoded by the HIS175 allele, suggesting that anterior gradient-2 can function as a survival factor. Deletion of the C-terminal 10 amino acids of anterior gradient-2 neutralizes the colony enhancing activity of the gene, suggesting a key role for this domain in enhancing cell survival. Constitutive overexpression of anterior gradient-2 does not alter cell-cycle parameters in unstressed cells, suggesting that this gene is not directly modifying the cell cycle. However, cells overexpressing anterior gradient-2 attenuate p53 phosphorylation at both Ser15 and Ser392 and silence p53 transactivation function in ultraviolet (UV)-damaged cells. Deletion of the C-terminal 10 amino acids of anterior gradient-2 permits phosphorylation at Ser15 in UV-damaged cells, suggesting that the C-terminal motif promoting colony survival also contributes to suppression of the Ser15 kinase pathway. These data identify anterior gradient-2 as a novel survival factor whose study may shed light on cellular pathways that attenuate the tumor suppressor p53.

Overexpression and poly-ubiquitylation of the DEAD-box RNA helicase p68 in colorectal tumours

The DEAD box RNA helicase, p68, is upregulated in exponentially growing cells and shows cell cycle-dependent changes in nuclear localization. Although some other DEAD box proteins have been implicated in cancer, there have been no reports of any link between p68 status and carcinogenesis. In the present study we have analysed specimens from 50 patients with colorectal adenocarcinomas, including cases in which an adenomatous polyp was also present, by immunohistochemistry and Western blotting. Our data indicate that p68 protein is consistently overexpressed in tumours as compared with matched normal tissue. Examination of the levels of p68 mRNA from both normal and tumour tissue showed no obvious specific increase in p68 mRNA levels in tumours nor any evidence of underlying mutations in the p68 coding region. Interestingly, however, the accumulated p68 appears to be poly-ubiquitylated, suggesting a possible defect in proteasome-mediated degradation in these tumours. This overexpression/ubiquitylation is observed in both pre-invasive and invasive lesions suggesting that the dysregulation of p68 expression occurs early during tumour development. Finally, we demonstrate that ubiquitylation of p68 occurs in cultured cells, thereby providing a model for the molecular analysis of this process and its potential role in tumorigenesis.

Elevated Levels of Oncogenic Protein Kinase Pim-1 Induce the p53 Pathway in Cultured Cells and Correlate with Increased Mdm2 in Mantle Cell Lymphoma

Mutation of the p53 gene is a common event during tumor pathogenesis. Other mechanisms, such as mdm2 amplification, provide alternative routes through which dysfunction of the p53 pathway is promoted. Here, we address the hypothesis that elevated expression of pim oncogenes might suppress p53 by regulating Mdm2. At a physiological level, we show that endogenous Pim-1 and Pim-2 interact with endogenous Mdm2. Additionally, the Pim kinases phosphorylate Mdm2 in vitro and in cultured cells at Ser166 and Ser186, two previously identified targets of other signaling pathways, including Akt. Surprisingly, at high levels of Pim expression, as would occur in tumors, active, but not inactive, Pim-1 or Pim-2 blocks the degradation of both p53 and Mdm2 in a manner that is independent of Mdm2 phosphorylation, leading to increased p53 levels and, proportionately, p53-dependent transactivation. Additionally, Pim-1 induces endogenous ARF, p53, Mdm2, and p21 in primary murine embryo fibroblasts and stimulates senescence-associated β-galactosidase levels, consistent with the induction of senescence. Immunohistochemical analysis of a cohort of 33 human mantle cell lymphomas shows that elevated expression of Pim-1 occurs in 42% of cases, with elevated Pim-2 occurring in 9% of cases, all of which also express Pim-1. Notably, elevated Pim-1 correlates with elevated Mdm2 in MCL with a p value of 0.003. Taken together, our data are consistent with the idea that Pim normally interacts with the p53 pathway but, when expressed at pathological levels, behaves as a classic dominant oncogene that stimulates a protective response through induction of the p53 pathway.

Phenformin as prophylaxis and therapy in breast cancer xenografts

Background:Observations that diabetics treated with biguanide drugs have a reduced risk of developing cancer have prompted an enthusiasm for these agents as anti-cancer therapies. We sought to determine the efficacy of the biguanide phenformin in the chemoprophylaxis and in the treatment of oestrogen receptor (ER)-positive MCF7 and receptor triple-negative MDAMB231 xenografts in immunocompromised mice. We also compared the efficacy of phenformin and metformin in the treatment of MDAMB231.Methods:Immunocompromised mice were divided into groups: (1) phenformin administered for 2 weeks prior to cell injection; (2) established tumours treated with phenformin; (3) established tumours treated with metformin (only for MDAMB231 tumours); (4) untreated controls. Post-treatment tumours, liver and spleen were harvested for further analysis.Results:Phenformin significantly inhibited both the development and growth of MCF7 and MDAMB231 tumours, and for MDAMB231 at greater efficacy than metformin without murine toxicity. The number of mitotic figures was significantly fewer in xenografts treated with phenformin compared with controls. Results suggested that the mechanism of action of phenformin in vivo is consistent with AMPK activation.Conclusion:Phenformin has clinical potential as an antineoplastic agent and should be considered for clinical trials both in ER-positive and triple-negative breast cancer.

C-MYC translocation in t(14;18) positive follicular lymphoma at presentation: An adverse prognostic indicator?

Follicular lymphoma (FL) is a common subtype of low grade B-cell non-Hodgkin lymphoma (NHL). Although this form of lymphoma often pursues an indolent course, in some cases it may behave in a more aggressive manner. Clinical and histological parameters have been shown to correlate with an adverse prognosis but a number of cytogenetic abnormalities may also be associated with aggressive disease. Although, the t(14;18) in itself does not affect outcome in cases of FL, secondary abnormalities that occur in a complex polyploid karyotype may identify cases with a poor prognosis. It is unusual to find both t(14;18) and C-MYC translocation in the same tumour; those cases in which it has been described include examples of high-grade B-cell NHL (either de novo or transformed FL) or B-cell acute lymphoblastic lymphoma. In this report, three cases of FL are described in which both t(14;18) and a C-MYC translocation were identified at presentation. We also summarize four further cases from the literature. This is a small series but one which raises the possibility that the presence of a C-MYC translocations at presentation may identify a particularly aggressive subtype of FL. Further studies are required to investigate the true incidence of this aberration, the impact on C-MYC regulation, clinical course and response to treatment.

Seliciclib (CYC202, R-roscovitine) enhances the antitumor effect of doxorubicin in vivo in a breast cancer xenograft model

We sought to determine whether seliciclib (CYC202, R‐roscovitine) could increase the antitumor effects of doxorubicin, with no increase in toxicity, in an MCF7 breast cancer xenograft model. The efficacy of seliciclib combined with doxorubicin was compared with single agent doxorubicin or seliciclib administered to MCF7 cells and to nude mice bearing established MCF7 xenografts. Post‐treatment cells and tumors were examined by cell cycle analysis, immunohistochemistry and real‐time PCR. Seliciclib significantly enhanced the antitumor effect of doxorubicin without additional murine toxicity. MIB1 (ki67) immunohistochemistry demonstrated reduced proliferation with treatment. The levels of p21 and p27 increased after treatment with doxorubicin or seliciclib alone or in combination, compared to untreated controls. However, no changes in p53 protein (DO1, CM1), survivin or p53 phosphorylation (SER15) were observed in treated tumors compared with controls. In conclusion, the CDK inhibitor seliciclib (R‐roscovitine) enhances the antitumor effect of doxorubicin in MCF7 tumors without increased toxicity with a mechanism that involves cell cycle arrest rather than apoptosis.

BCL2 gene abnormalities define distinct clinical subsets of follicular lymphoma

Aims:  Follicular lymphoma (FL) arising primarily in the skin has recently been proposed as a distinct entity on the basis of a low incidence of t(14;18)(q32;q21) and bcl‐2 expression, with a very high percentage of patients surviving more than 5 years. However, cases of t(14;18)(q32;q21)‐positive primary cutaneous FL (PCFL) and examples of t(14;18)(q32;q21)‐negative FL at nodal and other extranodal sites, are well documented. The aim of this study was to test the hypothesis that there is a subtype of FL lacking t(14;18)(q32;q21), which preferentially involves certain sites but is not restricted by anatomical location.

Gene expression in colorectal neoplasia: modifications induced by tissue ischaemic time and tissue handling protocol

Bray S E, Paulin F E M, Fong S C, Baker L, Carey F A, Levison D A, Steele R J C & Kernohan N M
(2010) Histopathology56, 240–250

Chromosomal imbalances in gastric and esophageal adenocarcinoma: specific comparative genomic hybridization-detected abnormalities segregate with junctional adenocarcinomas.

The incidence of adenocarcinoma arising at the esophagogastric junction (EGJ) is increasing at a rate greater than that for any other form of solid malignancy. Commensurate with this, the incidence of histologically similar tumors arising in the gastric body and antral mucosa is declining. The increased incidence of the proximal group of tumors may reflect, in part, the higher prevalence of Barrett esophagus. These epidemiological features suggest that histologically similar tumors arising at the EGJ and from the distal stomach are different, which may be reflected in the genetic abnormalities that characterize the two groups of tumors. The purpose of this study was to screen genomic DNA from adenocarcinomas of the esophagus and stomach for regions of chromosomal imbalance, using comparative genomic hybridization to determine whether tumors at the EGJ (junctional tumors) have a different profile compared with tumors of the distal stomach. Tumor samples were derived from a series of 48 gastroesophageal adenocarcinomas (20 junctional and 28 distal) that were acquired prospectively from patients undergoing esophagogastrectomy. These tumors are characterized by several regions of chromosomal imbalance with no obvious correlation between most regions of abnormal copy number and tumor type. However, our study shows for the first time cytogenetic abnormalities (5p+ and 18q−) that identify statistically significant differences (P < 0.02 and < 0.05, respectively) between junctional and distal gastric tumors. These differences are gain of 5p (55% [11/20] of junctional tumors vs. 21% [6/28] of distal gastric tumors) and loss of 18q (25% [5/20] cases of junctional tumors vs. 4% [1/28] of distal tumors) segregating with tumors of the EGJ. These abnormalities may distinguish distinct tumor subtypes that are recognized in epidemiological and clinical studies but that are otherwise histologically identical.

The calcium‐binding domain of the stress protein SEP53 is required for survival in response to deoxycholic acid‐mediated injury

Stress protein responses have evolved in part as a mechanism to protect cells from the toxic effects of environmental damaging agents. Oesophageal squamous epithelial cells have evolved an atypical stress response that results in the synthesis of a 53 kDa protein of undefined function named squamous epithelial‐induced stress protein of 53 kDa (SEP53). Given the role of deoxycholic acid (DCA) as a potential damaging agent in squamous epithelium, we developed assays measuring the effects of DCA on SEP53‐mediated responses to damage. To achieve this, we cloned the human SEP53 gene, developed a panel of monoclonal antibodies to the protein, and showed that SEP53 expression is predominantly confined to squamous epithelium. Clonogenic assays were used to show that SEP53 can function as a survival factor in mammalian cell lines, can attenuate DCA‐induced apoptotic cell death, and can attenuate DCA‐mediated increases in intracellular free calcium. Deletion of the highly conserved EF‐hand calcium‐binding domain in SEP53 neutralizes the colony survival activity of the protein, neutralizes the protective effects of SEP53 after DCA exposure, and permits calcium elevation in response to DCA challenge. These data indicate that the squamous cell‐stress protein SEP53 can function as a modifier of the DCA‐mediated calcium influx and identify a novel survival pathway whose study may shed light on mechanisms relating to squamous cell injury and associated cancer development.