AACR Centennial Conference: Translational Cancer Medicine-- Nov 4-8, 2007; Singapore

PL02-05 

All cancers are due to abnormalities in DNA. The availability of the human genome sequence has led to the proposal that resequencing of cancer genomes will reveal the full complement of somatic mutations and hence all the cancer genes. To explore the nature of the information that will be derived from cancer genome sequencing we have sequenced the coding exons of the family of 518 protein kinases, ~1.3Mb DNA per cancer sample, in 210 cancers of diverse histological types. Despite the screen being directed toward the coding regions of a gene family that has previously been strongly implicated in oncogenesis, the results indicate that the majority of somatic mutations detected are “passengers”. There is considerable variation in the number and pattern of these mutations between individual cancers, indicating substantial diversity of processes of molecular evolution between cancers. The imprints of exogenous mutagenic exposures, mutagenic treatment regimes and DNA repair defects can all be seen in the distinctive mutational signatures of individual cancers. This systematic mutation screen and others have previously yielded a number of cancer genes that are frequently mutated in one or more cancer types and which are now anticancer drug targets (for example BRAF , PIK3CA , and EGFR ). However, detailed analyses of the data from our screen additionally suggest that there exist a large number of additional “driver” mutations which are distributed across a substantial number of genes. It therefore appears that cells may be able to utilise mutations in a large repertoire of potential cancer genes to acquire the neoplastic phenotype. However, many of these genes are employed only infrequently. These findings may have implications for future anticancer drug development.

Patterns of Somatic Mutation in Human Cancer Genomes

One of the main engines that drives cellular transformation is the loss of proper control of the mammalian cell cycle. The cyclin-dependent kinase inhibitor p21 (also known as p21WAF1/Cip1) promotes cell cycle arrest in response to many stimuli. It is well positioned to function as both a sensor and an effector of multiple anti-proliferative signals. This Review focuses on recent advances in our understanding of the regulation of p21 and its biological functions with emphasis on its p53-independent tumour suppressor activities and paradoxical tumour-promoting activities, and their implications in cancer.

p21 in cancer: intricate networks and multiple activities

Commensal and pathogenic microorganisms must resist the deleterious actions of bile in order to survive in the human gastrointestinal tract. Herein we review the current knowledge on the mechanisms by which Gram-positive and Gram-negative bacteria contend with bile stress. We describe the antimicrobial actions of bile, assess the variations in bile tolerance between bacterial genera and examine the interplay between bile stress and other stresses. The molecular mechanisms underlying bile tolerance are investigated and the relationship between bile and virulence is examined. Finally, the potential benefits of bile research are briefly discussed.

The interaction between bacteria and bile

Over the past three decades, molecular genetic studies have revealed some critical mutations underlying the pathogenesis of the sporadic and inherited forms of colorectal cancer (CRC). A relatively limited number of oncogenes and tumor-suppressor genes—most prominently the APC, KRAS, and p53 genes—are mutated in a sizeable fraction of CRCs, and a larger collection of genes that are mutated in subsets of CRC have begun to be defined. Together with DNA-methylation and chromatin-structure changes, the mutations act to dysregulate conserved signaling networks that exert context-dependent effects on critical cell phenotypes, including the regulation of cellular metabolism, proliferation, differentiation, and survival. Much work remains to be done to fully understand the nature and significance of the individual and collective genetic and epigenetic defects in CRC. Some key concepts for the field have emerged, two of which are emphasized in this review. Specifically, the gene defects in CRC often target protein...

Molecular Genetics of Colorectal Cancer

Reactive oxygen species (ROS) are generated as by-products of normal cellular metabolic activities. Superoxide dismutase, glutathione peroxidase, and catalase are the enzymes involved in protecting cells from the damaging effects of ROS. ROS are produced in response to ultraviolet radiation, cigarette smoking, alcohol, nonsteroidal anti-inflammatory drugs, ischemia-reperfusion injury, chronic infections, and inflammatory disorders. Disruption of normal cellular homeostasis by redox signaling may result in cardiovascular, neurodegenerative diseases and cancer. ROS are produced within the gastrointestinal (GI) tract, but their roles in pathophysiology and disease pathogenesis have not been well studied. Despite the protective barrier provided by the mucosa, ingested materials and microbial pathogens can induce oxidative injury and GI inflammatory responses involving the epithelium and immune/inflammatory cells. The pathogenesis of various GI diseases including peptic ulcers, gastrointestinal cancers, and inflammatory bowel disease is in part due to oxidative stress. Unraveling the signaling events initiated at the cellular level by oxidative free radicals as well as the physiological responses to such stress is important to better understand disease pathogenesis and to develop new therapies to manage a variety of conditions for which current therapies are not always sufficient.

Oxidative Stress: An Essential Factor in the Pathogenesis of Gastrointestinal Mucosal Diseases

Publisher Summary Genomic fingerprinting by arbitrarily primed polymerase chain reaction (AP-PCR) is a sensitive and efficient method for generating a large number of molecular markers. Based on the selective amplification of DNA sequences flanked, by chance, by sequences matching an arbitrarily chosen primer, AP-PCR reveals sequence polymorphisms between different template DNAs. The ability to detect differences between fingerprints of closely related organisms made this approach a useful tool for studying genetic diversity, population biology, epidemiology, and genetic mapping. The more recent application of AP-PCR fingerprinting to RNA, differential display, or RNA-arbitrarily primed PCR (RAP-PCR) has resulted in an interesting tool for the detection of differential gene expression. This chapter focuses on the use of RAP-PCR fingerprinting. It describes the method and provides the present protocols for the different steps from initial RNA preparation through sequence analysis and confirmation of differential expression of the transcripts identified.

Identification of differentially expressed genes using RNA fingerprinting by arbitrarily primed polymerase chain reaction.

Neoplasia of the Gastrointestinal Tract

The underlying molecular mechanisms of the tumor-promoting activity of bile acids such as chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) and the protective effect of ursodeoxycholic acid (UDCA) remain largely unclear. Using RNA arbitrarily primed PCR (RAP-PCR) for differential display, we identified, cloned and sequenced differentially expressed transcripts after treating gastric carcinoma cells (St 23132) with the bile acids CDCA, DCA and UDCA. One of these transcripts was identified to be an estrogen-responsive RING finger protein (efp) mRNA. The differential expression of efp in gastric cancer cells was confirmed by low stringency RT-PCR. efp mRNA levels were induced 3-fold in gastric carcinoma cells after CDCA and DCA treatment, whereas no change in expression was detected after UDCA treatment. Finally, treatment of the colon carcinoma cell line HT 29 with DCA resulted in a 2- to 5-fold induction of efp mRNA levels whereas UDCA did not induce efp. As expected, efp expression was also increased after 24 h of estrogen treatment. In summary, a synergy or a common pathway of tumor enhancement of bile acids and estrogen via efp in gastrointestinal carcinogenesis can be envisioned.

Estrogen-responsive RING finger mRNA induction in gastrointestinal carcinoma cells following bile acid treatment

Here, we report a case of a young man who presented with a significant upper gastrointestinal bleed treated by endoscopic removal of multiple hyperplastic polyps. Gastric hyperplastic polyps are a relatively uncommon cause of overt gastrointestinal bleeding. While most hyperplastic gastric polyps are asymptomatic, they may present with abdominal pain, iron deficiency anemia or gastric outlet obstruction. These polyps are associated with conditions such as Helicobacter pylori gastritis and atrophic autoimmune gastritis, which predispose the epithelium to chronic inflammation and epithelial repair. The patient presented to Northwestern Memorial Hospital in July 2011. The polyps were resected by clip-assisted snare polypectomy. Histopathologic assessment of the resected polyps demonstrated multiple, non-ulcerative hyperplastic polyps measuring 1.3-1.8 cm in size, without evidence of dysplasia or malignancy. This case describes a young adult patient with multiple, large gastric polyps causing overt gastrointestinal bleeding. This is a rare presentation in a young individual, as these polyps are typically identified in patients older than 60 years of age and less commonly, pediatric populations.

Gastric hyperplastic polyps causing upper gastrointestinal hemorrhage in a young adult.

Reactive oxygen species such as H2O2 are believed to play a prominent role in the injury and loss of transport function that affect the intestinal epithelium in inflammatory conditions such as inflammatory bowel diseases. Defects in intestinal epithelial ion transport regulation contribute to dysbiosis and inflammatory phenotypes. We previously showed that H2O2 inhibits Ca2+-dependent Cl- secretion across intestinal epithelial cells (IECs) via a phosphatidylinositol 3-kinase (PI3K)- and extracellular signal-regulated kinase (ERK)-dependent mechanism that occurs, at least in part, through inhibition of the basolateral Na+-K+-2Cl- cotransporter NKCC1. NKCC1 governs Cl- entry into crypt IECs and thus plays a critical role in maintaining the driving force for Cl- secretion. Electrolyte transport consumes large amounts of cellular energy, and direct pharmacological activation of the cellular energy sensor AMP-activated protein kinase (AMPK) has been shown to inhibit a number of ion transport proteins. Here, we show that H2O2 activates AMPK in human IEC lines and ex vivo human colon. Moreover, we demonstrate that the inhibitory effect of H2O2 on Ca2+-dependent Cl- secretion and NKCC1 activity is AMPK-dependent. This inhibitory effect is associated with a physical interaction between AMPK and NKCC1, as well as increased phosphorylation (Thr212,217) of NKCC1, without causing NKCC1 internalization. These data identify a key role for AMPK-NKCC1 interaction as a point of convergence for suppression of colonic epithelial ion transport by inflammatory reactive oxygen species.NEW & NOTEWORTHY H2O2 inhibition of intestinal epithelial Ca2+-dependent Cl- secretion involves recruitment of AMP-activated protein kinase (AMPK) downstream of ERK and phosphatidylinositol 3-kinase signaling pathways, physical interaction of AMPK with the Na+-K+-2Cl- cotransporter NKCC1, and AMPK-dependent suppression of NKCC1-mediated electrolyte influx without causing NKCC1 internalization. It is intriguing that, in human intestinal epithelial cell lines and human colon, H2O2 activation of AMPK increased phosphorylation of NKCC1 residues required for promoting, not inhibiting, NKCC1 activity. These data identify an elevated complexity of AMPK regulation of NKCC1 in the setting of an inflammatory stimulus.

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Barbara H. Jung

Papers

Identification of differentially expressed genes using RNA fingerprinting by arbitrarily primed polymerase chain reaction.

Neoplasia of the Gastrointestinal Tract

Estrogen-responsive RING finger mRNA induction in gastrointestinal carcinoma cells following bile acid treatment

Gastric hyperplastic polyps causing upper gastrointestinal hemorrhage in a young adult.

AMPK mediates inhibition of electrolyte transport and NKCC1 activity by reactive oxygen species