Pathways In Cancer Development Research Articles

Expression and mutation analysis of epidermal growth factor receptor in head and neck squamous cell carcinoma

The epidermal growth factor receptor (EGFR)-RAS-RAF-mitogen-activated protein kinase signaling cascade is an important pathway in cancer development and recent reports show that EGFR and its downstream signaling molecules are mutated in a number of cancers. We have analyzed 91 Japanese head and neck squamous cell carcinomas (HNSCC) and 12 HNSCC cell lines for mutations in EGFR, ErbB2, and K-ras. Exons encoding the hot-spot regions in the tyrosine kinase domain of both EGFR (exons 18, 19, and 21) and ErbB2 (exons 18-23), as well as exons 1 and 2 of K-ras were amplified by polymerase chain reaction and sequenced directly. EGFR expression was also analyzed in 65 HNSCC patients using immunohistochemistry. Only one silent mutation, C836T, was found in exon 21 of EGFR in the UT-SCC-16A cell line and its corresponding metastasic cell line UT-SCC-16B. No other mutation was found in EGFR, ErbB2, or K-ras. All tumors showed EGFR expression. In 21 (32%) tumors, EGFR was expressed weakly (+1). In 27 (42%) tumors it was expressed (+2) moderately, and in 17 (26%) tumors high expression (+3) was detected. Overexpression (+2, +3) was found in 44 tumors (68%). A worse tumor differentiation and a positive nodal stage were significantly associated with EGFR overexpression (P = 0.02, P = 0.032, respectively). Similar to patients from western ethnicity, mutations are absent or rare in Japanese HNSCC. Protein overexpression rather than mutation might be responsible for activation of the EGFR pathway in HNSCC.

Hedgehog signaling overrides p53-mediated tumor suppression by activating Mdm2

The hedgehog (Hh) signaling pathway regulates the development of many organs in mammals, and activation of this pathway is widely observed in human cancers. Although it is known that Hh signaling activates the expression of genes involved in cell growth, the precise role of the Hh pathway in cancer development is still unclear. Here, we show that constitutively activated mutants of Smoothened (Smo), a transducer of the Hh signaling pathway, inhibit the accumulation of the tumor suppressor protein p53. This inhibition was also observed in the presence of Hh ligand or with the overexpression of the transcription factors Gli1 and Gli2, downstream effectors of Smo, indicating that this inhibition is specific for the Hh pathway. We also report that Smo mutants augment p53 binding to the E3 ubiquitin-protein ligase Mdm2 and promote p53 ubiquitination. Furthermore, Hh signaling induced the phosphorylation of human Mdm2 protein on serines 166 and 186, which are activating phosphorylation sites of Mdm2. Smo mutants enhanced the proliferation of mouse embryonic fibroblasts (MEFs) while inducing a DNA-damage response. Moreover, Smo partially inhibited p53-dependent apoptosis and cell growth inhibition in oncogene-expressing MEFs. We also found that accumulation of p53 is inhibited by Hh signaling in several human cancer cell lines. Therefore, the Hh pathway may be a powerful accelerator of oncogenesis by activating cell proliferation and inhibiting the p53-mediated anti-cancer barrier induced by oncogenic stress.

Activated neutrophils induce an hMSH2-dependent G2/M checkpoint arrest and replication errors at a (CA)13-repeat in colon epithelial cells

Objective: Chronic inflammation in ulcerative colitis is associated with increased risk for colorectal cancer. Its molecular pathway of cancer development is poorly understood. We investigated the role of neutrophil-derived cellular...

Demethylation of (Cytosine-5-C-methyl) DNA and regulation of transcription in the epigenetic pathways of cancer development

Cancer cells and tissues exhibit genome wide hypomethylation and regional hypermethylation. CpG-methylation of DNA ((Me)CpG-DNA) is defined as the formation of a C-C covalent bond between the 5'-C of cytosine and the -CH(3) group of S-adenosylmethionine. Removal of the sole -CH(3) group from the methylated cytosine of DNA is one of the many ways of DNA-demethylation, which contributes to activation of transcription. The mechanism of demethylation, the candidate enzyme(s) exhibiting direct demethylase activity and associated cofactors are not firmly established. Genome-wide hypomethylation can be obtained in several ways by inactivation of DNMT enzyme activity, including covalent trapping of DNMT by cytosine base analogues. Removal of methyl layer could also be occurred by excision of the 5-methyl cytosine base by DNA glycosylases. The importance of truly chemically defined direct demethylation of intact DNA in regulation of gene expression, development, cell differentiation and transformation are discussed in this contribution.

Differential expression of α-methylacyl-coenzyme A racemase in colorectal carcinoma bears clinical and pathologic significance

alpha-methylacyl-coenzyme A racemase (AMACR) is a recently discovered biomarker that is shown to be overexpressed in some prostatic carcinomas and associated with prostatic cancer progression. Given that AMACR plays an important role in peroxisomal beta-oxidation of branched-chain fatty acids from red meat and dairy products, and that consumption of red meat may increase risk of developing colon cancer as suggested by epidemiological studies, it is plausible to explore the function of AMACR in colorectal carcinoma. A few previous studies have indeed observed overexpression of AMACR in 45% to 69% of the colorectal carcinomas. However, the clinical and pathologic characteristics of such AMACR expressers have not been investigated. In this study, the immunohistochemical expression pattern of AMACR of 163 patients with primary colorectal carcinoma treated primarily with surgical resection was analyzed and correlated with tumor pathologic features (tumor location, histologic type, grade, pathologic stage, lymph node and distant metastasis) and patient outcome (disease-specific survival). The results showed variable positive staining for AMACR in 123 (75%) of 163 tumors, and moderate to strong staining in 63 (39%) of 163. Lack of staining or low-intensity staining appeared to correlate significantly with mucinous histology (P < .001), poor tumor differentiation (P = .021), and presence of lymphovascular invasion (P = .032). Patients whose tumors showed lack of staining or low-intensity staining also had a significantly worse 5-year disease-specific survival (P < .012), as did patients whose tumors had lymphovascular invasion, or were of high American Joint Committee on Cancer (AJCC) stage. On multivariate analysis, AMACR staining and AJCC staging remained independent predictors for patient outcome. Thus, our data suggest that AMACR expression in colorectal carcinoma correlates with certain tumor pathologic characteristics (histologic type, differentiation, and lymphovascular invasion) and patient outcome. Additional confirmatory studies are needed to establish the significance of AMACR as a prognostic marker for colorectal carcinoma. Further investigation on interaction between AMACR and other known colorectal cancer development pathways may provide new insights on colorectal carcinogenesis.

Human HIF‐3α4 is a dominant‐negative regulator of HIF‐1 and is down‐regulated in renal cell carcinoma

A universal response to changes in cellular oxygen tension is governed by a family of heterodimeric transcription factors called hypoxia-inducible factor (HIF). Tumor hypoxia, as well as various cancer-causing mutations, has been shown to elevate the level of HIF-1alpha, signifying a critical role of the HIF pathway in cancer development. The recently identified third member of the human HIF-alpha family, HIF-3alpha, produces multiple splice variants that contain extra DNA binding elements and protein-protein interaction motifs not found in HIF-1alpha or HIF-2alpha. Here we report the molecular cloning of the alternatively spliced human HIF-3alpha variant HIF-3alpha4 and show that it attenuates the ability of HIF-1 to bind hypoxia-responsive elements located within the enhancer/promoter of HIF target genes. The overexpression of HIF-3alpha4 suppresses the transcriptional activity of HIF-1 and siRNA-mediated knockdown of the endogenous HIF-3alpha4 increases transcription by hypoxia-inducible genes. HIF-3alpha4 itself is oxygen-regulated, suggesting a novel feedback mechanism of controlling HIF-1 activity. Furthermore, the expression of HIF-3alpha4 is dramatically down-regulated in the majority of primary renal carcinomas. These results demonstrate an important dominant-negative regulation of HIF-1-mediated gene transcription by HIF-3alpha4 in vivo and underscore its potential significance in renal epithelial oncogenesis.

Dominant-Negative Notch3 Receptor Inhibits Mitogen-Activated Protein Kinase Pathway and the Growth of Human Lung Cancers

Notch3 is a member of an evolutionarily conserved family of cell surface receptors important in cell-fate determination in both vertebrates and invertebrates. Significant data support the role of Notch pathway in cancer development, although the conflicting role of Notch signaling pathways in tumorigenesis suggests that its action is highly context-dependent. Furthermore, although Notch receptors signal primarily through the regulation of hairy enhancer of split (HES) and HES-related (HRT) genes, they are known to crosstalk with other signaling pathways, including the epidermal growth factor (EGF) and the mitogen-activated protein kinase pathways. Whereas much is known about the role of Notch1 in human cancer, the role of Notch3 in epithelial tumors, such as lung carcinomas, has not been well established. In this study, we show that Notch3 is expressed in 80 of 207 (39%) resected human lung tumors and that its expression is positively correlated with EGF receptor expression. Inhibition of the Notch3 pathway using a dominant-negative receptor dramatically reduces growth in soft agar and increases growth factor dependence. We also find that Notch inhibition increases sensitivity to EGF receptor tyrosine kinase inhibition and decrease in phosphorylation of the mitogen-activated protein kinase. These observations support a role for Notch3 signaling in lung cancer, and one potential mechanism of maintaining the neoplastic phenotype is through the modulation of the EGF pathway.

Principles of Molecular Oncology: Second Edition

This second edition of Principles of Molecular Oncology comes just 4 years after the first, and considering that we are now in the ‘postgenomics’ era, it does well to keep pace with the advances of this rapidly evolving field. The text catalogues an amazing array of molecular biology-inspired techniques and approaches for the diagnosis, treatment, and understanding of cancer, which is even more astounding when weighed against the fact that when DNA was discovered 50 years ago, ‘cancer knowledge was based on epidemiology and pathology, and treatment consisted of surgery and radiation therapy.’ This text offers the reader a comprehensive overview of how 25 years of molecular biology have been brought to bear on the problems of this complex disease. The opening chapter introduces the current state of affairs in cancer biology, including its epidemiology, diagnosis and treatment, the ‘older’ and ‘newer’ hypotheses surrounding its causes, and the contributions of diet, viruses, nicotine/tobacco, and hereditary predisposition to its development. Despite the vast increase in our knowledge, the existing therapies have had a relatively minor effect on mortality. As such, the anticipated dawn of the ‘era of targeted therapies' is eagerly awaited by all. One kind of targeted or tailored approach is addressed in the first section of the book, where molecular markers of cancers are discussed. Realising that earlier detection is a major advantage in cancer treatment, scientists are searching for more and better markers to be used as both diagnostic and prognostic factors to tailor the utility and efficacy of treatments. The identification of markers in sporadic and heritable tumours, and in solid tumours is discussed. Also, the hunt for circulating tumour markers to assess tumour burden is detailed. Traditional approaches of histological diagnosis and karyotyping are presented side by side with the newer approaches of gene expression and protein profiling. The middle section of the book explains our molecular and mechanistic understanding of the deregulated cellular pathways in cancer development, including signal transduction pathways, cell proliferation, angiogenesis, invasion/metastasis, drug resistance, and genomic instability. As these are each robust areas of research, there is a large amount of information here, and each of the chapters is comprehensive and detailed in its dissection of these recognised abnormalities of cancerous cells. Although some chapters are more readable than others, in general the information presented is concise and points the interested reader towards the primary sources of information. The final section deals with the therapies arising from molecular biology. As researchers try to benefit from all this new information, the hope is to move on from ‘scientifically interesting to clinically useful.’ Although the approaches being taking are research-driven, spanning suicide gene therapy, monoclonal antibody therapies, and assisted drug-design and development, understanding the function of a gene does not necessarily mean it will be a good target for cancer treatment. This is a major deficiency facing molecular oncologists today: with so many possible genes, which represent the best, ‘druggable’ target on which to focus our energies. Nevertheless, a trickle of next-generation drugs, whose mechanisms of action are understood, are emerging. These successes are hailed repeatedly and inspire the hope that pervades this textbook and the field in general. Despite the fact that there are 65 contributors to this edition of Principles from Europe and America, it is highly readable, with most of the chapters being well written and up-to-date, even listing the newest compounds and their ongoing clinical trials. Each chapter is well referenced, although the text is not very well indexed, making it difficult to find particular genes or drugs of interest within the chapters. As there are many authors, the book is repetitive in places, but this only serves to underscore the fundamental importance of certain pathways, like p53, in cancer. Overall, this is a thorough treatment of a fast-moving and exciting field. As such, it will appeal to clinicians and scientists, and it should spur on young medical students or budding researchers to the coming era of molecular approaches to human diseases.

Loss of hMSH2 expression in primary breast cancer with p53 alterations

Inactivation of DNA mismatch repair genes (MRG) is a recently described pathway of cancer development and progression resulting in genetic instability. Germline mutations in MRG have been studied predominantly in patients with hereditary non-polyposis colorectal cancer (HNPCC) where it is associated with microsatellite instability (MSI). The expression of MRG in primary breast cancer is still largely unexplored. The hMSH2 MRG encodes a protein that recognizes and binds to mismatch sequences of DNA. We investigated the relation-ship between hMSH2 expression and clinicopathological and biological characteristics, including p53 and p185 expression, in 44 primary invasive breast cancers. hMSH2 was not expressed in 11 cases (25%). Interestingly, p53 (p=0.05), p185 and steroidal receptor expression (p=0.07) were more frequent in tumors without hMSH2 expression. Furthermore, in 30 of 44 cases we analyzed hMSH2 expression in relation to MSI at 9 dinucleotide loci, and found that MRG expression was not significantly related to MSI. The presence of hMSH2 and p53 alterations in the same tumor suggests that the two oncoproteins act through a common mutational pathway, whereas the absence of a correlation between hMSH2 and MSI suggests that oncogenetic mechanisms of progression in primary breast cancer differ from those in HNPCC.

The Wnt signaling pathway and its role in tumor development.

Cancer development depends on the aberrant activation of signal transduction pathways that control cell growth and survival and play important roles in normal embryonic development. This review will focus on one of the most powerful pathways, the canonical Wnt signal transduction cascade, which has been originally described in vertebrate and non-vertebrate embryogenesis and subsequently associated with the development of a multitude of different tumor types, mainly of gastrointestinal origin. In recent years, a variety of novel interacting components and functions have been identified in the Wnt pathway revealing not only the complexity of Wnt signaling but also its potency. Here we will concentrate on the role of the Wnt pathway in cancer development with emphasis placed on the molecular defects known to promote neoplastic transformation in humans and in animal models.

Immunohistochemical analyses of colon cancer in I1307K APC mutation carriers compared with noncarriers.

The I1307K APC germline mutation is associated with an increased risk to colorectal cancer (CRC). Whether and to what extent the somatic features and the molecular pathways of cancer development in mutation carriers differ from colorectal cancer in noncarriers remains unknown. To gain insight into this issue, 52 Israeli patients with CRC, 24 of whom were I1307K APC mutation carriers, were analyzed. The expression pattern of genes known to be involved in the pathogenesis of sporadic CRC was assessed immunohistochemically: E-cadherin, beta-catenin, deleted in colon cancer (DCC), and p53. In addition, tumors were genotyped for somatic activating mutations in Ki-ras oncogene. Mutation carriers and noncarriers were comparable in age at diagnosis (64.3 +/- 10.1 years for carriers and 60.8 +/- 14.1 years for noncarriers), tumor location in the colon, and disease stages. Tumors of I1307K mutation carriers displayed positive p53 immunostaining and loss of beta-catenin, E-cadherin, and DCC expression more often compared with noncarriers, although none of these differences reached statistical significance. Mutation frequencies in the Ki-ras gene were similar in both groups. In conclusion, the molecular pathways in CRC in I1307K APC mutation carriers are seemingly similar to those of sporadic cases, but a larger study is clearly needed.

Immunohistochemistry for hMLH1 and hMSH2: a practical test for DNA mismatch repair-deficient tumors.

Inactivation of deoxyribonucleic acid (DNA) mismatch repair genes, most commonly human mutL homologue 1 (hMLH1) or human mutS homologue 2 (hMSH2), is a recently described alternate pathway in cancer development and progression. The resulting genetic instability is characterized by widespread somatic mutations in tumor DNA, and is termed high-frequency microsatellite instability (MSI-H). Although described in a variety of tumors, mismatch repair deficiency has been studied predominantly in colorectal carcinoma. Most MSI-H colorectal carcinomas are sporadic, but some occur in patients with hereditary nonpolyposis colorectal cancer (HNPCC), and are associated with germline mutations in mismatch repair genes. Until now, the identification of MSI-H cancers has required molecular testing. To evaluate the role of immunohistochemistry as a new screening tool for mismatch repair-deficient neoplasms, the authors studied the expression of hMLH1 and hMSH2, using commercially available monoclonal antibodies, in 72 formalin-fixed, paraffin-embedded tumors that had been tested previously for microsatellite instability. They compared immunohistochemical patterns of 38 MSI-H neoplasms, including 16 cases from HNPCC patients with known germline mutations in hMLH1 or hMSH2, with 34 neoplasms that did not show microsatellite instability. Thirty-seven of 38 MSI-H neoplasms were predicted to have a mismatch repair gene defect, as demonstrated by the absence of hMLH1 and/or hMSH2 expression. This included correspondence with all 16 cases with germline mutations. All 34 microsatellite-stable cancers had intact staining with both antibodies. These findings clearly demonstrate that immunohistochemistry can discriminate accurately between MSI-H and microsatellite-stable tumors, providing a practical new technique with important clinical and research applications.