René Lambert, MD
Considerable progress occurred in recent decades in the early diagnosis of digestive cancer or its precursors. The 3 major companies now produce electronic video-endoscopes equipped with high-resolution imaging, magnification, techniques for image processing of which Narrow Band Imaging is the more reliable. Moreover, abdominal and endoscopic ultrasonography and radiological imaging with CTscan or MRI detect with high sensitivity tumors in biliary ducts, pancreas and liver. Nowadays, the genetic study of primary alterations in the DNA structure as well as the epigenetic study of alterations superimposed on this basic structure offers a revolutionary perspective in the diagnosis and treatment of digestive cancer. The analysis of the cancer genome 1-6 opens a wide perspective for new “filter tests” in the selection of persons at risk. The persons so selected still deserve to be explored by endoscopic or radiological imaging for the direct localization and for histopathologic confirmation. Epigenetic mechanisms also open a new field in the therapy of early or advanced cancer.
The human genome is stored on 23 chromosomes The major part of the 3 billion base-pairs of DNA (desoxyribonucleic acid) is non coding and there are only around 23,000 genes coding for proteins and containing the genetic instructions used in the development and function of the cells The tissue stability is ensured by the balanced rhythm of mitosis and apoptosis. Extensive sequencing of the nucleotides in the polymeric helix is now possible through hybridization when using microarray probes. The DNA stored in the nucleus of the cell is a double helix of nucleotides, with backbones made of sugars (deoxyribose) and phosphate groups, joined by ester bonds. Attached to each sugar is one of four types of bases: adenine, guanine, cytosine, and thymine. The two helices are linked together by hybridization between the 4 bases. The sequence of these four bases along the backbone encodes information for the building of proteins. The double DNA helix is stored in the nucleus of the cell and compacted with proteins, histones, in the nucleosomes. The histones, which ensure the stability of DNA, may interfere in the organization of DNA.
RNA and messenger-RNA
RNA or ribonucleic acid, is a long chain of nucleotide units. RNA is usually single-stranded. Each nucleotide with a backbone of sugar (ribose) and phosphate is linked to one of 4 nitrogenous bases (Adenine, Guanine, Cytosine, and Uracil instead of Thymine). RNA is transcribed in the nucleus from DNA by RNApolymerases and then localized to the ribosomes of the cytoplasm where it receives information transferred from the DNA by way of the messenger-RNA (mRNA). The mRNA carries the genetic instructions for proteins and is a copy of a segment of the DNA in which the introns have been removed. The coding sequence of the mRNA determines the amino acid sequence in the protein that will be produced.
MicroRNA (miRNA) are short RNA molecules, on average 22 nucleotides long, that regulate gene expression. For that purpose, miRNA is complementary to a part of messenger RNAs (mRNAs), which can be exported from the cell in macromolecular complexes with proteins: the exosomes. Tumor cells excrete exosomes containing tumor born miRNA into the microscopic vesicle of their surrounding microenvivronment. Circulating tumor miRNAs are very stable and can therefore be targeted by non-invasive tests for the detection of early tumors. The miRNAS can also be used for monitoring treatment of the tumor and its prognosis.
Molecular alterations in the cancer genome
Cancer arises in cells of a tissue as a result of genetic changes in the basic sequence of the nucleic acid helix. There is a distinction between somatic mutations, acquired under the influence of environmental factors, and germline mutations transmitted by the ascendants. Cancer can also arise through epigenetic mechanisms during DNA duplication; molecular alterations are then superimposed upon the basic structure of the DNA helix. Genomics and epigenomics are new disciplines devoted to the study of those mechanisms. The cancerous cell develops after successive alterations on 15 to 20 genes. A double mechanism is frequently involved in the development of a cancer, when the initial genetic mutation in the basic structure of DNA leads to epigenetic mutations.
Alterations in the basal sequence of nucleic acids occur through 3 distinct mechanisms:
Epigenetic mechanisms in cancer
Cancer is a genetic disease and the basic structure of the genetic code, with the information contained in the genes, can be modified by point mutations in a single site of the sequence, either under the influence of environmental factors or through transmission from the parents of a heritable germline mutation, as it occurs in familial colorectal cancer. Epigenetic alterations with modification (activation or inhibition) in the function of the genes occur independently of an alteration in the basic DNA structure, and play a major role in the molecular mechanisms of sporadic cancer. The molecular basis of epigenetics is complex, there are 3 mechanisms:
1 - DNA hypermethylation, the best known epigenetic mechanism, concerns the CpG islands in the “promoter“ part of many genes; it is characterized by addition of a methyl group to the Cytosine base linked to Guanine in the CpG dinucleotide under the influence of DNA-methyltransferase. The hypermethylation of gene promoters may induce the silencing of suppressor genes and plays a role in carcinogenesis when this silencing function is suppressed. Hypermethylation of the promoter in genes is easily detected in tissue specimens by autofluorescence and is also detected in the plasma with potential application to screening tests.
2 - remodeling through translational modification of the amino acids in the histone protein component of DNA in the nuclear chromatin: this is a potential cause of cancer when there is histone deacetylation.
3 - Micro-RNA: this is the third mechanism that controls the expression of genes. MiRNA deregulation is highly targeted on oncogenes and tumor suppressors as compared to hypermethylation and histone modifications. Silencing of miRNA suppressor function will activate oncogenes resulting in malignant transformation. The expression of various mi-RNA has been shown to be altered in digestive cancer (pancreas and colon); they could act as biomarkers of cancer in blood or body fluids.
There is a relationship between the 3 major epigenetic mechanisms. The histone modifying enzymes are under direct control of miRNAS while the expression of certain miRNA depends on methylation of their promoters. In addition, hypermethylation has an impact on the interaction of histones with the DNA expression.
In a normal adult tissue a small contingent of stem cells perpetuate themselves through “self-renewal”. They give birth to other stem cells that have the same capacity to proliferate as the parent cells and will differentiate into specialized cell types. Normal stem cells maintain the turnover of regeneration and also act as a repair system for the body. Epigenetic modifications play an important role in the function of stem cells. The genes that regulate renewal are inactivated by epigenetic modification in normal cells whereas these self-renewal pathways are reactivated in stem cells.
The cancerous cells that constitute the bulk of the tumor mass have limited capacity to proliferate, while a small population of cells is able to self-renewal and sustaining of the tumor growth. These cells share certain common characteristics with the stem cells ensuring the stability of normal tissues and are often called cancer stem cells (CSC). On the other hand the doubt on their origin justifies their denomination as tumor-initiating cells (TICs) because their development could also result from reactivation of normal cells in the tissue. The tumor initiating cells (or cancer stem cells) differ from the large mass of the other cancerous cells by their ability to renewal; in addition they may resist chemotherapy or radiotherapy, and cause recurrence or metastasis when they are not destroyed. These cells also have the capacity to transport various substrates across cellular membranes because they strongly express the ABC transporters, which rely on the energy of ATP hydrolysis.
Detection of cancer stem cells in a tumor, based on expression of epithelial cell adhesion molecules and surface markers like CD 44, CD24, EpCAM, CD133, could help in estimation of its aggressiveness and in delineating treatment options. It is critical to identify these cells in tumors, because they are a privileged target in the treatment and justify new therapeutic approaches with dormancy induced by miRNA decoys. As an example, when the miRNA (miR34) linked to p53 gene is introduced in a preparation of p53 deficient human gastric cancer cells, apoptosis of the cells occurs in relation to restoration of the p53 function in the preparation.
The role of genetic factors has been extensively reviewed in the detection of the most frequent form of heritable digestive cancer i.e. colorectal cancer. In this situation the study of epigenetic factors is not helpful and the identification of the point mutation by genetic testing is proposed after the initial questionnaire on family antecedents. However when digestive cancer occurs as a sporadic disease epigenetic factors play a major role and their study may contribute to diagnosis. There are two potential targets in epigenomics for the diagnosis of digestive cancer: 1. DNA tumor biomarkers for CpG hypermethylation in the promoter of various genes offering a global methylation profile. 2. Overexpression of specific categories of miRNA, which can transit to plasma as well. Such bio-markers have been first tested in stools, and then in plasma to obtain increased compliance of the tested persons. When DNA markers of multiple genes in stools or in plasma, detect a tumor further procedures of localization (endoscopic diagnostic or radiological imaging), as well as histopathologic control are required. The efficacy of DNA biomarkers in the early detection of digestive cancer or precancerous lesions has been confirmed for esophagus, stomach, colon, pancreas, bile duct or liver across many recent publications. Most biomarkers are still in a phase of assessment in pilot studies, however a new colorectal cancer single biomarker in blood has been licensed in 2009 in the USA offering the Septin 9 methylated DNA test as a commercialized kit for colorectal cancer screening (Epigenomics AG). Biomarkers based on DNA and miRNAS are not yet cost/ effective, but the route is now open for developments in efficacy and lowering of costs.
The contribution of epigenetics factors to the management of digestive cancer is not limited to diagnosis and new therapeutic strategies are suggested. MiRNA may have a specific role in the control of cancer as stressed by the Nobel laureate David Baltimore in the symposium on biological complexity recently (October 27-29) held at La Jolla in a joint action of Salk Institute, Nature, and Fondation IPSEN. The concept that only a subset of cells drives tumor formation justifies treatments that specifically target the tumor initiating cells. In the future, the treatment of cancer resistant to chemotherapy, and of relapsing or metastatic cancer, should benefit from the still experimental studies with epigenomics.