Genetic alteration and gene expression modulation during cancer progression

Abstract

Cancer progresses through a series of histopathological stages. Progression is thought to be driven by the accumulation of genetic alterations and consequently gene expression pattern changes. The identification of genes and pathways involved will not only enhance our understanding of the biology of this process, it will also provide new targets for early diagnosis and facilitate treatment design. Genomic approaches have proven to be effective in detecting chromosomal alterations and identifying genes disrupted in cancer. Gene expression profiling has led to the subclassification of tumors. In this article, we will describe the current technologies used in cancer gene discovery, the model systems used to validate the significance of the genes and pathways, and some of the genes and pathways implicated in the progression of preneoplastic and early stage cancer.

Figure 1

Laser capture microdissection of prostate tissue sections. Panel A shows hematoxylin & eosin (H&E) stained prostate section. Black arrow indicates stromal cells. Red arrows indicate epithelial cells. Panel B shows laser outline of the cells to be collected. Panel C shows the remaining cells after laser capture. Panel D shows the cells collected from the outlined area. Images provided by Dr. J R. Vielkind.

Figure 2

Cytogentic analysis. Panel A shows an image of fluorescent in situ hybridization. The metaphase chromosome spread is hybridized with two locus specific probes labeled with FITC (green) and SpectrumRed (red). Panel B Spectral karyotype (SKY) analysis of immortalized human prostate epithelial cells. The line was derived from cells previously described [18]. Images provided by Dr. J. Squire.

Figure 3

Schematic representation of randomly amplified polymorphic DNA PCR analysis of tumor DNA. Panel A shows chromosome with multiple binding sites for selected primers (red arrows). Panel B is a representation of a given locus in six different patients. Patient 1 contains both primer sites, patients 2, 4, and 5 contain neither sites, and patients 3 and 6 contain only one of the primer sites. Solid line represents normal chromosome, while the dotted line represents regions of chromosomal loss. Panel C displays the DNA fingerprints for paired normal and tumor DNA from these six individuals. The blue bands correspond to fragments indicated in Panel B.

Figure 4

Principle of array CGH. This figure shows the steps in BAC array CGH. (A) BAC clones are selected from a physical map of the genome. (B) DNA samples are extracted from selected BAC clones and their identity is confirmed by DNA fingerprinting or sequence analysis. (C) A multi-step amplification process generates sufficient material from each clone for array spotting. Each clone is spotted in replicate onto a solid support. (D) Reference DNA and test DNA are differentially labeled with cyanine 3 and cyanine 5 respectively. (E) The two labeled products are combined and hybridized onto the spotted slide. (F) Images from hybridized slides are obtained by scanning in two channels. Signal intensity ratios from individual spots can be displayed as a simple plot (G) or by using more complex software such as SeeGH, which can display copy number alterations throughout the whole genome (H) [82].

Figure 5

SeeGH display of whole genome array comparative genomic hybridization. SeeGH translates spot signal ratio data from array CGH experiments to give high resolution chromosome profiles (see Fig 4). Signal ratios are plotted as a log2 scale. This figure shows a whole genome profile of a squamous non-small cell lung carcinoma. Vertical green and red lines are scale bars indicating log₂ ratios. Copy number losses are indicated by a shift in ratio to the left of zero, while gains are reflected by a shift to the right. Red and green arrows highlight examples of copy number deletions or gains respectively.

Figure 6

Serial analysis of gene expression (SAGE) library construction. This figure shows the steps in SAGE profiling. (A) An RNA population is reverse transcribed to cDNAs using oligo-T primers attached to magnetic beads. (B) cDNAs are collected and digested with the restriction endonuclease Nla III. (C) Linkers containing sequence recognized by BsmF I are ligated to the digested cDNAs. Sequence tags are released from the beads by BsmF I digestion (BsmF I cuts at a fixed distance downstream from its recognition site). (D) Released DNA tags are ligated together to form ditags. (E) Ditags are amplified and then digested with Nla III to remove the linkers. (F) Ditags are ligated together to form a concatemer which is then clones into a plasmid vector to generate a SAGE library. The identity and abundance of tags is deduced from DNA sequence analysis of plasmid clones of concatemated ditags. (G) Relative abundance of gene expression – between genes within the same RNA population or between samples – is deduced by counting sequence tags. Diagrams provided by Dr. K. Lonergan.

Figure 7

Progression model of ductal breast cancer. Histopathological stages of the most common form of breast cancer include atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS), and invasive ductal carcinoma. This figure highlights the changes that occur in breast cancer throughout the histopathological stages of the disease.

Figure 8

Progression model of prostate cancer. The prostate cancer progression model suggests that normal prostatic epithelium changes to prostatic intraepithelial neoplasm (PIN), which in turn becomes localized invasive cancer, metastatic, and, finally, hormone refractory disease with increasing severity reflected in a higher Gleason grade. This figure outlines the changes that occur in the progression of prostate cancer.

Figure 9

Fluorescence bronchoscopy. Panel A shows a white light bronchoscopy image obtained using the LIFE LUNG device from the upper left lobe of the lung. Panel B shows the detection of a carcinoma in situ lesion due to abnormal autofluorescence. The lesion is observed as a brownish area in a background of green fluorescence. Images provided by Dr. S. Lam.

Figure 10

Progression model of squamous non-small cell lung carcinoma. Squamous cell carcinoma of the lung progresses from normal, metaplasia, dysplasia, carcinoma in situ to invasive carcinoma. The alterations present in the various stages of the disease are outlined in this figure.

Figure 11

Progression model of colorectal cancer. Colorectal cancer typically progresses from normal epithelium through dysplasia and adenoma stages to carcinoma in situ and finally to invasive cancer. The changes that occur in the progression of colorectal cancer are outlined in this figure.