Briefings in Functional Genomics Advance Access published online on May 24, 2007
Briefings in Functional Genomics, doi:10.1093/bfgp/elm007
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Molecular analysis of deletions in human chromosome 3p21 and the role of resident cancer genes in disease
Corresponding author. Debora Angeloni. Scuola Superiore Sant’Anna and Institute of Clinical Physiology – National Research Council (IFC-CNR), Via Moruzzi, 1 - 56124 Pisa, Italy. Tel: +39 050 315-3092, Fax: +39 050 315-3327, E-mail: angeloni{at}ifc.cnr.it
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ABSTRACT |
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ABSTRACT
DELETION MAPPING OF HUMAN...Epithelial cancers inflict a heavy human and social burden. It was estimated [Tyczynski JE, Bray F, Parkin DM. Lung cancer in Europe in 2000: epidemiology, prevention, and early detection. Lancet Oncol 2003;4:45–55 (Review)] that in Europe, in the year 2000, 347 000 persons died of lung cancer alone, the deadliest cancer disease.
Loss of heterozygosity and large homozygous deletions of the human chromosome region 3p21 are especially frequent in epithelial cancers of several organs. In fact, 3p21 is a very peculiar region of the genome harbouring, tightly clustered, several types of cancer-causing genes (CCG) (Lerman MI, Minna JD. The 630 kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumour suppressor genes. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium. Cancer Res 2000;60:6116–33).
Current results show that, unlike it was thought initially, many tumour suppressor genes (TSG) are located close by even in small genomic regions. They may be involved, perhaps with varying role, in different types of tumour, and may be influenced by the genetic background of different human populations as well as by environmental pollutants (cigarette smoking, professional exposure).
This review will discuss methods of molecular analysis of genomic deletions to uncover CCG at 3p21, will summarize the present knowledge regarding the TSGs located in this region, and will describe a possible model of epithelial cancer pathogenesis.
Keywords: 3p21, cancer stem cell (CSC), epithelial cancer, haploinsufficiency, promoter hypermethylation; loss of heterozygosity (LOH), tumour suppressor gene(s) (TSG)
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DELETION MAPPING OF HUMAN CHROMOSOME 3 |
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Allelic loss (AL) is a significant sign of somatically acquired genetic events in the evolution of many tumours, and is a useful tool to discover the location of new tumour suppressor genes (TSGs).
Cytogenetic and allelotyping studies of fresh tumours and tumour cell lines have shown that AL from several distinct regions on chromosome 3p, including 3p25, 3p21–22, 3p21.3, 3p12–13 and 3p14, are the earliest and most frequent genomic abnormalities involved in a wide spectrum of major epithelial cancers of lung [3–8], breast [9, 10], kidney [11], head and neck [12], ovary [13], cervix [14, 15], colon [16], pancreas [17], esophagous [18], bladder [19] and other organs [20], (Figure 1).
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3p deletions are detected in almost 100% of small-cell lung cancer (SCLC), renal cell carcinoma (RCC), more than 80% of breast carcinomas and more than 90% of non-small-cell lung cancer (NSCLC) cell lines [20–24]. In addition, 3p losses are found as clonal lesions in the smoking-damaged respiratory epithelium, including histologically normal, and in hyperplasia, the earliest detectable premalignant lesion [25, 26]. This shows that genetic changes precede histological changes and affect a so-called cancerous field predisposing the onset of cancer [27]. Such field defects were found in current and former smokers and persisted for years after smoking cessation, but were reversible.
The discovery of those deletions, on one hand suggested that 3p harbours a number of genes whose inactivation is strongly associated with the initiation and/or development of lung and other tumour diseases, some genes being cancer specific and others common to different cancer types [7, 20, 22–24, 28, 29]. On the other hand, it prompted several efforts for positional cloning of the putative resident TSGs.
Deletion mapping was performed with a variety of experimental techniques using polymorphic markers of different types: restriction-fragment length polymorphisms (RFLPs), microsatellites and single-nucleotide polymorphisms (SNPs).
A polymorphic marker is essentially a short sequence of DNA located in a precisely defined genomic region that allows distinguishing the paternal from the maternal allele. Assuming that an individual is heterozygous for a given marker, this DNA is then tested for the presence of both alleles in the tumour specimen. If the tumour lacks one of the alleles (that is shows loss of heterozygosity, LOH), it is deduced that some form of loss has taken place in the specific region, therein suggesting the presence of a TSG. Such loss might derive from mitotic recombination or actual physical deletion. Physical loss can be distinguished from a translocation event with fluorescent in situ hybridization (FISH), using specific fluorescent probes that hybridize with and highlight the chromosome region of interest.
RFLPs allow differentiating alleles by analysis of patterns derived from DNA cleavage. Two alleles that differ for base changes in the target sequence of a given restriction endonuclease, will produce digestion fragments of different length. The patterns generated can be used to differentiate alleles from one another.
Microsatellite DNA polymorphisms are short DNA fragments (usually within 100 bp) of repeated units of 1–5 bp, are uniformly distributed along the genome, abundant (around 5 x 105), and can show more than 10 alleles for a given locus.
SNPs (single-base polymorphisms whose frequency exceeds 1% in at least one population) are the most common form of genetic variation in the human genome (one every 400–1000 bp in average). About 5.6 million SNPs have been identified (dbSNP Build ID 126p) and they are suitable for microarray analysis.
This variety of tools allowed the discovery of 3p deletions varying in magnitude from interstitial to large terminal, however technical difficulties made hard the identification of resident TSGs. In fact, even the LOH frequency of a given marker in the same type of tumour could vary from one study to another. For example, the D3S1284 polymorphic marker located in 3p13, was found deleted in 8% of NSCLC in one study [29] but in 63% of SCLC in another study [23]. A mix of normal tissue, connective stroma and blood vessels, together with the tumour specimen examined, represented the possible source of error. Therefore, the introduction of laser-assisted microdissection of tumour samples coupled with PCR techniques represented a very useful way for more accurate separation of the cancerous specimen from the normal tissue and the histologically different surrounding areas [reviewed in 30]. PCR-based techniques allowed a very detailed molecular analysis of a small number of cells (500–1000). Using a panel of 28 3p-markers on microdissected material from 97 lung cancer samples and 54 preneoplastic, microdissected respiratory epithelium samples, a high-resolution LOH study showed AL in 96% of lung cancers and 78% of preneoplastic/preinvasive lesions [23]. Interestingly, AL was in some cases multiple and discontinuous, that is areas of LOH were interspersed with areas of retention of heterozygosity. The 91% SCLC and 95% squamous cell carcinomas (SCC) demonstrated larger segments of 3p loss, whereas 71% of adenocarcinomas and preneoplastic lesions showed smaller 3p deletions. Moreover, there was a progressive increase in the frequency and size of loss with increasing severity of histopathological changes (histologically normal epithelium < hyperplasia < moderate dysplasia < severe dysplasia < carcinoma in situ < invasive cancer).
Interestingly, the same parental allele was lost in 88% of samples in different foci, similarly with what observed in the full-blown tumour. This phenomenon, caused by a yet unknown molecular mechanism, might be relevant to determine a parental-inherited predisposition to cancer.
A systematic bias was shown consisting in the high-molecular weight allele being less sensitive to normal cell contamination than the low-molecular weight allele, so that in 100 papers published between 1994 and 1999, the loss of L allele in tumours was detected at the 52% frequency of loss of the H allele [31]. These artifacts were solved, at least in part, with the introduction of multiplex PCR-based polymorphisms together with rules to account for this bias [31].
Multiple regions of 3p loss indicated by integrated data analysis are the following: telomere-D3S1597, D3S1111-D3S2432, D3S2432-D3S1537, D3S1537, D3S1537-D3S1612, D3S4604/Luca19.1-D3S4622/Luca4.1, D3S4624/Luca2.1, D3S4624/Luca2.1-D3S1582, D3S1766, D3S1234-D3S1300 (FHIT/FRA3B) region centered on D3S1300), D3S1284-D3S1577 (U2020/ROBO1-DUTT1 region centered on D3S1274) and D3S1511-centromere [32].
A set of six genes in the 600-kb deleted region at 3p21.3 showed loss in 77% of lung cancers, 70% of preneoplastic/preinvasive lesions associated with lung cancer and 49% of mildly abnormal, preneoplastic/preinvasive lesions found in cancer-free smokers. Significantly, loss of this region was never found in 18 epithelial samples of people that never smoke [2].
It was recently shown that the pattern of chromosomal aberrations hitting 3p21 in epithelial cancers is not at all simple but involves, together with homozygous and hemizygous deletions, also duplications and amplifications, with frequency varying between 15% and 42.5% as shown by quantitative-PCR [33]. This might have a very complex effect on the phenotype. An example is given by the putative oncogene tyrosine-kinase receptor Ron [34] and the gene for its ligand, macrophage stimulating protein (MSP) [35], both mapping at 3p21 [PDB] .3. Amplification of Ron might have a direct effect on cell proliferation. However it is also possible that the extensive hypermethylation of this region in cancer has an additional effect on modulating Ron activity. Ron transcription is driven by two promoters: a distal one controlling the full-length transcript, encoding the ligand-binding pocket and the tyrosine kinase domain, and an intragenic one, controlling the transcription of the kinase domain only which, contextually, is constitutively active [36, 37]. In SCLC primary tumours and cell lines, and in erythroleukaemia cell lines, hypermethylation of the proximal promoter seems to facilitate the intragenic promoter activity leading to a higher abundance of the active kinase, and possibly an unbalanced ratio between the two forms of Ron. This results in the net positive control of cell proliferation and inhibition of differentiation [37].
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HIGH-THROUGHPUT TECHNOLOGIES TO INVESTIGATE THE GENOME |
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Sequencing of the human genome, progress in bioinformatics and technology, and the development of several methods of molecular gene profiling, provide the opportunity to interrogate the genome and transcriptome for the various genetic (including point mutations, small deletion/insertion, translocations, copy-number changes, LOH) and epigenetic (e.g. methylation) somatic alterations that underlie cancer, promoting a non-reductionist approach to cancer biology. Microarrays of various types are now available.
SNP microarrays allow describing at once LOH and copy-number changes on a genome-wide scale [38]. LOH is not always equivalent to copy-number losses, as it may arise after hemizygous deletion alone or followed by gene duplication resulting in copy-neutral LOH. Loss of a single chromosome in hyperploid cells, although looks like a deletion, it leaves the remaining chromosomes with retention of heterozygosity. Conversely, high amplification level of a single allele results in allelic imbalance also giving the appearance of LOH. SNPs on a small chromosomal segment tend to be inherited as a block: the haplotype. A common method to determine LOH consists of comparing the haplotype between paired tumour and normal tissues (here only SNPs heterozygous in the germline are informative). Current methods allow simultaneous genotyping of over 5000 000 SNPs across the genome, with two major technological platforms: oligonucleotide probes spotted on gene chips (Affymetrix) or adsorbed on beads (Illumina).
SNP arrays allow also identifying LOH with no need of comparison with normal paired DNA, although at lower resolution [39]. In this case, statistical analyses are applied to identify strings of consecutive homozygous SNPs (here every SNP is informative) that are longer than expected by chance alone.
SNP arrays can also determine the parental origin of an amplified or deleted chromosome region. Recently it was shown [40] that when a region is amplified, the extra copies tend to derive from a single parent, which may reflect the preferential amplification of a given parental allele harbouring an activating mutation. Certain copy-number alterations correlate strongly with the tissue of origin [41]. Such so-called lineage-restricted alterations may harbour novel cancer-causing genes directing onset or progression of tissue-type specific cancers. Combining genome-wide data from the NCI60 cancer cell lines, several melanoma cell lines were clustered on the basis of increased dosage of the 3p-resident gene MITF [41]. These results suggest that, like oncogene addiction, ‘lineage addiction’ may represent a fundamental tumour survival mechanism with possible important therapeutic implications.
Many studies are now published based on the use of SNP arrays, some with highly sophisticated technological means (see SNP-mass spectrometry genotyping [42]). Comparative genomic hybridization (CGH) has been extensively used to map DNA copy number changes to a chromosomal position [43]: differentially labeled and denatured genomic DNA pools are simultaneously hybridized to immobilized and denatured chromosome methaphase spreads. The fluorescence ratio is then measured along each chromosome, creating a genome-wide profile of the relative copy number. In this way, the whole genome can be interrogated for DNA copy number in a single experiment, initially with a resolution of one clone per megabase [38] and now with a tiling resolution of approximately one clone per 100 kb. After analyzing large numbers of tumours, regions harbouring putative cancer-causing genes (CCG) can be highlighted thanks to recurrent copy-number changes. CGH microarrays, with DNA fragments of known genomic location immobilized on glass slides, are also available and are currently the most used method for high-resolution screening of genomic copy-number changes. [44–46]. The methaphase spreads have been replaced by other hybridization substrates, such as bacterial artificial chromosomes, cDNAs, oligonucleotides and exon-specific PCR products, to allow direct linking of aberrations to sequences of known genomic location.
For a contextual, genome-wide analysis study of methylation, deletions and amplifications of cancer genes, Dr Zabarovsky's group proposed the use of NotI-clone microarrays and genomic subtraction [47], as NotI recognition sites are closely associated with CpG islands and gene promoters. A method for cloning deleted sequences, named CODE (Cloning Of DEleted sequences) genomic subtraction procedure, can be adapted to NotI flanking sequences and to CpG islands [47]. A NotI-CODE method can be used to prepare NotI representations containing 0.1–0.5% of the total genome. The NotI representations contain 10-fold less repetitive sequences than the whole human genome and can be used as probes for hybridization to NotI microarrays. This procedure, combining NotI arrays and NotI representations, allows an advantageous simultaneous detection of copy-number changes and methylation, an epigenetic phenomenon relevant to cancer pathogenesis.
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MOLECULAR ANALYSIS OF 3P21 |
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3p21.1–p21.2
Various studies discovered LOH located here [4] and reported cancer-cell growth suppression activity of this region [48], suggesting the presence of resident TSGs.
Three resident candidate TSGs are discussed in Table 1: DRR1 (downregulated in renal cell carcinoma 1, TU3A), BAP1 (BRCA1-associated protein 1, KIAA0272) and ARP (arginine-rich, mutated in early stage tumours; ARMET).
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3p21.3C (CENTROMERIC): the LUCA region
The localization and identification of the several potential TSG harboured at 3p21.3 has represented one of the most exciting progress in recent cancer genetics and molecular biology research [2, 8, 31, 49].
The difficulty of performing genetic linkage studies of lung cancer families [50, 51] has prompted the search for resident TSGs with methods of AL mapping in tumours, cell lines, and pre-malignant lesions of breast and lung [7, 26, 52–55].
As discussed in the first paragraph of this review, the regions of 3p loss in lung and breast cancer and pre-malignant lesions are multiple and often large. To make reasonable a positional cloning effort of the resident putative TSGs (working name: LUng CAncer TSGs), a minimal deleted region was identified by overlapping several large homozygous deletions discovered in the lung cancer cell lines NCI-470, H1450 and GLC20 and in one breast cancer specimen (HCC1500), [5, 56–60], Figure 2. In this way, the critical region was narrowed to 120 kb [57] on the assumption that lung and breast cancers would arise from mutations inactivating the same TSG(s).
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An international consortium was created that physically mapped, cloned and annotated the genomic DNA surrounding the 3p21.3 locus [2].
A 630-kb clone contig was initially constructed, containing a minimal set of 23 cosmids and one PI phage covering the 370-kb deletion overlap. The overlapping deletions breakpoints were defined and precisely located; the transcriptional map encoded in the contig was generated based on identifying transcribed sequences, genes and CpG islands [58].
Subsequentely, the 630-kb clone contig was sequenced jointly by The Washington University and The Sanger Human Genome Sequencing Centers. The cloning effort proceeded through the integration of bioinformatics and experimental efforts, in that some genes were identified with ‘wet biology’ methods such as screening of cDNA libraries with cosmid DNA probes and using the cosmids for exon capture, while others were predicted in silico, using a variety of algorithms [2].
Briefly, after masking DNA repeats and low-density regions, the contig sequence fragments were used for BLASTN searches against EST, Unigene and non-redundant nucleotide databases to identify potential transcripts (ESTs) and build EST clusters. Next, genomic sequences assembled from cosmid clones were analyzed with gene prediction programs to identify putative coding sequences and predicted protein sequences. These were then used in BLASTN and TBLASTN searches to identify cDNAs and ESTs already present in databases, therefore independently cloned. A decisive ‘proof of existence’ of a candidate gene was considered the presence of orthologous genes in model organisms. The genes’ expression territories were studied with Northern blotting in normal and lung cancer specimens. Possible rearrangements were studied with Southern blotting in a large panel of SCLC and NSCLC cell lines. The mutation status was analyzed extensively by RT-PCR single strand conformation polymorphism (SSCP), PCR-SSCP and resequencing.
In total, 25 genes were mapped in the extremely gene-dense region represented in the 630-kb contig. Nineteen genes were found within the deleted overlap region of 370-kb (Figure 2): eleven genes in the distal 250-kb region, eight genes in the 120-kb proximal, minimally deleted region. These eight candidate TSGs were designated: CACNA2D2 (AF042792 [GenBank] , AF042793 [GenBank] ), PL6 (U09584 [GenBank] ), 101F6 (AF040704 [GenBank] ), NPRL2 (AF040707 [GenBank] ), BLU (U70880 [GenBank] ), RASSF1 (AF102770 [GenBank] , AF040703 [GenBank] ), FUS1 (AF055479 [GenBank] ) and HYAL2 (U09577 [GenBank] ).
They are briefly described in a further paragraph.
The candidate TSGs frequently undergo AL, none showed a frequent mutation rate in lung cancer samples; however several showed absent or reduced expression level due to promoter hypermethylation [2].
Since the prognosis for lung cancer patients strongly correlates with the stage of the disease at the time of presentation, it is important to identify sensitive markers for early diagnosis. With regard to this, TSG DNA methylation may prove a useful target. It was shown that aberrant methylation of TSGs can be detected in 100% of patient with SCC as early as 3 years before clinical diagnosis [61]. Moreover, it was shown that hypermethylation of TSGs can be evaluated in circulating blood instead of requiring an invasive biopsy, and can predict the course of disease in patients of breast [62] and gastric cancer [63], and SCC [64]. Recently, RASSF1A hypermethylation was demonstrated in the blood of patients with cutaneous melanoma [65].
So, on one hand TSG hypermethylation is likely the initiating event of many cancers but as such its detection may also become a tool for early, non-invasive diagnosis.
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FUNCTIONAL STUDIES OF TUMOUR SUPPRESSOR ACTIVITY |
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The candidate TSGs at 3p21.3 affect many biological processes such as cell proliferation, differentiation, cycle kinetics, signal transduction, ion exchange and cell death. These activities must be correlated, indirectly or directly, with TS properties.
One parameter to discover the TS potential of a gene is to demonstrate that the tumour phenotype, characterized by inactivation of the gene, can be rescued by replacing the mutated or deleted genes with the wt alleles. If carcinogens cause inactivation of 3p21.3 genes through various mechanisms (LOH, homozygous deletion, promoter silencing) leading to human cancer onset and development, one can predict that replacing 3p21.3 genes would result in tumour suppression, just like it was observed with the classical Rb or p53 TSGs. Several methods were developed to functionally test the putative TS activity of the 3p21.3 resident genes [32].
Tetracycline-regulated gene expression vectors
It was previously observed that mouse tumour–human normal cell microcell hybrids show non-random loss of human 3p21–p22 fragments when they undergo progressive growth in immune suppressed SCID mice, permissive to human tumour xenografts growth, suggesting that a growth-suppressing gene was located in the 3p expelled fragments [66]. When new retroviral and episomal vectors were engineered, suited for tight regulation of genes that cause suppression of cell growth, it became possible to generate cell lines for studying the effect of inducible transgenes in human cancer cells [67]. The gene inactivation test (GIT) was developed to characterize candidate TSGs, based on the reversible, functional inactivation of the analysed genes. The GIT test would mimic the natural process of TSG inactivation as it happens in patients. A panel of 14 cancer cell lines was used in which candidate genes were transfected and expressed under the control of tetracycline [67]. The experiment was based on the hypothesis that when a candidate TSG expression is allowed, the tumour growth should be arrested. Implanting the transfected cells, xenograft tumours were developed in mice with the gene expressed or not expressed depending on tetracycline administration in the drinking water. Several 3p21.3 candidate TSGs were studied, at least four can be now considered as true lung TSGs: NPRL2/G21, RASSF1A, SEMA3B and HYA22/RBSP3 (located in the more telomeric AP20 region).
Adenoviral transduction of candidate TSGs
Six genes (101F6, NPRL2/G21, BLU, FUS1, HYAL2 and HYAL1) were analysed for their candidate growth-suppression properties using recombinant adenovirus-mediated gene transfer in vitro and in vivo [68].
Forced expression of wild-type FUS1, 101F6 and NPRL2/G21 genes significantly inhibited cell growth by induction of apoptosis and alteration of cell cycle processes in 3p-deficient human lung cancer cell lines NCI-H1299 and A549 NSCLC, but not in the 3p-normal NCI-H358 NSCLC and normal human bronchial epithelial cells. Intra-tumoural injection of Ad-101F6, Ad-FUS1, Ad-NPRL2 and Ad-HYAL2 vectors, or systemic administration of protamine-complexed vectors significantly suppressed growth of H1299 and A549 xenografts and inhibited A549 experimental lung metastases in nu/nu mice [68].
These results showed that several genes in the 3p21.3 chromosomal region can be considered tumour suppressors in vitro and in vivo, and paved the way to translational approaches. The FUS1 gene is currently being given by systemic administration in nanoparticles, in a clinical trial of relapsed metastatic lung cancer at the M.D. Anderson Cancer Center [69].
Transfection of PAC clones
The genes from the 3p21.3 critical region were also introduced in cancer cells as genomic sequences, so as to avoid the possible gene over-expression effects caused by viral promoters [70]. The P1 artificial clones (PACs), containing one or several of the candidate TSGs accompanied by their natural promoters and regulatory sequences, were transfected in GLC45, a SCLC cell line that normally causes tumours in nude mice. The authors found that the integrated PAC most effective at reducing tumour growth in mice was one containing two genes, PL6 and 101F6/CYB561D2 [70].
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THE 3p21.3 MINIMAL DELETION-RESIDENT CANDIDATE TUMOUR SUPPRESSOR GENES |
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The eight genes mapped in this region were designated: CACNA2D2, PL6, 101F6, G21, BLU, RASSF1A, FUS1, HYAL2 (Figure 2). Some of them are now proved TSGs.
CACNA2D2
(Calcium Channel, Voltage-Dependent, Alpha-2/Delta Subunit 2; Kiaa0558) Gene locus: about 140 kb, with at least 40 exons. mRNA: 5.5–5.7 kb, with three splice forms [2]. The corresponding 1146-amino acid protein is a functional calcium channel auxiliary regulatory subunit [71]. No mutations were detected in detailed analysis of 100 lung cancers, however, CACNA2D2 is epigenetically inactivated in a large fraction of NSCLCs and in some SCLCs and can suppress the malignant phenotype of lung cancer cells in vitro and in vivo. Cacna2d2–/– mice exhibited growth retardation, reduced life span, ataxic gait with apoptosis of cerebellar granule cells and Purkinje cell depletion, enhanced susceptibility to seizures and cardiac abnormalities. CACNA2D2 is indispensable for the central nervous system function, may be involved in hereditary cerebellar ataxias and epileptic disorders in humans and is involved in cardiac function sympathetic regulation [72].
PL6
Gene locus: 4.5 kb, with two exons. mRNA: 2.2 kb, widely expressed in normal tissues and in most lung cancer cell lines analysed [2]. The corresponding 351-amino acid protein is 92% identical to the mouse protein. It contains 6–8 transmembrane helices. The cytoplasmic portions contain an OMP-decase domain, possibly involved in protein-protein interactions, and a bipartite nuclear localization signal [2]. No mutations were detected in cancer cell lines tested. PL6 is an unlikely candidate TSG [2].
101F6
(CYB561D2, cytochrome b-561 domain containing 2, TSP10)
Genomic locus: 3.2 kb, four exons. mRNA: 1.5 kb, widely expressed in normal tissues and also in most lung cancer cell lines tested [2].The 222-amino acid integral protein is 95% identical to the mouse protein. It contains 6 transmembrane helices with cytoplasmatic N- and C-termini [2]. No mutations were detected [2]. Wt-101F6 forced expression inhibited tumour cell growth and induced apoptosis in 3p21.3-homozygously deficient cell lines. Intra-tumoural injection or systemic administration of Ad-101F6 vectors significantly suppressed tumour xenograft growth and inhibited experimental lung metastases in null mice [70, 73].
G21
(NPR2L; NPRL2; TUMOUR SUPPRESSOR CANDIDATE 4, TUSC4)
Gene locus: 3.3 kb, 11 exons. mRNA: 1.8 kb, expressed with several splicing variants in all normal tissues tested [2]. The corresponding protein contains a bipartite nuclear localization signal and a granulin protein-binding domain. It is possibly involved in mismatch repair and apoptosis induction. It has highly conserved orthologs [2]. Inactivating mutations were found in lung, cervical, breast and clear cell renal carcinoma [73]. Transfection with lipid nanoparticles of NPRL2 significantly increased therapeutic efficacy of cisplatin, a most commonly used drug in cancer treatment, and overcame cisplatin-induced resistance [74], in a mouse model. G21 was suggested as a possible molecular therapeutic-agent for enhancing response and resensitizing non-responders to cisplatin treatment [74].
BLU
(FLU, ZINC FINGER MYND DOMAIN-CONTAINING PROTEIN 10; ZMYND10)
Gene locus: about 4.5 kb, Transcribed into a 2 kb mRNA [2].The 441-amino acid protein has sequence homology with the MTG/ETO family of transcription factors and to suppressins (cell-cycle entry regulator), [2]. BLU is an E2F-regulated, stress-responsive protein. Only three missense mutations in BLU were found in 61 lung cancer cell lines but, with variable frequency depending to the tumour type, BLU was found silenced in lung cancer [75–77], nasopharyngeal carcinoma [78–80], gliomas [81], liver tumours [82], esophageal squamous cell carcinomas [83], neuroblastomas [84], gallbladder carcinoma [85] and cervical neoplasias [86]. Promoter hypermethylation of BLU was reversed after 5-aza-2' deoxycytidine treatment. Over-expression of BLU inhibited colony formation in kidney cancer and neuroblastoma cell lines [75].
RASSF1, working name 123F2
(RDA32; NORE2A; REH3P21)
Genomic locus: 7.6 kb, with five exons [2]. MRNA: 2 kb, with at least six splicing isoforms [2, 58]. Most common and probably the most relevant to cancer are RASSF1A and RASSF1C [87–89]. They are driven from separate CpG-type promoters and share four terminal exons. The mRNA for both genes is well expressed in different normal tissues. RASSF1C but not the RASSF1A mRNAs are well expressed in most lung cancers. Missense mutations are rare but, noticeably, loss of RASSF1A expression is caused by promoter hypermethylation in over 90% of SCLCs and SCCs and in about 50% NSCLCs. RASSF1 suppresses lung cancer cells growth in vitro and in vivo [88, 89]. This gene is silenced in many other human cancers including kidney [90, 91], breast [89, 92], nasopharyngeal [93], prostate [94], bladder [95, 96] and other cancers [97–100]. RASSF1A TS activity was demonstrated using a tetracycline-regulated system [67, 90, 94]. Ectopic expression of RASSF1A in a breast cancer cell line enhanced apoptosis [89]. RASSF1A induces cell-cycle arrest by engaging the Rb-family [101, 102] and inhibits the accumulation of cyclin D1. RASSF1A acts at the level of G1 to S phase cell cycle progression [101]. RASSF1 interacts with E4F1 [103], a phosphoprotein involved in cell cycle progression, to enhance G1 cell-cycle arrest and S-phase inhibition. RASSF1A is also a mitosis-specific inhibitor of the APC/C (anaphase-promoting complex/cyclosome) [104, 105]. RASSF1 gene contributes to the spatiotemporal regulation of mitosis through a novel mechanism (reviewed in [105]). RASSF1A is now considered an important TSG involved in the onset or progression of perhaps about 75% of human cancer diseases. In lung cancer it is likely to play an early ‘gatekeeper’ role, whose loss of function leads to pre-malignant lesions and then invasive cancer.
Fus1
(TUSC2 tumour suppressor candidate 2; PAP; PDAP2; C3orf11)
Genomic locus: about 3.4 kb. The 1.8 kb mRNA is well expressed in all analysed human tissues, and in 20 lung cancer cell lines [2]. The FUS1 protein (110-amino acid) is probably a soluble cytoplasmic protein, with no known domains or motifs. Recently wt-Fus1 was characterized as an N-myristoylated protein [106]. A myristoylation-deficient mutant Fus1 protein, discovered with a SELDI-TOF approach in human primary lung cancer and cancer cell lines, was readily exposed to rapid proteasome-dependent degradation. Myristoylation seems required for FUS1 TS function and suggests that deficient post-translational modifications might play a role in TSG-mediated carcinogenesis [107]. Three mutations were discovered in 79 lung cancer cell lines DNA leading to truncated products [2]. No evidence of promoter methylation in cancer cells was found, however, the protein was undetectable in several lung cancer specimens [108]. FUS1 over-expression resulted in 60–80% inhibition of colony formation for NSCLC lines lacking endogenous FUS1 expression. In contrast, a similar level of expression of a tumour-acquired mutant form of FUS1 protein did not significantly suppress colony formation. In other experiments, Fus1 over-expression decreased colony formation of the 75% and increased 2-fold the doubling time, arresting H1299 cells in G1 [108]. FUS1 introduced with an adenovirus vector induced apoptosis in p53 wild-type lung cancer cells and suppressed the xenograft growth both with a local tumour injection and after systemic administration in immunodeprived mice [68]. These data suggest that FUS1 functions as a 3p21.3 TSG, probably in a haploinsufficient manner.
HYAL2 (HYALURONIDASE 2), working name LUCA2
Gene locus: 2.8 kb, with three exons [2]. The 2 kb mRNA is well expressed in all analysed normal human tissues, and in lung cancer cell lines except where homozygously deleted (SCLC line NCI-H524), [2]. The 474-amino acid protein is a member of a large family of hyaluronidases [109]. It is a cell-surface glycosylphosphatidylinositol (GPI)-anchored receptor for jaagsiekte sheep retrovirus (JSRV) cell entry [110]. JSRV is an ovine retrovirus that causes in sheep a contagious form of lung cancer arising from epithelial cells of the lower airway [111]. Antiserum directed against the JSRV capside protein cross-reacted with 30% of human pulmonary adenocarcinoma samples but not with normal lung tissue, or adenocarcinomas from other tissues [112], suggesting that related viruses may be involved in human lung cancer. While there is no known oncogene in JSRV, the Env protein could sequester HYAL2 and thus liberate an oncogenic factor negatively controlled by HYAL2, possibly the RON receptor tyrosine kinase, thereby mediating epithelial cells transformation by jaagsiekte retrovirus [113]. No mutations were detected in 40 lung cancer cell lines tested [2].
DISTAL 3p21.3, CER1
Distally to the 3p21.3 LUCA region, between markers D3S32 and D3S2354, is located another region that was found frequently deleted in lung and other cancers [5, 20, 114]. GIT showed that this region was consistently eliminated from mouse–human microcell hybrids containing human chromosome 3 [66, 115, 116]. Chromosome 3 transfer into the human RCC KH39 cell line, containing uniparental disomic chromosome 3, highlighted growth-suppression properties of this region, because the xenografts subsequently produced resulted in fewer and smaller tumours. Fine mapping of the region deleted in tumour xenografts led to identify the so-called common eliminated region1 (CER1), that spans 1 Mb between the markers D3S2 and D3S3582 [117]. A detailed map showed that this region contains 19 genes [117]. Three of them, RIS1, LIMD1 and LTF are bona fide TSGs, [reviewed in 118], (Table 2). A second eliminated region, not always consistently deleted in tumour xenografts (ER2) was also identified at 3p21.1.
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A potential CCG located toward the telomeric end of this region is CTNNB1 (catenin beta cadherin-associated protein). The gene locus spans about 23 kb and encodes for a 781-amino acid protein that participates in a protein complex including GSK, APC and axin. CTNNB1 is involved in regulating cell adhesion and participtes at the wnt signaling pathway. This gene is not frequently mutated in lung cancer, however a large body of studies has now implicated dominant oncogenic mutations of beta catenin in a variety of major tumours among which malignancies of the colon, endometrium, thyroid, hepatoblastoma and uterus [reviewed in 119].
3p21.3T (TELOMERIC), THE AP20 REGION
Together with the centromeric border of 3p21.3, the other region most frequently hit in major epithelial cancers is the telomeric border of 3p21.3, called the AP20 region (Alu-PCR clone 20), [120]. AP20 was also found strongly methylated in RCC cell lines studied, suggesting a mechanism of TSGs inactivation similar to that operating in 3p21.3C.
Precise mapping of 19 homozygous deletions in this region resulted in the localization of the minimal overlap to the interval flanked by markers D3S1298 and D3S3623. In this interval four genes were discovered: APRG1, ITGA9, HYA22/RBSP3 (Table 3) and VILL [32]. Functional studies to evaluate their TS potential are ongoing.
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DELETION OF CHROMOSOME ARM 3P IN HAEMATOLOGIC MALIGNANCIES |
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While cytogenetic aberrations resulting in deletion of 3p are common in solid tumours, these deletions are rare in haematologic malignancies, with frequencies of 2.9% of acute myeloid leukaemias (AML), 0.7% of myelodysplastic syndromes (MDS), 1.0% of chronic myeloid leukaemia (CML) with changes in addition to t(9;22), 1.5% of acute lymphoblastic leukemias (ALL), 4.2% of chronic lymphoproliferative disorder (CLD) and 1.1% of non-Hodgkin's lymphomas (NHL), the majority occurring together with other abnormalities [121].
The frequencies of 3p loss did not differ significantly among the MDS, ALL and CLD morphologic subgroups, between B-and T-cell ALL, CLD and NHL, among low-, intermediate- and high-grade NHL, or between therapy-related MDS and de novo MDS. On the other hand, the incidence of 3p deletions was higher in treatment-associated AML than in de novo AML. The most frequently deleted chromosome bands were 3p25 in AML, 3p26 in MDS, 3p14 in CMD, 3p25, 3p23 and 3p21 in CML, 3p26 and 3p25 in ALL, 3p26 and 3p25 in CLD, 3p26 in HD and 3p26 in NHL. These deletion hot spots are more distal than those reported in most solid tumour types, suggesting that haematologic malignancies and solid neoplasms involve different TSGs [121].
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A GENE MODEL FOR 3p21.3-DRIVEN CANCER PATHOGENESIS |
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Based on all acquired data, a model of lung cancerogenesis may be proposed as follows [7, 24, 122], (Figure 3).
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Epithelial tumours rarely consist of a single clone, but rather of a mixture of different clones with an acquired characteristic genetic instability [123, 124]. Mutations (although rare in 3p21.3 genes), gene dosage effect and aneuploidy cooperate in determining the full-blown tumour phenotype.
Consequent to smoking damage, thousands of different sites throughout the respiratory epithelium are hit by genetic aberrations, creating clones of initiated cells, a ‘cancerization field’ [27]. Initiated clones undergo a phase of Darwinian evolution that selects those more fit to circumvent environmental stress factors (hypoxia, malnutrition, immune system attacks, etc.), [125]. The clones that survive selection carry on invasion. Distinct clonal tumours may fuse and develop further into polyclonal tumours: from a clinical perspective, it is not unusual finding synchronous primary tumours with dissimilar hystology and distinct genetic signatures [126].
From a genomic point of view, as reviewed in the first paragraph, the earliest and most frequent damage is most likely represented by hemizygous inactivation of TSGs at 3p21.3. In fact, these changes were found perhaps in all types of lung cancer, in their pre-malignant precursor lesions and even in the histologically normal bronchial epithelium of current and former smokers. Since cancer development is a very complex process that requires the participation of several CCGs, other genomic hits are required and they are possibly represented by either the inactivation of the second allele of one of the hemizygous 3p21.3 TSGs, or other TSGs elsewhere. Noticeably, Rb, p53, p16 are usually found deleted in lung and other epithelial cancers, at later stages of carcinogenesis, when histologically dysplastic lesions or carcinoma in situ become evident [23, 127, 128].
It is likely that complete loss of 3p, or the entire chromosome 3, probably due to mitotic non-disjunction, or to the presence of a fragile site at 3p14.2, are secondary events in carcinoma development, whereas interstitial deletions or promoter hypermethylation are earliest.
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THE ‘CANCER STEM CELL’ HYPOTHESIS |
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Stem cells are characteristically defined by their capacity for self-renewal, extended life span, multipotency and resistance to potentially toxic compounds. Recently, a ‘cancer stem cell’ (CSC) hypothesis was proposed, according to which cells with tumour regenerating capacity would represent the driving force of tumour formation [129]. In fact, several theoretical requirements for a cell to be able to generate an expanding tumour fit the definition of a stem cells, provided that mutations are introduced to account for the aberrant growth and invasiveness characteristics.
Especially important for the possible therapeutic implications, is the stem cells inherent resistance to metabolize potentially toxic drugs that, if on one hand may help the stem cell to escape environmentally inflicted airway injuries, on the other hand may help the tumour cell survive chemotherapeutic drugs [130, 131].
While the relevance of CSC in tumours of the haematopoietic system has been shown, similar evidence for lung cancer is still missing. However, animal models of pulmonary stem cells biology have shown that different niches [132], harbouring different stem cells populations do exist throughout the conducting airways (the tracheobronchial and bronchiolar airways) and the gas-exchanging spaces (alveoli) of the lungs. Those are localized to the proximal airway submucosal glands, intercartilagineous rings, neuroepithelial bodies, terminal bronchioles and bronchoalveolar duct junctions [133].
The phenotypically distinct tumour subclasses collectively referred to as ‘lung cancer’, follow a proximal-to-distal distribution. Moving distally from the trachea, major tumour types are represented by SCC, SCLC and adenocarcinomas/bronchoalveolar carcinomas. Most originating sites of SCC, SCLC and adenocarcinomas appear to coincide with recently identified stem cell niches [133], strongly suggesting that tumour growth is supported by different cell types activated by permissive oncogenic mutations or epigenetic TSGs inactivation, in different local microenvironments.
Due to slow airways cellular turnover, methods for in vivo analysis of stem cells have required prior injury to the lung. Methods developed for the characterization of adult airway stem cells include localization of label-retaining cells, retroviral tagging of epithelial cells seeded into xenografts, air-liquid interface cultures to track clonal proliferative potential and multiple transgenic mouse models [134]. Ex vivo and in vitro models have used genetic markers to track lineage relationships, together with reconstitution systems that mimic airway biology.
Table 4 summarizes data regarding different lung cancers from the perspective of the stem cell niches putatively involved in their onset [see references in Table 4 and 135].
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Recently, it was shown that an epigenetic stem cell signature is preserved in cancer [136], where promoter hypermethylation studies were suggestive of a crosstalk between embryonic stem (ES) cell-specific transcription repressors and de novo DNA methyltransferases in an early cancer precursor cell with repressor-targets similar to those of ES cells. These results provide a mechanistic basis for predisposition of certain promoter CpG islands to cancer-associated DNA hypermethylation, imply that the first predisposing steps towards malignancy may occur very early in the cell lineage, and are consistent with reports of field changes in normal tissue surrounding malignant lesions [137]. The crosstalk between a specific subset of repressors and DNA methyltransferases would halt the proper differentiation of a given stem or progenitor cell and could predispose to further malignant development, explaining the diversity of DNA hypermethylation targets observed in different types of cancer [136].
All the previous observations are making clear that a whole new strategy of therapeutic interventions must be orchestrated because therapies that target the main tumour mass but spare the stem cells are bound to fail, leaving the patient at risk of cancer recurrence.
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GLOBAL GENE PROFILING IN LUNG CANCER |
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On a general basis, the great clinical success of small-molecule inhibitors of tyrosine kinases [138], reinforced by the observation that the ‘addiction’ of a cancer cell to the relevant mutated oncogene creates a therapeutic window of access to the given tumour type [139], has created a paradigm that represent now the basis for cancer genomics projects: identify the cancer-specific genetic lesion and find an agent active against it [140]. These projects have two main possible outcomes: first, they allow a subcategorization of cancers, correlating gene expression patterns with prognosis and suggesting therapeutic tools [141, 142]. Second, they may provide a molecular portrait of individual tumours, ideally helping clinicians in tailoring the best therapy to fit individual patients’ needs.
Global gene expression profiling has succeeded in revealing reproducible human lung adenocarcinoma subtypes in multiple patient cohorts, which differ significantly in important clinical behaviors such as stage-specific survival and metastatic patterns [143]. Moreover, specific subsets of cancer genes (‘signatures’) with strong prognostic value have already been correlated positively with lung cancer subtypes [144].
However, two very recent studies have also shown that indeed the complexity of cancer genomes exceeds expectations.
A large-scale resequencing study, in which the coding exons of 518 protein kinase genes were analyzed in 210 diverse human cancers, has shown that patterns of somatic changes in cancer are highly variable [145]. This study discovered a larger than anticipated number of cancer genes and highlighted the evolutionary diversity of cancers. Dealing with this great variability, the authors classified the somatic mutations identified as ‘driver’ (i.e. primarily responsible of oncogenesis) and ‘passenger’ (i.e. simply accumulated in the cancer cell, without providing any growth benefit).
High-throughput, mass-spectrometric genotyping of 238 known oncogene mutations in 1000 samples of 17 different tumour types, besides confirming known data, also allowed the discovery of previously unrecognized oncogene mutations and found an unexpectedly high number of co-occurring mutations. These results suggest that alterations in multiple oncogenic pathways may elicit complementary rather than redundant effects on tumourigenesis [146], and imply that a cost-effective, high-throughput screening may discover rare but potentially useful drug targets.
In a broader perspective, cancer genes (CAN) could be categorized as CCG, with high mutation rate (about 100%) in relevant cancers, and cancer-promoting genes (CPG), with lower mutation rate (about 5-10%) but able to drive tumour survival, invasion and metastasis. Specific subsets of CPGs are relevant to specific cancer types.
Finally, it is worth noting that also microRNA, a class of small non-coding RNA genes representing a new class of genes involved in human tumourigenesis [147], have been profiled in lung cancer patients. MicroRNA expression was found extensively altered in 104 pairs of primary lung cancers and matched normal lung tissue [148]. Furthermore, patients’ survival rate was found to correlate with microRNA molecular signatures of lung adenocarcinoma subtypes [148].
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Acknowledgements |
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The author thanks Dr Michael I. Lerman for critical reading of the manuscript.
The author is presently supported by the Italian Ministry of Education and Research (Ministero dell’Universita’ e della Ricerca).
Hunc terrorem animi tenebrasque necessest non radii solis neque lucida tela diei discutiant, sed naturae species ratiosque. Lucretius - De Rerum Natura 2.59-61
Now it is the time to dissipate terror and darkness not by sunbeams or the bright darts of daylight, but by virtue of knowledge and understanding of nature.
Key Points
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FOOTNOTES |
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Debora Angeloni, during her post-doctoral fellowship at the Laboratory of Immunobiology, NCI-NIH, participated in the effort of The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium. She is now Assistant Professor of Molecular Biology at the Scuola Superiore Sant’Anna, Pisa, Italy.
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