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On the elucidation of a tumour suppressor role of 3p in lung cancer Elst, Arja ter

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Download date: 27-09-2021 Chapter 1

Candidate lung tumour suppressor regions at the short arm of chromosome 3. What evidence is there?

Arja ter Elst Charles H.C.M. Buys

Department of Medical Genetics, University Medical Center Groningen, Groningen, The Netherlands LUNG CANCER AND THE SHORT ARM OF CHROMOSOME 3

Lung cancer is the leading cause of cancer death among both men and women in the western world. Consistent chromosomal aberrations occurring in lung tumours may provide a clue to the somatic genetic events leading to tumour development. Deletions of the short arm of chromosome 3 are a most common abnormality in lung cancer. They have been reported to occur in approximately 75% of non small cell lung cancer (NSCLC) tumours and in up to 100% of small cell lung cancer (SCLC) tumours (reviewed in Kok et al., 1997; Zabarovsky et al., 2002). Such deletions have also been found in the histological normal tissue surrounding tumours and in preneoplastic and preinvasive lesions (reveiwed in Kok et al., 1997). In addition, 3p deletions have been found in histologically normal tissue of about 50% of smokers and former smokers, not in control individuals (Wistuba et al., 1997). This suggests that losses at the short arm of chromosome 3 represent an early chromosomal change in the development of lung tumours. Moreover, introduction by microcell- mediated chromosome transfer of a normal human chromosome 3 into a lung adenocarcinoma cell line, A549, resulted in a suppression of growth of the transfected cell line in nude mice compared to the growth of the parental cell line (Satoh et al., 1993). The region of interest could be confined by the discovery of overlapping homozygous deletions in three different SCLC cell lines, NCI-H740, GLC20 and NCI-H1450, with a smallest overlap of 370 kb (Daly et al., 1993; Kok et al., 1994; Roche et al., 1996). When a cosmid contig was constructed for the 370 kb smallest region of overlap, a search for genes by cDNA library screening and CpG island identification revealed that the region was gene-rich (Wei et al., 1996). Sequencing of the whole region led by experimental and informatics methods to the identification of 19 genes (Lerman and Minna, 2000). These genes will be discussed later in this chapter. Chapter 1

LOSS OF SEGMENTS OF THE SHORT ARM OF CHROMOSOME 3 IN A VARIETY OF TUMOUR TYPES

Hemizygous and homozygous deletions of the short arm of chromosome 3 are also found in a multitude of other epithelial cancers, including renal cell carcinoma, head and neck carcinoma, nasopharyngeal carcinoma, malignant mesothelioma, and uterine cervix carcinoma (Kok et al., 1997). For lung cancer, three non-overlapping deletion regions were described 3p25, 3p21.3 and 3p12-p14 (Hibi et al., 1992). The number of distinct regions on the short arm of chromosome 3 that have been implicated in the development of tumours, has been expanded to seven in recent years. These include the regions indicated in the following as 3p22 AP20, 3p21.3 CER1 and CER2, 3p21 D3F15S2 region, 3p21.3 LUCA, 3p14 FHIT and 3p12 ROBO1 (Fig. 1).

The 3p22 AP20 region In small cell lung cancer (SCLC) homozygous deletions at the AP20 region have been reported to occur in the large majority of cases (Senchenko et al., 2004). The smallest region of overlap of homozygous deletions in this region was found in a breast cancer cell line and in a renal cancer cell line (RCC) and mapped between the markers D3S3623 and D3S1298 (Fig.1). This region contains four genes: APRG1, coding for AP20 region protein; ITGA9, coding for integrin alpha 9; CTDSPL, encoding CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase-like and VILL, coding for villin-like. Altered expression of CTDSPL was detected in a panel of epithelial cancer biopsies and cell lines (Kashuba et al., 2004). In addition, clones from a CTDSPL-transfected RCC cell line and a CTDSPL- transfected SCLC cell line showed an inhibition of tumour growth in nude mice in comparison to the growth of the non-transfected parental cell lines (Kashuba et al., 2004).

The 3p21.3 CER1 and CER2 regions SCID tumours caused by cell lines carrying a human chromosome 3 on a mouse fibrosarcoma background show non-random elimination of 3p21.3 sequences that are supposed to contain tumour suppressor genes (Kholodnyuk et al., 1997). Szeles et al. (1997) defined the genetic length of the eliminated region designated CER1, common elimination region 1, as 1.6 cM flanked by D3S1029 and D3S643. The

8 Introduction

Figure 1. A map of the short arm of chromosome 3 giving the position of the genes and markers located in the region corresponding with the deletion. physical size of CER1 was further restricted to approx. 1 Mb after the region was covered with a PAC contig (Yang et al., 1999). Seventeen genes are located in CER1 (Fig. 1), whose telomeric border is at D3S3582, while its centromeric border is in the first intron of LRRC2 (Kiss et al., 2002). One of these 17 genes, LTF, coding for lactoferrin, was tested for tumourigenicity by injecting SCID mice with clones from a mouse fibrosarcoma cell line transfected with a PAC containing the gene. The LTF

9 Chapter 1 promoter sequences appeared to become hypermethylated and expression of LTF was lost in derived tumours (Yang et al., 2003). Expression of LTF was also found absent in 18 of 37 SCLC cell lines, 11 of 43 NSCLC cell lines and 7 of 13 primary NSCLC tumours (Iijima et al., 2005). When a human nonpapillary renal cell carcinoma cell line was used as recipient of the human chromosome 3, CER1 appeared to become also eliminated on a human background. The elimination region is flanked by D3S3582 and CCR5, i.e. about 250 kb shorter than CER1 on a mouse background (Kholodnyuk et al., 2002). In addition, a second common elimination region was found, CER2, located between marker RH94338 and marker SHGC154057. In CER2 (Fig.1), seven genes have been identified, including two genes coding for chemokine receptors (Kholodnyuk et al., 2002). It may be noted that in CER1 seven of the 17 genes are encoding chemokine receptors.

The 3p21.3 D3F15S2 region UBE1L coding for ubiquitin-activating enzyme E1-like, was isolated from a region considered by Kok et al. (1987) as most consistently reduced to hemizygosity in SCLC, the D3F15S2 locus. The gene was picked up by hybridisation of a lung cDNA library with DNA from a human 3p21 fragment in a Chinese hamster-human hybrid (Carritt et al., 1992). UBE1L spans about 8.5 of genomic DNA. It has 26 exons and an open reading frame of 1009 nucleotides. The gene encodes a member of the E1 ubiquitin-activating enzyme family, which are involved in the modification of proteins with ubiquitin in order to target abnormal or short-lived proteins for degradation. The mRNA concentration of UBE1L in SCLC cell lines was found to be 0.5%-3% of that in normal lung tissue. No mutations or rearrangements of the remaining allele were, however, found in SCLC (Kok et al., 1993). UBE1L expression was found to be enhanced after treatment of an acute promyelocytic leukemia cell line with all-trans- retinoic acid (RA), which induces remission in acute promyelocytic leukemia. UBE1L might mediate degradation of the oncogenic PML/RA receptor of the t(15:17) rearrangement found in acute promyelocytic leukemia (Kitareewan et al., 2002). Treatment of immortalised human bronchial cells with RA also resulted in a higher expression of UBE1L and cotransfection of UBE1L with CCND1 in these cells resulted in a repression of CCND1 in a UBE1L dosage dependent manner (Pitha- Rowe et al., 2004). Since overexpression of Cyclin D1 is frequently observed in tumours and may contribute to tumourigenesis, this is an interesting observation.

10 Introduction

The 3p21.3 LUCA region As already discussed this region contains 19 genes (Fig.1). Further homozygous deletions of 3p21.3 were found by FISH in three uncultured lung squamous cell carcinoma tumours, at marker D3S2968 (Todd et al., 1997) and by real-time PCR in four uncultured RCC tumours and four uncultured breast cancer tumours at marker D3S3874 (Senchenko et al., 2004). A mouse fibrosarcoma cell line, A9, containing 2 Mb of the human chromosome 3 which included the 370-kb critical region, showed reduction of tumour growth in a tumourigenicity test (Killary et al., 1992).

Figure 2. A map of the LUCA region giving the positions of the genes located in the region. The map at the right is a magnification of part of the map.

11 Chapter 1

A similar effect was observed for A9 cells containing a P1-phage with 80 kb from the 370-kb critical region, but not for A9 cells containing P1 phages with the flanking DNA sequences (Todd et al., 1996).

Loss of 3p14.2, FHIT in lung cancer Homozygous deletions of 3p14.2 were frequently detected in several cancer cell lines including renal cancer cell lines and lung cancer cell lines (Lisitsyn et al., 1995). Using exon amplification from cosmids covering the deleted region allowed identification of the human FHIT gene, a member of the histidine triad gene family (Ohta et al., 1996). In approximately 50% of esophageal, stomach, and colon carcinomas an abberrant transcript of FHIT was detected. To determine the role of FHIT in lung cancer, cDNA of 59 primary lung tumours was sequenced. Aberrant transcripts were found in 80% of SCLC and 40% of NSCLC. In addition, loss of FHIT alleles was found in 76% of lung tumours (Sozzi et al., 1996). Since then a number of papers have described aberrant transcripts or loss of FHIT alleles in SCLC and NSCLC (Sozzi et al., 1998; Tseng et al., 1999; Zochbauer-Muller et al., 2000; Ho et al., 2002). FHIT promoter hypermethylation was detected in 36% of 120 primary NSCLC tumours (Tomizawa et al., 2004). Presence of abnormal transcripts, in terms of frequency and variety, is, however, not cancer-specific, since abnormal transcripts have also been found in normal lung tissue with the same frequency and variety (Tokuchi et al., 1999). In addition, the normal transcript of FHIT appeared to occur in renal cell cancer- and lung cancer-derived cell lines, including a cell line with a homozygous deletion in the FRA3B region (van den Berg et al., 1997). Nevertheless, a strong anti-tumourigenic effect of FHIT-transfected NSCLC cell lines has been reported after injection into nude mice (Roz et al., 2002). In addition, a significant suppression of both primary and metastatic lung tumour growth in nude mice was observed when treating the tumours with a DOTAP-FHIT complex (Ramesh et al., 2001).

Loss of 3p12, ROBO1 A sub-microscopic deletion was found in the SCLC cell line U2020 (Rabbitts et al., 1990). This region was further characterised by (Latif et al., 1992) and was estimated to be in the range of 4-7 MB. By cDNA library screening a gene called DUTT1 (Deleted in U Twenty Twenty) was isolated from this homozygous deletion. (Sundaresan et al., 1998). The same gene was independently isolated, ROBO1, as

12 Introduction the human homologue of the Drosophila gene Roundabout (Kidd et al., 1998). Cytogenetic analysis of the homozygous deletion revealed that genetic loss had occurred by complex rearrangements rather than a simple interstitial deletion of chromosome 3 (Heppell-Parton et al., 1999). Mutations and promoter hypermethylation of ROBO1 appeared to be rare in NSCLC and SCLC primary tumours (Dallol et al., 2002). Mice homozygous for a deleted form of ROBO1, frequently die at birth due to respiratory failure because of delayed lung maturation (Xian et al., 2001), whereas heterozygous mice develop lymphomas and carcinomas in their second year of life with a 3-fold increase in incidence compared with controls (Xian et al., 2004). In addition to the large homozygous deletion found in the SCLC cell line U2020, a much smaller 3p12 homozygous deletion was found in the SCLC cell line GLC20 (Angeloni et al., this thesis), as a second deletion next to a homozygous deletion in the LUCA region. By means of fiber-fluorescent in situ hybridisation experiments by P1-clones from the region, the length of deletion was found to be approximately 110 to 130 kb. This 3p12 homozygously deleted region of GLC20 affects exon 2 of ROBO1, causing the loss of amino acids 19-128 from the encoded protein. Two novel transcripts located in the second intron of ROBO1 were discovered in this homozygous deletion. Based on their characteristics both transcripts do not seem to encode proteins, but represent non-coding RNAs. Possible miRNA target sites were discovered in the sequence of these RNAs. In addition, a computational identified miRNA (cand893 HS3 78768573-78768661 R) is located in the 3p12 homozygously deleted region of GLC20. Target genes for this miRNA have not yet been discovered.

Multiple regions on the short arm of chromosome 3 have been found deleted. This might indicate that deletions or mutations of a combination of genes from several of these regions are responsible for the development of lung cancer. In this review we focus on genes or regulatory sequences in the 3p21.3 critical region (LUCA region).

GENES IN THE 3P21.3 CRITICAL REGION.

RBM6 RBM6 codes for RNA binding motif 6. Its cloning, structure, expression in normal tissue and function have been reviewed by Lerman and Minna et al., (2000) and Zabarovsky et al., (2002). More recently it was found that exon 5, which is excluded

13 Chapter 1 in a shorter transcript of RBM6, contains a RNP-1 RNA binding motif which might be important for tumour suppression (Sutherland et al., 2005). This doesnot seem in agreement with the results of Timmer et al. (1999), who found that in SCLC cell lines the transcript with exon 5 was higher expressed than the shorter transcript, whereas in normal tissue the expression of both the shorter and the longer transcripts had similar levels. That would attribute a tumour suppressor function to the shorter product lacking the RNP-1 RNA binding motif. Since RBM6 is well expressed in lung cancer cell lines and no mutations have been found RMB6 is not a likely tumour suppressor candidate.

RBM5 RBM5, codes for RNA binding motif 5. Its cloning, structure, expression in normal tissue and function have been reviewed by Lerman and Minna et al. (2000) and Zabarovsky et al. (2002). Although RBM5 appeared to be well expressed in lung cancer cell lines, more recently Oh et al. (2002) reported a lower expression of RMB5 in primary NSCLC tumours than in adjacent normal tissue in 9 out of 11 (82%) of the analysed cases. RBM5 has an alternative splice variant, which lacks exon 6 and results, like the alternative splice variant of RMB6, in a truncated protein. This shorter variant appeared to be widely expressed at a low level in normal tissue, but is expressed at increased levels in T-leukaemic cell lines (Mourtada-Maarabouni et al., 2003). Overexpression of this variant resulted in inhibition of CD95-mediated apoptosis, whereas overexpression of the full-length form suppressed cell proliferation by inducing apoptosis and by extending the G1 phase of the cell cycle. RBM5 has been tested for tumour suppressor activity in several functional assays. Overexpression of the gene in two breast cancer cell lines (MCF-7 and HBL-100) and one human fibrosarcoma cell line (HT1080) resulted in suppression of colony formation (Edamatsu et al., 2000; Oh et al., 2002). Overexpression of RBM5 in a mouse fibrosarcoma cell line (A9), resulted in a significant suppression of tumourigenicity of the cells when injected into nude mice (Oh et al., 2002). Overexpression of RBM5 in Jurkat T lymphoblastic leukaemia cells rendered the cells more susceptible to the death-inducing ligand TRIAL in TNF-α and FAS- mediated apoptosis (Rintala-Maki and Sutherland, 2004). Also for the breast cancer cell line MCF-7, a positive correlation was found for RBM5 overexpression and TNF- α susceptibility (Rintala-Maki et al., 2004). So far, RBM5 has never been tested for a tumour suppressor function in lung cancer-derived cell lines.

14 Introduction

SEMA3F SEMA3F codes for Semaphorin 3F. Its cloning, structure, expression in normal tissue and function have been reviewed by Lerman and Minna et al. (2000) and Zabarovsky et al. (2002). Apart from their role in the guidance of nerve growth cone migration, semaphorins may play a role in the cardiovascular system. It has been suggested that SEMA3F can inhibit angiogenesis in vitro by competition with VEGF165 for binding to the neuropilin-1 receptor (Miao et al., 1999; Kessler et al., 2004). It has also been suggested that SEMA3F and VEGF have opposing effects on the motility of primary tumour cells. By exposing two breast cancer cell lines to cultured supernatant of COS-7 cells transfected with SEMA3F, it was found that SEMA3F inhibits cell spreading and membrane ruffling, activities associated with increased cell migration and metastasis, whereas VEGF promotes these changes (Nasarre et al., 2003). For the same two breast cancer cell lines, it was demonstrated that SEMA3F inhibited migration of one of them, while in the other breast cancer cell line intercellular contacts were disrupted, and delocalisation of E- cadherin and beta-catenin took place (Nasarre et al., 2005). A SEMA3F transcript was detected in about 80% of the tested lung cancer cell lines. The typical membrane staining of the SEMA3F protein got, however, lost in 50% of the tested high-grade neuroendocrine tumours (see by Zabarovsky et al., (2002)). More recently, Lanteujoul et al., (2003) investigated the immunostaining of SEMA3F and VEGF in 50 preneoplastic lesions and 112 lung tumours. Immunostaining of SEMA3F got lost in 88% of preneoplastic lesions analysed. The degree of loss appeared to correlate with tumour stage, whereas VEGF staining increased with tumour grade. Since both SEMA3F and VEGF are deregulated in lung cancer, a common pathway might be involved in both breast cancer and lung cancer. Kusy et al. (2005b) characterised the SEMA3F promoter and tested 22 cancer cell lines, including 8 lung cancer cell lines, for promoter hypermethylation. This was found in all the lung cancer cell lines. In 15 primary lung tumours mostly partial methylation was observed. Treatment with demethylating agents had no effect on SEMA3F expression. Treatment with a histone deacetylate inhibitor stimulated SEMA3F expression. Contradicting results have been found with respect to a role of SEMA3F in tumour suppression. Mouse fibrosarcoma (A9) cells and ovarian adenocarcinoma cells transfected with SEMA3F showed a reduced tumour growth upon injection into nude mice, as well as an arrest in G2 in response to drugs such as taxol and

15 Chapter 1 adriamycin in vitro (Xiang et al., 2002). In addition, overexpression of SEMA3F in highly metastatic melanoma cells (A375SM) blocked metastases after injection into nude mice (Bielenberg et al., 2004). In contrast, however, SEMA3F transfected into NCI-H1299 or GLC45 lung cancer cell lines did not affect cell growth in vitro or tumour growth in vivo (Tomizawa et al., 2001; Xiang et al., 2002). When transfected in a squamous carcinoma cell line (NCI-H157), SEMA3F inhibited tumour growth after the transfected cells were transferred into the of nude rats. When SEMA3F was transfected into a large cell carcinoma cell line (NCI-H460), the cells readily formed tumours in the trachea of in nude rats (Kusy et al., 2005a). The ability of SEMA3F to reduce tumour growth seems thus to be cell line-dependent. Altogether a role of SEMA3F in lung cancer development is not yet clear.

GNAT1 GNAT1 codes for a guanine nucleotide binding protein (G protein) alpha transducing activity polypeptide. Its cloning, structure and what was known about its expression and function has been reviewed by Lerman and Minna et al. (2000). Since GNAT1 is not expressed in normal lung tissue (not in lung cancer cell lines) and no mutations have been found, GNAT1 is not a likely tumour suppressor candidate.

SLC38A3 SLC38A3 encodes solute carrier family 38, member 3. Its cloning, structure and what was known about its expression and function has been reviewed by Lerman and Minna et al. (2000). Since SLC38A3 is well expressed in normal lung tissue and lung cancer cell lines and no mutations have been found, SLC38A3 is not a likely tumour suppressor candidate.

GNAI2 GNAI2 encodes guanine nucleotide binding protein (G protein) alpha inhibiting activity polypeptide 2. Its cloning, structure and what was known about its expression in normal tissue and function has been reviewed by Lerman and Minna et al. (2000) and Zabarovsky et al. (2002). Two GNAI2 activating mutations have been found in human endocrine tumours (Lyons et al., 1990). Transfection of the activated form of GNAI2, also known as Gip2, into rat fibroblast (Rat-1A) cells induced oncogenic transformation, but this was not the case in mouse embryonic fibroblast (NIH3T3) cells (Pace et al., 1991). In contrast, Hermouet et al., (1991) found that transfection

16 Introduction of an activated mutant of GNAI2 in NIH 3T3 cells resulted in a reduced doubling time, a decreased serum requirement and a somewhat anchorage-independent growth proliferation. Transfection of an inactivating mutant of GNAI2 slowed down the growth of NIH3T3 cells. Nude mice injected with murine melanoma (K-1735) cells expressing this inactivating mutant showed a delayed tumour formation (Hermouet et al., 1996). Mice deficient for GNAI2, however, developed adenocarcinoma of the colon (Rudolph et al., 1995). Since GNAI2 is well expressed in lung cancer cell lines and no mutations have been found in lung cancer cell lines, it is an unlikely tumour suppressor candidate for lung cancer. The activating mutations of GNAI2 found in endocrine tumours and the induced growth after transfection of this constitutively active GNAI2 rather classify it as a possible proto-oncogene.

SEMA3B SEMA3B codes for semaphorin 3B. Its cloning, structure and what was known about its expression and function has been reviewed by Lerman and Minna et al. (2000) and Zabarovsky et al. (2002). SEMA3B is the second semaphorin coding gene in the 3p21.3 critical region. Like for SEMA3F, its receptors include neuropilin 1 and 2 (Npn-1 and Npn-2) and plexins (Tamagnone et al., 1999; Takahashi et al., 1999; Rohm et al., 2000). Apart from its role in axon guidance, SEMA3B has been suggested to be a direct transcriptional target of p53. SEMA3B might be involved in p53-dependent suppression of cell growth (Ochi et al., 2002). The observed loss of expression in lung cancer cell lines and primary tumours as compared to normal lung tissue might be caused by hypermethylation of the SEMA3B promoter. Although in some reports a correlation was found between loss of expression, loss of heterozygosity in 3p21.3 and promoter hypermethylation of SEMA3B (Tomizawa et al., 2001; Kuroki et al., 2003), this could not be confirmed in a recent study of 138 primary non small cell lung tumours (Ito et al., 2005). In addition, in a study of 64 bronchial aspirates promoter hypermethylation of SEMA3B was found in bronchial aspirates of both tumour cases and non-tumour cases (Grote et al., 2005), suggesting non-tumour specific promoter hypermethylation of SEMA3B. A tumour suppressor function of SEMA3B has been claimed based upon results obtained in several cell lines. Wildtype SEMA3B transfected into the lung cancer cell line NCI-H1299 induced growth inhibition and apoptosis in vitro (Tomizawa et al., 2001; Castro-Rivera et al., 2004). This pro-apoptotic effect was significantly decreased by vascular endothelial growth factor (VEGF165), competing with SEMA3B

17 Chapter 1 for their common receptor neuropilin (Castro-Rivera et al., 2004). In addition, overexpression of SEMA3B in the ovarian adenocarcinoma cell line HEY exhibited a diminished tumourigenicity upon injection into BALB/c nu/nu mice (Tse et al., 2002). Homozygous SEMA3B null mice of 12 months of age did not show any pathological abnormalities after histological analysis of several tissues including lung (van der Weyden et al., 2005). This might suggest a level of redundancy between class 3 semaphorins. In lung cancer, the common heterozygous loss of a large part of 3p will eliminate one copy of both SEMA3F and SEMA3B. Occurrence of mutations in the second copy is, however, rare for both genes.

IFRD2 IFRD2 codes for an interferon-related developmental regulator 2. Its cloning, structure and what was known about its expression and function has been reviewed by Lerman and Minna et al. (2000). The relatively low expression of IFRD2 in normal lung tissue as well as in lung cancer cell lines and the lack of mutations make it an unlikely tumour suppressor candidate for lung cancer.

NAT6 NAT6 codes for N-acetyltransferase 6. Its cloning, structure and what was known about its expression and function has been reviewed by Lerman and Minna et al. (2000). More recently, a highly complex organisation structure was found in humans and in mice for NAT6 together with HYAL1, HYAL3 and IFRD2. In mice, the second exon of nat6 is located within the first intron of hyal3. Ifrd2 was found immediately 3’ to hyal3. This dense organisation was accompanied by significant levels of cotranscription of hyal1, nat6 and hyal3, leading to bicistronic mRNA products for hyal1 with nat6 and hyal1 with hyal3 (Shuttleworth et al., 2002). NAT6 expression has never been determined in lung cancer. But only four missense mutations were detected in 78 lung cancer cell lines. A tumour suppression function for NAT6 in lung cancer is therefore not very likely.

Hyaluronidases Within the 3p21.3 critical region three different hyaluronidase genes HYAL1, HYAL2 and HYAL3, have been identified. Hyaluronidases are a family of enzymes crucial for the spread of bacterial infections, for toxins present in various venoms and possibly for cancer progression (Lokeshwar et al., 2002). Hyaluronidases degrade hyaluronic

18 Introduction acid (HA), which is present in body fluids, tissues and the extracellular matrix. It is synthesised by cells of mesenchymal origin in response to different stimuli (Li et al., 2000). HA keeps tissues hydrated and maintains osmotic balance and cartilage integrity (Tammi et al., 2002). It can also actively regulate cell adhesion, migration, and proliferation by interacting with specific cell surface receptors (Turley et al., 2002). Hyaluronan chains have size-specific biological activities. High molecular weight HA has anti-inflammatory, immunosuppressive (McBride and Bard, 1979; Delmage et al., 1986) and anti-angiogenic (Feinberg and Beebe, 1983) properties. HYAL2 digestion of hyaluronan results in lower molecular weight hyaluronan of 20 kDa which is a potent stimulator of inflammatory cytokines (Noble, 2002). Angiogenesis is also stimulated by these smaller fragments (Rooney et al., 1995; Trochon et al., 1997; Slevin et al., 1998). Dendritic cells are activated by 6-20 kDa sized hyaluronan oligomers (Termeer et al., 2000; Termeer et al., 2003) HYAL1 digestion of hyaluronan results in tetrasaccharides, which have an anti-apoptotic effect and suppress cell death in cell cultures undergoing hyperthermia or serum starvation (Xu et al., 2002). In tumour development, hyaluronan was found to expand upon hydration and opens up spaces for tumour cell migration (Lokeshwar et al., 2001). In concordance, hyaluronan was found upregulated in several cancers including breast cancer, colorectal cancer and lung cancer (Auvinen et al., 1997; Auvinen et al., 2000; Pirinen et al., 2001). Upregulation has been shown to correlate with grade and metastatic potential (Zhang et al., 1995; Jojovic et al., 2002). Normal connective tissue cells immediately adjacent to an invasive tumour could also be responsible for the production of tumour-associated hyaluronan (Knudson et al., 1989). For hyaluronidase expression contradictory data has been described. In most studies hyaluronidase expression was found elevated in tumour tissue (Wilkinson et al., 1996; Patel et al., 2002; Franzmann et al., 2003). In one study, however, hyaluronidase expression in tumour tissue was found to be reduced (Fiszer Szafarz and Szafarz, 1973).

HYAL3 HYAL3 codes for hyaluronidase 3. Its cloning, structure and what was known about its expression and function has been reviewed by Lerman and Minna et al. (2000). For HYAL3 no precise function is, however, known yet. The lack of expression of HYAL3 in lung tissue as well as in lung cancer cell lines make HYAL3 a less likely tumour suppressor candidate for lung cancer.

19 Chapter 1

HYAL1 HYAL1 was found using a partial sequence derived from a protein purified from serum to screen an EST database (Frost et al., 1997). The structure of HYAL1 and what was known about its expression and function has been reviewed by Lerman and Minna et al. (2000) and Zabarovsky et al. (2002). More recently, murine Hyal1 and Hyal2 overexpression was found to enhance the sensitivity of murine L929 fibroblasts to tumour necrosis factor mediated cell death (Chang, 2002), implicating HYAL1 and HYAL2 in the TNF pathway. Although Lerman and Minna et al. (2000) found no expression of HYAL1 in 18 out of 20 lung cancer cell lines tested, including 10 SCLC cell lines, Junker et al., (2003) found HYAL1 expression in all twenty SCLC cell lines tested. Hyaluronidase activity could, however, not be detected in these cell lines, possibly due to aberrant splicing of the pre-mRNA, described earlier for head and neck squamous cell carcinomas (see review by Zabarovsky et al. (2002)). HYAL1 seems to have both a tumour-suppressive and a tumour-inducing effect. There are several reports on a tumour-suppressive effect in functional tests. When transfected into a subclone of the colon cancer cell line DHD-K12, HYAL1 showed a significant growth reduction upon injection in BD-IX rats in comparison with the mock transfected cell line (Jacobson et al., 2002). In addition, hyaluronidase administration to SCID mice bearing human breast tumour xenografts, caused eradication of hyaluronan and rapid reduction in tumour size (Shuster et al., 2002). Earlier, hyaluronidase was found to act as an anticarcinogenic agent in BALB/C mice (Pawlowski et al., 1979). Other authors have reported, however, a tumour-inducing effect of HYAL1. Expression of hyaluronidase by tumour cells was found to induce angiogenesis in vivo (Liu et al., 1996). More recently, overexpression of HYAL1 was shown to enhance the metastatic behaviour of a prostate cell line (Patel et al., 2002). HYAL1 secretion has been suggested to correlate with prostate cancer progression (Lokeshwar et al., 2001). Blocking of HYAL1 in a bladder cancer cell line resulted in a reduced tumour growth in nude mice and a reduced infiltration in skeletal muscle was observed compared with the bladder cancer cells transfected with vector only (Lokeshwar et al., 2005b). No effect was seen in NSCLC xenografts after intratumoural injection of recombinant adenoviral vectors containing HYAL1 or HYAL2 (Ji et al., 2002). This has so far the only experiment in which HYAL1 has been tested for a role in lung cancer. The contradicting findings about the role of HYAL1 in cancer as resulting from these experiments were recently explained in a study of Lokeshwar et al. (2005a) who describe that HYAL1 can function as either a

20 Introduction tumour suppressor or a tumour promoter depending on the concentration of the gene product.

HYAL2 HYAL2 codes for hyaluronidase 2. Its cloning, structure and what was known about its expression has been reviewed by Lerman and Minna et al. (2000). Hyaluronidase 2 is a glucosylphosphatidylinositol (GPI) anchored cell-surface protein (Rai et al., 2001), with lysosomal hyaluronidase activity, degrading high molecular mass hyaluronan (8000 kDa) to products of about 20 kDa (Lepperdinger et al., 1998) (see review by Zabarovsky et al. (2002)). More recently, (2005) it was found that digestion of hyaluronan with increasing concentrations of soluble HYAL2 at the optimal pH of 5.5 resulted in increasing degradation of the 20 kDa intermediate. In SV40- immortalised human bronchial cells HYAL2 is associated with the MST1 receptor, thereby negatively regulating MST1R signalling (Fig. 3). Hyal2 was shown to be a virus entry receptor of Jaagsiekte sheep retrovirus (JSRV), a virus which causes cancer of the lower airways and alveoli in sheep (Rai et al., 2001). It mediates the entry of the highly oncogenic retrovirus JSRV into the cell (see review by Zabarovsky et al. (2002)). Hyal2 has also been shown to be the receptor for enzootic nasal tumour virus, which induces nasal epithelial cancer in sheep (Dirks et al., 2002). The retrovirus binds to HYAL2, which is consequently degraded. The release of HYAL3 from MSTR1 activates the MSTR1 pathway and leads to constitutive activation of AKT and MAPK proteins and thereby to proliferation and promotion of cell survival (Danilkovitch-Miagkova et al., 2003). As for HYAL1, contradicting findings have also been published for a role of HYAL2 in tumour suppression. HYAL2 is well expressed in all lung cancer cell lines tested (Lerman and Minna, 2000). In non-Hodgkin lymphomas, however, the lowest level of HYAL2 expression was found in the most aggressive type of lymphoma, diffuse large cell lymphomas (Bertrand et al., 2005). Since the gene is well expressed in lung cancer cell lines and no mutations have been detected in 40 lung cancer cell lines tested, HYAL2 is not a likely tumour suppressor candidate for lung cancer.

21 Chapter 1

Figure 3. Model of JSRV-mediated transformation of human bronchial epithelial cells adapted from Danilkovitch-Miagkova et al. (2003). A: Cells expressing MST1R as an inactive dimer, association of HYAL2 with MST1R prevents MST1R activation. B: JSRV interacts with HYAL2 via the Env protein, which leads to viral entry. C: Intracellular degradation of HYAL2. D: Possible MST1R conformational changes may cause constitutive activation of oncogenic pathways.

TUSC2 TUSC2 codes for tumour suppressor candidate 2. Its cloning, structure and what was known about its expression and function has been reviewed by Lerman and Minna et al. (2000) and Zabarovsky et al. (2002). Although some mutations have been found in TUSC2 these did not seem to impair the expression of the gene. More recently it

22 Introduction was postulated, however, that deficient post-translational modification could have an effect on TUSC2 function. In human primary tumours and lung cancer cell lines a defect was observed in N-myristoylation of TUSC2 (Uno et al., 2004). N- myristoylation appears required for TUSC2 mediated tumour suppressor activity, since TUSC2 which is not myristoylated, is readily degraded. Some experimental evidence has been obtained supporting a tumour suppressor function of TUSC2. TUSC2 transfection into NSCLC cell lines NCI-H1299 or NCI-H322 resulted in a dramatically reduced colony formation compared to the mock transfected cell line (Kondo et al., 2001). Also the proliferation of several different NSCLC cell lines (A549, NCI-H1299, NCI-H358 and NCI-H460) transfected with TUSC2 was significantly reduced. Growth inhibition was also seen in vivo after intratumoural injection of human xenografts with a TUSC2 adenoviral vector construct, as was a significant inhibition of the development of A549 pulmonary metastases after intravenous injection of the same construct (Ji et al., 2002). Growth inhibition and inhibition of pulmonary metastases could also be established by intratumoural administration of a TUSC2 liposomal gene complex or intravenous injection of this complex, respectively (Ito et al., 2004).

RASSF1 RASSF1 codes for ras-association domain family 1 protein. Its cloning, structure and what was known about its expression has been reviewed by Lerman and Minna et al. (2000) and Zabarovsky et al. (2002). RASSF1 function has been extensively studied and many proteins have been suggested to interact with the protein product of the major transcript, RASSF1A. This protein product was suggested to block the cell cycle at the level of G1/S-phase transition by negatively regulating the accumulation of cyclin D1 (Shivakumar et al., 2002). More recently, it was suggested that RASSF1A blocking of cyclin D1 accumulation was mediated through suppression of the c-jun-NH2-Kinase (JNK) pathway (Whang et al., 2005). RASSF1A was also suggested to interact with E4F1 (Fenton et al., 2004), an E1A-regulated transcription factor which is associated with a number of cell cycle regulating proteins including cyclin A, cyclin E and cyclin B (Fajas et al., 2000; Sandy et al., 2000). Overexpression of RASSF1A together with E4F1 resulted in a greater increase of cells in the G1-phase than overexpression of RASSF1A alone (Fenton et al., 2004). The binding of RASSF1A to E4F1 has been suggested to enhance the inhibitory effect of E4F1 on Cyclin A2 expression (Ahmed-Choudhury et al., 2005). RASSF1A

23 Chapter 1 has also been suggested to interact with several different microtubulus associated proteins including MAP1B and its close homologue VCY2IP1 (Dallol et al., 2004). Furthermore, RASSF1A seems to colocalise with the microtubuli and to stabilise the microtubuli, which at mitosis results in metaphase arrest (Liu et al., 2002; Liu et al., 2003a). Stabilisation of microtubuli by RASSF1A was found to be mediated through the inhibitory effect of RASSF1A on the anaphase-promoting complex (APC). RASSF1A binds to Cdc20, thereby inhibiting binding of Cdc20 to APC, which leaves APC inactive and the cells arrested in promethapase (Song et al., 2004; Castro et al., 2005). In addition, RASSF1A cotransfected with activated K-RAS blocks the ability of oncogenic K-RAS to promote genomic instability (Vos et al., 2004). RASSF1A is therefore suggested to be an inhibitor of DNA synthesis, mitosis and cytokinesis. RASSF1A has also been suggested to heterodimerise with RASSF5 and thereby to bind indirectly to RAS-GTP (Khokhlatchev et al., 2002; Ortiz-Vega et al., 2002). In addition, it is proposed that RASSF5 and RASSF1A keep STK4 in an inhibited state, but have the ability to direct STK4 to specific cellular sites and/or co- localise it with upstream activators and/or substrates, thereby augmenting STK4 apoptosis (Praskova et al., 2004). Furthermore, RASSF1A has been suggested to associate with connector enhancer of kinase suppressor of Ras 1 (CNKSR1), a multidomain scaffold protein discovered in Drosophila, in which it is necessary for RAS activation of Raf kinase. Rabizadeh et al. (2004) have shown that coexpression of CNKSR1 with RASSF1A greatly enhances CNKSR1-induced apoptosis, probably mediated by recruitment of STK4, which had already been shown to be associated with RASSF1A. A physical interaction was also found between RASSF1A and PMCA4b which encodes a plasma membrane calmodulin-dependent calcium ATPase. Coexpression of RASSF1A and PMCA4b was shown to inhibit the EGF- dependent activation of the ERK pathway (Armesilla et al., 2004). One of the other RASSF1 transcripts, RASSF1C, has been suggested to interact with IGFBP-5, coding for insulin-like growth factor binding protein 5. More recently, IGFBP-5 was suggested to be able to stimulate cell proliferation mediated by activation of the p38 MAP kinase and extracellular signal-regulated kinase (ERK)-1/2 pathways. SiRNA- mediated knockdown of RASSF1C was found to block the IGFBP-5-induced ERK1/2 phosphorylation, thereby blocking cell proliferation (Amaar et al., 2005). Recently, a model was proposed where RASSF1A and MOAP1 associate upon TNFα stimulation. Subsequently the complex promotes a conformational change of BAX, which mediates insertion of BAX into the mitochondrial membrane and ultimately

24 Introduction cytochrome c release and apoptosis (Baksh et al., 2005). Figure 4 summarises the interactions and pathways of RASSF1 transcripts. The expression of RASSF1A is reduced in several cancers, including SCLC (Dammann et al., 2000), NSCLC and breast cancer (Burbee et al., 2001), gastric cancer (Byun et al., 2001), bladder cancer (Lee et al., 2001), thyroid cancer (Schagdarsurengin et al., 2002) and osteosarcoma (Lim et al., 2003). Only two confirmed somatic mutations have been found in over 200 samples of different tumour tissues, including SCLC and breast cancer (Dammann et al., 2003). In contrast, a high mutation rate, 17/23 (74%), was found for RASSF1A in primary nasopharyngeal carcinomas by the PCR-cloning-sequencing strategy (Pan et al., 2005). A rare polymorphism was found in codon 133 of exon 3 (alanine-serine) in the germline of two cervical cancers, one nasopharyngeal cancer and in lung and breast cancer cell lines (Yu et al., 2003). This polymorphism was found in 21% of patients with breast carcinoma and 24% of patients with fibroadenoma, whereas it was only found in 3% of the controls (Schagdarsurengin et al., 2005). An alternative mechanism for loss of expression was found for RASSF1A. The promoter of RASSF1A, but not of RASSF1C, is frequently hypermethylated in several cancers including lung cancer, breast cancer and renal cell cancer (Burbee et al., 2001; Dammann et al., 2001; Yoon et al., 2001). Table 1 summarises the methylation profiles found for tumours tested for promoter hypermethylation. A tumour suppressor function of RASSF1A has been tested in vitro in lung cancer cell lines, kidney cell lines and prostate cell lines. RASSF1A reinsertion led to reduced colony formation and/or anchorage-independent growth in soft agar (Dammann et al., 2000; Burbee et al., 2001; Dreijerink et al., 2001; Kuzmin et al., 2002; Chow et al., 2004; Li et al., 2004b). In addition, tumour suppressor activity has also been suggested by results obtained in several human lung cancer cell lines in vivo (Burbee et al., 2001; Li et al., 2004b). In a mouse RASSF1A knockout model, heterozygous RASSF1A+/- mice and homozygous RASSF1A-/- mice were significantly more tumour prone for spontaneous tumour formation as well as for chemical induction of tumours (Tommasi et al., 2005). In contrast, Suzuki et al. (2004) could not detect any growth effect in soft agar colony formation of NCI-H1299 (NSCLC) cells 14 days after DNA methyltransferase 1 (DNMT1) knockdown, although they showed that RASSF1A promoter methylation was reduced by 80% and RASSF1A expression was 60% increased.

25 Chapter 1

Figure 4. A summary of RASSF1 transcripts interactions and pathways adapted from Agathanggelou et al. (2005).

26 Introduction

Table 1. RASSF1A promoter hypermethylation in primary tumours

Tumour type primary tumours noncancerous reference tissue Small cell lung cancer 79% 22(28) (Dammann et al., 2001) 72% 21(29) (Agathanggelou et al., 2001) ~ 80% 34(43) (Toyooka et al., 2001) 50% 4(8) (Honorio et al., 2003) Non-small cell lung cancer 38% 22(58) (Dammann et al., 2000) 34% 14(41) (Agathanggelou et al., 2001) 30% 32(107) (Burbee et al., 2001) ~ 30% 34(115) (Toyooka et al., 2001) 32% 35(110) (Tomizawa et al., 2002) 32% 65(204 (Kim et al., 2003) 21% 5(21) (Honorio et al., 2003) 43% 32(75) (Yanagawa et al., 2003) Adenocarcinoma 47% 7(15) (Ramirez et al., 2003) 39% 49(125) (Kim et al., 2003) 55% 18(33) (Li et al., 2003) 39% 28(72) (Tomizawa et al., 2004) 38% 80(209) (Divine et al., 2004) 50% 50(100) (Marsit et al., 2004) 31% 31(101) (Ito et al., 2005) 43% 13(30) (Ito et al., 2005) Stage I ac 32% 35(110) (Tomizawa et al., 2002) Squamous cell carcinoma 24% 6(25) (Ramirez et al., 2003) 30% 32(107) (Zochbauer-Muller et al., 2003) 30% 25(84) (Kim et al., 2003) 25% 5(20) (Li et al., 2003) 41% 51(124) (Maruyama et al., 2004) 13% 6(45) (Tomizawa et al., 2004) 31% 22(71) (Marsit et al., 2004) Large cell carcinoma 40% 4(10) (Ramirez et al., 2003) 25% 3(12) (Li et al., 2003) Lung cancer 60% 12(20) (Guo et al., 2004) Carcinoid cancer 50% 20(40) (Toyooka et al., 2001) Sputum from chronic 30% 4(13) (Honorio et al., 2003) smokers 4% 3(73) (Zochbauer-Muller et al., 2003) Sputum from former smokers 50% 1(2) (Honorio et al., 2003) Breast cancer 62% 28(45) (Dammann et al., 2001) 9% 4(44) (Agathanggelou et al., 2001) 49% 19(39) (Burbee et al., 2001) 56% 20(36) (Lehmann et al., 2002) 58% 56(97) (Chen et al., 2003) 81% 122(147) (Shinozaki et al., 2005) 95% 38(40) 93% 37(40) (Yeo et al., 2005) 68% 13(19) 7% 2(28) (Fackler et al., 2004) Invasive breast cancer (IDC) 65% 11(17) (Honorio et al., 2003) 70% 19(27) (Fackler et al., 2003) 64% 29(45) (Pu et al., 2003) Ductal carcinoma in situ 42% 5(12) (Honorio et al., 2003) (DCIS) 75% 33 (44) (Fackler et al., 2003)

27 Chapter 1

Table 1 continued

Tumour type primary tumours noncancerous Reference tissue In situ lobular carcinoma 62% 8(13) (Fackler et al., 2003) (LCIS) ILC 84% 16(19) (Fackler et al., 2003) benign 34% 12(36) (Pu et al., 2003) In situ 62% 13(21) (Pu et al., 2003) primary 23% 6(23) (Muller et al., 2003) recurrent 80% 8 (10) (Muller et al., 2003) 56% 14(25) (Mehrotra et al., 2004) Metastasis bone 78% 7(9) (Mehrotra et al., 2004) Metastasis brain 67% 4(6) (Mehrotra et al., 2004) Metastasis lung 100% 10(10) (Mehrotra et al., 2004) Ovarian cancer 10% 2(21) (Agathanggelou et al., 2001) 40% 8(20) (Yoon et al., 2001) 49% 20(41) (Toyooka et al., 2001) 50% 25(50) (Ibanez de Caceres et al., 2004) Invasive 30% 14(46) (Makarla et al., 2005) Not invasive 0% 0(92) 13% 2(16) (Makarla et al., 2005) Cervical cancer 0% 0(22) (Agathanggelou et al., 2001) 17.5% 9(51) (Cohen et al., 2003) 30% 11(33) SCC (Yu et al., 2003) 12% 2(17) AC (Yu et al., 2003) 10% 4(42) SCC (Kuzmin et al., 2003) 24% 8(34) AC (Kuzmin et al., 2003) 21% 4(19) ASC (Kuzmin et al., 2003) 12.2% 10 (82) 41.2% 7(17) (Kang et al., 2005) Malignant mesothelioma 32% 21(66) (Toyooka et al., 2002) 0% 0(1) (Seidel et al., 2004) phaeochromocytoma 22% 5(23) (Astuti et al., 2001) 50% 13(26) Renal cell carcinoma 56% 18(32) (Yoon et al., 2001) 91% 39(43) (Dreijerink et al., 2001) Papillary RCC 44% 12(27) (Morrissey et al., 2001) 70% 14(20) (Dulaimi et al., 2004) 100% 9(9) (Gonzalgo et al., 2004) Clear Cell 46% 23(50) (Dulaimi et al., 2004) 90% 9 (21) (Gonzalgo et al., 2004) Wilm’s tumour 0% 0(1) (Dulaimi et al., 2004) 88% 15(17) (Hoque et al., 2004) CC-RCC 23% 32(138) (Morrissey et al., 2001) Chromophobe 17% 1(6) (Dulaimi et al., 2004) oncocytoma 14% 1(7) (Dulaimi et al., 2004) Collecting duct 60% 3(5) (Dulaimi et al., 2004) RCC unclassified 20% 1(5) (Dulaimi et al., 2004) TCC renal pelvis 33% 2(6) (Dulaimi et al., 2004) oncocytoma 25% 2(8) (Gonzalgo et al., 2004) Nasopharyngeal cancer 67% 14(21) (Lo et al., 2001) 50% 8(16) (Tong et al., 2002) 67% 20(30) (Chang et al., 2003) Mouth and throat rinsing fluid 33% 10(30) (Chang et al., 2003) Nasopharyngeal swab 37% 11(30) (Chang et al., 2003) Head and neck cancer 8% 6(80) (Hasegawa et al., 2002)

28 Introduction

Table 1 continued

Tumour type primary tumours noncancerous Reference tissue 17% 4(24) (Hogg et al., 2002) 15% 7(46) (Dong et al., 2003) 10% 3 (32) 0% 0(32) (Maruya et al., 2004) Betel-associated oral carcinoma Squamous cell carcinoma 93% 25(27) (Tran et al., 2005) Verrucous carcinoma 100% 9(9) (Tran et al., 2005) Gastric cancer 43% 39(90) (Byun et al., 2001) 67% 14(21) (Kang et al., 2002) 4% 2(56) (Kang et al., 2002) 7,5% 6(80) (Kang et al., 2003) MSI-H ~ 3% 1(36) (Kim et al., 2003) MSI-S 0% (Kim et al., 2003) Gastric stromal 40% 15(38) (House et al., 2003) Chronic gastritis 0.4% 1(268) (Kang et al., 2003) Bladder cancer 60% 33(55) (Lee et al., 2001) 35% 34(98) (Maruyama et al., 2001) 48% 19(40) (Chan et al., 2003) Low grade transitional cell 47.5% 19(40) (Chan et al., 2003) carcinoma (TCC) Carcinoma in situ 0% 0(6) (Chan et al., 2003) 60% 76(127) 42% 16(37) (Friedrich et al., 2004) 32% 116(351) (Marsit et al., 2005) Biliary tract carcinomas 27% 10(37) (Tozawa et al., 2004) Prostate cancer 53% 54(101) (Maruyama et al., 2002) 100% 11(11) (Kuzmin et al., 2002) 71% 37(52) (Liu et al., 2002) 67% 59(90) (Woodson et al., 2004) 96% 70(73) (Yegnasubramanian et al., 2004) 83% 20(24) (Woodson et al., 2004) 78% 88 (113) 53% 19(36) (Florl et al., 2004) 99.2% 117(118) (Jeronimo et al., 2004) (high grade intraepithelial 100% 38(38) (Jeronimo et al., 2004) neoplasia 30% 3(10) (Woodson et al., 2004) (beging prostatic hyperplasia) 93.3 28(30) (Jeronimo et al., 2004) Colon cancer 12% 3(26) (Yoon et al., 2001) 20% 45(222) (van Engeland et al., 2002) 45% 13(29) (Wagner et al., 2002) High methyl donor intake 15% 9(61) (van Engeland et al., 2003) Low methyl donor intake 25% 15(61) Flat-type 81.3% 39(48) 49% 19(39) (Sakamoto et al., 2004) 3% 2(65) (Xu et al., 2004) Thyroid cancer 71% 27(38) (Schagdarsurengin et al., 2002) Follicular 75% 9(12) (Xing et al., 2004) Follicular thyroid carcinoma 100% 4(4) (Nakamura et al., 2005) Follicular adenoma 33% 1(3) (Nakamura et al., 2005) papillary 20% 6(30) (Xing et al., 2004) Papillary thyroid carcinoma 26% 11(34) 0% 0(27) (Nakamura et al., 2005) Benign adenomas 44% 4(9) (Xing et al., 2004)

29 Chapter 1

Table 1 continued

Tumour type primary tumours noncancerous Reference tissue Medullary thyroid carcinoma 40% 2(5) (Nakamura et al., 2005) Anaplastic thyroid carcinoma 33% 4(12) (Nakamura et al., 2005) Hyalinizing trabecular 25% 5(20) (Nakamura et al., 2005) Pituitary adenomas 38% 20(52) (Qian et al., 2005) Salivary adenoid cystic 42% 25(60) (Li et al., 2005) Pediatric cancer 40% 70(175) (Harada et al., 2002) 10 different 67% 16(24) 31% 4(13) (Wong et al., 2004) Neuroblastoma 55% 37(67) (Astuti et al., 2001) 70% 39(56) (Yang et al., 2004) Hodgkin’s lymphoma 65% 34(52) (Murray et al., 2004) Wilms tumour 73% 22(30) (Ehrlich et al., 2002) 54% 22(30) (Wagner et al., 2002) Esophageal squamous cell 52% 25(48) (Kuroki et al., 2003) carcinoma (slokdarm) 51% 24(47) (Kuroki et al., 2003) Malignant melanoma cutaneous 55% 24(55) (Spugnardi et al., 2003) 22% (Reifenberger et al., 2004) 36% 9(25) (Furuta et al., 2004) Cholangiocarcinoma 69% 9(13) (Wong et al., 2002) Extrahepatic 85% 28(33) (Chen et al., 2005) cholangiocarcinoma Hepatocellular carcinoma 85% 70(82) (Zhang et al., 2002) 93% 14(15) (Schagdarsurengin et al., 2003) 100% 29(29) 83% 24(29) (Yu et al., 2002) 95% 41(43) 70% 16(23) (Zhong et al., 2003) 67% 40(60) (Lee et al., 2003) Testicular germ cell 71% 17(24) (Honorio et al., 2003) tumours 0% 0(25) (Kawakami et al., 2003) Testicular malignant 100% 3(3) lymphomas Male germ cell tumours 21.7% 20(92) (Koul et al., 2002) 83 primary Differentiated nonseminoma 35.7% 25(70) (Koul et al., 2004) Primary brain tumour Medullablastoma 80% 22(27) (Lusher et al., 2002)

100% 5(5) 93% 41(44) (Lindsey et al., 2004) Glioblastoma multiforme 57% 12(21) (Balana et al., 2003) Serum 50% 13(26) (Balana et al., 2003) Gliomas (invasive, invariably 54% 25(46) (Horiguchi et al., 2003) fata intracerebral tumours) astrocytoma 69.8% 37(53) (Yu et al., 2004) ependymoma 85% 30(35) (Hamilton et al., 2005) Multiple myeloma 28% 9(32) (Ng et al., 2003) Muliple myeloma 15% 4(29) (Seidl et al., 2004) Monoclonal gammopathies 14% 17 (113) Pancreatic tumours

30 Introduction

Table 1 continued

Tumour type primary tumours noncancerous Reference tissue PEN 75% 36(48) (House et al., 2003) 83% 10(12) (Dammann et al., 2003) adenocarcinoma 64% 29(45) (Dammann et al., 2003) Soft tissue sarcomas (Seidel et al., 2004) Liposarcoma 18% 4(22) (Seidel et al., 2004) Leiomyosarcoma 39% 7(18) (Seidel et al., 2004) MFH 6% 1(18) (Seidel et al., 2004) Rhabdomyosarcoma 0% 0(6) (Seidel et al., 2004) Neurogenic sarcoma 50% 3(6) (Seidel et al., 2004) Synovial sarcoma 33% 2(6) (Seidel et al., 2004) Fibrosarcoma 0% 0(3) (Seidel et al., 2004) Malignant 0% 0(3) (Seidel et al., 2004) hemangiopericytoma

ZMYND10 ZMYND10, codes for zinc finger, MYND-type containing 10. Its cloning, structure and what was known about its expression and function has been reviewed by Lerman and Minna et al. (2000) and by Zabarovsky et al. (2002). The gene contains a Zinc finger MYND domain that is involved in specific protein-protein interactions (Lerman and Minna, 2000). More recently, Liu et al. (2003b) found that the domain may play a role in transcription regulation. The functional promoter of ZMYND10 was found to become activated by environmental stresses such as heat shock and was found to be regulated by E2F, a transcription factor involved in the control of the cell cycle (Qiu et al., 2004). Although loss of expression of ZMYND10 was detected in 70% of SCLC and NSCLC cell lines (Lerman and Minna, 2000) and in 78% of primary nasopharyngeal carcinomas (Liu et al., 2003b) only three missense mutations were found in 61 lung cancer cell lines (Lerman and Minna, 2000) and no pathogenic mutations were found in 45 primary nasopharyngeal tumours and five nasopharyngeal cell lines (Liu et al., 2003b). Since the second allele of ZMYND10 is rarely inactivated by mutations, hypermethylation of the promoter might be an alternative mechanism explaining the frequently found loss of expression. Methylation of the ZMYND10 promoter region CpG island was detected in 21/54 (39%) of lung cancer cell lines, 3/7 (42%) of breast cancer cell lines, 3/6 (50%) of kidney cancer cell lines, 6/7 (86%) of neuroblastoma cell lines and 4/5 (80%) nasopharyngeal carcinoma cell lines (Agathanggelou et al., 2003). In addition,

31 Chapter 1

ZMYND10 methylation was also detected in primary tumours: in SCLC 4/29 or 14% of cases (Agathanggelou et al., 2003), in NSCLC 26/145 or 19% (Agathanggelou et al., 2003) 41/138 or 30% (Ito et al., 2005) 68/160 or 43% (Marsit et al., 2005), in neuroblastomas 20/49 or 41% (Agathanggelou et al., 2003) and in nasopharyngeal carcinomas 17/23 or 74% (Liu et al., 2003b), 8/26 or 30.8 % (Chow et al., 2004) and in 19/29 or 66% (Qiu et al., 2004). Promoter hypermethylation of ZMYND10 seemed to be inversely correlated with smoking and occurred more often in adenocarcinoma than in squamous cell carcinoma (Marsit et al., 2005). The highest percentage of promoter hypermethylation was found in nasopharyngeal carcinomas, 74%. In addition, 7/29 (24%) of primary tumours of ZMYND10 were found homozygously deleted for ZMYND10, implying that in total up to 83% of the nasopharyngeal tumours showed some aberration of ZMYND10 (Qiu et al., 2004). ZMYND10 seemed to show tumour suppressor activity in vitro, since transfection of ZMYND10 resulted in a 49% and 75% reduced colony formation in an NSCLC cell line (NCI-H1299) and a neuroblastoma cell line (SK-N-SH), respectively (Agathanggelou et al., 2003). No tumour suppressor activity was found in vivo, since no significant growth inhibition was detected after intratumoural injection of an adenoviral vector containing ZMYND10 into a human NSCLC xenograft (Ji et al., 2002). Also after injection into a nude mice of a esophageal squamous cell carcinoma cell line (SLMT-1) transfected with ZMYND10 no reduction of tumour growth was found as compared to the parental cell line (Yi Lo et al., 2005).

TUSC4 TUSC4 codes for tumor suppressor candidate 4. Its cloning, structure and what was known about its expression has been reviewed by Lerman and Minna et al. (2000). The main spliceform of TUSC4 encodes a soluble protein with a bipartite nuclear localisation signal, a protein-binding domain, similarity to the MutS core domain and a newly identified nitrogen permease regulator 2 domain with unknown function (Li et al., 2004a). TUSC4 appeared to be expressed in lung cancer, although the 3’end of TUSC4 was found homozygously deleted in 6 of the 19 cancer cell lines of different origin, including SCLC cell lines (U2020 and A549), a non-SCLC cell line (H647) and renal cell carcinoma cell lines (HN4 and ACHN) (Li et al., 2004a). In five of the six cell lines the homozygous deletion included RASSF1. Promoter hypermethylation was not observed in an analysis of six nasopharyngeal carcinoma cell lines (Chow et al., 2004). There is evidence of tumour suppressor activity of this gene. After

32 Introduction transfection of NSCLC cells with TUSC4 in an adenoviral vector, in vitro cell proliferation was significantly reduced. Growth inhibition of TUSC4 was also seen in human xenografts after intratumoural injection (Ji et al., 2002). In addition, Li et al. (2004a) using a gene inactivation test with a tetracycline inducible vector system, found a reduced colony formation in vitro and a delayed growth rate in vivo of a TUSC4-transfected RCC cell line (KRC/Y) and two SCLC cell lines (U2020 and A549).

CYB561D2 CYB561D2 codes for cytochrome b-561 domain containing 2. Its cloning, structure and what was known about its expression and function has been reviewed by Lerman and Minna et al. (2000). There is evidence of tumour suppressor activity of CYB561D2, since cell proliferation of NSCLC cells transfected with CYB561D2 in an adenoviral vector, was significantly reduced and also after intratumoural injection of the same clones into xenografts tumour growth appeared inhibited (Ji et al., 2002). Perhaps, the abundant expression of CYB561D2 found in lung cancer cell lines must be explained by posttranslational modification at the protein level.

PL6 PL6 codes for placental protein 6. Its cloning, structure and what was known about its expression has been reviewed by Lerman and Minna et al. (2000). Since PL6 expression was reduced in SCLC cell lines, PL6 might be involved in small cell lung cancer.

CACNA2D2 CACNA2D2 codes for calcium channel, voltage-dependent, alpha 2/delta subunit 2 gene. Its cloning, structure and what was known about its expression and function has been reviewed by Lerman and Minna et al. (2000) and Zabarovsky et al. (2002). Although over 50% of lung cancer cell lines tested showed a reduced or absent expression of CACNA2D2, particularly in NSCLCs, no mutations could be detected in 60 lung cancer cell lines and 40 primary tumours (Lerman and Minna, 2000). Loss of CACNA2D2 expression is probably not mediated by promoter hypermethylation, since in only one primary glioma tumour promoter hypermethylation of CACNA2D2 could be detected (Hesson et al., 2004). Promoter hypermethylation has, however,

33 Chapter 1 not been tested in lung tumours. CACNA2D2 was shown to possess tumour suppressor activity. Cell proliferation of the NSCLC cell lines NCI-H358, NCI-H460, NCI-1299 and A549 transfected with CACNA2D2 in an adenoviral vector, was reduced significantly. A significant suppression of tumour growth compared to control groups treated with PBS was found in NCI-H460 tumours treated with CACNA2D2 in an adenoviral vector. CACNA2D2 was also found capable of inducing apoptosis in three out of four NSCLC cell lines tested. It was shown that the apoptotic effect is associated with the regulation of cystosolic Ca2+ contents and the activation of the mitochondrial pathway (Carboni et al., 2003).

A number of genes, RBM5, SEMA3F, SEMA3B, HYAL1, TUSC2, RASSF1, ZMYND10, TUSC4, CYB561D2, PL6 and CACNA2D2 from the 3p21.3 critical region have been implicated in lung cancer. Convincing evidence for an involvement of any of these genes in the development of lung cancer is, however, lacking. In the functional tests for these genes cDNAs have mostly been cloned behind strong viral promoters that cause an abundant production of a single protein which may well disturb essential cellular processes and thereby affect growth and proliferation of the tumour cells, i.e. mimic tumour suppression. To demonstrate a tumour suppressor role, an approach would be needed in which a functional effect is achieved at a low gene dosage.

34 Introduction

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