KAI1 tetraspanin and metastasis suppressor

The International Journal of Biochemistry & Cell Biology 37 (2005) 530–534

Molecules in focus

KAI1 tetraspanin and metastasis suppressor Paul Jacksona,b,∗ , Alexandra Marreirosa,b , Pamela J. Russella,b a b

Oncology Research Centre, Level 2 Clinical Sciences Building, Prince of Wales Hospital, Barker Street, Randwick, NSW 2031, Australia Faculty of Medicine, The University of New South Wales, Kensington, NSW 2052, Australia Received 30 July 2004; received in revised form 3 August 2004; accepted 9 August 2004

Abstract KAI1 is a widely expressed transmembrane glycoprotein of the tetraspanin family. Substantial experimental evidence suggests that KAI1 is an important regulator of cell behaviour. A loss of KAI1 expression is also associated with the advanced stages of many human malignancies and results in the acquisition of invasive and metastatic capabilities by tumour cells, yet the underlying mechanisms responsible for this down-regulation of KAI1 expression remain to be resolved. The recent identification of signalling pathways downstream of KAI1, and proteins that specifically interact with KAI1, are beginning to elucidate the biological pathways involving KAI1. © 2004 Elsevier Ltd. All rights reserved. Keywords: KAI1; TM4SF; Tetraspanin; Human cancer; Metastasis suppressor

1. Introduction KAI1 (CD82/C33/R2/IA4) was first identified as a gene on human Chromosome 11 responsible for specific inhibition of tumour metastasis, but not the incidence or growth rate of tumours, in a rat model of prostate cancer (Rinker-Schaeffer et al., 1994; Dong et al., 1995). The discovery that a loss of KAI1 expression occurred in advanced stages of human prostate cancer, but not breast cancer (Dong et al., 1996), led to the proposal that KAI1 might be a prostate-specific metas∗

Corresponding author. Tel.: +61 2 9382 2616; fax: +61 2 9382 2629. E-mail address: [email protected] (P. Jackson). 1357-2725/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2004.08.009

tasis suppressor. Subsequent studies, in many different tumour types, have now emphasised the importance of KAI1 as a general suppressor of the metastatic phenotype (reviewed in Jackson & Marreiros, in press). The molecular basis for down-regulation of KAI1 expression in advanced tumours and identification of the normal functional role of KAI1, are two areas under intensive investigation.

2. Structure The KAI1 gene is located on human chromosome 11p11.2, composed of 10 exons and 9 introns, and spans a total of about 80 kb of DNA (Dong, Isaacs,

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Fig. 1. Structure of the KAI1 gene and protein. Coding exons are indicated by black boxes and non-coding exons by white boxes. Hatched areas in bar protein structure show location of transmembrane domains. Green circles in the protein conformation indicate residues potentially glycosylated; yellow circles represent cysteines available for disulphide bonding. Abbreviations: TM1–4, transmembrane regions 1–4; ED1–2, extracellular domains 1 and 2. Illustration modified from Bienstock and Barrett (2001), and Jackson and Marreiros (in press).

Barrett, & Isaacs, 1997; Fig. 1). Starting in exon 3 and ending in exon 10, translation results in a 267 amino acid protein belonging to a large family of transmembane glycoproteins called tetraspanins (TM4SF; Dong et al., 1995). The KAI1 amino acid sequence is identical to that of a previously characterised TM4SF antigen designated CD82 (Dong et al., 1995). TM4SF proteins have cytoplasmic N- and C-termini, and traverse the cell membrane four times forming one small and one large extracellular loop with residues susceptible to post-translational phosphorylation and/or Nlinked glycosylation (Rubinstein & Boucheix, 1999; Fig. 1). Due to glycosylation, KAI1 has a molecular mass of 46–60 kDa, rather than 28 kDa based on

amino acid content (White, Lamb, & Barrett, 1998). N-glycosylation of KAI1 is associated with decreased cell motility and increased apoptotic cell death (Ono, Handa, Withers, & Hakomori, 1999). 3. Synthesis and degradation 3.1. Cellular expression KAI1 protein is mainly localised to the cell membrane. KAI1 is widely expressed in normal human tissues, with highest levels in prostate, lung, liver, kidney, bone marrow and placenta (Dong et al., 1995). Down-regulation of KAI1 mRNA and protein levels

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is common to advanced stages of many human cancer types but appears unlikely to involve loss of heterozygosity at the KAI1 locus or mutations within the KAI1 gene (reviewed in Jackson & Marreiros, in press).

3.2. Transcription The DNA sequence surrounding the KAI1 transcription initiation site is GC-rich (68% GC). Ratios of observed/expected CpG are 0.64 for 1 kb of sequence including 735 bp of promoter sequence, exon 1 and the first 272 bp of intron 1, rising to 0.77 for the 450 bp upstream of the transcription initiation site (Dong et al., 1997). Thus, the KAI1 promoter is associated with a CpG island. Although suggesting that methylation might be involved in KAI1 transcription, there is no evidence to-date that this region of the KAI1 promoter is methylated in either normal or tumourigenic tissues and cells (reviewed in Jackson & Marreiros, in press). The KAI1 promoter lacks typical TATA- and CAATbinding motifs, but has binding sites for many transcription factors including p53, AP2, AP1 and Sp1 (Dong et al., 1997; Fig. 1). Recent studies indicate that the minimal promoter consists of two elements; −197 to the transcription initiation site, and +1 to +351, which provide strong but not epithelial-specific activity (Gao et al., 2003). In addition, there is a negative acting element between −735 and −197. Transcription factors responsible for basal activity and the negative acting element are not yet identified. Following identification of a p53-binding motif in the KAI1 promoter, evidence for p53 as a major factor regulating KAI1 transcription has been contradictory (reviewed in Jackson & Puisiuex, 2000). A recent study supports the idea that binding of p53, AP2 and AP1 is required to mediate the effect of an enhancer-like element between −922 and −846 (Marreiros, Czolij, Yardley, Crossley, & Jackson, 2003). Upstream factors regulating KAI1 transcription are not clearly defined, however, phorbol ester elevates KAI1 mRNA levels in prostate cancer cell lines which normally express little or no KAI1, suggesting a role for protein kinase C (Akita et al., 2000). Likewise, nerve growth factor up-regulates KAI1 mRNA levels in prostate cancer cells (Sigala et al., 1999). In each case, however, it is unclear if effects on KAI1 expression are directly due to added agents or indirectly as a result of altered cell behaviour.

4. Biological function KAI1 interacts with integrin ␣4␤1, other TM4SF proteins, and cell surface molecules including, CD4, CD8, CD19, CD21 and MHC class I and class II in lymphocytes, forming what is now known as ‘the tetraspanin web’ (reviewed in Hemler, 2001). In a lymphocyte context, KAI1 is involved in pathways leading to monocyte (Lebel-Binay et al., 1995a) or Tcell activation (Lebel-Binay et al., 1995b; LaguadriereGesbert, Lebel-Binay, Hubeau, Fradelizi, & Conjeaud, 1998; Shibagaki et al., 1998), suggesting that KAI1 plays a role in regulating signalling pathways. In nonlymphoid cells, in vitro studies in which KAI1 is over-expressed or down-regulated have provided considerable evidence for the importance of KAI1 to tumour cell behaviour. Reduced KAI1 expression is associated with altered adhesion to specific components of the extracellular matrix such as fibronectin, reduced cell–cell interactions and increased cell motility, leading to a more invasive and metastatic ability (reviewed in Jackson & Marreiros, in press). The molecular basis for these effects is not yet clear, but several recent reports suggest a complex combination of altered signalling pathways and reduced interactions between KAI1 and specific proteins involved in these processes (summarised in Fig. 2). A direct interaction between KAI1 and the epidermal growth factor receptor (EGFr) attenuates EGFr-induced lamellipodia formation and migration signalling, by regulating dimerisation and internalisation of EGFr (Odintsova, Sugiura, & Berditchevski, 2001; Odintsova, Voortman, Gilbert, & Berditchevski, 2003). KAI1 also suppresses motility by decreasing signalling via the FAK-Lyn-p130CAS -CrkII pathway which regulates organisation of the actin cytoskeleton, by inhibiting formation of active p130CAS CrkII complexes (Zhang, He, Zhou, & Liu, 2003b). KAI1/CD82 associated protein (KASP) and KAI1 COOH-terminal interacting tetraspanin (KITENIN) are two newly identified KAI1-binding proteins. KASP (also known as EW12/PGRL) is a member of the immunoglobulin superfamily and inhibits cell migration (Zhang, Lane, Charrin, Rubinstein, & Liu, 2003a). Binding of KAI1 to KASP enhances this effect in a synergistic manner. In contrast, KITENIN (originally identified as a TM4SF protein called VANGL1, the human homologue of Drosophila VANG which negatively regulates the non-canonical Frizzled signalling

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Fig. 2. Schematic illustration of possible functional interactions between KAI1 and binding proteins to inhibit metastatic behaviour in cells.

pathway) enhances invasion and metastatic behaviour of tumour cells, and binding to KAI1 inhibits this effect (Lee et al., 2004). Our understanding is very limited, but KAI1 may act, in part, to control cellular adhesion and limit migration by modulating reorganisation of the actin cytoskeleton in response to pro-migratory signals. An investigation of the relationship between EGFr signalling, the p130cas -Crk pathway and both KASP and KITENIN function, will be important areas to pursue.

5. Possible therapeutic applications A loss of KAI1 expression occurs in the advanced stages of many cancers. It is associated with poor patient prognosis in disease such as non-small cell lung (Adachi et al., 1996) and ovarian (Schindl, Birner, Breitenecker, & Oberhuber, 2001) cancers, and recurrence in breast (Huang et al., 1998) and bladder (Su et al., 2004) cancers. Identification of patients whose tumour cells show reduced KAI1 expression may allow them to be targeted for tailored therapy to inhibit metastasis. Strategies to restore KAI1 expression might also be explored to limit the spread of tumour cells.

Acknowledgements The authors wish to apologise to all authors whose work could not be adequately described or discussed due to space considerations.

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