Identification of regulatory regions within the KAI1 promoter: a role for binding of AP1, AP2 and p53

Abstract

The mechanism underlying loss of KAI1 gene expression in invasive and metastatic tumour cells is unknown. A possible scenario could involve altered expression or function of protein factors normally involved in regulating KAI1 transcription. To explore this possibility, we have initiated a study to characterise regulatory elements of the KAI1 promoter, using as a model, two bladder cancer cell lines (BL13 and HT1376) expressing high levels of endogenous KAI1 messenger RNA (mRNA). Transfection experiments using reporter plasmids with progressive KAI1 promoter deletions, identified a 76 bp region upstream of the transcription initiation site which contained putative binding motifs for AP2, p53 and AP1, as essential for reporter activity. DNA-binding studies using nuclear extracts from both cell lines, showed that AP1 and AP2 formed specific complexes with oligonucleotides containing KAI1 promoter motifs. Mutation of either motif abrogated reporter activity and abolished specific complex formation. In BL13 cells (endogenous wildtype p53), but not in HT1376 cells (endogenous mutant p53), mutation of the p53-binding motif also abrogated reporter activity and abolished specific complex formation in gel shift assays. These data suggested that a combination of AP2, p53 and AP1 binding to specific motifs within the KAI1 promoter might be required for high level promoter activity and that loss of expression or function of these factors might contribute to loss of KAI1 expression in invasive tumours and tumour cell lines. To explore this possibility, we examined levels of these proteins in nuclear extracts of BL13 and HT1376, as well as three bladder cancer cell lines which expressed little or no KAI1 mRNA. Our data suggested that a loss of KAI1 mRNA was not simply due to absence of AP2, AP1 or p53 expression.

Introduction

Through a combination of chromosome transfer and transfection experiments in a rat model of prostate cancer, the protein encoded by the KAI1 gene on human chromosome 11p11.2 was initially described as a prostate-specific metastasis suppressor (Dong et al., 1995). Subsequently, reduced expression of KAI1 has been demonstrated in the invasive and metastatic stages of many human cancer types (Ow et al., 2000 and references therein). An absence of KAI1 is also associated with poor patient prognosis in non-small lung cancer (Adachi et al., 1996) and with breast cancer recurrence (Huang et al., 1998a, Huang et al., 1998b), suggesting that loss of KAI1 function confers an important advantage on tumour cells. This hypothesis is supported by studies of tumour cell lines, showing that loss of KAI1 is associated with increased in vitro invasive and in vivo metastatic behaviour (Phillips et al., 1998, Takaoka et al., 1998, Jackson et al., 2000a, Yang et al., 2001). Given the potential importance of altered KAI1 expression to a wide range of malignancies, a major goal of current research is to identify the underlying basis. In spite of much effort, available evidence suggests that promoter hypermethylation, gene mutation, and loss of heterozygosity at the KAI1 locus (Dong et al., 1996, Kawana et al., 1997, Tagawa et al., 1999, Jackson et al., 2000b) are not involved in the process. Other research has focussed on factors that might regulate KAI1 transcription and which might be lost or have altered function in tumours and tumour cell lines. A p53-binding motif is present within the KAI1 promoter (Mashimo et al., 1998) but results from studies by several groups, including our own, have thus far provided little evidence to support the notion that p53 is the major determinant of KAI1 expression (reviewed in Jackson and Puisieux, 2000). The proximal KAI1 promoter contains potential binding sites for several other protein transcription factors, including AP2, GATA-1, TCF-1, AP1 and Sp1 (Dong et al., 1997). These factors may be responsible for epithelial – and tissue – specific expression of KAI1, but to-date there is no evidence that the binding sites in the promoter are functional, and their role in regulating KAI1 transcription is unclear.

As part of studies investigating possible mechanisms for loss of KAI1 expression in bladder cancer and bladder cancer cell lines, we have initiated a functional characterisation of the KAI1 promoter, using as a model, two bladder cancer cell lines expressing high levels of KAI1 messenger RNA (mRNA), and which should contain all transcription factors required for KAI1 transcription. We identify a sequence between promoter residues −922 and −846 essential for activity of the KAI1 promoter in these cell lines, and that binding of AP2α, p53 and AP1 (c-jun) to this region is required for promoter activity. We also explored the possibility that a loss of expression of these three proteins is responsible for low levels of KAI1 mRNA expression in certain bladder cancer cell lines.