The Role of HRK Gene in Human Cancer

M Nakamura, K Shimada and N Konishi

Department of Pathology, Nara Medical University School of Medicine, Nara, Japan

Apoptosis regulators play one of the most critical roles in Especially, members of the Bcl-2 gene family play tumorigenesis, and an imbalance between cell proliferation critical roles in apoptotic regulation in cancer cells, not and apoptosis may contribute to tumor progression. HRK only in tumorigenesis, but also in chemotherapy- was itself originally identified as a proapoptotic gene induced cell death (Reed, 1995). induced by diminished levels of cytokine in hematopoietic The proapoptotic Bcl-2 family can be separated into cells and cultured neurons and repressed by the expression two subgroups. The first subgroup includes members of of death-repressor proteins. A few analyses of HRK multiple Bcl-2 homology (BH) domain proteins, such as protein expression in primary central nervous system Bax; these gene proteins contain several BH regions. lymphomas have been performed, and little is known The second subgroup includes proteins that share only about the epigenetic or post-transcriptional mechanisms the homologous BH3 domain, an amphipathic a-helical that may participate in HRK inactivation. Here we show segment that is necessary for triggering cell death the data on the 50-CpG methylation status, loss of (Chittenden et al., 1995). The BH3 domain inactivates heterozygosity on 12q13.1 and its association with HRK apoptosis, but apparently does not interact with the expression in human malignancies, including prostate proapoptoic proteins, Bak or Bax (Inohara et al., 1997). cancers, astrocytic tumors and primary central nervous Members of this newly described group of BH3-only system lymphomas. Aberrant methylation of CpG islands proteins—including HRK/DP5, Bim, Noxa, Bbc3/ within the promoter is an epigenetic event largely Puma, Bmf, Bad, Bik/Blk/Nbk and Bid—all share the responsible for the silencing of the HRK gene and biological function of promoting Bax-dependent apop- subsequent low apoptotic counts in our series of malig- tosis through mitochondrial release of cytochrome c and nancies. Inactivation of HRK apparently occurs in a initiation of downstream cell death programs (Puthala- substantial proportion of all tumor phenotypes and, as a kath and Strasser, 2002). Among these factors, a novel potential proapoptotic gene, HRK may contribute to the regulator of cell death, Human Hrk, and its mouse development and progression of many human cancers. homolog (DP5) (Imaizumi et al., 1997), regulates Bcl-2 Oncogene (2009) 27, S105–S113; doi:10.1038/onc.2009.48 and Bcl-xL through selective interaction with the BH3 domain (Inohara et al., 1997). Keywords: HRK; prostate cancer; glioblastoma; primary HRK, discovered by Inohara et al. (1997), was itself central nervous system lymphoma originally identified as a proapoptotic gene located at 12q13.1 and is transcriptionally regulated in various cell types (Imaizumi et al., 1999; Sanz et al., 2000), being induced within an hour after administration of cell Introduction death stimuli (Harris and Johnson, 2001). HRK also selectively interacts with survival-promoting proteins, Apoptosis plays roles in both organ development and Bcl-2 and Bcl-xL. Common to the BH3 proteins, the tissue homeostasis. Its deregulation, together with product of HRK localizes to the membranes of defective control of proliferation, contributes to critical intracellular organelles in a pattern similar to that tumor progression (Evan and Vousden, 2001; Steinbach reported earlier for Bcl-2 and Bcl-xL. HRK is preva- et al., 2002; Debatin et al., 2003). Analyses of cell lently expressed in hematopoietic tissues and cultured turnover relating to apoptosis and proliferation provide neurons (Inohara et al., 1997; Sanz et al., 2000), but has insights into tumor doubling time, prognosis and also been shown in the pancreas, liver, lung, kidney and treatment response (Zhang et al., 1998) in that high prostate (Inohara et al., 1997; Higuchi et al., 2008). The apoptotic counts have been associated with a high role of HRK in apoptosis has been described mainly in histological grade and shortened survival in a number of hematopoietic tissues and cultured neurons; transcrip- studies (Vakkala et al., 1999). This cell death program is tion of HRK is specifically induced at the mRNA known to be activated or inactivated by a variety of and protein levels after growth factor withdrawal in genes, such as Bcl-2 and Bcl-xL, Bak and Bax. these cells, leading to cell death (Sanz et al., 2000; Harris and Johnson, 2001). Levels of HRK are high in embryonic neuronal tissues that undergo apoptosis Correspondence: Professor N Konishi, Department of Pathology, (Imaizumi et al., 1997), and in cultured neurons, its Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan. expression is increased after nerve growth factor with- E-mail: [email protected] drawal or exposure to b-amyloid (Imaizumi et al., 1999). HRK inactivation in human malignancy M Nakamura et al S106 Interestingly, b-amyloid-induced apoptosis of cultured much clinically useful information for tumor develop- neurons was shown to be mediated by E2F1 (Giovanni ment, prognosis and treatment response (Nakamura et al., 2000). et al., 1997). Some limited studies have focused on Some studies have also shown that transcription of critical regulators of apoptosis (Steinbach et al., 2002; the HRK gene is under the control of the c-Jun Debatin et al., 2003), and high apoptotic counts have N-terminal kinase pathway (Harris and Johnson, been associated with high histological grade and 2001) as well as the transcriptional repressor DREAM shortened survival (Shimada et al., 2008). Activated (Sanz et al., 2002) and that apoptosis by HRK requires regulation of mitogen-activated protein kinases and the expression of Bax (Harris and Johnson, 2001). Fas-associated death domain-containing proteins affects Sunayama et al. (2004) have shown that HRK appears cell growth, proliferation, survival and progression in to be located in the mitochondria associated with p32. prostate cancers (Shimada et al., 2006). This suggests that physical and functional interaction HRK seems to be an important component in the with p32 could have a crucial role in HRK-mediated regulation of apoptosis on tumor cells and the inactiva- apoptosis. In addition, the overexpression of Bcl-2 or tion of HRK, leading to loss of HRK expression and Bcl-xL inhibits the induction of HRK and prevents subsequent low apoptotic counts—events that may be apoptosis after growth factor withdrawal (Sanz et al., relevant to the pathogenesis of prostate cancer. 2000). As enforced coexpression of HRK and the antiapoptotic protein, Bcl-2 or Bcl-xL, inhibits the Aberrant methylation of HRK in the gene promoter and death-promoting activity of HRK (Inohara et al., exon 1. DNA methylation patterns in the promoter 1997), it appears that the balance between the steady- region can be determined by methylation-specific PCR state levels of Bcl-xL or Bcl-2 and those of HRK either (Herman et al., 1996). In addition to that of many tumor suppresses or induces apoptosis. suppressor genes, transcription of HRK might be The expression of HRK in hematopoietic progenitor regulated by the promoter that is rich in CpG cell lines can be induced not only by growth factor dinucleotides. The question, however, arises as to withdrawal but also by treatment with etoposide, a whether or not a reduced HRK expression in the tumor chemotherapeutic drug that induces apoptosis. On samples can be explained solely by promoter methyla- growth factor withdrawal or treatment with certain tion of the gene. CpG islands of HRK are relatively chemotherapeutic drugs, the repressor mechanism may large, and it is not clear whether they are coordinately be released (that is, by post-translational modification or regulated with respect to hypermethylation. To address binding to a specific factor), allowing the expression of the issue, we originally designed methylated and HRK. Thus, alterations in protein form/expression may unmethylated methylation-specific PCR primers that also be a critical feature for the survival or cell death of spanned the transcription start site and translation start cancer cells, enabling them to escape apoptosis resulting site for both promoter and exon 1 sequence (Nakamura from the DNA damage caused by chemotherapeutic et al., 2005b, 2006; Higuchi et al., 2008). Methylation of drugs and/or radiotherapy (Zhang et al., 2000). The loss the HRK promoter was detected in 0, 14, 38, 100 and of HRK expression and the underlying mechanism(s) of 25% of cancers graded as Gleason score (GS), downregulation, however, have not been widely exam- respectively (Table 1 and Figure 1). Detectable exon 1 ined in human malignancies. methylation was marginally more frequent in lower- In this text, we reviewed the 50-CpG methylation grade lesions; however, methylation of exon 1 occurred status, loss of heterozygosity (LOH) on 12q13.1 and its at the same frequency as promoter methylation in association with HRK expression in human malignan- higher-grade cancers. In comparison, none of the 30 cies, including prostate cancer, astrocytic tumor and samples of normal epithelial or stromal tissues from primary central nervous system lymphoma. areas adjacent to the tumors showed methylation in either gene region probed.

HRK alteration in human malignancies Detection of HRK mRNA by reverse transcription PCR and immunohistochemistry. Although transcripts for Prostate cancer HRK were retrieved in the majority of normal controls In western countries, prostate cancer is the most and lower-grade prostate cancers, PCR products corre- common malignancy and the second leading cause of sponding to HRK were less infrequently found in high- cancer death in men (Nelson et al., 2003). Various grade cancers (Table 1 and Figure 1). Cytoplasmic studies have been performed investigating the genetic expression of HRK by immunohistochemistry was factors related to prostate carcinogenesis (Konishi et al., observed in epithelial cells in peritumoral prostate 2005a, b, 2008), but as yet, critical specific molecular tissues (Figure 2). Among tumor tissues, 88% of lesions markers that clearly identify malignant potential have with GS 5 and 6 were immunopositive for HRK, with not been found. Apoptosis regulators play one of the more than 20% of cells counted showing staining. In most critical roles in tumorigenesis, and an imbalance contrast, higher-grade lesions tended to show less HRK between cell proliferation and apoptosis contributes to expression overall (Table 1). There was significant tumor progression (Steinbach et al., 2002; Debatin et al., positive correlation between HRK transcription and 2003). Analyses of apoptosis and proliferation provide HRK immunoreactivity, specifically in GS 6 tumors in

Oncogene HRK inactivation in human malignancy M Nakamura et al S107 Table 1 Correlation between gene status, protein expression and apoptosis in prostate cancers Gleason score (GS) GS 5 (n ¼ 2) GS 6 (n ¼ 22) GS 7 (n ¼ 24) GS 8 (n ¼ 2) GS 9 (n ¼ 4)

HRK expression IHC (À) 0 3/22 (14%) 10/24 (42%) 1/2 (50%) 1/4 (25%) RT–PCR (À) 0 2/17 (12%) 8/21 (38%) 2/2 (100%) 1/3 (33%)

HRK methylation Promoter 0 3/22 (14%) 9/24 (38%) 2/2 (100%) 1/4 (25%) Exon 1 0 5/22 (23%) 12/24(50%) 2/2 (100%) 1/4(25%)

LOH at HRK locus 0 4/22 (18%) 7/24 (29%) 0/2 (0%) 1/4 (25%) Mean apoptotic index 1.40±1.62 2.58±0.62 1.79±0.82 1.60±0.28 2.15±0.25

Abbreviations: IHC (À), negative immunohistochemical staining; LOH, loss of heterozygosity; RT–PCR, reverse transcription PCR; RT–PCR (À), negative expression by RT–PCR.

NC PC 1 6 8 7

UMUMUMUMUMUM

promoter 122 bp 116 bp

128 bp exon 1 112 bp

HRK 141 bp

G3PDH 111 bp N2 3 4 78 91011

Case 8

D12S112 D12S245 Figure 1 (a) Results of methylation-specific PCR of CpG islands of the HRK promoters in prostate cancers. In case 1, who assigned a Gleason score of 6 (GS 6), methylation occurred in exon 1 of HRK, rather than in the promoter region. Cases 6 (GS 8), 7 and 8 (GS 7), however, were HRK methylated in both promoter and exon 1 (U, PCR product amplified by unmethylation-specific primers; M, PCR product amplified by methylation-specific primers; NC, negative control; PC, positive control). (b and c) RT–PCR analysis of HRK mRNA expression in prostate cancer. HRK expression was evident in cases 3 (GS 5), 4, 9 (both GS 6), 10 and 11 (both GS 7) despite apparent lack of 12q13.1 LOH. However, both of this were positive for both LOH and HRK promoter and exon 1 methylation in case 8 (N, normal control). This figure is modified from Higuchi et al. (2008). RT–PCR, reverse transcription PCR. that a majority of lesions showed transcripts in both (Sanz et al., 2000), whereas protein expression of other analyses. BH3 family members is regulated by post-transcrip- HRK expression was readily detectable in GS 5 and 6 tional mechanisms (Inohara et al., 1997). For example, cancers without apparent HRK methylation. In con- although HRK activity is mediated by its methylation trast, the expression of HRK was not detected in higher- status at the transcription start site of the gene, the grade cancers that showed methylation in both regions proapoptotic activity of BAD is controlled by the level of exon 1 and promoter (Table 1). Abnormal methyla- of its phosphorylation (Downward, 1999). tion may be a specific feature of HRK in various tumor cells that is not found in any other member of the BH3 family (Obata et al., 2003). Expression of HRK is HRK methylation and expression and 12q13.1 LOH. regulated at the transcriptional level by cellular stress Could reduced HRK expression be determined solely by apparently caused by the absence of growth factors methylation at the transcription start site of the gene?

Oncogene HRK inactivation in human malignancy M Nakamura et al S108 DA 1 DA 2 AA 1 PGB1 PGB2 SGB3 SGB1 UM UM UM UM UM UM UM 122 bp HRK promoter 112 bp 148 bp HRK Exon 1 132 bp

Figure 2 (a) In patient 1, the diffuse astrocytoma DA 1 is unmethylated across promoter and exon 1. In patient DA 2, promoter and exon 1 hypermethylation is found. AA 1 is an anaplastic astrocytoma showing a methylated exon 1, but unmethylated promoters in HRK, whereas this case showed methylation in both promoter and exon 1 in the corresponding secondary glioblastoma SGB 1. In secondary glioblastoma SGB 3, both promoter and exon 1 of HRK are methylated, but only exon 1 is methylated in primary glioblastoma PGB1 (U, PCR product amplified by unmethylation-specific primers; M, PCR product amplified by methylation-specific primers). (b) HRK immunohistochemistry showing membrane and cytoplasmic immunoreactivity in the majority of tumor cells from anaplastic astrocytoma (AA 1) (hematoxylin counterstain, Â 250). (c) In the same case, the progressive secondary glioblastoma (SGB 1) exhibits diffuse tumor cell clusters with loss of HRK expression (hematoxylin counterstain, Â 250). This figure is modified from Nakamura et al. (2005b). Another possibility is that, in some samples, only one stained nuclei or nuclear fragments with a cytoplasmic halo allele of the HRK gene is hypermethylated. In these were recognized as TUNEL-positive apoptotic cells. cases, the specific methylated and unmethylated bands A significant difference in the incidence of prostate would be expected to be of equal intensity. The HRK epithelial cell apoptosis was detected between tumors gene is located in 12q13.1, where LOH has frequently with a GS of 5–6 and those with a GS of 7 or higher been detected in human tumors (Kim et al., 2001; (Table 1). We have also shown a close inverse Wistuba et al., 2001); thus, HRK may be inactivated not correlation between loss of HRK expression by im- only by DNA methylation, but also by gene deletion. munohistochemistry and decreased apoptotic indices A significant relationship between 12q13.1 LOH and (AIs) in cancers of each grade. The association between repression of both HRK mRNA and protein levels has apoptosis and HRK inactivation was uncovered, indi- been shown, although LOH at 12q13.1 was detected in cating that both genetic hypermethylation and allelic only 23% of the prostate cancers analysed (Table 1). In loss of apoptotic-associated genes can contribute to the cohort of GS 6 cancers, two of four cases showing aberration of apoptotic regulation in prostate cancer. LOH also showed methylation of both the promoter and exon 1 of the gene. In contrast, all seven GS 7 Detection of multiple methylated genes and p53 muta- cancers showing allelic loss in this region exhibited tions. It is noteworthy that prostate cancer exhibiting concurrent gene methylation in both regions. However, HRK methylation tends to show methylation in the two cases with LOH still yielded detectable HRK panel of genes. Co-methylation of HRK and p14ARF expression. Therefore, LOH alone may not completely occurred in 13% of evaluated lesions; 47% showed suppress HRK expression because many genes can be methylation of both HRK and p16INK4a; O6-MGMT and expressed in a monoallelic fashion. Some DNA methy- HRK were methylated in 60% of samples; and co- lation-independent mechanisms, including mutations or methylation of GST-P and HRK was detected in 87% of loss of one or more allele, may also be assumed. informative lesions. Prostate carcinomas could be grouped with other cancers displaying CpG island Apoptotic indices. When evaluating the apoptotic cell methylator phenotype (CIMP). CIMP was initially number, we confirmed the presence of standard mor- described in colorectal cancers, and tumors exhibiting phological characteristics of apoptosis. Moreover, we CIMP have a global promoter hypermethylation pattern employed the transferase-mediated digoxigenin-tagged that contributes to tumorigenesis (Toyota et al., 1999). 16-desoxy-uridine-triphosphate nick end-labeling (TU- It remains unclear why HRK and one or more of these NEL) assay to detect and quantify apoptosis in the specific apoptosis-related genes are more likely to tumor specimens. Only those cells showing darkly undergo structural alterations, but genetic instability

Oncogene HRK inactivation in human malignancy M Nakamura et al S109 might be closely associated with multiple epigenetic 2000). However, there have been no reports on the effects, such as methylation, which occur during the expression and/or the actual role of HRK distinctive course of tumor progression. from astrocytic tumor. In colorectal cancers, cases without p53 mutations have frequently shown aberrant methylation of HRK Aberrant methylation of HRK. In our series of astro- (Obata et al., 2003). Mutation of p53 was detected in a cytic tumors, methylation of the HRK promoter was low number of GS 6 and 7 cancers (9 and 13%, observed in 19% (7/36) of low-grade diffuse astrocyto- respectively), but not in other tumors with higher GS. mas, in 22% (7/32) of anaplastic astrocytomas, in 27% p53 mutations and p14ARF methylations or deletions are (17/64) of primary glioblastomas and in 43% (12/28) of rare in both GS 6 and 7 cancers, a finding that leads us secondary glioblastomas (Table 2), whereas 25, 25, 31 to believe that aberrations in the p14ARF/p53/HRK and 50% of these individual subtypes, respectively, apoptotic pathway are not necessarily related to showed exon 1 methylation. Neither the promoter nor prostate tumorigenesis. It has also been shown that exon 1 was methylated in normal cells from areas reduced phosphorylation of Fas-associated death do- adjacent to the tumors. main-containing protein is associated with progression in high-grade prostate cancer (Shimada et al., 2005). A Detection of HRK expression. Both membrane and GS of 7 is thought to be a borderline score for a good cytoplasmic immunoreactivity to HRK were detected in prognosis in prostate cancers (Konishi et al., 2005b; peritumoral neurons and glial cells in the brain tissue Shimada et al., 2006); perhaps significantly linked HRK specimens. There was a significant increase in the inactivation and decreased AIs were detected in cancer frequency of loss of HRK expression in secondary samples graded GS 7. Therefore, assays based on glioblastomas compared with other types of tumors; evaluating a combination of genetic alterations, includ- more than half of the secondary glioblastoma cases ing HRK hypermethylation, LOH, dephosphorylation showed little to no immunohistochemical response. In and mutation, might be useful in the prognosis of contrast, HRK immunoreactivity was consistently prostate cancers. higher in the other tumor subtypes in that 86% of diffuse astrocytomas, 78% of anaplastic astrocytomas Astrocytic tumors and 72% of primary glioblastomas showed HRK High-grade astrocytic tumors, especially glioblastoma expression in more than 20% of cells (Table 2 and (WHO grade IV), are the most frequent and most Figure 2). This reflects a clear trend toward decreasing malignant tumors of the human nervous system. From a level of HRK expression with increasing tumor grade. clinical and biological point of view, glioblastomas are mRNA transcripts were retrieved in the majority of divided into two subtypes: primary or de novo glioblas- normal controls, but 18% of diffuse astrocytomas, 30% tomas develop rapidly, without clinical or histopatho- of both primary glioblastomas and anaplastic astro- logical evidence of less-malignant precursor lesions, and cytomas and 57% of secondary glioblastomas yielded constitute the majority of diagnosed cases, whereas no detectable reverse transcription PCR products secondary glioblastoma develops more slowly and corresponding to HRK. This loss of HRK mRNA progressively from low-grade diffuse (WHO grade II) transcription significantly correlated with HRK immu- or anaplastic (WHO grade III) astrocytoma (Kleihues noreactivity in each category of tumor. et al., 2002). Glioblastomas typically are very resistant Reduced or lost HRK expression was significantly to induction of apoptosis and, consequently, do not associated with CpG island methylation in the promoter respond well to conventional chemotherapy. HRK is region in each type of astrocytic tumor. However, some reportedly an important proapoptotic in cultured few tumors showed exon 1 methylation, yet retained neurons and is repressed by the expression of death- HRK expression. This suggests that methylation of the repressor proteins (Inohara et al., 1997; Sanz et al., first exon is only partially related to gene expression in

Table 2 Correlation between HRK gene status and apoptosis in astrocytic tumors Variables Diffuse astrocytoma Anaplastic astrocytoma Primary glioblastoma Secondary glioblastoma (n ¼ 36) (n ¼ 32) (n ¼ 64) (n ¼ 28)

HRK expression IHC (À) 5/36 (14%) 7/32 (22%) 18/64 (28%) 17/28 (61%) RT–PCR (À) 2/11 (18%) 3/10 (30%) 8/27 (30%) 4/7 (57%)

LOH at HRK locus 2/36 (6%) 7/32 (22%) 16/64(25%) 13/28 (46%)

HRK methylation Promoter 7/36 (19%) 7/32 (22%) 17/64(27%) 12/28 (43%) Exon 1 9/36 (25%) 8/32 (25%) 20/64(31%) 14/28(50%)

TUNEL (mean) 0.007±0.011 0.404±0.361 1.022±1.315 0.945±0.409

Abbreviations: IHC, immunohistochemistry; LOH, loss of heterozygosity; RT–PCR, reverse transcription PCR; TUNEL, transferase-mediated digoxigenin-tagged 16-desoxy-uridine-triphosphate nick end-labeling.

Oncogene HRK inactivation in human malignancy M Nakamura et al S110 astrocytic tumors at least, whereas methylation of the secondary glioblastomas than in those with p53 muta- promoter region correlated more significantly with gene tions. This significant inverse association strongly expression. Thus, our data have first suggested that the suggests that HRK and p53 act within the same aberrant methylation of HRK is associated with loss of pathway. Epigenetic inactivation of HRK may thus lead protein and mRNA expression, and is probably the to loss of apoptosis in secondary glioblastomas still main mechanism of transcriptional silencing of HRK in expressing wild-type p53. astrocytic tumors. HRK methylation was detected exclusively in tumors A clear agreement between decreased mRNA level showing a simultaneous methylation of multiple genes, and reduced HRK expression also indicates that HRK that is p14ARF and p16INK4a. The astrocytic tumors expression very likely stems from an altered transcrip- included in our series may also belong to the CIMP tion of the gene. This could be mediated by the group and have downregulation of these genes through difference in high-grade tumor heterogeneity, in which aberrant 50-CpG methylation (Toyota et al., 1999). one region is methylated, the other regions being Noting the relation between INK4a/ARF gene altera- unmethylated and expressing the gene. Hypermethyla- tion, as well as high frequencies of the p53 mutation, and tion of HRK appeared to become more frequent as the LOH with HRK inactivation, we believe that structural lesion progressed as this epigenetic alteration was genetic alterations involved in the ‘p14ARF–p53–HRK’ significantly more frequent in secondary glioblastomas apoptosis pathway are likely to be present in secondary than in other types of tumors analysed. In addition, if glioblastomas. Although it remains unclear why HRK hypermethylation was detected in earlier lesions, it was and p16INK4a and p14ARF are prone to undergo such usually also observed in the corresponding secondary structural alterations, genetic instability might be closely glioblastoma. associated with one or more particular regions in such cancer-linked genes, especially in secondary glioblasto- Correlation of expression and LOH at 12q13.1 with HRK ma (Baylin et al., 2001; Ehrlich, 2002). It is also methylation status. There were significant relationships noteworthy that a majority of genes reported to be between 12q13.1 LOH and repression of both HRK methylated in tumors also tend to be methylated in protein and mRNA. Twenty-five samples lacking HRK regions associated with lower-grade malignancy (Toyota expression, but exhibiting aberrant methylation and et al., 1999; Nakamura et al., 2001a, b, 2005a). In LOH, suggest that the biallelic inactivation of the gene agreement with these reports, HRK methylation was resulted from the loss of one allele and 50-CpG detected less frequently in low-grade diffuse astrocyto- methylation in the remaining allele. Of the 38 astrocytic ma, suggesting that inactivation of the gene may be tumors detected with 12q13.1 LOH, six tumors showed involved in a critical step in malignant progression. low expression of mRNA and protein without promoter hypermethylation. In these cases, the HRK gene might Primary central nervous system lymphoma have been silenced by another genetic or epigenetic Primary central nervous system lymphoma (PCNSL) is mechanism, such as specific mutations or histone defined as a diffuse lymphoma presenting in the brain or deacetylation. There were few other tumors with no spinal cord in the absence of systemic lymphoma. There demonstrable HRK expression and no evidence of 50- are no lymph nodes or lymphatics within the nervous CpG methylation and LOH. Alternatively, negative system and, therefore, the pathogenesis of PCNSL in expression could result from several pathways of gene immunocompetent patients is still poorly understood. inactivation, such as loss of transcription factors or Apoptosis regulators play one of the most critical roles dysfunction of the regulatory proteins. in tumorigenesis in many tissues, including malignant lymphomas (Debatin et al., 2003). HRK, which was Evaluation of AI. Higher apoptotic counts in situ have itself originally identified as a proapoptotic gene, was been strongly associated with a higher histological induced by diminished levels of cytokine in hemato- grade. A pattern of HRK expression was indicated poietic cells and cultured neurons and repressed by the specific to secondary glioblastoma and correlated with expression of death-repressor proteins (Inohara et al., the incidence of apoptosis. The mean AI in those 1997; Sanz et al., 2000). Transcription of HRK is secondary glioblastomas with hypermethylated promo- specifically induced after growth factor withdrawal in ter sequences was also significantly lower than in those hematopoietic tissues (Inohara et al., 1997). without promoter methylation, but the AI showed no correlation with HRK expression and hypermethylation Methylation status of HRK vs HRK expression and in the other subtypes of tumors. The diversity of LOH at 12q13.1. Methylation of the HRK promoter mechanisms involved in the regulation and implementa- and of exon 1 was detected in 10 cases in 35 PCNSLs tion of programmed cell death, which is indicative of the (Table 3), but in none of the normal cells from areas rich variety of targets for inhibition of apoptosis during adjacent to the tumors. The concurrent methylation of tumorigenic development, must be considered. both the promoter region and exon 1 occurred in six cases. Hypermethylation and loss of HRK expression Association with other genes. Similar to colorectal appeared to become more frequent as the lesions cancers (Obata et al., 2003), methylation of the gene progressed. In two patients with recurring tumors, if was more frequently detected in p53-competent hypermethylation and HRK loss were detected in earlier

Oncogene HRK inactivation in human malignancy M Nakamura et al S111 Table 3 Correlation of HRK methylation, expression and LOH at tumors may result in the positive mRNA/protein 12q13.1 in PCNSLs expression observed. In the same vein, we saw a clear HRK alteration HRK expression P-value correlation between methylation þ LOH and silencing of HRK in all samples except for 2 cases; in these ( þ )(À) tumors, HRK expression was lost despite the lack of gene methylation and LOH. Methylation status Promoter Methylated 2 8 Unmethylated 23 2 o0.0001 Correlation with apoptosis and therapy. Overall, the ± Exon 1 Methylated 5 5 mean AI of cases was 2.83 2.06%. There was a close Unmethylated 20 5 0.107 correlation between the loss of HRK expression and a decreased AI. In addition, the mean AI of methylated LOH detection tumors was significantly lower than that of unmethy- 12q13.1 þ 26 lated lesions, suggesting the involvement of HRK À 23 40.003 apoptosis pathway. Abbreviations: LOH, loss of heterozygosity; PCNSL, primary central As described in the section of astrocytic tumors, an nervous system lymphoma. inverse correlation was found between the methylation of the HRK gene and p53 mutations in secondary lesions, it was also observed in later lesions. In addition, glioblastomas, suggesting the involvement of structural we saw HRK promoter methylation in three recurrent genetic alterations in the ‘p14ARF/p53/HRK’ apoptosis tumors in which the primary lesions were unmethylated, pathway. Our analysis of PCNSLs failed to corroborate suggesting that HRK methylation may occur in latter that hypothesis. However, we did detect a p53 mutation stages of PCNSL tumorigenesis or may be related to in a single tumor sample, indicating that p53 cannot be recurrence/malignancy. ruled out in the development of some PCNSLs. It has Immunohistochemically, 29% of PCNSLs showed a also been reported that tumors expressing wild-type p53 loss of expression, whereas the remaining cases all tend to be resistant to gene therapy using adenovirus- showed immunoreactivity in more than 20% of cells. mediated transfer of the p53 gene (Sasaki et al., 2001). The reduction in gene expression revealed by the Epigenetic therapies using 50-aza-20-deoxycytidine may immunohistochemical analysis correlated highly with represent a useful alternative strategy for treating such the amount of mRNA transcript, which was further cases, although further studies are required to determine confirmed by the reverse transcription PCR analysis. whether cells expressing HRK are susceptible to There was a clear correlation between aberrant promo- apoptosis induced through chemotherapeutic drug- ter methylation and silencing of HRK as in similar activated pathways. prostate cancer or astrocytic tumors. We showed a significant association between reduced HRK expression and promoter hypermethylation; however, some Methylation status of HRK and other genes. HRK cases showing exon 1 methylation were also positive for methylation was detected exclusively in our series of HRK expression. This indicates that the promoter PCNSLs showing simultaneous methylation of multiple ARF INK4a 6 Kip1 region, including the upstream transcription-starting genes (p14 , p16 , RB1, O -MGMT and p27 )as site, may strongly regulate HRK expression and relate well as prostate cancers and astrocytic tumors. Ten cases to biological significance in PCNSLs, but it may not be showed methylation in more than three genes, and the only important target for methylation in all tumors. among those, 80% cases showed HRK methylation, When compared with LOH at the gene locus, a whereas only 3 of the 21 cases showing methylation in significant positive association was found between less than two genes, did so. It remains unclear why HRK concurrent promoter methylation/LOH and repression and one or more of these specific genes are more likely of both HRK mRNA and protein levels (Table 3). A to undergo structural alterations, but genetic instability total of five cases without HRK expression showed both might be closely associated with multiple epigenetic methylation and LOH, whereas all 19 tumors with effects, including methylation, that occur during the expressing HRK showed an unmethylated promoter course of tumor progression. region and retention of both alleles at 12q13.1. Biallelic inactivation of the gene potentially resulted from the Patient outcome. Mean overall survival for patients loss of one allele and methylation of the other. It should with methylated HRK was not significantly different also be pointed out that two cases with LOH still from that for those patients whose primary tumors did showed detectable HRK expression. As many genes can not show HRK methylation, whereas the relapse-free be expressed monoallelically, LOH alone may not survival time in patients having HRK methylation- completely suppress HRK expression. Another possibi- positive tumors was significantly shorter than that for lity is that HRK expression is dependent on the presence those whose tumors were HRK methylation-negative of several distinct tumor sub-populations, one of which (Figure 3, P ¼ 0.028, log-rank test). In a number of might have lost the expression due to methylation, instances, the presence of CIMP in a tumor has been whereas others remained unmethylated and protein correlated with clinicopathological features and/or expressive. Alternatively, partial methylation at only prognosis (Toyota et al., 1999; Esteller et al., 2001). In some sites or methylation of only one allele in these our investigation, we found that methylation of multiple

Oncogene HRK inactivation in human malignancy M Nakamura et al S112

1.0 1.0

0.8 0.8 P =0.028 P = 0.784 0.6 0.6 HRK-M (–) 0.4 0.4 HRK-M (–)

0.2 0.2 Overall survival Overall Relapse-free survival HRK-M (+) 0 0 HRK-M (+)

10 20 30 10 20 30 40 Months Months

1.0 1.0 P<0.001 0.8 0.8 P =0.008

0.6 0.6 3M (–) 3M (–) 0.4 0.4

0.2 survival Overall 0.2 Relapse-free survival 0 3M (+) 0 3M (+)

10 20 30 10 20 30 40 Months Months Figure 3 (a and b) Kaplan–Meier curves reflecting the probability for 35 patients of having no recurrence with PCNSL in relation to the methylation status. A statistically significant difference in recurrence rate was observed between the promoter methylation-positive and promoter methylation-negative groups. There is no statistical difference for overall survival in relation to the methylation status. HRK-M ( þ ), methylated HRK group; HRK-M (À), unmethylated HRK group. (c and d) Kaplan–Meier curves for relapse-free survival (c) and overall survival (d) in relation to the methylation status of all 35 patients with PCNSL. 3M ( þ ), methylated more than three genes group; 3M (À), methylated less than three genes group. This figure is modified from Nakamura et al. (2006). PCNSL, primary central nervous system lymphoma.

genes (>3 genes) in PCNSLs was an independent and tumors and PCNSL, particularly when coupled with strong predictor of disease-specific recurrence and alleleic LOH. Inactivation of HRK in combination with survival (Figure 3). Epigenetic tumor profiling by the methylation profile of other multiple genes apparently methylation status might be a more powerful biomarker occurs in a substantial proportion of all tumor phenotypes of risk prediction in PCNSLs than are other clinico- and, as a potential proapoptotic gene, may contribute to pathological features and may reflect the silencing of the development and progression of these tumors. It is critical genes affecting growth control, proliferation, also noteworthy that PCNSLs exhibiting CIMP have a apoptosis and cell differentiation that can lead to significant correlation with shorter progression-free survi- selective growth advantage, tumor progression, recur- val and overall survival. In addition, we provide some rence and decreased survival. evidence that HRK may be involved in the progression from low-grade astrocytoma to secondary glioblastoma, but does not play a major role in the evolution of primary Conclusions glioblastoma.

Aberrant methylation of CpG islands within the promoter is an epigenetic event largely responsible for silencing of Conﬂict of interest the HRK gene and subsequent low apoptotic counts in our series of malignancies, including prostate cancer, astrocytic The authors declare no conﬂict of interest.

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