SASH1: a Candidate Tumor Suppressor Gene on Chromosome 6Q24. 3 Is

SASH1: a candidate tumor suppressor gene on chromosome 6q24.3 is downregulated in breast cancer

Constanze Zeller*,1, Bernd Hinzmann2, Susanne Seitz1, Helmuth Prokoph1, Elke Burkhard- Goettges1,Jo¨ rg Fischer3, Burkhard Jandrig1, Lope-Estevez Schwarz4, Andre´ Rosenthal2 and Siegfried Scherneck1

1Department of Tumor Genetics, Max Delbrueck Center for Molecular Medicine, Robert Roessle Str. 10, 13125 Berlin, Germany; 2MetaGen Pharmaceuticals GmbH, Oudenarder Str. 16, 13347 Berlin, Germany; 3Department of Medical Informatics, Robert Roessle Hospital, Lindenberger Weg 80, 13122 Berlin, Germany; 4Department of Surgery and Surgical Oncology, Robert Roessle Hospital, Lindenberger Weg 80, 13122 Berlin, Germany

Loss of heterozygosity (LOH) and in silico expression Introduction analysis were applied to identify genes significantly downregulated in breast cancer within the genomic Breast cancer (BC) represents the most common interval 6q23–25. Systematic comparison of candidate malignant neoplasm in Western countries affecting one EST sequences with genomic sequences from this interval in eight to one in ten woman (Casey, 1997). The revealed the genomic structure of a potential target gene knowledge of molecular genetic mechanisms underlying on 6q24.3, which we called SAM and SH3 domain breast tumorigenesis has increased since the discovery of containing 1 (SASH1). Loss of the gene-internal marker the breast cancer susceptibility genes BRCA1 and D6S311, found in 30% of primary breast cancer, was BRCA2 (Miki et al., 1994; Wooster et al., 1995). significantly correlated with poor survival and increase in Germline mutations in one of those genes account for tumor size. Two SASH1 transcripts of approximately 4.4 about 30% of hereditary BC in the German population and 7.5 kb exist and are predominantly transcribed in the (German Consortium for Hereditary Breast and Ovar- human breast, lung, thyroid, spleen, placenta and thymus. ian Cancer, 2002). However, not all patients with a In breast cancer cell lines, SASH1 is only expressed at strong family history of this malignancy display altera- low levels. SASH1 is downregulated in the majority tions in these high penetrance genes. Moreover, there is (74%) of breast tumors in comparison with corresponding no evidence of a correlation between mutations in normal breast epithelial tissues. In addition, SASH1 is BRCA1/BRCA2 and cases of sporadic BC, which make also downregulated in tumors of the lung and thyroid. up to 90–95% of all breast cancers. The search for yet Analysis of the protein domain structure revealed that uncharacterized genes commonly involved in the devel- SASH1 is a member of a recently described family of opment and/or progression of BC is complicated SH3/SAM adapter molecules and thus suggests a role in because of the great inter- and intratumor heterogeneity signaling pathways. We assume that SASH1 is a new of the disease (Devilee and Cornelisse, 1994). To date, a tumor suppressor gene possibly involved in tumorigenesis number of variations in different loci were found to of breast and other solid cancers. We were unable to find contribute to an increase in the lifetime breast cancer mutations in the coding region of the gene in primary risk. These genes are associated with rare genetic breast cancers showing LOHwithin the critical region. syndromes (TP53, ATM, PTEN, LKB1), metabolic or We therefore hypothesize that other mechanisms as for immunomodulatory pathways (such as GSTM1, instance methylation of the promoter region of SASH1 CYP19) or proto-oncogenic loci (for instance HRAS1) are responsible for the loss of expression of SASH1 in (De Jong et al., 2002). Genomewide loss of hetero- ONCOGENOMICS primary and metastatic breast cancer. zygosity (LOH) and comparative genome hybridization Oncogene (2003) 22, 2972–2983. doi:10.1038/sj.onc.1206474 (CGH) studies showed that large fractions of the human genome are aberrant in primary breast tumors and may Q1 Keywords: breast cancer; LOH analysis; 6q24.3; tumor harbor additional breast cancer-associated genes. suppressor genes; SAM; SH3 In our studies, we focused on the chromosomal region 6q23–25, which is a common site of allelic loss in BC (Fujii et al., 1996; Rodriguez et al., 2000) and also in other cancers like cervical (Acevedo et al., 2002), ovarian (Shridhar et al., 1999; Wan et al., 1999), pancreatic (Barghorn et al., 2001) and prostate tumors *Correspondence: C Zeller; E-mail: [email protected] (Srikantan et al., 1999). As LOH is an important Received 19 September 2002; revised 12 February 2003; accepted 12 hallmark in the process of tumor suppressor gene (TSG) February 2003 inactivation (Knudson, 1971), the 6q23–25 region SASH1 downregulation in breast cancer C Zeller et al 2973 appears to be a hot spot for several solid tumors and defined SROs were sites of common allelic loss in may contain one or more candidate genes involved in tumors showing more complex deletion patterns. We tumor suppression. This hypothesis was strongly sup- observed 31% LOH around marker D6S310, 30% at the ported by microcell-mediated chromosome transfer marker D6S311 and 33% at the D6S1654 locus. studies performed by our group, which showed that Furthermore, we looked for associations between the introduction of a normal human chromosome 6 LOH and selected clinicopathological parameters, fragment encompassing the microsatellite marker including survival time, histological type, estrogen D6S310 can suppress the neoplastic phenotype of the receptor (ER) status and tumor size. A statistically BC cell line CAL51 (Theile et al., 1996). significant correlation was observed for LOH at the In this report, we narrowed down the location of the D6S311 locus with survival time of patients (P- putative TSG candidates by allelotyping primary breast value ¼ 0.034). Four of 14 patients with a heterozygous tumors. Simultaneously, an in silico approach was deletion of D6S311 did not survive the clinical follow-up applied to obtain information about expressed genes (of up to 53 months). In the group without LOH of within the defined interval and about their in silico D6S311, however, one of 33 patients died within this expression profile. Combination of these methods interval of time. Also, LOH at the D6S311 locus was revealed two target sequences showing a significantly significantly associated with growing tumor sizes (P- reduced expression in breast tumor versus normal tissue. value ¼ 0.028). The rate of LOH was lowest among The first candidate gene was located next to the marker patients with small tumors (To2 cm). LOH increased D6S310, and matched the putative TSG LOT-1/ZAC linearly (P-value ¼ 0.041) with tumor size in patients (Abdollahi et al., 1997; Spengler et al., 1997). The and occurred most frequently in patients with tumors second candidate gene encompassing the expressed >5 cm. Except for D6S311, there was no further sequence tag (EST) Z28844 was located adjacent to significant correlation between LOH at the remaining D6S311. This marker is part of the gene KIAA0790 eight loci and any of the investigated clinicopathological (Nagase et al., 1998). We describe here the complete features. genomic structure of the gene which we called SAM and SH3 domain containing 1 (SASH1). We show that D6S311 resides within SASH1, a gene with potential SASH1 expression is downregulated in both breast signaling properties tumors and BC cell lines. An attempt to detect mutations in tumor specimens failed. However, various The association between LOH at D6S311 and reduced sequence variations in the coding region of the SASH1 survival time prompted us initially to search for TSG gene were identified. SASH1 encodes a protein contain- candidates around this marker. Searching public data- ing sterile a module (SAM) and Src homology domain 3 bases revealed that the marker D6S311 was located in (SH3) domains that are predominantly seen in signaling the first intron of a novel gene, known as KIAA0790 molecules, adapters and scaffold proteins. Since the derived from its cDNA entry. No other genes in the functional domains of the protein suggested a role in immediate vicinity to D6S311 were identified by either signaling pathways, we further characterized the expres- gene prediction methods from genomic sequence nor by sion of SASH1 in primary BC and other solid tumors. comparison of EST/cDNA sequences with genomic sequence entries. The SASH1 cDNA was assembled from a combination of ESTs including EST Z28844 and KIAA0790 Results mRNA (REFSEQ Accession number. XM_044015). Refined deletion mapping within 6q23–25 and association The complete SASH1 cDNA of 7709 bp, which we with clinicopathological parameters submitted to EMBL (Accession number BN000088), displays two polyadenylation signals at positions 4407 Nine polymorphic microsatellite markers covering the and 7685 bp, respectively (Figure 3). The human gene genomic region 6q23–25 were selected for LOH analysis comprises 20 exons (Figure 3) and contains an open (Figure 1) in a panel of 61 sporadic breast tumors reading frame of 1247 amino-acid residues with a (Table 1). The deletion pattern of tumors with partial molecular weight of approximately 140 kDa. Determi- allelic loss (Figure 2) allowed the identification of three nation of functional properties of the putative SASH1 independent smallest regions of overlapping deletions gene product using protein databases suggested a role in (SRO). Samples S186, S183A and S193 carried an signal transduction. The deduced protein harbors two interstitial deletion of the marker D6S310 which defined sterile a module (SAM) domains and a Src homology the SRO I of 560 kb, delimited by D6S1648 and domain 3 (SH3) indicative of adapter or scaffolding AFM269zg5. A second SRO (SRO II) of 1.98 Mb was functions. Furthermore, a proline-rich sequence span- defined by the samples S237, S189 and S210 showing ning 25 residues is located in the immediate amino- loss of the marker D6S311 and retention of hetero- terminal portion of the human protein. These findings zygosity at the adjacent markers D6S978 and D6S1637. supported its putative function as a TSG. Thus, we The third subregion SRO III encompassing 4.92 Mb was subjected the gene to further characterization. Based on marked by interstitial loss of the D6S1654 locus in the functional domains, we named the gene SAMand samples S200 and S209 while both alleles of markers SH3 domain containing 1 (SASH1). Alignment of D6S1637 and D6S441 were retained. Moreover, the SASH1 mouse (ENSMUSP00000015449) and human

Oncogene SASH1 downregulation in breast cancer C Zeller et al 2974

Figure 1 Schematic representation of LOH frequencies in sporadic breast tumors at the chromosomal region 6q23–25. On the left, the surveyed microsatellite markers are listed in their relative order from centromer (cen) to telomer (tel). Horizontal bars indicate LOH frequencies (% LOH). On the right, LOH frequencies (% LOH), the number of tumors tested (T), the number of tumors being informative (I) and the absolute number of tumors showing LOH (L) are documented

amino-acid sequences revealed high conservation Although the observed size of approximately 7.5 kb for throughout the conceptual protein (85% homology). the longer transcript did not directly correspond to a full-length transcript of 7.7 kb, we supposed that the EST cluster around Z28844 was part of an extended Distribution of SASH1 in normal tissues reveals two 30UTR of SASH1. Therefore, we performed Northern transcripts of 4.4 and 7.5 kb blot analysis using Z28844 as a probe (Figure 4b). A To elucidate the relationship between KIAA0790 and single transcript of about 7.5 kb could be detected in all the consensus sequence around Z28844, we looked for tissues but not in brain. We also looked for the existence corresponding transcription products. Determination of of a long SASH1 transcript using reversed transcrip- KIAA0790 expression in a variety of normal human tion–polymerase chain reaction (RT–PCR) with primers tissues by Northern blotting showed two transcripts of located in exon 4 and at the end of the assembly in cell approximately 4.4 and 7.5 kb (Figure 4a). Tissue lines derived from normal and tumor breast tissues. RT– distribution suggested ubiquitous expression of the PCR analysis of MCF10A, HBL100 and CAL51 4.4 kb transcript, which was most abundant in lung, confirmed the presence of a 6.8 kb transcript (data not placenta, spleen and thymus. The larger transcript was shown). Sequencing of the cloned 6.8 kb products expressed to a lesser extent in all tissues surveyed except revealed exactly the assembled SASH1 cDNA. Thus, for brain. The finding of two transcripts by Northern we verified that the cluster around Z28844 is part of an analysis is in agreement with the use of both poly- alternatively processed SASH1 mRNA with a long adenylation sites predicted from the SASH1 mRNA. 30UTR. In addition, comparison between the 30UTR of

Oncogene SASH1 downregulation in breast cancer C Zeller et al 2975 Table 1 Clinicopathological features mouse (EMBL Accession number. AJ507736) and Number of human SASH1 showed remarkable similarity (data not Characteristics cases shown). Moreover, we found no evidence of alternative splicing of SASH1 transcripts by program-based pre- Tumor size (cm) diction, EST sequences and RT–PCR experiments. o2 (pT1) 18 2–5 (pT2) 25 >5 (pT3/pT4) 16 (8/8) Unknown 2 In silico profiling and expression analysis shows significant downregulation of the SASH1 transcript in Histological type primary breast cancer and other solid tumors Invasive ductal with a predominant intraductal 7 component We then used an in silico approach to analyse the Invasive ductal 31 expression pattern of the SASH1 transcript in compari- Invasive lobular 15 Other 8 son with other transcripts mapped within the genomic region between D6S453 (141.0 Mb) and D6S442 Grade (155.3 Mb). This approach consists of two steps. First, Well differentiated (G1) 5 150 different ESTs that were identified to map between Moderately differentiated (G2) 26 Poorly/not differentiated (G3/G4) 27 (26/1) markers D6S453 and D6S442 (GeneMap’98) were Unknown 3 automatically extended using the software program ‘AUTEX’ (Schmitt et al., 1999). In a second step, the Estrogen receptor status ER positive 30 resulting extended EST consensus sequences were then ER negative 29 subjected to an electronic profiling method which we call Unknown 2 electronic Northern analysis or eNORTH. eNORTH Lymph node metastases allows to determine the in silico expression pattern of a Node-positive 28 cDNA fragment between normal and tumor tissues by Node-negative 17 counting the distribution of ESTs between these tissues, Unknown 16 and normalizing counts for each tissue in respect to the Distant metastases total number of ESTs available for each tissue. This Yes 18 analysis can be performed easily in silico as the tissue No 31 origin for each EST is available in the header informa- Unknown 12 tion. We used several millions of ESTs obtained by Age at diagnosis (years) sequencing from 30 human tissues and provided either o45 13 by the CGAP project at NCI or by Incyte Genomics. In 45–55 12 total, we identified 62 differentially expressed cDNA >55 36 sequences in 16 different tissues across the 14 MB. We Living status then focused on those transcripts that were differentially Alive 52 expressed only in breast cancer tissue versus breast Dead 6 Unknown 3 normal epithelial cells. With this approach, we were able to narrow down the number of candidate cDNAs down

Figure 2 Schematic representation of independently deleted smallest regions of overlap (SRO I-III) in 22 breast tumors showing partial deletions at 6q23–25 and candidate TSGs identiﬁed by expression analysis in silico. On the left, microsatellite markers and their relative map positions (http://www.ncbi.nlm.nih.gov/mapview/maps.cgi?orq ¼ hum&chr ¼ 6) are listed. Numbers on top identify the tumor samples. On the right, candidate genes are shown which were signiﬁcantly downregulated in breast tumors versus normal breast tissue. LOT-1/ZAC is located in SRO I while candidate SASH1 was found to be situated in SRO II. The region spanning SASH1 is outlined by a gray box. Black circles denote LOH, white circles retention of heterozygosity, gray circles homozygous loci, speckled circles represent untested tumors and hatched circles indicate microsatellite instability

Oncogene SASH1 downregulation in breast cancer C Zeller et al 2976

Figure 3 Schematic view of the genomic organization of SASH1. Genomic structure of SASH1 showing the 20 exons (boxes) to scale; sizes of introns (horizontal lines) are represented by vertical numbers. Gene regions encoding the SH3 domain (striped) and the two SAM domains (gray shaded) are shown. Hatched areas mark the 50UTR and 30UTR, respectively. Exon 1 contains the translation start site and the stop codon is situated in exon 20 followed by the polyadenylation signal (black circle). Alternatively, transcripts with an expanded 30UTR can be transcribed via use of a second polyadenylation signal located in the region of the EST cluster around Z28844. The BAC containing the 50 orientated region of the gene and the PAC clone spanning main parts of SASH1 are denoted by the lines on top. The accession numbers of the corresponding clone sequences are given above the line

Figure 4 Expression of SASH1 and Z28844 in human tissues. Northern blots containing 1 mg of poly(A)+RNA per lane from adult tissues (Clontech) were hybridized with (a)aSASH1 cDNA fragment encompassing nucleotides 834–4726 and designated SASH12-18. One major transcript of approximately 4.4 kb was observed in all tissues, while an additional transcript of about 7.5 kb was present in all tissues to a lower extent, (b) a cDNA probe (J12417) for the EST cluster around Z28844 comprising the 1.7 kb EcoRI/PacI insert within the IMAGE clone 212843 (GenBank Accession number H70389). The same ﬁlters were probed with a cDNA fragment of b- actin (Clontech) as a loading control

to 10 sequences. Eight sequences were upregulated in TSG candidate LOT-1/ZAC (12 ESTs from breast breast tumor versus normal tissue, while the remaining normal, one EST from breast tumor tissue, P-value ¼ two cDNAs showed a signiﬁcantly reduced expression in 0.0406, EST pool sizes: 120738 and 67596, respectively). breast tumor versus normal tissue. One was the known The other extended target cDNA sequence was

Oncogene SASH1 downregulation in breast cancer C Zeller et al 2977 assembled around the EST Z28844 containing the In silico expression analysis indicated that SASH1 marker D6S1118E and represented parts of the SASH1 transcripts are significantly reduced in breast tumor transcript. The electronic northern profile shows that tissues. In order to confirm this finding, we employed an SASH1 is downregulated in breast tumors as 15 ESTs RT–PCR approach using nine different BC cell lines were identified from normal breast tissue but only two and primers specific for exons 14 and 15. As controls we ESTs from breast tumor tissue (EST pool sizes: 120 738 used two immortalized mammary epithelial cell lines and 67 596, respectively). The P-value of 0.0425 for MCF10A and 184A1 (Figure 5). SASH1 expression is this observation is significant. The EST cluster around completely lost in the two of the nine BC cell lines Z28844 showing downregulation in breast tumors MDA-MB-231 and CAMA-1. In MCF7, BRC230, was localized 1 kb downstream of KIAA0790.As MT-1 and CAL51 the mRNA level is significantly this EST assembly did not show a significant open reduced while no change of the transcript level was reading frame, we assumed that it was part of a observed in MT-3, R193 and BT-20. These observations longer KIAA0790 transcript and represented its 50 or partly correspond to FISH analysis data on deletions of 30UTR. This was later confirmed by RT–PCR 6q in BC cell lines. All BC cell lines with deletion of the experiments. region 6q24.3 (Zhang et al., 1998) had reduced or

Figure 5 Expression of SASH1 in a panel of human cell lines (two normal breast (BN), nine breast cancer (BC) cell lines) detected by RT–PCR. A SASH1 cDNA fragment of 321 bp generated from exons 14 and 15 and the positive control for RNA integrity, a 126 bp portion of PBGD coding region, were ampliﬁed in a multiplex RT–PCR and visualized on a polyacrylamid gel stained with ethidium bromide (size marker (M))

Figure 6 Expression ratio of SASH1 in 15 primary breast tumors as compared to matched normal breast epithelia. Horizontal bars show the expression ratio of the matched samples which were normalized to PBGD. The codes of the tumors analysed are given on the left

Oncogene SASH1 downregulation in breast cancer C Zeller et al 2978 missing levels of SASH1 transcripts (BRC230, MT-1, sponding normal control tissue. SASH1 expression was MDA-MB-231 and CAMA-1). However, decreased examined by hybridization with a probe covering 697 bp expression was also observed in cell lines without loss from exons 4 to 10. SASH1 transcripts were significantly of 6q24.3 (MCF7, CAL51). reduced in 74% (37/50) of all breast tumors as compared We then asked the question if downregulation of to the corresponding normal tissue (Figure 7). The gene SASH1 transcripts was also a relevant event in vivo is also strongly downregulated in the three breast tumor during tumorigenesis. To address these questions, we metastasis samples spotted onto the Clontech Array. quantified the level of SASH1 mRNA in a panel of 15 The array also allowed us to study SASH1 expression primary breast tumors matched to normal breast tissues profiles across matched pairs from a number of other from the same patient using a highly sensitive fluores- solid tumors. SASH1 transcripts were strongly reduced cence-based RT–PCR assay. Primary breast tumors that in 19 of the 21 spotted lung tumor samples (90%) and in we had previously used for the LOH study at the five of the six spotted thyroid tumors. SASH1 expres- SASH1 locus could not be re-analysed because of sion was also downregulated in a few colon carcinoma shortage of tissue. Comparison of the relative mRNA samples (Figure 7). levels in the matched samples showed a reduced expression in all tested tumors. In nine of the 15 samples Mutation analysis in sporadic and familial BC we observed at least a twofold reduction of SASH1 transcripts as compared to the corresponding normal To investigate whether sequence alterations of SASH1 breast tissues (Figure 6). could be responsible for its downregulation in primary To confirm these results, we investigated a larger breast tumors and BC cell lines, we analysed 66 primary panel of tumors using a Cancer Expression Profiling breast tumors for somatic mutations in the entire coding Array (Clontech), which contained cDNAs from 50 region and splice junctions of the gene. The panel different breast tumor tissues matched with the corre- included tumors subjected to LOH analysis at the region

Figure 7 SASH1 expression in normalized cDNAs from normal and tumor tissues. The Cancer Proﬁling Expression Array (Clontech) was hybridized with a SASH1 cDNA probe (SASH1-Ex4-10) covering nucleotides 834 – 1531. To verify equally spotted amounts of cDNA, the array was also hybridized with a cDNA probe for ubiquitin (Clontech) (data not shown). Cell lines: HELA (cervix cancer), Daudi (Burkitt’s lymphoma), K562 (chronic myelogenous leukemia), HL-60 (promyelocytic leukemia), G361 (melanoma), A549 (lung carcinoma), MOLT-4 (lymphobic leukemia), SW480 (colorectal adenocarcinoma), Raji (Burkitt’s lymphoma). Controls in vertical order from top to bottom: ubiquitin cDNA, yeast total RNA; yeast tRNA; Escherichia coli DNA; poly(A); human C0t-1 DNA; human genomic DNA. N, normal tissue; T, tumor tissue. The outlined groups of dots represent normal, metastatic (spotted side by side) and tumor tissue from the same patient. The arrow indicates one sample of matched normal and tumor colon tissue in the column of stomach samples

Oncogene SASH1 downregulation in breast cancer C Zeller et al 2979 Table 2 Frequencies of sequence variations in SASH1 in sporadic BC, familial BC and controls Frequency Amino-acid in sporadic Frequency in Frequency SNP reference Location Nucleotide change1 change2 Allele BC familial BC in controls cluster ID Exon 1 529-530insCCCGAG 18 19ins PE À/À 0.76 0.82 0.68 À/ins 0.19 0.18 0.30 ins/ins 0.05 0.00 0.02 Intron 4 861+64C>A CC 0.51 0.37 0.40 CA 0.34 0.43 0.38 AA 0.15 0.20 0.22 Exon 6 967A>G AA 0.20 0.26 n.d. rs1883625 AG 0.51 0.37 n.d. GG 0.29 0.37 n.d. Intron 7 1102+114T>C TT 0.76 0.86 0.70 TC 0.16 0.14 0.30 CC 0.08 0.00 0.00 Exon 10 1558C>A CC 1.00 0.96 0.96 CA 0.00 0.04 0.04 AA 0.00 0.00 0.00 Exon 10 1666C>T CC 0.97 0.82 0.90 CT 0.00 0.00 0.10 TT 0.03 0.18 0.00 Intron 15 2210-37G>C GG 0.88 0.77 0.74 GC 0.09 0.18 0.22 CC 0.03 0.05 0.04 Exon 15 2287G>A GG 0.74 0.91 0.70 rs2294787 GA 0.23 0.09 0.30 AA 0.03 0.00 0.00 Intron 17 2684+24G>A GG 0.74 0.75 n.d. GA 0.23 0.21 n.d. AA 0.03 0.04 n.d. Exon 18 3126G>A R884Q GG 0.41 0.36 0.30 rs208696 GA 0.42 0.48 0.38 AA 0.17 0.16 0.32 Exon 18 3559C>A H961N CC 0.99 1.00 0.98 CA 0.01 0.00 0.02 AA 0.00 0.00 0.00 Exon 18 3640A>G AA 0.99 1.00 1.00 AG 0.01 0.00 0.00 GG 0.00 0.00 0.00 Exon 18 3748G>A GG 0.99 1.00 1.00 GA 0.01 0.00 0.00 AA 0.00 0.00 0.00 Exon 18 3802G>A GG 0.99 1.00 1.00 GA 0.01 0.00 0.00 AA 0.00 0.00 0.00 Exon 19 3895C>T CC 1.00 0.98 0.98 CT 0.00 0.02 0.02 TT 0.00 0.00 0.00 Exon 19 3896G>A V1141I GG 0.99 1.00 1.00 GA 0.01 0.00 0.00 AA 0.00 0.00 0.00 Exon 19 3932C>T L1153F CC 1.00 0.98 1.00 CT 0.00 0.02 0.00 TT 0.00 0.00 0.00 Exon 20 4435T>C TT 0.72 0.77 n.d. rs8641 TC 0.22 0.18 n.d CC 0.06 0.05 n.d.

1Sequence numbering follows the published sequence for human SASH1 cDNA (EMBL Accession number BN000088); 2The amino-acid positions are based on the protein sequence for human SASH1 protein (EMBL Accession number CAD47811) n.d: not detected

6q23–25, as described above. We also investigated the tions include four missense, nine silent and four intronic SASH1 gene for mutations in blood samples from 44 changes as well as one insertion of two amino acids. The patients with familial breast cancer as the region 6q23– substitutions in intronic sequences of SASH1 did not 25 may also be prone to harbor a gene involved in affect splice sites. With the exception of the 6 bp inherited BC (Zuppan et al., 1991). insertion in exon 1, three silent mutations in exon 18 As shown in Table 2, we observed a total of 18 (3640A>G, 3748G>A, 3802G>A) and two missense sequence alterations in the SASH1 gene. These altera- mutations in exon 19 (3896G>A, 3932C>T), the

Oncogene SASH1 downregulation in breast cancer C Zeller et al 2980 observed variations represent single-nucleotide poly- et al., 1999). In the absence of loss-of-function muta- morphisms (SNPs) with similar frequencies in patients tions, inactivation of the putative TSG was suggested to and controls. Interestingly, an insertion of six nucleo- be mediated via promoter methylation and imprinting tides (exon 1) causing an in-frame insertion of two status (Bilanges et al., 1999; Fagotto et al., 2000). amino acids was found in various patients and controls. Here we report on the identification and initial This insertion predicts a new variant of the protein with characterization of a novel TSG candidate for BC, an additional proline (P) and glutamic acid (E) residue which we refer to as SASH1. This gene was identified by in the proline-stretch region (PE-region). a combination of LOH mapping in primary BC and Upon mutation screening we did not detect tumor- expression profiling analysis in silico within the critical specific mutations in sporadic BC. In detail, substitu- region of 6q23–25 flanked by the markers D6S1648 and tions in exon 18 (i.e. 3559C>A, 3640A>G, 3748G>A, D6S442, proximal and distal of D6S310, respectively. 3802G>A) and exon 19 (3896G>A) were not only Fine mapping revealed three regions of smallest over- found in the tumor but also in the corresponding normal lapping deletions. These were located next to the breast tissue of the patients. No mutations were revealed markers D6S310 (SRO I spanning 560 kb, 31% LOH), within the functional domains of the gene. As the D6S311 (SRO II encompassing 1.98 Mb, 30% LOH) homozygous G>A substitution at position 2287 (SH3 and D6S1654 (SRO III encompassing 4.92 Mb, 33% domain) observed in two BC samples and missing in LOH). We performed correlation studies in order to controls does not cause an amino-acid change, it was show association between LOH around microsatellite not regarded to be critical. markers and specific clinicopathological parameters. Across the set of hereditary BC we observed a Loss of D6S311 was significantly associated with shorter heterozygous mutation (3932C>T) which did not occur survival time and greater tumor size of BC patients in in sporadic BC and controls. The substitution 3932C>T comparison with patients with retention of both alleles. leads to a heterozygous amino-acid change from leucin This finding suggested that loss of a novel gene located to phenylalanine. Although this position is conserved in in the vicinity of D6S311 may be responsible for a mouse and human, both amino acids display a hydro- growth advantage to breast tumor cells and may phobic character and therefore the influence of this contribute to the progression of BC. In this respect, variant seems to be moderate. D6S311 might serve as a prognostic marker as LOH of In summary, we could not find inactivating events D6S311 would indicate a poor outcome of the disease. such as premature stop codons in sporadic and familial On the basis of the above findings we initiated an in BC. Strikingly, we could neither demonstrate mutations silico expression analysis of all ESTs anchored around the in patients with LOH at D6S311/D6S1637 nor in BC marker D6S311 and identified a novel TSG candidate in samples showing reduced expression of SASH1. These the vicinity of this marker. This TSG candidate contains data suggest mechanisms other than mutations in EST Z28844 and is downregulated in primary breast coding sequences to be involved in downregulation of cancers. Detailed sequence analysis showed that this TSG SASH1. candidate is identical with the long 7.5 kb transcript of a novel gene which we named SASH1 and which was derived and annotated from the KIAA0790 cDNA Discussion sequence. The SASH1 gene harbors the microsatellite marker D6S311 and is located in 6q24.3. Allelic losses in the chromosomal region 6q23–25 have The SASH1 gene shows a high degree of conservation frequently been associated with BC and many other among mouse and human on the nucleotide and protein solid tumors, which supports the hypothesis that this level. Based on the presence of an SH3 and two SAM region harbors one or more TSG possibly involved in domains, the putative protein may have a function in tumor formation and progression (Tirkkonen et al., signal transduction cascades. SH3 domains mediate 1998; Huang et al., 2000; Stallmach et al., 2002). In protein–protein interactions by binding to polyproline addition, we previously showed that microcell-mediated motifs and are involved in a broad spectrum of transfer of a small chromosomal fragment around the processes such as tyrosine kinase signaling and cytosce- marker D6S310 on 6q23 causes loss of the tumorigenic letal organization (Pawson, 1995; Buday, 1999). A phenotype in CAL51 breast cancer cells (Theile et al., similar function is attributed to SAM domains that 1996). However, we failed to detect any candidate TSG not only show the ability to self-associate (Carroll et al., after complete sequence analysis of the PAC dJ181M7, 1996) but can also heterotypically interact with other which contains marker D6S310 and covers a large proteins (Serra-Pages et al., 1995). Moreover, Kim et al. genomic region around this marker (unpublished (2002) recently suggested that polymerization of SAM results). To date, the gene LOT-1/ZAC located near domains in transcriptional repressers yields repressive the marker D6S310 has been suggested as a potential chromatin structure and is involved in transcriptional TSG for ovarian and breast cancer. LOT-1/ZAC control. Based on the functional domains SASH1 may encodes a zinc-finger protein which was shown to belong to the previously described novel family of exhibit antiproliterative activities by regulating apopto- putative adapter and scaffold proteins. This family sis and cell cycle arrest. Expression of the gene is includes the SAM-and SH3-containing genes HACS1 reduced or lost in ovarian and breast cancer cell lines (Claudio et al., 2001), NASH1 (Uchida et al., 2001) and and primary tumors (Abdollahi et al., 1997; Bilanges SLY (Beer et al., 2001).

Oncogene SASH1 downregulation in breast cancer C Zeller et al 2981 The functional features of SASH1 support the idea proteins or with the SASH1-internal SH3 domain, we that the gene is possibly involved in tumor suppression. speculate that the binding properties of the mutant protein Therefore, we analysed the expression of the gene in variant may be changed because of altered conformation. vitro and in vivo. SASH1 expression was verified in a Our results show that reduced expression of SASH1 variety of tissues, preferentially in breast, lung, thymus, may not be attributed to somatic mutations in the thyroid, placenta and spleen. Northern analysis showed coding sequences of SASH1. Other mechanisms are that two mRNA transcripts of approximately 4.4 and likely responsible for the loss of expression of SASH1 at 7.5 kb that differ only in the length of the 30UTR exist. this locus. Transcriptional mechanisms including epige- The length of the shorter 4.4 kb transcript was in netic changes, genetic variations in the promoter as well agreement with the annotated KIAA0790 cDNA. The as negative or positive expression of binding factors may 7.5 kb isoform is expressed at lower levels in the be responsible for the inactivation of SASH1. Further- examined tissues except for brain. more, alterations in the 30UTRs of the different In six of nine breast cancer cell lines, SASH1 is transcripts might change post-transcriptional regulation significantly reduced or completely lost at the mRNA of gene expression. On the other hand, inactivation of level. Most of these cell lines displayed chromosomal SASH1 may provide cancer cells with an advantage in loss of the region 6q24.3, which suggested that down- microevolution, as proposed for the TSG E-cad (Cheng regulation may partially be caused by loss of the gene. et al., 2001). SASH1-specific polyclonal and monoclonal Using a sensitive RT–PCR assay and expression antibodies may be versatile tools to provide more insight analysis with a panel of spotted primary breast cancers into the gene’s function. As SASH1 belongs to a novel and corresponding normal controls, we proved a strong family of putative adapter and scaffold molecules and decrease of SASH1 mRNA in about 70% of total 65 has been shown by us to be downregulated in primary primary BC samples. In addition, SASH1 was also breast cancer and other solid tumors, it will be significantly reduced in primary tumors of lung and interesting to investigate if other members of this family thyroid. Loss of the long arm of chromosome 6 is a also possess a similar potential to act as tumor frequent event in lung tumors and cell lines (Wurster- suppressors. Hill et al., 1984; Virmani et al., 1998). Interestingly, Goeze et al. (2002) suggested that deletion of 6q21–qter is associated with the metastatic phenotype in primary Materials and methods lung adenocarcinomas. This implies that a putative TSG Materials from this area could be involved in more aggressive stages of lung tumors when lost. We did not observe a A total of 78 tumor specimens and corresponding nontumor decreased expression of SASH1 in most of the primary epithelial tissues from breast cancer patients who had under- tumors although high frequencies of LOH have been gone surgery were collected at the Robert Roessle Hospital reported for SASH1-internal markers D6S311 and (Berlin, Germany). In total, 61 samples were utilized for LOH studies, 66 for mutation analysis and 12 were selected for D6S1637 in ovarian cancer (Shridhar et al., 1999). semiquantitative RT–PCR. Additionally, blood samples from To further investigate whether SASH1 may function 44 breast cancer patients with a family history of BC, collected as a TSG in BC, we searched for the ‘second hit’ at the Max Delbrueck Center (Berlin, Germany), were according to Knudson’s ‘two hit hypothesis’. Characteri- investigated for SASH1 mutations. Blood samples (50) from zation of genetic alterations in 66 sporadic breast tumors German patients, who had attended Berlin hospitals, served as and lymphocytes from 44 BC patients with a strong family controls. The study was performed with the approval of the history of breast cancer did not reveal a loss-of-function local ethics committee. mutation. We identified 14 new sequence alterations, six of The cell lines used in this study were obtained from the which were silent substitutions, and four were detected in American Type Culture Collection (ATCC) (Rockville, MD) intronic regions. We found three new missense substitu- (MCF10A, 184A1, MDA-MB-231) or were kindly provided by J Gioanni (Centre Antoine-Lacassagne, Nice, France) tions and one in-frame insertion of two amino acids. The (CAL51), D Amadori (Morgagni-Pierantoni Hospital, Forli, observed silent (3748G>A, 3802G>A) and missense Italy) (BRC230), I. Fichtner (MT-1, MT-3) and M. Theile mutations (3896G>A) were not specific to the sporadic (MDC, Berlin, Germany) (CAMA-1, MCF7, BT-20) and Dr tumors and did not occur in tumors with LOH of the wild- Arnold (atugen, Berlin, Germany) (R193). type allele. In hereditary BC, the role of the missense To confirm their identity, the cell lines were screened with mutation 3932C>T needs further determination in microsatellite markers D16S539, D5S818 and D7S820 and the additional affected family members. The frequency of resulting fingerprints were compared with the corresponding the observed sequence variations did not significantly entities in the ATCC database of DNA fingerprints of STR differ in BC and controls. Although functional studies (http//:www.atcc.org/). have not been performed, we assume that most of these variations do not alter the function of SASH1 and, in DNA/RNA extraction consequence, do not play an important role in BC. DNA was extracted using standard phenol/chloroform meth- However, the observed polymorphism in the first exon ods. Total RNA was prepared using the Perfect RNA, results in a new variant of the protein carrying an Eukaryotic, Mini Kit (Eppendorf) or the TRIZOL reagent additional proline and glutamic acid residue in the PE- (Life Technologies) following the manufacturer’s instructions. region. Since the PE-region of SASH1 could be of Frozen tissue samples were sectioned with a cryostat functional importance regarding interactions with other microtome. The tumor cell content, which was assessed in an

Oncogene SASH1 downregulation in breast cancer C Zeller et al 2982 adjacent hematoxylin–eosin stained section, was estimated to cDNA sequencing. Primers used for SASH1 were U1824 (50- be more than 50%. CGGGAAAGCGTCAAGTCGGA-30) and L2145 (50-CCAG- GAGATCCTCCACAGAC-30). RT–PCR was carried out in a LOH analysis one-step reaction using QIAGEN One-Step RT–PCR Kit or in a two step-reaction using QIAGEN Omniscript RT Kit Primers for the nine polymorphic markers were synthesized according to the manufacturer’s instruction. In one-tube according to published primer sequences (Genome Data Base, multiplex RT–PCR, 100 ng RNA were used in a 25 ml reaction 1998). The order of the markers was derived from existing volume. In the two-tube RT–PCR, 400 ng RNA were subjected consensus maps (Cedar Genetics, 1999; http://cedar.genetics.- to randomly primed RT in a 25 ml PCR reaction. Initial soton.ac.uk/pub/chrom6). denaturation was performed at 951C for 5 min, followed by 40 PCR were carried out with 40 ng DNA in PCR buffer cycles of 30 s at 951C, 30 s at 621C and 1 min at 721C, with a m (Perkin-Elmer), 0.25 m of each primer (one primer of each final extension at 721C for 10 min. pair was fluorescence labeled), 375 mm of dNTPs each and 0.25 U Taq-Polymerase (Perkin-Elmer). Denaturation at 941C for Semiquantitative fluorescent RT–PCR analysis Expression 5 min was followed by 30–35 cycles of 30 s at 941C, 30 s at analysis of primary breast tumors and matched tissues was annealing temperature (between 52 and 651C) and 30 s or 1 min performed with fluorescent dye-labeled primers as described at 721C, with a final extension of 10 min, 721C. above (U1824-TET, PBGD-HEX). For quantification, PCR The PCR products were mixed with an internal standard products were applied to an ABI 377 DNA sequencer. Data size marker and fractionated on a denaturating 6% poly- were collected automatically and analysed by the Genescan 3.1 acrylamide gel using an ABI 377 DNA sequencer. LOH data software (ABI), which provides quantification based on peak were automatically analysed by comparing nontumor and size and peak area. tumor tissue allele peak sizes, heights and area ratios. Intensity The reproducibility of the method was confirmed by two or signal ratio differences of 35% or more were considered independent PCRs for two matched samples. The PCR sufficient for LOH assignment. products were subjected to quantification six times. Means and s.d.’s were evaluated and found within a reproducible Statistical analysis range. All statistical analyses were done using the statistical program SPSS. To evaluate associations between LOH and clinico- Cancer profiling array Expression analysis of SASH1 was pathological variables, we applied Fisher’s exact test (Cross- performed using a Cancer Profiling Array (Clontech), which tabs statistics) in the case of categorical variables and the contained matched cDNA pairs of individual patients. In Mann–Whitney test or the H-test of Kruskal and Wallis in the addition, cDNA from cancer cell lines and several positive and case of noncategorical variables. Survival curves calculated by negative controls were immobilized on the array. All cDNAs Kaplan–Meier estimations were compared according to the were normalized based on the expression of four housekeeping log-rank test (Sachs, 1992). For all statistical tests, we used a genes. SASH1 (SASH1-Ex4-10) and ubiquitin (Clontech) comparison related significance level of Po0.05. cDNA fragments were 32P-random-primer labeled (Random Labeling Kit, Roche) and used to probe the same filter. Expression analysis Prehybridization, hybridization and washing were performed following the manufacturer’s protocol. Expression analysis in silico Data were obtained following the procedure decribed by Schmitt et al. (1999) using EST information from public (NCI) and proprietary sources (Incyte Mutation analysis Genomics). The set of data comprised a total of 2 017 017 Exons 1–20 of SASH1 were amplified by PCR using intronic ESTs, which included 120 738 ESTs from breast normal and primer sequences (available upon request) and examined by 67 596 ESTs from breast tumor tissue. single-strand conformation polymorphism analysis (SSCP). PCR was carried out with 100 ng DNA, 15 ml Taq PCR Master Northern blots Multiple tissue northern (MTN) filters were Mix (QIAGEN), 15 pmol primer each in a final volume of purchased from Clontech, which were loaded with 1 mgof 30 ml. Cycling parameters included an initial denaturation step poly(A)+RNA per lane. Probes corresponding to cDNA at 951C for 5 min, 35 cycles of denaturation at 951C for 30 s, fragments SASH12-18, J12417 and b-actin (Clontech) annealing at optimal temperature (ranging from 51 to 681C) were labeled with 32P-dCTP using Prime-a-Gene system (Pro- for 40 s and extension at 721C for 1 min 30 s, followed by a mega). Each blot was stripped and reprobed with final extension at 721C for 10 min. PCR products showing b-actin after successful hybridization with SASH1-specific mobility shifts in the SSCP-Gel were purified with a QIAquick fragments, respectively. Prehybridization, hybridization and PCR purification Kit (QIAGEN) and then sequenced on ABI washing were performed as recommended by the manufacturer. 377 sequencers.

RT–PCR Gene expression was measured determining the exponential range of amplification of the PCR for SASH1 and Acknowledgements the normalization control gene porphobilinogen deaminase/ We thank K Poppe and S Werner for the excellent technical hydroxymethylbilane synthase (PBGD) (Yoo et al., 1993). To assistance. This work was supported by: Deutsche Krebshilfe confirm product specificity, PCR products were subjected to (Grant number: 10-1249).

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