Identification of Smyd4 As a Potential Tumor Suppressor Gene Involved in Breast Cancer Development

Published OnlineFirst April 21, 2009; DOI: 10.1158/0008-5472.CAN-08-4097 Published Online First on April 21, 2009 as 10.1158/0008-5472.CAN-08-4097

Research Article

Identification of Smyd4 as a Potential Tumor Suppressor Gene Involved in Breast Cancer Development

Liping Hu, Yiwei Tony Zhu, Chao Qi, and Yi-Jun Zhu

The Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois

Abstract different chromosomes, the recombination catalyzed by Cre leads to the translocation. If loxP sites are integrated into DNA in the To identify genes involved in breast tumorigenesis, we same direction on same chromosome, the recombination leads to applied the retroviral LoxP-Cre system to a nontumorigenic the deletion of the interval DNA between two loxP sites. The mouse mammary epithelial cell line NOG8to create random proviral integration sites of loxP carrying vectors can be chromosome deletion/translocation. We found that the determined through inverse PCR and sequencing (4, 5). disruption of one allele of Smyd4 (SET and MYND domain The loss of heterozygosity or DNA methylation changes at containing 4) gene through chromosome translocation led to human chromosome 17p13.3, in absence of any p53 genetic tumorigenesis. The expression of Smyd4 was markedly alterations at 17p13.1, seems to be frequently involved in various decreased in tumor cells. Re-expression of Smyd4 resulted types of human malignant tumors including sporadic breast in growth suppression of tumor cells and inhibition of tumor cancers, ovarian cancers, medulloblastomas, and small cell lung formation in nude mice. Furthermore, the RNA interference– carcinomas (6–10). These results suggest that one or more tumor mediated suppression of Smyd4 expression in human suppressor genes located at 17p13.3 could be involved in MCF10A mammary epithelial cells caused their growth in tumorigenesis. Among more than hundreds of genes residing in soft agar. Microarray studies revealed that platelet-derived this region, previous studies have resulted in the identifications of growth factor receptor A polypeptide (Pdgfr-A) was highly several tumor suppressors, including MAX binding protein, hyper- expressed in tumor cells compared with NOG8cells. Re- methylated in cancer 1, and ovarian cancer gene 1 (11–14). Loss of expression of Smyd4 significantly reduced the expression of MAX binding protein severely disrupts mammary gland involution Pdgfr-A in tumor cells. In human breast cancers, reverse and leads to hyperplastic ducts (15), but no mutations in transcription-PCR results revealed that Smyd4 expression MAX binding protein have been described in human breast tumors was totally silenced in 2 of 10 specimens. These findings (16–18). Hypermethylated in cancer 1 is hypermethylated and indicate that Smyd4, as a potential tumor suppressor, plays a transcriptionally silent in many types of human cancers including critical role in breast carcinogenesis at least partly through breast cancer (13, 19). Heterozygous hypermethylated in cancer inhibiting the expression of Pdgfr-A, and could be a novel 1 mutant mice do not develop mammary gland tumors, although target for improving treatment of breast cancer. [Cancer Res they are predisposed to other malignant tumors including 2009;69(9):4067–72] pulmonary carcinomas, lymphomas, and sarcomas (20). There could be additional breast tumor suppressor genes located at 17p13.3. Introduction SET domain often carries lysine methyltransferase activity for Breast cancer is one of the leading causes of cancer deaths in histone modification. Among over 50 SET domain containing women in the United States. Over the past decades, the intensive proteins in the mammalian genome (21), Smyd family consisting of efforts on breast cancer to identify cancer genes and understand 5 members is defined by a SET domain that is separated into how they contribute to tumorigenesis have lead to a better 2 fragments by an MYND domain, followed by a cysteine-rich post– understanding of molecular characterization. Identification of SET domain (22, 23). Smyd1 possesses histone H3K4 methyltrans- cancer genes has been greatly facilitated by analyzing recurring ferase activity (24). Targeted disruption of Smyd1 gene results in chromosome rearrangements (1) that are found in the malignant impaired cardiomyocyte maturation, defective cardiac morpho- cells of most patients with leukemia, lymphoma, and solid tumors genesis, and embryonic lethality (25). Smyd2 was reported to carry (2). Chromosomal engineering in the mouse genome has been histone H3K36–specific methyltransferase, inhibit transcription, achieved using Cre-loxP technique (3). The Cre recombinase and suppress cell proliferation (23). Recent findings reveal that the belongs to the E integrase family and catalyzes recombination substrate of Smyd2-mediated methylation also includes nonhistone between 2 identical 34-bp sites called loxP. Recently, to identify protein such as tumor suppressor p53 (26). Like Smyd1, Smyd3 also additional breast cancer genes, we developed Cre-loxP–based possesses histone H3K4 methyltransferase activity (27). The Smyd3 method to generate random chromosome rearrangement such as expression is up-regulated in colorectal and hepatocellullar translocation and deletion by integrating two loxP sites into the carcinoma (27). Increased expression of Smyd3 is essential for mouse genome (4). If two loxP sites are integrated into DNA on two the proliferation of breast cancer cell line via its regulating expression of proto-oncogene WNT10B (28). Besides modifying histone, Smyd3 also methylates lysine 831 of VEGFR1 protein (29). Note: Supplementary data for this article are available at Cancer Research Online The functions and enzyme activities of Smyd4 and Smyd5 remain (http://cancerres.aacrjournals.org/). Requests for reprints: Yi-Jun Zhu, Department of Pathology, Feinberg School of to be defined. Medicine, Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611-3008. Here, we report our effort to identify and characterize the role of Phone: 312-503-3113; Fax: 312-503-8249; E-mail: [email protected]. I2009 American Association for Cancer Research. Smyd4 as a potential tumor suppressor in breast cancer, which is doi:10.1158/0008-5472.CAN-08-4097 located at human 17p13.3. Smyd4 was found to significantly www.aacrjournals.org 4067 Cancer Res 2009; 69: (9). May 1, 2009

Cancer Research suppress the breast tumorigenesis at least partly through inhibiting expression of platelet-derived growth factor receptor a (Pdgfr-a)in breast tumor cells.

Materials and Methods Constructs and cell culture. Plib-Smyd4-Bla was constructed by inserting EcoRI/XhoI fragment of Smyd4 cDNA into EcoRI/SalI sites of Plib-Bla. Plib-Bla was generated by inserting SV40-directed blasticidin resistance gene into ClaI/SalI sites of PliB (Clontech). NOG8 and the tumor cell line were grown in DMEM, supplemented with 10% fetal bovine serum (FBS) at 37jC in a humidified atmosphere of 5% of CO2. MCF10A were maintained in DMEM/F12 containing 5% horse serum, 10 Ag/mL human insulin, 0.5 Ag/mL hydrocortisone, and 20 ng/mL epidermal growth factor (Sigma-Aldrich). The NOG8 cells with recombination carried an intact puromycin- resistant gene were selected and cultured in the regular medium containing 2 Ag/mL of puromycin (Invitrogen). The NOG8 cells carrying recombination are injected s.c. into the nude mice for tumor formation. The tumor cells were harvested from the primary tumor dissected out of mouse and cultured in the medium containing puromycin. Chromosome engineering and tumor development. The chromosome engineering on NOG8 cells was performed as described (4). Phoenix-Eco packaging cells (5 Â 106) were transfected with 15 Ag of LacZ/Neo-5¶Pur- LoxP-TK or Hyg-3¶Pur-LoxP vector through the calcium phosphate precipitation method. Forty-eight hours after the transfection, LacZ/Neo- 5¶Pur-LoxP-TK viral supernatant was added to NOG8 cells (5 Â 105) with 8 Ag/mL of polybrene. Hyg-3¶Pur-LoxP retrovirus was used for infection on the second day. The cells were subjected to G418 and hygromycin selection 48 h after retroviral infection. One week after selection, cells from one 10- cm plate were infected with 108 adenovirus expressing Cre recombinase. Puromycin (1 Ag/mL) was added to the medium 24 h later, and the cells were selected with puromycin for 5 d. The cells were then injected s.c. into the nude mice for tumor formation. Two months later, the tumor was harvested for further analysis. Inverse PCR and localization of retroviral integration site. Ten micrograms of tumor DNA was digested with BamHI /ApoI for localization of the 3¶ long terminal repeat (LTR) or SSPI/Afl III for localization of the 5¶LTR. The digested DNA was blunt with T4 polymerase and then circularized by ligation using T4 ligase (New England Bio Labs) in a total volume of 100 AL at room temperature overnight. Circular DNA was purified with phenol/chloroform extraction, precipitated with ethanol, and dissolved in 50 AL of water. The 100 AL of PCR reaction mixture contains Figure 1. Disruption of one allele of Smyd4 gene caused markedly reduced A, 0.2 mmol/L each of deoxynucleotide triphosphates, 10 pmol of each forward expression of Smyd4. chromosome rearrangement (translocation or deletion) A Â A can be accomplished using cre-loxP systems. An artificial intron with loxP or reverse primer, 30 Lof3.3 buffer, 4.8 L of Mg(OAc)2 (25 mmol/L), sequence was introduced into the puromycin-resistant gene. The intact and 2 AL of rTh polymerase (Roche). One microliter of primary PCR product puromycin-resistant gene were divided into two parts (5¶ part with loxP and was used as template in second PCR reaction. The second PCR products 3¶ part with loxP) and cloned into PLIB vector to generate two vectors: ¶ ¶ ¶ were separated on 1% of agarose gel, and purified with the QIA quick gel LacZ/Neo-5 Pur-LoxP-TK and Hyg-Loxp-3 Pur, respectively. For 1 tumor, the 5 Pur-LoxP-TK was inserted into the 10th intron of Smyd4 gene on mouse extraction kit (Qiagen). The purified second PCR products were sequenced chromosome 11, whereas 3¶ Hyg-Pur-LoxP was inserted into 6th intron of Msra at Northwestern University Biotech Facility. The retroviral integration site gene on chromosome 14. The recombination between the two loxP sites by Cre was determined through blasting the sequence against National Center for led to translocation between chromosome 11 and 14, creating a fusion gene. B, Biotechnology Information mouse genome database. The primary and confirmation of the translocation between chromosome 11 and 14. The recombination was further confirmed by PCR with one primer located in the nested primers used were as follows:. Smyd4 on chromosome 11 and another primer specifically for Hyg-3¶Pur-loxP For locating the 5¶LTR: 5¶-CATGGTCAGGTCATGGATGA-3¶ and 5¶- vector (lane 1, 6.7-kb), and another PCR with one primer located in the Mrsa on AGGAACAGCGAGACCACGATT-3¶; its nested primers: 5¶-ACGATGGTGCA- chromosome 14 and another primer specifically for LacZ/Neo-5¶Pur-LoxP-TK lane 2, M, C, GGATATCCT-3¶ and 5¶-GATGCAAACAGCAAGAGGCT-3¶. vector ( 2.2 kb). DNA 1 kb plus Marker (Invitrogen). the reduced ¶ ¶ ¶ ¶ expression of Smyd4 in mammary tumor cells was revealed by quantitative real- For locating the 3 LTR: 5 -CCGCTAAAGCGCATGCTCCA-3 and 5 - time RT-PCR. TGCAAGAACTCTTCCTCACG-3¶; its nested primers: 5¶-CTGCCTTGGGAA- AAGCGCCT-3¶ and 5¶-CTCGACATCGGCAAGGTGT-3¶. Generation of tumor cell line re-expressing Smyd4. The retrovirus Reverse transcription-PCR and real-time RT-PCR. RNAs from packaging cells, 5 Â 106 of Phoenix cells, were transfected with 15 Ag of Plib- cultured cells were prepared using TRIzol (Invitrogen). Reverse trans- Smyd4-Bla or Plib-Bla through calcium phosphate precipitation method ciption-PCR (RT-PCR) was performed using SuperScript III One-Step RT- (Clontech). Forty-eight hours after transfection, the viral supernatant was PCR System (Invitrogen) with 1 Ag of total RNA. PCR products were used to infect tumor cells established from the original tumor. The cells analyzed on 1% agarose gels and visualized by ethidium bromide staining. were selected with 10 Ag/mL of blasticidin (Invitrogen) 48 h later. In 1 wk, For real-time RT-PCR, cDNAs were synthesized with 2 Ag total RNA using a the clones emerged and were combined for further analysis. Superscript first-strand cDNA synthesis kit (Invitrogen). The real-time PCR

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Smyd4 Is a Putative Tumor Suppressor was performed using Applied Biosystems 7300 Thermal cycler and a SYBR further analysis. The four shRNA constructs showed similar degree of Green PCR kit (Applied Biosystems). The amplification was performed suppression. The results presented here are from experiments using using 40 cycles. SYBR Green fluorescence was monitored in each cycle for V2HS_88307. the reference and marker gene. Samples were analyzed in triplicate, and expression was compared with h-actin. Cell proliferation assay and anchorage-independent growth in soft Results agar. The cell proliferation was monitored by cell counting using trypan Decreased expression of Smyd4 gene due to chromosome blue exclusion. For soft agar growth, 5,000 cells from each stable cell line or translocation. We previously reported our effort on efficient mock (empty vector) cells were mixed with 1.5 mL of 0.3% of agar (Sigma) in 10% FBS/DMEM, then overlaid onto 1.5 mL of 0.5% agar/10% FBS/DMEM generation of random chromosome rearrangement with Cre-loxP in 1 well of a 6-well plate. The medium was changed every 3 d, and colonies system (4). In this study, Cre-loxP recombination system was used were grown for 4 wk. Colony formation was counted and repeated in to create chromosome deletion/translocation in nontumorigenic triplicate. mouse mammary epithelial cell line NOG8. NOG8 cells were Tumor formation assay in vivo. The nu/nu athymic mice were used sequentially infected with LacZ/Neo-5¶Pur-LoxP-TK virus, Hyg- under Institutional Animal Care and Use Committee approval. A total of 3¶Pur-loxP virus, and Cre adenovirus. Recombinant between these 6 1 Â 10 of Smyd4 re-expressing cancer cells or control cancer cells in 200 AL two vectors can generate an intact puromycin-resistant gene with A of media were combined with 200 L of Matrigel (BD Biosciences) and correct reading frame. The cells with combination carrying an injected s.c. into the dorsal neck region of nu/nu athymic mice. Tumor intact puromycin-resistant gene would survive against puromycin. growth was monitored weekly. The NOG8 cells with chromosome rearrangement were injected s.c. Small interfering RNA. Four of Smyd4 shRNA constructs (V2HS_88307, V2HS_88308, V2HS_88309, and V2HS_255847) and one mock shRNA (pSM2 into nude mice for tumor formation. The tumor cells were empty vector) constructs were purchased from Open Biosystems. Retrovirus harvested from the primary tumor and subcultured. was produced by transfecting Ampho 293 cells with shRNA vectors for 48 h. For one tumor, inverse-PCR and sequencing revealed that the 5¶ MCF10A cells were infected for 24 h with viral supernatant and selected for Pur-LoxP-TK was inserted into the 10th intron of Smyd4 gene on 6 to 8 d with 0.5 Ag/mL of puromycin. The cells were then combined for mouse chromosome 11, whereas 3¶ Hyg-Pur-LoxP was inserted into

Figure 2. Smyd4 functions as a tumor suppressor. A, cell proliferation assay shows that Smyd4 suppressed the growth of tumor cells in vitro. Exponentially grown breast tumor cells infected with retroviruses expressing Smyd4 or control retroviruses were monitored by cell counts using trypan blue exclusion. The expression of Smyd4 mRNA was determined by real-time RT-PCR. B, Smyd4 suppressed anchorage-independent growth in soft agar growth. Both the size and number of colony were decreased with Smyd4 re-expression. C, Smdy4 inhibited tumor growth in vivo. Two million breast tumor cells re-expressing smyd4 (R1, R2) or control tumor cells (L1, L2) were injected into 5-wk-old athymic nude mice for tumor formation. Tumor growth was monitored weekly. Smdy4 re-expression dramatically suppressedthe tumor growth in nude mice compared with control tumor cells with disrupted Smyd4 expression. www.aacrjournals.org 4069 Cancer Res 2009; 69: (9). May 1, 2009

Cancer Research

we assessed if Smyd4 plays a role in inhibiting cell proliferation. The tumor cells were infected with retroviruses expressing Smyd4 or with control retroviruses. The re-expression of Smyd4 was confirmed by real-time RT-PCR (Fig. 2A). Cell proliferation assay revealed that Smyd4 re-expression dramatically decreased the growth of cancer cells (Fig. 2A). The effect of Smyd4 re-expression on the anchorage-independent growth of cells in soft agar was tested. Compared with the tumor cells infected with control viruses, the numbers of colonies formed in soft agar were dramatically reduced in Smyd4 re- expression tumor cells (Fig. 2B), indicating that Smyd4 suppresses the anchorage independent growth of tumor cells. To further show that Smyd4 is a tumor suppressor, we tested whether the recovery of Smyd4 expression in tumor could suppress tumor growth in vivo in athymic nude mice. Cells (2 Â 106) were injected s.c. into the dorsal neck region of nu/nu athymic mice. Four weeks later, control tumor cells formed tumors, whereas Smyd4 re-expressing tumor cells were incapable of forming tumors (Fig. 2C). Both in vitro and in vivo data conclusively indicate that Smyd4 suppressed the tumor growth. Transformation of human breast epithelial cell MCF10A by

Figure 3. Suppression of Smyd4 expression promotes anchorage-independent Smyd4 RNA interference. To determine if Smyd4 knockdown growth of human MCF10A mammary epithelial cells. MCF10A cells were could lead to transformation, we knocked down Smyd4 expression infected with retroviruses expressing Smyd4-shRNA or control nonspecific in human mammary gland epithelial cell MCF-10A using RNA shRNA and seeded in 0.3% agar containing MEM medium with 5% horse serum. The colony formation was monitored weekly. The colony numbers were counted interference. MCF10A cells are of mammary origin and exhibit 4 wk later, after which colonies were stained with 0.005% crystal violet staining anchorage-dependent growth property in vitro (30). Smyd4-shRNA solution (Sigma-Aldrich). A, real-time RT-PCR showed the suppression of vectors or control vector were integrated and expressed in Smyd4 mRNA in cells expressing Smyd4-shRNA. B, representative picture demonstrating the colonies formed by cells with Smyd4 knockdown or control MCF10A cells using standard retroviral infection. Real-time RT- cells. C, the markedly increased number of colonies formed by MCF10A cells PCR revealed that Smyd4 expression was efficiently suppressed with Smyd4 knockdown compared with control MCF10A cells. Columns, mean from two independent triplicate experiments; bars, SD. in MCF10A cells expressing Smyd4 shRNA (Fig. 3A). Smyd4 knockdown cells and the control cells were seeded into soft agar and allowed to grow for 4 weeks. As a result, the control MCF10A 6th intron of Msra (NM_026322, mouse methionine sulfoxide cells were incapable of forming foci in soft agar, whereas the reductase A) gene on chromosome 14 (The inverse PCR sequence MCF10A cells with Smyd4 knockdown readily formed foci, and blast results were provided in the Supplementary Data). Two indicating that Smyd4 knockdown promotes the transformation loxP sites recombination led to translocation between chromo- of breast epithelial cells (Fig. 3B and C). some 11 and 14, creating a fusion gene including the promoter, The expression of Pdgfr-A is repressed by Smyd4. To identify exon 1 to 10 of Smyd4 gene, plus the promoter and exon 1 to 5 of the down-stream genes of Smyd4, microarray analysis were Msra gene (Fig. 1A). After determining the integration sites, the performed with the tumor cells and NOG8 cells. By comparing recombination between the two vectors integrated into different expression profiles of tumor cells and NOG8 cells, we selected four sites was further confirmed by PCR using primers specific for genes (Pdgfr-a, semaphorin 5A, myogenic factor 6, and nerve growth recombination (Fig. 1B). One 6.7-kb band were amplified by PCR factor receptor) that were known for their potential roles in with one primer (5¶-TGACAAGAAGCTCTCAGCAG-3¶) located in tumorigenesis and up-regulated in tumor cells for further studies. the Smyd4 on chromosome 11 and another primer (5¶-CTCGA- CATCGGCAAGGTGT-3¶) specifically for Hyg-3¶ Pur-loxP vector. Another 2.2-kb band were amplified by PCR with 1 primer (5¶-CCTTGTCACCTAAGAGGGG-3¶) located in Mrsa on chromosome 14 and another primer (5¶-TCCTTCGGGCACCTCGACG-3¶) specifically for LacZ/Neo-5¶Pur-LoxP-TK vector. The promoters from these two genes act in opposite direction. As the result of translocation, one allele of Smyd4 gene is disrupted but no fusion protein is created. Given that Msra, as a regulator of antioxidant defense, is mainly involved in neurodegenerative diseases and unlikely plays a critical role in tumorigenesis, we focused on Smyd4 gene. At first, we examined if Smyd4 expression is altered due to translocation. Real-time RT-PCR revealed that the level of Smyd4 mRNA from tumor cells was less than one third of that from NOG8 cells Figure 4. The expression of Pdgfr-a was suppressed by Smyd4. The Pdgfr-a expression (Fig. 1B). expression was quantitated by real-time RT-PCR. A, the expression of Pdgfr-a was dramatically increased in tumor cells in comparison with NOG8 cells. Growth suppression of tumor cells through Smyd4 re- B, re-expression of Smyd4 significantly suppressed the expression of Pdgfr-a in expression. As Smyd4 expression was reduced in the tumor cells, tumor cells. Expression of h-actin served as a quantitative control.

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Smyd4 Is a Putative Tumor Suppressor

Real-time RT-PCR confirmed that Pdgfr-a expression was dramatically elevated in tumor cells (Fig. 4A) but not detectable in NOG8 cells during 40 cycles of amplification. In addition, the microarray data (data not shown here but in the Supplementary Data) indicates that the NOG8-derived tumors highly express Pdgfa and Pdgfb, which are the ligands of Pdgfr-a homodimer (31). Furthermore, real-time RT-PCR revealed that Pdgfr-a expression was significantly suppressed by re-expressing exogenous Smyd4 in tumor cells (Fig. 4B). For the rest of the three genes, their expression was increased in tumor cells but the re-expression of Smyd4 could not suppress their expression (data not shown). As Pdgfr-a overexpression was reported in breast cancer and associated with tumor progression (32), the finding indicates that Smyd4 acts as a tumor suppressor at least partly through controlling Pdgfr-a expression. Smyd4 expression is silenced in some of human breast cancers. Human Smyd4 gene is located at chromosome 17p13.3, which is a region commonly exhibiting loss of heterozygosity in breast cancers. After having shown that Smyd4 is a potential tumor suppressor, we asked if Smyd4 is involved in human breast cancers. We analyzed the expression of Smyd4 gene in 10 of human breast carcinomas. RT-PCR results reveal that Smyd4 gene expressions were completely silenced in two breast cancer cases and markedly decreased in 3 additional cases (Fig. 5A). To find if there is correlation between the expression of Smyd4 and Pdgfr-a, we also examined the Pdgfr-a expression. For the human breast cancers in which Smyd4 expression were totally silenced or markedly decreased, the concordance was found in two of human breast cancers (HBT-5, HB-10) but not found in the others (HBT-1, HBT-9; Fig. 5B). In addition, the expression of Pdgfr-a was not suppressed in some breast tumors expressing Smyd4 (HBT-3). These findings indicated that Smyd4 is not the only factor determining the expression of Pdgfr-a in breast cancers.

Figure 5. Smyd4 expression was silenced in some human breast cancers. Discussion Loss of Smyd4 expression was associated with up-regulation of Pdgfr-a expression in some primary human breast tumors. A, RT-PCR was performed In present work, we found that disruption of Smyd4 expression is with total RNA prepared from primary breast tumors. Tumor HBT-1 and HBT-5 do associated the tumorigenesis of a nontumorigenic mammary cell not express Smyd4, whereas tumor HBT-6, HBT-9, and HBT-10 markedly reduced Smyd4 expression. Expression of h-actin served as a control. B, the line. Re-expressing Smyd4 in tumor cells suppressed the prolifer- quantitative real-time PCR analyses of Pdgfr-a and Smyd4 expression in human ation of cancer cells, the anchorage-independent growth of tumor breast cancers. Human MCF10A mammary epithelial cells served as a normal cells, and tumor formation in nude mice. RNA interference–mediated control. Smyd4 knockdown in MCF10A led to anchorage-independent growth. Smyd4 was found to suppress Pdgfra expression. From 10 human breast cancer cases, Smyd4 gene expressions were revealed to be lysine residues, Smyd4 could suppress tumor growth through completely silenced in 2 breast cancer cases and markedly modifying histone or nonhistone proteins. The study on the activity decreased in 3 additional cases. These findings suggest that Smyd4 of Smyd4 in the modification of histone or nonhistone proteins is a potential novel breast tumor suppressor. is undergoing. Chromosome deletion of 17p13.3 is frequently involved in The tumor developed after the chromosome translocation was various types of human malignant tumors (6–10). Although likely to undergo additional genetic changes, as evidenced by the previous studies has identified three tumor suppressor genes finding that the expression of three of four genes that are including MAX binding protein, Hic1, and ovarian cancer gene 1 up-regulated in the tumor could not be suppressed by Smyd4 (11–14) in this region, there is evidence suggesting the presence of re-expression. Nevertheless, the decreased expression of Smyd4 is additional novel tumor suppressors. Our studies reveal that Smyd4 critical for the tumorigenesis as the re-expression of Smyd4 is another potential novel tumor suppressor from 17p13.3. The abolished the tumor formation. involvement of Smyd4 in other types of tumors needs to be further Pdgfr-a is involved in the proliferation and survival of a variety investigated. of tumors (33, 34). Pdgfr-a–activating mutations were detected in Haploinsufficiency of Smyd4 could be an explanation as to why gastrointestinal stromal tumor and brain tumors. High expression the loss of one allele of Smyd4 gene led to the tumorigenesis. of Pdgfr-a is detected in many types of tumors including breast However, the detail of pathway through which Smyd4 is involved in cancers. Autocrine activation of Pdgfr-a promotes the progression breast cancer development needs to be further studied. Consider- or aggressiveness of ovarian cancer and breast cancer (32, 35). ing the potential methyltransferase activity of Smyd family on The importance of Pdgfr-a signaling was corroborated by the www.aacrjournals.org 4071 Cancer Res 2009; 69: (9). May 1, 2009

Cancer Research observation that disrupting Pdgfr-a–mediated signaling results in expression of factors that control Pdgfr-a. Given that Smyd4 significant inhibition of tumor growth (36). Recent findings carries a conserve SET domain that often possesses methyltrasfer- revealed that inhibition of Pdgfr-a restricts the growth of human ase activity, Smyd4 is likely to act as a repressor for certain breast cancer in the bone of nude mice (37, 38), indicating its role transcription factor that regulates the expression of Pdgfr-a gene. in organ specific metastasis. We found that Pdgfr-a was highly expressed in the mammary cancer cells with reduced expression of Disclosure of Potential Conflicts of Interest Smyd4. Re-expressing Smyd4 in the beast cancer cells significantly suppressed the expression of Pdgfr-a. In our present study, the No potential conflicts of interest were disclosed. concordance between the loss of Smyd4 expression and up- regulation of Pdgfr-a RNA was found in some of our human breast Acknowledgments cancer samples but not in all human breast cancers with loss of Received 10/23/08; revised 2/24/09; accepted 3/2/09; published OnlineFirst 4/21/09. Smyd4 expression. Our present study provides a novel mechanism Grant support: NIH grant CA 88898 (Y-J. Zhu) and Department of Defense Breast through which abnormal expression of Pdgfr-a occurs in breast Cancer Research Program (C. Qi). The costs of publication of this article were defrayed in part by the payment of page cancers and possibly in other cancers. Smyd4 may modify the charges. This article must therefore be hereby marked advertisement in accordance expression of Pdgfr-a directly or indirectly through affecting the with 18 U.S.C. Section 1734 solely to indicate this fact.

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Cancer Res 2009; 69: (9). May 1, 2009 4072 www.aacrjournals.org

Identification of Smyd4 as a Potential Tumor Suppressor Gene Involved in Breast Cancer Development

Liping Hu, Yiwei Tony Zhu, Chao Qi, et al.

Cancer Res Published OnlineFirst April 21, 2009.

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