Frequent Mutation of rs13281615 and Its Association with PVT1 Expression and Cell Proliferation in Breast Cancer

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ScienceDirect Journal of Genetics and Genomics 41 (2014) 187e195

JGG ORIGINAL RESEARCH

Frequent Mutation of rs13281615 and Its Association with PVT1 Expression and Cell Proliferation in Breast Cancer Zhiqian Zhang a, Zhengmao Zhu a, Baotong Zhang a, Weidong Li b, Xin Li a, Xiao Wu a, Lijuan Wang a, Liya Fu a, Li Fu b, Jin-Tang Dong a,c,* a

Department of Genetics and Cell Biology, Nankai University College of Life Sciences, Tianjin 300071, China Key Laboratory of Breast Cancer Research, Department of Breast Cancer Pathology and Research Laboratory, Cancer Hospital of Tianjin Medical University, Tianjin 300060, China c Department of Hematology and Medical Oncology, Emory University School of Medicine, Emory Winship Cancer Institute, Atlanta 30322, USA b

Received 14 November 2013; revised 18 March 2014; accepted 27 March 2014 Available online 5 April 2014

ABSTRACT The q24 band of chromosome 8 (8q24) is frequently amplified in human cancers including breast cancer, and several SNPs (single nucleotide polymorphisms) at 8q24, including rs13281615, have been identified for their association with cancer risks. These SNPs are in a “gene desert” region, and their functions in cancer development remain to be illustrated, although several of the SNPs appear to influence the genes in the “desert” in a long-range manner, including the v-myc avian myelocytomatosis viral oncogene homolog (MYC ) and the nonprotein coding plasmacytoma variant translocation 1 (PVT1), both of which have been implicated in human cancers. In the current study, we examined rs13281615 for its potential role in breast cancer using normal and cancer tissues from 121 Chinese women with breast cancer. In addition to confirming the association of the GG genotype of rs13281615 with breast cancer risk, we found that germline GG genotype was significantly associated with estrogen receptor (ER) positivity, higher tumor grade and higher proliferation index. We also found frequent somatic mutations (22/121 or 18.2%) of this SNP in breast cancer. Interestingly, the majority of the mutations (17/22 or 77%) involved a G/A change, resulting in a decrease in the number of cancers with the GG risk genotype and subsequent loss of GG association with higher tumor grade and proliferation index in cancers. Furthermore, PVT1 expression was increased in cancers, and the increase was associated with the GG genotype of rs13281615. These results suggest that the GG genotype of SNP rs13281615 plays a role in breast cancer likely by influencing PVT1 expression, and that during oncogenesis, “protective” mutations could occur. KEYWORDS: rs13281615; Mutation; PVT1; Breast cancer

INTRODUCTION The development and progression of human cancer is a complex, multi-step process that usually takes several decades

Abbreviation: ER, estrogen receptor; miRNA, microRNA; MYC, v-myc avian myelocytomatosis viral oncogene homolog; PVT1, plasmacytoma variant translocation 1; SNP, single nucleotide polymorphism. * Corresponding author. Tel: þ1 404 712 2568, fax: þ1 404 712 2571. E-mail addresses: [email protected], [email protected] (J.-T. Dong).

(Hanahan and Weinberg, 2000; Knudson, 2001). A large number of genetic and epigenetic alterations occur during tumorigenesis, resulting in the activation of oncogenes and inactivation of tumor suppressor genes that drive tumorigenesis (Munger, 2002). Both germline and somatic mutations occur in oncogenes and tumor suppressor genes. Whereas somatic mutations in these genes appear to be more common (Futreal et al., 2004), more and more germline variations in both coding and noncoding regions of genes, including single nucleotide polymorphisms (SNPs), have been linked to

1673-8527/$ - see front matter Copyright Ó 2014, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved. http://dx.doi.org/10.1016/j.jgg.2014.03.006

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increased risks of various human cancers by genome-wide association studies (Easton et al., 2007; Gold et al., 2008; Thomas et al., 2009; Turnbull et al., 2010). Some cancer-associated SNPs are located in chromosomal regions that do not encode genes. One such SNP is rs13281615 at 8q24.21, whose association with breast cancer risk has been confirmed in a number of studies in different ethnic groups including Chinese women (Easton et al., 2007; Fletcher et al., 2008; Garcia-Closas et al., 2008; Ghoussaini et al., 2008; McInerney et al., 2009; Long et al., 2010; Odefrey et al., 2010; Turnbull et al., 2010; Zheng et al., 2010; Chan et al., 2012). At this SNP, the G-allele is the risk allele for breast cancer while the AA genotype has a protective effect (Gong et al., 2013). The GG genotype at SNP rs13281615 was associated with estrogen receptor (ER) positivity, progesterone receptor (PR) positivity, lower tumor grade, and improved patient survival in previous studies (Garcia-Closas et al., 2008; Broeks et al., 2011), further suggesting a function of this SNP in breast cancer development. Cancer-prone SNPs located in the 8q24 “gene desert” region may affect the transcription of genes next to the region, even if the distance between a SNP and a gene is large (Jia et al., 2009; Pomerantz et al., 2009b; Tuupanen et al., 2009; Ahmadiyeh et al., 2010; Dworkin et al. 2010). One example is SNP rs6983267, which, also at 8q24, has been associated with increased risks in colorectal, prostate and breast cancers. Although quite distant from the v-myc avian myelocytomatosis viral oncogene homolog (MYC ) gene at 8q24, different genotypes of the SNP had different effects on the transcription of MYC, with GG increasing MYC transcription via the transcription factor TCF-4 (Sotelo et al., 2010). Although rs13281615 is only 50 kb centromeric to SNP rs6983267, several studies indicated that the region surrounding this SNP does not contain a cis-regulatory element for MYC expression in human tumors (Ahmadiyeh et al., 2010; Wasserman et al., 2010). The other gene in the 8q24 “desert region” is the non-protein coding plasmacytoma variant translocation 1 (PVT1), which is 55 kb telomeric to the MYC gene and thus further away from rs13281615 (Feo et al., 1994). PVT1 is a non-coding RNA whose transcription results in several miRNAs (microRNAs). PVT1 also plays a role in tumorigenesis, as it is a translocation site in variant Burkitt’s lymphomas and multiple myeloma (Boehm and Rabbitts, 1989; Nagoshi et al., 2012), and is overexpressed and mutated in T cell lymphomas (Beck-Engeser et al., 2008). In addition, PVT1 is amplified and overexpressed in ovarian and breast cancers (Guan et al., 2007), and appears to affect chemotherapy sensitivity (You et al., 2011). PVT1 can be highly expressed in some tumors where MYC is not expressed (Carramusa et al., 2007). A specific genotype of a SNP can be associated with PVT1 expression change in cancers, such as SNP rs378854, which is in imperfect linkage disequilibrium with the prostate cancer risk SNP rs620861, where the GG genotype of rs378854 decreased the binding of transcription factor YY1 thus leading to increased PVT1 expression (Meyer et al., 2011). In this study, we determined the genotype distribution of SNP rs13281615 in both normal and cancer tissues of the

breast, analyzed the expression of PVT1, and correlated genotypes of the SNP with PVT1 expression and clinicopathological features of breast cancer. In addition to confirming the GG genotype association with breast cancer risk, we also found that the germline GG genotype was significantly associated with ER positivity, higher tumor grade and increased cell proliferation. The SNP underwent frequent somatic G/A mutations that decreased the number of tumors with GG risk genotype, and the GG genotype was significantly associated with higher PVT1 expression in cancers. These results suggest that the GG genotype of SNP rs13281615 plays a role in breast cancer, likely by influencing PVT1 expression. These G/A mutations also suggest that during oncogenesis, “protective” mutations could occur. RESULTS Frequent somatic mutations of rs13281615 in breast cancer We genotyped SNP rs13281615 in 32 breast cancer cell lines, five of which had matched noncancerous blood or mammary epithelial cells as normal samples (HCC38, HCC1143, HCC1599, HCC1937 and Hs 578T). We also genotyped both normal breast tissues and matched cancer tissues from 121 women. Among the five breast cancer cell lines with genomic DNA from matched normal cells, three (HCC1143, HCC1599 and HCC1937) had somatic mutations, and all three mutations were GA/AA changes (Fig. 1AeC and H). Of the 121 breast cancer samples, 22 (18.2%) showed somatic mutations, including 8 (6.6%) GG/GA (Fig. 1D and I), 5 (4.1%) GG/AA (Fig. 1E and I), 5 (4.1%) GA/GG (Fig. 1F and I), and 4 (3.3%) GA/AA (Fig. 1G and I). Among the 22 mutations in primary tumors, 17 involved a G/A mutation whereas only 5 involved an A/G mutation (Fig. 1I), consistent with the findings from cell lines. According to findings from association studies, the G allele was the risk allele while the A allele was the protective allele in breast cancer (Easton et al., 2007; Fletcher et al., 2008; Garcia-Closas et al., 2008; Ghoussaini et al., 2008; McInerney et al., 2009; Long et al., 2010; Odefrey et al., 2010; Turnbull et al., 2010; Zheng et al., 2010; Chan et al., 2012). Therefore, the majority of mutations led to increased protective alleles. We also compared frequencies of three rs13281615 genotypes between the 121 normal tissues in this study and a group of healthy Chinese women reported in previous studies (Gong et al., 2013). Consistent with previous findings, the GA and GG genotypes were significantly more frequent in breast cancer patients than in healthy controls (Table 1). We also sequenced a 473-bp fragment that contains three SNPs in or surrounding PVT1-encoded miR-1208, including rs2648861, rs10956412 and rs56863230, in both normal and tumor DNA samples from the same patients. These SNPs are about 800 kb telomeric to rs13281615. Only two samples, neither of which had somatic change at SNP rs13281615, had a somatic change at one of the three SNPs, i.e., rs2648861. None of the three SNPs in miR-1208 had somatic changes in

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the 22 samples that had somatic mutations at SNP rs13281615, and at least one of them showed a homozygous genotype in each of the normal and tumor samples. In the 119 samples that had no somatic changes in any of the three SNPs, genotype distributions of the three SNPs were as follows: rs2648861, GG (46/119, 38.7%), GT (50/119, 42.0%), and TT (23/119, 19.3%); rs10956412, CC (9/119, 7.5%), CA (41/119, 34.5%), and AA (69/119, 58.0%); and rs56863230, GG (93/119, 78.2%), GC (26/119, 21.2%), and CC (0/119, 0%) (Table S1).

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status, and the expression of the proliferative marker Ki67 (Table 2). The GG genotype in normal tissues had a significant association with higher tumor grade, increased Ki67-positive cells, and a positive ER status (Table 2). However, the GG genotype in breast cancers, which had 17 G/A mutations, was not associated with higher tumor grade or higher Ki67 index, although an association with ER positivity was still significant (Table 2). DISCUSSION

Association of the GG genotype of rs13281615 with increased PVT1 expression in breast cancer Considering that a risk SNP at 8q24 could be associated with abnormal expression in one of the two genes at 8q24, MYC and PVT1, such as rs6983267 with MYC, rs378854 with PVT1, or lack of association of rs13281615 with PVT1 (Pomerantz et al., 2009a; Tuupanen et al., 2009; Wasserman et al., 2010; Wright et al., 2010; Meyer et al., 2011; Sur et al., 2012; Takatsuno et al., 2013) (Fig. 2A), we measured PVT1 expression by quantitative PCR in 27 normal breast tissues and 43 breast cancer tissues, 27 of which had matched normal tissues. We then associated different genotypes of rs13281615 with PVT1 expression in both normal and cancer tissues. In the 27 normal tissues, PVT1 expression was detectable, but there was no detectable difference in PVT1 expression between the GG genotype and the GA or AA genotype (Fig. 2B). In the 43 breast cancer tissues, PVT1 expression was significantly higher in those with the GG genotype than that in the GA or AA genotype (Fig. 2B). Compared to normal tissues with any of the genotypes, PVT1 expression was also higher in the tumors with the GG genotype (Fig. 2B). Given that 27 of the tumors had matched normal tissues, we also calculated the ratio of PVT1 expression between normal and tumor tissue of the same patient, and compared the frequencies among the three genotypes. Consistent with increased PVT1 expression for the GG genotype in tumors, the tumor to normal ratio of PVT1 expression was significantly higher for the GG genotype than that for the GA or AA genotype (Fig. 2C). We also examined PVT1 expression in 31 breast cancer cell lines. Although the average level of PVT1 expression appeared to be higher in cell lines with the GG genotype than those with the GA and AA genotype, the differences were not statistically significant, likely due to the smaller number of samples (Fig. S1). Association of the GG genotype of rs13281615 with higher tumor grade, ERpositivity, and higher Ki67 index To further evaluate the role of SNP rs13281615 in breast cancer, we explored correlations between different genotypes of the SNP in both normal and cancer tissues of the 121 patients with various clinicopathological characteristics of breast cancer, including age at diagnosis, tumor size, tumor grade, tumor stage, lymph node metastasis, ER/PR/HER2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2)

The GG genotype of SNP rs13281615 is associated with breast cancer risk. Based on previous studies that have identified risk alleles of several SNPs at 8q24 and that demonstrated their association with altered expression of MYC or PVT1 in human cancers, we evaluated whether a specific genotype of SNP rs13281615 plays a role in breast cancer by analyzing its genotype distribution, somatic mutations, and associations of its genotypes with tumor characteristics and PVT1 expression. Although the GG genotype of rs13281615 has been repeatedly shown to increase breast cancer risk in Caucasian women, the findings in Chinese women have been inconsistent with some studies showing a similar association (Chan et al., 2012; Gong et al., 2013) while others not (Long et al., 2010; Jiang et al., 2011). We noticed that the genotype distribution of rs13281615 is different among different ethnic groups. In European women, the GG genotype is less frequent than the AA genotype (GG, 17.5%; GA, 47.0%; AA, 35.5%) (Gong et al., 2013). In Chinese women, however, both the GG and AA genotypes have a similar frequency (GG, 24.9%‒ 27.2%; GA, 47.4%‒50.0%; AA, 23.5%‒27.8%) (Gong et al., 2013). Comparison of genotype distribution between breast cancer patients in our study and the controls in the previous study (Gong et al., 2013) confirmed a significant association between the GG or GA genotype with breast cancer risk. The GG genotype of SNP rs13281615 is associated with ER positivity, higher tumor grade and higher proliferation index. With regard to the effect of different genotypes of rs13281615 on various clinicopathologic characteristics of breast cancer, previous studies showed an association of the GG risk genotype with ER positivity, PR positivity and lower tumor grade in European women (Garcia-Closas et al., 2008; Broeks et al., 2011). In our study of Chinese women, whereas the GG genotype of rs13281615 was also associated with ER positivity, it was not associated with PR status (Table 2). Furthermore, the GG genotype was associated with a higher tumor grade, which is opposite to the association observed in European women. We also found a significant association between the GG risk genotype and a higher Ki67 index (Table 2). Ki67 index is a component of histopathological grade of breast cancer (Pathmanathan and Balleine, 2013). Therefore, the association between the GG risk genotype of rs13281615 and both tumor grade and Ki67 further indicates a role of the GG genotype of rs13281615 in breast cancer development. SNP rs13281615 undergoes frequent somatic mutations in breast cancer, as revealed by sequencing analysis of 121

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Fig. 1. Somatic mutations of SNP rs13281615 in both primary tumors and cell lines from breast cancer. A‒ ‒C: Detection of a G/A mutation in breast cancer cell line HCC1143 (A) but not in breast cancer cell lines Hs 578T and HCC38 (B and C) by DNA sequencing. D‒ ‒G: Detection of different somatic mutations of rs13281615, including GG/GA (D), GG/AA (E), GA/GG (F) and GA/AA (G), in different breast cancer specimens, with matched normal breast tissues as controls. Lower part in each figure panel shows another sequencing result for SNP rs10956412, which does not show mutation in the same sample. T, tumor; N, normal. Patient number is shown at the top, whereas SNP name and sequence shown at the bottom, in each panel. Vertical arrows point to the subject SNPs. H: Distribution of genotypes of rs13281615 (GG, GA and AA) in breast cancer cell lines that did not have matched normal DNA for mutation detection. I: Frequencies of somatic mutations of rs13281615 in 121 primary breast cancers.

primary tumors and five cell lines of breast cancers and their matched normal DNA samples (Fig. 1). Somatic mutation of rs13281615 has not been reported in previous studies, and in general, somatic mutations of SNPs are rare in cancer. For example, we examined three other SNPs that are close to SNP rs13281615 at 8q24, rs2648861, rs10956412 and rs56863230, for their mutations in the same 121 primary breast cancers, and only one of them, i.e., rs2648861, had a somatic mutation in 2 of the 121 (1.7%) tumors (Fig. 1). This mutation frequency is much less than that for rs13281615 (22/121 or 18.2%).

Gene mutation is essential for tumors to occur, and mutations usually activate oncogenes or inactivate tumor suppressor genes. Considering that the GG genotype of rs13281615 confers higher risk, we expected that mutations of this SNP should result in more tumors with the GG genotype. Unexpectedly, most (17/22 or 77%) mutations of rs13281615 involved a G/A change, leading to a conversion of GG or GA in normal tissue to AA in tumors in 9 patients. These mutations appeared to have functional consequence, as G/A mutations decreased the number of tumors with the GG genotype and subsequently eliminated the association of GG

Z. Zhang et al. / Journal of Genetics and Genomics 41 (2014) 187e195 Table 1 Association of germline GG genotype of SNP rs13281615 with increased risk of breast cancer Genotype/allele

Case (%)

Control (%)

OR (95% CI)

P value*

AA

13 (10.7)

1428 (25.6)

1 (Reference)

e

GA

67 (55.4)

2752 (49.2)

2.67 (1.47e4.86)

0.0008

GG

41 (33.8)

1409 (25.2)

3.20 (1.70e5.99)

0.0001

108 (89.3)

4161 (74.4)

3.12 (1.71e5.68)

0.0002

A

93 (38.4)

5608 (50.2)

1 (Reference)

e

G

149 (61.6)

5570 (49.8)

1.61 (1.24e2.10)

0.0003

GA þ GG

*P value was calculated using the Chi-square test.

genotype with higher tumor grade and higher Ki67 index in tumors (Table 2). The association of the GG genotype with ER positivity was still significant in tumors though, which could be due to the specific patient population used in this study. These results suggest that the G/A mutation could be a “protective” event in ER-positive breast cancer. The “risk” GG genotype of SNP rs13281615 is associated with increased PVT1 expression in breast cancer. Only two genes, MYC and PVT1, are currently known to exist in the 1-Mb 8q24 region spanning the risk SNPs (Beck-Engeser et al., 2008), and a risk SNP could affect the expression of

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one of the genes. For example, the GG genotype of rs6983267 is associated with increased MYC expression, while the GG genotype of rs378854 is associated with increased PVT1 expression in prostate and colon cancers (Pomerantz et al., 2009a; Tuupanen et al., 2009; Wasserman et al., 2010; Wright et al., 2010; Meyer et al., 2011; Sur et al., 2012; Takatsuno et al., 2013). For SNP rs13281615, its GG genotype did not show an association with PVT1 expression in prostate cancer. Whether an association exists in breast cancer is unknown. We found that PVT1 expression was significantly increased in breast cancers with the “risk” GG genotype (Fig. 2), suggesting that the GG genotype could lead to a more active chromatin environment for PVT1 transcription in breast cancer, while the GA or AA genotype could not. The role of PVT1 in tumorigenesis has been indicated by translocation in Burkitt’s lymphomas and multiple myeloma (Boehm and Rabbitts, 1989; Nagoshi et al., 2012), mutation in T cell lymphomas (Beck-Engeser et al., 2008), amplification in breast cancer (Guan et al., 2007), and modulation of chemosensitivity in pancreatic cancer (You et al., 2011). PVT1 is highly expressed in some tumors where MYC is not expressed (Carramusa et al., 2007). With RNA samples of 27 normal tissues and 43 breast cancers, which is a relatively small sample for statistical analysis, we did not find any significant

Fig. 2. Association of the GG genotype of SNP rs13281615 with higher PVT1 expression in breast cancer tissues. A: A schematic illustrating the relative location of risk SNPs rs13281615, rs6983267 and rs378854, genes MYC and PVT1, and non-risk SNPs rs2648861, rs10954612 and rs56823230 within 1-Mb genomic DNA at 8q24. Established association between the GG genotype and increased expression of MYC or PVT1 in prostate and/or colon cancer is indicated by a line with arrow. B and C: The GG genotype of rs13281615 in both normal and cancer tissues is significantly associated with increased PVT1 expression in breast cancer tissues, as indicated by the relative expression levels of PVT1 (B) or the PVT1 expression ratio between cancer tissue and matched normal tissue (C), as detected by real-time PCR. Expression levels are grouped to different genotypes (GG, GA or AA). P values are for the comparison between the GG genotype in cancer samples and GA or AA genotype in cancer samples or GG, GA or AA genotype in normal samples. * and ** indicate P values smaller than 0.05 and 0.01, respectively. T, tumor; N, normal.

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Table 2 Association of the GG genotype with higher tumor grade, ER positivity and increased cell proliferation in breast cancer Characteristics

Samples for each genotype (N/T) GG

GA

AA

50

19/15

28/27

5/10

>50

22/18

39/39

8/12

Age at diagnosis (years)

P value* (N/T)

0.845/0.881

Tumor size (cm)

0.907/0.370

2

7/4

16/17

3/5

>2

19/18

35/32

8/12

I

0/0

3/3

2/2

II‒ ‒III

33/29

49/46

7/14

L

18/12

31/34

7/10

þ

21/20

33/28

6/12

I

10/8

14/15

4/5

II

14/12

24/23

5/8

III/IV

9/7

20/18

3/7

L

11/7

35/34

6/11

D

29/26

31/30

7/11

L

11/9

21/18

3/8

þ

29/24

45/46

10/14

Histological grade

0.042/0.121**

Lymph node metastasis

0.890/0.269

Tumor stage (pTNM)

0.910/0.972

ER

0.036/0.009

PR

0.777/0.728

HER-2

MATERIALS AND METHODS 0.784/0.312

L/þ

26/20

46/44

10/18

þþ/þþþ

13/12

20/20

3/4

L

4/4

7/7

6/6

D

36/29

59/57

7/16

Ki67

For SNP rs6983267 at 8q24, previous studies showed that its GG genotype increases the binding of the TCF-4 transcription factor to MYC enhancers to elevate its transcription (Tuupanen et al., 2009). For another SNP at 8q24, rs378854, its GG genotype has been associated with increased PVT1 expression in prostate cancer, and the YY1 transcription factor was suggested to mediate PVT1 upregulation (Meyer et al., 2011). For SNP rs13281615, whereas its GG genotype was associated with increased PVT1 expression in breast cancer (Fig. 2), it is unknown whether and how the GG genotype directly influences PVT1 expression. In summary, in evaluating whether SNP rs13281615 plays a role in breast cancer, we found that the GG genotype of this SNP was significantly more common in breast cancer patients than in controls in Chinese women. The GG genotype was associated with ER positivity, higher tumor grade, and higher proliferation index. The SNP was frequently mutated in breast cancer, and the mutation appeared to be a “protective” event as it mostly mutated the risk G allele to the protective A allele, eliminating the association of GG with higher tumor grade and proliferation index in tumors. In addition, the GG genotype was significantly associated with increased PVT1 expression in breast cancers. These findings suggest that the GG genotype of SNP rs13281615 influences breast cancer development likely by modulating PVT1 expression, although more questions remain to be addressed. These findings also suggest that during oncogenesis, “protective” mutations could also occur.

0.002/0.154

*P values were for the comparisons between different genotypes (GG, GA or AA) and different readings of a characteristic (e.g., I vs. II‒III for grade;  vs. þ for ER status) in either tumor tissues (T) or matched normal tissues (N). All P values, except those for histological grade, were calculated by using the Chi-square test in patients who had data available. **P values for histological grade were calculated by using Fisher’s exact test (GG vs. AA). For ER or PR, cases were considered positive if nuclear immunoreactivity was present in 1% of tumor cells. For HER2, the immunohistochemical score was assigned according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) Guideline: 0, no staining; þ, weak incomplete membrane staining in any percentage of cells; þþ, weak or moderate and heterogeneous complete membrane staining in at least 10% of cells; þþþ, strong and complete homogeneous membrane staining in >30% of cells. For Ki67 positivity based on IHC staining, the cut off value was 15%. The characteristics associated with the genotypes of rs13281615 were marked in bold.

association between PVT1 expression and clinicopathological characteristics of breast cancer. Nevertheless, it remains to be determined whether the risk GG genotype of rs13281615 modulates PVT1 expression to affect breast cancer development.

Cell lines The breast cancer cell line BRF71T1 was purchased from Biological Research & Faculty (BRFF, Ijamsville, USA). Additional 31 breast cancer cell lines (HCC38, HCC1143, HCC1599, HCC1937, Hs 578T, BT-20, BT-474, BT-483, BT-549, CAMA-1, DU4475, HCC70, HCC202, HCC1500, HCC1806, HCC2218, HCC1395, MDA-MB-134, MDAMB-175, MDA-MB-231, MDA-MB-415, MDA-MB-453, MDA-MB-468, MDA-MB-157, MDA-MB-361, MCF7, SW527, T-47D, UACC893, ZR-75-1 and ZR-75-30) and 5 matched peripheral blood or normal mammary epithelial cell lines (HCC38BL, HCC1143BL, HCC1599BL, HCC1937BL and Hs 578Bst) were purchased from the American Type Culture Collection (ATCC, Manassas, USA). Clinical specimens A total of 121 primary breast carcinomas and their adjacent normal tissues were obtained from surgically treated patients with breast cancer at the Cancer Hospital of Tianjin Medical University, Tianjin, China. Noncancerous tissues were harvested at least 5 cm away from corresponding tumors, and surgical margins were confirmed to be clear of residual cancers. Use of the materials was approved by the hospital’s

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Ethics Review Committee. Tissues were cut into small pieces, snap frozen in liquid nitrogen, and stored in a 80 C freezer until use.

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each sample, the relative expression of PVT1 was evaluated by the comparative 2DDCt method. Statistical analysis

Extraction of genomic DNA and total RNA Genomic DNA was extracted from cell lines and clinical samples using the DNeasy Tissue Kit (Qiagen, Germany) following the manufacturer’s instructions. DNA concentration was measured by following standard protocols, and the final concentration was 20 mg/mL. Total RNA from clinical specimens was isolated using the mirVanaÔ miRNA Isolation Kit (Life Technology, USA) by following the manufacturer’s protocol. RNA concentrations were measured according to standard methods. Genotyping SNPs rs13281615, rs2648861, rs10956412 and rs56863230 The following pairs of primers were used in genotyping: 50 -ACAAACAAGACTATTTCCTTTGAC-30 (forward) and 50 -CACAGAGAATATTGTCTGTGTATAGAG-30 (reverse) (rs13281615); 50 -TACTCTGGGAGCAAGCCGTACC-30 (for ward) and 50 -GTTACAATATTAAAACTTCAGCTCAATGG C-30 (reverse) (rs2648861, rs10956412 and rs56863230). Genomic DNA was amplified by PCR in a volume of 20 mL (2 mL of genomic DNA, 10 mmol/L primer and 10 mmol/L dNTPs). The PCR cycling profile consisted of an initial denaturing at 95 C for 10 min, followed by 36 cycles of 94 C for 30 s, 55 C for 30 s, and 72 C for 30 s, and an additional incubation at 72 C for 7 min. PCR products (about 400 bp) were directly sequenced using the Sanger method at RecalTech (Beijing, China). For rs13281615, 22 of 121 primary tumors and 3 of 5 breast cancer cell lines with paired normal DNA samples showed somatic mutations when compared to their matched normal samples, and these 25 samples and their matched normal samples were subjected to PCR and sequencing again. The second sequencing was done at SinoGenoMax (Beijing, China), and all somatic mutations were confirmed. Detection of PVT1 expression by real-time PCR Briefly, 0.5 mg of total RNA was reversely transcribed by using oligo-dT primers (TaKaRa, Japan), and 2 mL of the reverse transcription reaction mix were amplified by PCR with an initial denaturation at 95 C for 2 min and 50 cycles of 95 C for 30 s and 60 C for 60 s. GAPDH was used as an internal control. Each data point was in triplicate, and the SYBR green (TaKaRa) method was used with the IQ5 Real-time PCR detection system (Bio-Rad, USA). Primers used for PVT1 were 50 -CATATCCTTTCAGCACTCTGGAC-30 (forward) and 50 -CAACAGGAGAAGCAAACAGGG-30 (reverse), and those for GAPDH were 50 -GGTGGTCTCCTCTGACTTCAACA-30 (forward) and 50 -GTTGCTGTAGCCAAATTCGTTGT-30 (reverse). After determination of the threshold cycle (Ct) for

Statistical analysis was performed with IBM SPSS, version 21.0 (SPSS Inc., USA). Unpaired t test with Welch’s correction was used for group comparison of the PVT1 expression among tumor tissues and cell lines with GG genotype and other groups. Chi-square test was used for association analysis between the genotypes of rs13281615 and clinicopathological characteristics. ACKNOWLEDGEMENTS We thank Dr. Anthea Hammond for editing the manuscript. This work was supported by the National Nature Science Foundation of China (Nos. 30870980, 31171250, and 30625032) and the National Basic Research Program of China (No. 2007CB914802).

SUPPLEMENTARY DATA Fig. S1. Expression of PVT1 in breast cancer cell lines with different genotypes of rs13281615. Table S1. Genotype distribution of SNPs rs2648861, rs10956412 and rs56863230 in breast cancer tissues. Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jgg.2014.03.006. REFERENCES Ahmadiyeh, N., Pomerantz, M.M., Grisanzio, C., Herman, P., Jia, L., Almendro, V., He, H.H., Brown, M., Liu, X.S., Davis, M., Caswell, J.L., Beckwith, C.A., Hills, A., Macconaill, L., Coetzee, G.A., Regan, M.M., Freedman, M.L., 2010. 8q24 prostate, breast, and colon cancer risk loci show tissue-specific long-range interaction with MYC. Proc. Natl. Acad. Sci. USA 107, 9742e9746. Beck-Engeser, G.B., Lum, A.M., Huppi, K., Caplen, N.J., Wang, B.B., Wabl, M., 2008. Pvt1-encoded microRNAs in oncogenesis. Retrovirology 5, 4. Boehm, T., Rabbitts, T.H., 1989. The human T cell receptor genes are targets for chromosomal abnormalities in T cell tumors. FASEB J. 3, 2344e2359. Broeks, A., Schmidt, M.K., Sherman, M.E., Couch, F.J., Hopper, J.L., Dite, G.S., Apicella, C., Smith, L.D., Hammet, F., Southey, M.C., Van ’t Veer, L.J., de Groot, R., Smit, V.T., Fasching, P.A., Beckmann, M.W., Jud, S., Ekici, A.B., Hartmann, A., Hein, A., Schulz-Wendtland, R., Burwinkel, B., Marme, F., Schneeweiss, A., Sinn, H.P., Sohn, C., Tchatchou, S., Bojesen, S.E., Nordestgaard, B.G., Flyger, H., Orsted, D.D., Kaur-Knudsen, D., Milne, R.L., Perez, J.I., Zamora, P., Rodriguez, P.M., Benitez, J., Brauch, H., Justenhoven, C., Ko, Y.D., Hamann, U., Fischer, H.P., Bruning, T., Pesch, B., Chang-Claude, J., Wang-Gohrke, S., Bremer, M., Karstens, J.H., Hillemanns, P., Dork, T., Nevanlinna, H.A., Heikkinen, T., Heikkila, P., Blomqvist, C., Aittomaki, K., Aaltonen, K., Lindblom, A., Margolin, S., Mannermaa, A., Kosma, V.M., Kauppinen, J.M., Kataja, V., Auvinen, P., Eskelinen, M., Soini, Y., Chenevix-Trench, G., Spurdle, A.B., Beesley, J., Chen, X., Holland, H., Lambrechts, D., Claes, B., Vandorpe, T., Neven, P., Wildiers, H., FleschJanys, D., Hein, R., Loning, T., Kosel, M., Fredericksen, Z.S., Wang, X., Giles, G.G., Baglietto, L., Severi, G., McLean, C., Haiman, C.A.,

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