Gene Amplification Is a Mechanism of Six1 Overexpression in Breast Cancer

Research Article

Kelly J. Reichenberger,1 Ricardo D. Coletta,2,4 Aline P. Schulte,3 Marileila Varella-Garcia,3 and Heide L. Ford1,2

Departments of 1Biochemistry and Molecular Genetics, 2Obstetrics and Gynecology, and 3Medical Oncology, University of Colorado Health Sciences Center, Aurora, Colorado and 4Discipline of Pathology, University of Campinas Dental School, Piracicaba, Sa¨o Paolo, Brazil

Abstract as in DNA binding together with the homeodomain (10). Several of the Six family members, including Six1, Six3, and Six6, have been The Six1 homeoprotein plays a critical role in expanding progenitor populations during normal development via its implicated in the process of proliferation that precedes differen- stimulation of proliferation and inhibition of apoptosis. tiation (11–19), and it is this proliferative function that may in part Overexpression of Six1 is observed in several tumor types, contribute to their role in tumorigenesis (4, 5). Indeed, a number of suggesting that when expressed out of context, Six1 may the Six family members have been implicated in tumorigenesis. contribute to tumorigenesis by reinstating properties normally Six3 is expressed in human extraskeletal myxoid chondrosarcomas conveyed on developing cells. Indeed, Six1 contributes to (9) and Six5 in borderline ovarian tumors (8). We previously tumor cell proliferation both in breast cancer and in showed that Six1 is overexpressed in breast cancer and that its rhabdomyosarcomas, in which it is also implicated in overexpression leads to an increase in the proliferative capacity of metastasis. Whereas Six1 overexpression has been reported breast cancer cells via an up-regulation of the tissue-restricted cyclin A1 (4, 5). We further showed that overexpression of Six1 in several tumor types, the mechanism responsible for its overexpression has not previously been examined. Here we results in an increase in tumor burden in a nude mouse model of show that a change in gene dosage may contribute to Six1 breast cancer (4). Interestingly, overexpression of Six1 has since mRNA overexpression. Significant Six1 gene amplification and been observed in Wilms’ tumors (6), and in alveolar rhabdomyo- overrepresentation occurs in numerous breast cancer cell lines sarcomas (7), in which it has been shown to be critical for the as compared with normal mammary epithelial cells, and the metastatic process (20). Together, this suggests that overexpression changes in gene dosage correlate with increased Six1 mRNA of Six1 may contribute to the establishment or progression of levels. Of 214 human infiltrating ductal breast carcinomas numerous tumor types. examined for Six1 gene dosage, 4.7% show Six1 amplification/ Although the functional consequences of altered Six1 expression overrepresentation, and tumors that exhibit an increase in are being widely explored, the mechanism for Six1 overexpression Six1 gene dosage overexpress Six1 mRNA. These data implicate in breast cancer has not previously been determined. Here we Six1 gene amplification/overrepresentation as a mechanism report that gene amplification and overrepresentation of Six1 of Six1 mRNA overexpression in human breast cancer. occurs in breast cancer, and that overexpression of Six1 occurs in those tumors in which Six1 gene dosage is altered. (Cancer Res 2005; 65(7): 2668-75)

Introduction Materials and Methods Recent evidence suggests that many parallels exist between Cell culture. The 21T series of cell lines (16N, 21PT, 21NT, 21MT1, and normal development and tumorigenesis. Both processes require 21MT2) were cultured as previously described (21, 22). MCF-10A cells were changes in cellular proliferation, motility, invasion, apoptosis, and cultured in DMEM/F-12 medium supplemented with 5% fetal bovine serum neovascularization, and thus it is not surprising that genes critical (FBS), 0.1 Ag/mL cholera toxin, 10 Ag/mL insulin, 0.5 Ag/mL hydrocortisone, for normal development are often used by neoplastic lesions (1, 2). 0.02 Ag/mL epidermal growth factor, 2 mmol/L L-glutamine, and antibiotics. Indeed, many homeobox genes, which are known to be critical for MDA-MB-435 cells were cultured as previously described (23). MDA-MB-231 and ZR-75-1 cells were cultured in MEM supplemented with 5% FBS, 10 specifying cell fate during development, display altered expression mmol/L HEPES, 2 mmol/L L-glutamine, 0.1 mmol/L nonessential amino in cancer (2), and recent evidence suggests that their altered acids, 0.8 Ag/mL insulin, and antibiotics. MCF7 cells were cultured as expression is not merely correlative but can play a causal role in previously described (24). T47-D cells were cultured in DMEM supple- tumor progression (2–4). mented with 15% FBS, 2 mmol/L L-glutamine, and antibiotics. The Six family of homeobox genes is often misregulated in Quantitative real-time PCR and quantitative real-time RT-PCR. cancer (4–9). Members of the family encode proteins that are Genomic DNA was isolated using the DNeasy kit (Qiagen, Valencia, CA) and characterized by a divergent homeodomain, as well as a second total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) conserved domain, the Six domain, which is amino-terminal to the following the manufacturers’ protocols. Quantitative real-time PCR homeodomain and is involved in interactions with cofactors as well (qPCR) and quantitative real-time RT-PCR (qRT-PCR) experiments were accomplished using the Applied Biosystems model 7700 instrument (Foster City, CA). Amplified fragments were detected by using TaqMan fluorescence probes as previously described (25). Primers and probes for Six1 are as follows: Six1 forward primer, 5V-CACCTCCCCAAAGTCCAGAC-3V; Six1 Requests for reprints: Haide L. Ford, Obstetrics and Gynecology, Division of Basic reverse primer, 5V-CCTGGCGTGGCCCATA-3V; Six1 TaqMan probe, 5V-CGG Reproductive Sciences, University of Colorado Health Sciences Center, Fitzsimons TCCTTCTGCTGCAGGGCATA-3V. Standard curves were used to determine Campus, Mail stop 8309, PO BOX 6511, Aurora, CO 80045. Phone: 303-724-3509; Fax: 303-724-3512; E-mail: [email protected]. relative levels of DNA content or target gene expression. DNA samples were #2005 American Association for Cancer Research. normalized according to the levels of h-actin and RNA samples were

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2005 American Association for Cancer Research. Six1 Gene Amplification and Overexpression in Cancer normalized according to the levels of 18S rRNA. DNA or RNA isolations and 1.5 were considered overrepresented (29). In the few cases in which were done independently for each experiment. two tissue cores from the same patient had different patterns, the more Northern and Southern blot analysis. RNA (15 Ag) from each complex of the two patterns was used to define the patient. respective cell line was electrophoresed on a formaldehyde-agarose gel, RNA in situ hybridizations. Full-length Six1 was cloned into the transferred via capillary action, and probed with a radioactively labeled pZERO2 plasmid (Invitrogen), which includes SP6 and T7 promoter sites cDNA fragment of Six1, as previously described (5). Genomic DNA (10 Ag) to enable the generation of sense and antisense probes with the same isolated from the cell lines was digested with EcoR1, electrophoresed on a template DNA. Human Six1 sense and antisense probes were generated 1% agarose gel, transferred, and probed with a radioactively labeled cDNA using a DIG RNA labeling kit (Roche, Indianapolis, IN) following the fragment of Six1 or h-actin. manufacturer’s protocol. RNA in situ hybridizations were done as Fluorescence in situ hybridizations. DNA probes recognizing the Six1 previously described (30) on Ambion Landmark High Density Breast amplicon were derived from BAC clones RP11-1042B17, RP11-307P22, and Specific Array (Lot 013P09A). In cases in which two cores from the same RP11-246E14 and were generated using Vysis SpectrumRed-conjugated patient had different signal levels, the more complex of the two patterns dUTP and the Vysis Nick translation labeling kit following a modified was used to define the patient. version of the manufacturer’s protocol (Downer’s Grove, IL; ref. 26). A Statistical significance. One-way ANOVA was used to determine if centromeric chromosome 14 probe is not available; thus, the control cancer lines had differential Six1 expression as compared with normal lines. probe used was a near-centromeric chromosome 14 probe (14q11; RP11- Post hoc comparisons were made using the Kruskal-Wallis multiple 324B11) labeled with Vysis SpectrumGreen-conjugated dUTP. Fluores- comparison z test. In all cases, P < 0.05 was considered statistically cence in situ hybridization (FISH) was done on cell lines (26) and tissue significant, and all cases of significance are indicated with an asterisk. sections (27) as previously described. Breast cancer tissue arrays were obtained from the Cooperative Human Tissue Network [fifth-generation Tissue Array Research Program (TARP) multitumor tissue microarrays, Results specifically, the T-BO-1, TARP breast and ovarian cancer array] and from Ambion (LandMark High Density Breast Specific Array Lots 013P09A and Six1 is amplified, overrepresented, and overexpressed in 072P08A, Austin, TX). Approximately 100 nuclei were analyzed per tissue breast cancer cell lines. To identify a mechanism that may core. Analysis was done by dividing 14q23 (Six1) by 14q11 (near- contribute to Six1 overexpression in breast cancer, we examined centromere 14 control) signals in interphase FISH experiments. Ratios gene copy number in the 21T series of mammary carcinoma cell that were >1.5 were considered amplified (28), and ratios between 1.3 lines, a unique model for studying breast cancer progression. This

Figure 1. Six1 is overexpressed and amplified in the 21T series. A, Northern blot analysis of the 21T series using a radiolabeled Six1 cDNA probe shows an increase of Six1 mRNA in the tumor lines (21NT, PT, MT1, and MT2) as compared with the normal, immortalized cell line (16N). Bottom, ethidium bromide staining of 18s and 28s rRNA as a loading control. B, Southern blot analysis of the 21T series using a radiolabeled Six1 cDNA probe. Bottom, the same blot probed with radiolabeled actin cDNA as a loading control. C, qPCR on genomic DNA from the 21T series using Six1-specific primers/ probe normalized to actin shows an increase in gene dosage in the tumorigenic lines as opposed to the normal, immortalized 16N cell line. Columns, average of three different experiments. Bars, SE. D, FISH analysis done on metaphase spreads (top) or interphase nuclei (bottom) of the indicated cell lines shows 14q23 amplification in the tumor cell lines as opposed to the normal, immortalized 16N cell line. The SpectrumGreen-labeled probe was generated from a region close to the centromere of chromosome 14 as a copy number control (BAC clone RP11-324B11), whereas the SpectrumRed-labeled probe was generated from the chromosomal region that contains Six1 (BAC clone RP11-1042B17).

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Table 1. Six1 gene dosage in normal and cancerous mammary cell lines

Cell line Control (B11) Six1 (B17) Six1/control ratio

Range Average Range Average

21T series of cell lines 16N (normal) 2-8 3.9 2-8 3.9 1.0 21NT (primary) 3-6 3.9 3-20 13.1 3.4 21PT (primary) 3-4 4.0 1-14 11.0 2.8 21MT1 (met) 4-8 6.4 11-24 19.4 3.0 21MT2 (met) 3 3.0 10-12 10.8 3.6 Other normal and cancer mammary cell lines MCF-10A 2 2.0 2 2.0 1.0 MDA-MB-435 1-5 2.1 1-5 2.1 1.0 MDA-MB-231 2-3 2.2 2-4 2.4 1.1 ZR-75-1 2-3 2.1 2-4 2.5 1.2 MCF7 3-5 3.9 3-7 5.1 1.3 47-D 2 2.0 3-4 5.7 2.9

NOTE: Gene amplification was determined by comparing interphase ratios of the chromosomal region that contains Six1 (BAC clone RP11-1042B17, or B17) to a region representing the near centromere of chromosome 14 as a copy number control (BAC clone RP11-324B11, or B11). Range denotes the range of signals per cell observed in all cells counted from the cell line, and average denotes the average number of signals per cell. Gene amplification was noted if the ratio of Six1/control was >1.5; overrepresentation was noted if the Six1/control ratio was >1.3 but <1.5. 21T series: cell lines derived from a single patient with breast cancer who was diagnosed with an infiltrating ductal carcinoma. 16N was derived from the patient’s normal adjacent breast tissue, 21PT and 21NT from her primary tumor, and 21MT1 and 21MT2 from her metastatic pleural effusion. Other normal and cancerous mammary cell lines: MCF-10A, normal; all other listed lines are breast cancer cell lines.

series consists of normal and breast tumor cell lines derived from a To determine if the increase in Six1 gene dosage in the 21Tseries is single patient with breast cancer who had an infiltrating ductal specific for this series of cell lines (all derived from the same patient) carcinoma (21, 22, 31, 32). H16N2 (16N) was isolated and derived or whether it is a more general phenomenon in breast cancer, from the patient’s normal adjacent breast tissue (32), 21NT and Southern blot, qPCR, and FISH analyses were done on additional 21PT from her primary tumor, and 21MT1 and 21MT2 from normal, immortalized breast (MCF-10A) and cancer cell lines (MCF7, her metastatic pleural effusions (21, 22, 31). Interestingly, the MDA-MB-231, MDA-MB-435, T47-D, and ZR-75-1). As shown by all immortalized 16N mammary epithelial cell line expresses almost no three methods, MCF7, T47-D, and ZR-75-1 breast cancer cell lines Six1, whereas the primary and metastatic breast tumor lines express exhibit an increase in Six1 DNA content as compared with the high levels of Six1 (Fig. 1A), thus making this series of cell lines ideal normal breast cell line MCF-10A via (data not shown; Fig. 2A and B). for examining the cause of Six1 overexpression in an isogenic FISH analyses on both metaphase (Fig. 2B) and interphase cells (not background. Southern blot and qPCR analyses identified that the shown) further show that the mode of increasing Six1 DNA in these normal, immortalized 16N mammary epithelial cells have lower cell lines is both gene amplification, which is due to tandem levels of Six1 DNA than the breast tumor lines (21NT, 21PT, 21MT1, duplications, and overrepresentation, which is the result of and 21MT2), thus indicating that gene dosage is altered in the imbalanced genomic gains due to chromosomal rearrangements. tumorigenic lines and that gene amplification or overrepresentation Overrepresentation, like gene amplification, leads to an increase in may play a causal role in Six1 mRNA overexpression in this patient Six1 gene dosage, albeit at lower levels. Previous reports have (Fig. 1B and C). This was confirmed by FISH analysis on both suggested that interphase ratios (gene/control) between 1.3 and 1.5 metaphase spreads and interphase nuclei from each of the 21T represent overrepresentation (29), whereas those z1.5 generally series of cell lines using the control 14q11 probe (SpectrumGreen represent amplification (28). By these criteria, Six1 is amplified in labeled) versus the Six1-specific probe (SpectrumRed labeled; Fig. T47-D cells and overrepresented in MCF7 cells (Table 1), but is not 1D). FISH patterns observed in the metaphase spreads of the cancer amplified or overrepresented in ZR-75-1 cells, which fall just below lines 21NT, 21PT, 21MT1, and 21MT2 suggest that gene amplifica- the cutoff for overrepresentation (Six1/control = 1.2, Table 1). tion is the mechanism by which Six1 gene dosage is altered, because However, it should be noted that the low interphase ratio of Six1/ clusters of Six1 signals are present (Fig. 1D). FISH analyses done on control in ZR-75-1 cells occurs because of focal amplification, interphase nuclei was used to quantitate the number of Six1 gene because only 43% of the scored cells had increased copies of Six1, and copies per 14q11 copies per cell in the cell lines. An increased ratio the remaining 57% of cells had no gain in Six1 DNA. Thus, the actual of Six1 (red) to 14q11 (green) was observed in all of the tumorigenic ratio of Six1/control per cell, when examining only the cells in which lines as compared with the normal 16N cell line (Table 1). The Six1/ a gain in Six1 gene dosage occurs, is in fact considerably higher. This control ratio in each of the tumorigenic cell lines was greater than suggests that overrepresentation/amplification does occur in the the 1.5 defined to represent gene amplification (28), thus confirming ZR-75-1 mammary carcinoma cell line, although only focally. that in contrast to the 16N normal mammary epithelial cell line, To determine whether Six1 expression correlates with gene all four mammary tumor lines have amplified Six1. dosage in the above-mentioned normal and mammary carcinoma

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2005 American Association for Cancer Research. Six1 Gene Amplification and Overexpression in Cancer cell lines, qRT-PCR analyses were done (Fig. 2C). The breast cancer contribute to cancer, we walked down chromosome 14 in cell lines MCF7 and T47-D exhibit 8-fold and 3.7-fold increases in 21MT1 breast cancer cells (because Six1 is amplified in this cell Six1 expression, respectively, as compared with the normal line line) via FISH analysis with numerous BAC clones (Fig. 3). Using MCF-10A (Fig. 2C). In contrast, the ZR-75-1 cell line, in which a low SpectrumRed-labeled probes derived from various BAC clones, we level of overrepresentation occurs in a percentage of cells, did not mapped the 14q23 amplicon in the 21MT1 cell line and found have a significant increase in Six1 mRNA levels. MDA-MB-231 and that it extends f5 Mb and contains f35 genes, including Six1, MDA-MB-435 cells, in which Six1 gene dosage was not altered, also Six4, and Six6 (Fig. 3). do not exhibit statistically significant increases in Six1 expression 14q23 Status and Six1 expression in human breast tumors. as compared with MCF-10A cells (Fig. 2C). The correlation of Six1 To determine the prevalence of 14q23 amplification/overrepresen- overrepresentation with Six1 expression in numerous breast cancer tation in human breast cancer, we did FISH on multiple breast cell lines suggests that gene dosage is an important mechanism for tumor tissue arrays using the control 14q11 (green) and the RP11- inappropriately up-regulating Six1 in breast cancer. 1042B17 probe (red), which extends across the region of 14q23 Six4 and Six6, in addition to Six1, are amplified in containing Six1 (Fig. 3). Of the 214 infiltrating ductal carcinomas 21MT1 cells. The 14q23 chromosomal region contains a cluster examined, 4.7% (10/214) were positive for unbalanced 14q23 gains. of Six family members including Six1, Six4, and Six6 (ref. 33; Fig. Of the 10 cases where 14q23 gains were observed, 9 had Six1/ 3A). Because numerous Six family members have been implicated control ratios z1.5, and 6 had obvious clusters of Six1, in proliferation during normal development (11–19), it is possible demonstrating that most of the gains were due to gene that they may synergistically act to stimulate proliferation in a amplification (Table 2; Fig. 4A). Only 1 of 10 cases had a Six1/ tumorigenic setting. To determine whether the amplicon contains control ratio just under 1.5, falling in the category of overrepre- Six4 and Six6 in addition to Six1, as well as to identify other sentation as opposed to gene amplification (Table 2). However, this genes that may be in the 14q23 amplicon and that may patient exhibited clusters of Six1 DNA signals in some cells,

Figure 2. Six1 is amplified/overrepresented and overexpressed in MCF-7 and T47-D breast cancer cell lines. A, qPCR on genomic DNA from normal, immortalized breast (MCF-10A) and breast cancer (MCF-7, MDA-MB-231, MDA-MB-435, T47-D, and ZR-75-1) cell lines using Six1-specific primers/probe (normalized to h-actin) shows increased Six1 DNA content in MCF7, T47-D, and ZR-75-1 cells as compared with MCF-10A cells. Columns, average of three experiments; bars, SE. B, FISH analyses done (as in Fig. 1) on metaphase spreads of the indicated cell lines confirms Six1 gene gain in MCF7, T47-D, and ZR-75-1 cells. C, qRT-PCR of total RNA from normal breast (MCF-10A) and breast cancer (MCF-7, MDA-MB-231, MDA-MB-435, T47-D, and ZR-75-1) cell lines using Six1-specific primers/probe normalized to 18s rRNA shows an increase in Six1 mRNA levels in MCF7 and T47-D cells as compared with MCF-10A cells. Note that ZR-75-1 did not exhibit increased expression of Six1 (likely due to the low level of gain in overall Six1 gene dosage in this cell line, in which Six1 overrepresentation/amplification was focal). Columns, average of three experiments; bars, SE. Significance is indicated when the lines have statistically more Six1 mRNA expression than the MCF-10A line (*).

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Figure 3. The Six1 amplicon in 21MT1 cells is f5 Mb in size and includes Six1 family members Six4 and Six6. Ideogram of chromosome 14 (top) showing relative locations of Six1, Six4, and Six6 and BAC clones used for FISH analysis. BAC clones tested are listed under the ideogram and under each FISH figure in which the particular BAC clone was used. Clones outlined in red, amplification when hybridized to chromosome 14; clones outlined in yellow, not amplified. Chromosomes with amplification and interphase cells showing clusters of probe signals are both illustrated (red) as well as interphase cells without amplification (yellow). Clones are abbreviated as follows: RP11 clones: RP11 649E7, RP11 794A8 (794A8), RP11 409I10 (409I10), RP11 62H20 (62H20), RP11 246E14 (E14), RP11 1042B17 (B17), RP11 307P22 (P22), RP11 193F5 (193F5), RP11 22F2 (22F2), RP11 902B17 (902B17), RP11 355I22 (355I22), RP11 712C19 (712C19), RP11 125H8 (125H8). CTD clones: CTD 2184C24 (C24), CTD 2568P8 (P8). suggesting that focal gene amplification occurred in this patient’s greater) was observed in 47 (52%) of 90 primary infiltrating ductal tumor (not shown). Similar to the situation described in the ZR- carcinomas, and in 8 (89%) of 9 metastases resulting from 75-1 cell line above, the Six1/control ratio was thus reduced in this infiltrating ductal carcinomas. This correlates well with our patient due to tumor heterogeneity. previous study in which Six1 overexpression was first described in To further determine whether 14q23 amplification/overrepre- breast cancer (5). sentation correlates with Six1 gene expression in human breast tumors, in situ hybridization using a Six1-specific antisense probe was done on a second Ambion array (also used for the FISH Discussion analysis, Ambion 2) as this was the only array for which we had Previous studies have implicated Six1 in the etiology of several duplicate slides that were of high enough quality to perform both types of cancer, including breast cancer, Wilms’ tumor, and analyses. This array contained 90 high-quality, analyzable, primary rhabdomyosarcoma (4–7, 20), and it contributes to at least some infiltrating ductal carcinomas, 7 of which showed Six1 amplifi- of these cancers by controlling the cell cycle (4, 5) as well as by cation/overrepresentation (8%). Six (86%) of seven overexpressed controlling properties important for metastasis (20). Our data Six1f3-fold as compared with the average expression level in suggest that gene amplification/overrepresentation is one mecha- normal breast samples (n = 14), and the remaining 1 tumor nism responsible for Six1 overexpression in breast cancer. expressed Six1 at levels 1.4-fold above the normal breast control Amplification/overrepresentation of the region on 14q23 in which (Table 2; Fig. 4). No staining was observed in duplicate samples Six1 resides was found to correlate with Six1 expression in hybridized to the Six1-specific sense probe (not shown). Together, numerous breast cancer cell lines examined as well as in human this shows that the 14q23 genomic gain encompassing the Six1 breast infiltrating ductal carcinomas. Corroborating our finding, gene correlates with Six1 expression, and suggests that Six1 gene comparative genomic hybridization studies have shown that breast amplification/overrepresentation leads to Six1 overexpression. It cancers (34, 35), chondrosarcomas (36), prostate cancer (37), and should be noted that tumors that did not have 14q23 genomic fibrosarcomas (38) show increased 14q23 DNA content. In contrast, gains also showed Six1 overexpression, suggesting that other other studies have observed a loss of 14q23 DNA in breast cancer mechanisms are working to inappropriately increase expression of (39, 40), gastrointestinal stromal tumors (41), and neuroblastomas Six1. Altogether, Six1 overexpression (at a level of 3-fold or (42) and when taken together with the formerly mentioned

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2005 American Association for Cancer Research. Six1 Gene Amplification and Overexpression in Cancer comparative genomic hybridization studies, suggest that both (34, 55) and that, on the whole, as little as a 2-fold change in oncogenes and tumor suppressor genes may be found at 14q23. As DNA copy number is associated with a corresponding 1.5-fold 14q23 gains have not frequently and consistently been observed in change in mRNA levels (34). In this study, 52% of all primary human cancers, it may be necessary to use more precise analysis infiltrating ductal carcinomas overexpress Six1 (3-fold) as com- looking at specific genes to determine the role of this locus in pared with the average expression in normal mammary gland, breast and other cancers. As such, this report shows that when whereas 86% of primary infiltrating ductal carcinomas in which examined at a more refined location (encompassing the region on Six1 gene amplification is detected display a 3-fold overexpression 14q23 in which Six1 resides), 14q23 gains do indeed occur in a of Six1. This suggests that gene amplification is indeed an small, albeit significant, percentage of breast cancer cases. important mechanism for Six1 overexpression. Because f5% of infiltrating ductal carcinomas exhibit Six1 gene Interestingly, both Six4 and Six6 are amplified along with Six1 in amplification/overrepresentation, whereas about 50% of primary 21MT1 cells (Fig. 3) and therefore may be co-overexpressed with Six1 infiltrating ductal carcinomas overexpress Six1, additional mech- in a subset of breast cancers. Presently, it is known that over- anisms must exist that contribute to its overexpression in breast expression of Six1 in breast cancer cell lines can increase tumors. Such a phenomenon has been reported for other genes proliferation via the reactivation of the tissue-restricted cyclin A1 that are critical in breast tumorigenesis. For example, the (4); however, the role of Six4 and Six6 has not yet been examined. As chromosomal locus 11q13, in which cyclin D1 resides, is amplified numerous members of the Six family, including Six6, have been in up to 15% of breast cancers (43, 44), yet cyclin D1 is implicated in proliferative processes (4, 5, 10–19), the coordinate overexpressed in 35% to 45% of these cancers (45, 46). Additional overexpression of three Six family members in breast cancer may mechanisms that may contribute to the differential expression of a result in, among other things, a hyperproliferative phenotype gene in cancer include changes in histone acetylation or in DNA contributing to tumorigenesis. It will therefore be important to methylation (47–49), in mRNA stability (50), or in promoter activity determine whether Six4 and Six6 are co-overexpressed with Six1 in (51), among others. Six1 was recently identified as a target of E2F1 breast cancers that display 14q23 amplification/overrepresentation (52), a transcription factor critical for the G1-S transition whose and, furthermore, to determine whether these genes, along with activity is inhibited by Rb (53, 54). In many human cancers, some Six1, contribute to the tumorigenic process. aspect of the E2F/Rb pathway is altered (54). Thus, Six1 The amplicon on 14q23 in 21MT1 cells is f5 Mb long (see Fig. 3) overexpression in breast cancer may occur as a result of a and contains at least 35 genes in addition to Six family members, combination of gene amplification/overrepresentation and in- including several that are implicated in cancer. Genes present in the creased transcription due to an altered E2F/Rb pathway. amplicon that are associated with cancer include the hypoxia- Although multiple mechanisms may be involved in Six1 over- inducible factor 1 gene (HIF-1A), which encodes a transcription expression, the importance of gene amplification as a mechanism factor that is overexpressed in many types of cancer and whose of overexpression is clear. Recent studies show that gene transcriptional targets include several genes responsible for amplification is highly correlated with mRNA overexpression adaptation to reduced oxygen or hypoxic conditions, a common

Table 2. Descriptive statistics for 10 patients classified with an unbalanced gain of the Six1 gene

Tissue Patient Control (B11) SIX1 (B17) SIX1/Control SIX1 mRNA microarray (fold increase)* Range Average SD Range Average SD

c TARP 25 1-2 1.93 0.26 1-5 3.40 0.74 1.8 ND c Ambion 1 18 1-3 2.22 0.83 1-4 3.67 0.50 1.7 ND 63 1-3 1.67 0.65 2-4 2.91 0.70 1.7 ND c Ambion 2 14 1-6 2.74 1.31 3-8 4.68 1.63 1.7 3 c 17 1-6 2.23 1.41 1-7 3.03 1.52 1.4 3 c 31 1-3 1.93 0.62 3-8 4.07 0.72 2.1 3 c 34 1-2 1.64 0.49 3-8 3.87 0.64 2.4 3 42 2-4 2.22 0.67 3-6 3.56 0.73 1.6 3 80 2-3 2.40 0.52 2-5 3.50 0.85 1.5 1.4 107 1-3 2.35 0.50 3-6 4.09 0.80 1.7 3

NOTE: Results are summarized for FISH analyses (Six1/control) and Six1 in situ hybridization in 10 patient samples displaying Six1 amplification/ overrepresentation. The results for each different array are listed. The total number of high-quality, analyzable primary infiltrating ductal carcinomas on each array were as follows: TARP, 36; Ambion 1, 88; and Ambion 2, 90, to yield a total sample number of 214. For FISH experiments, amplification was determined by comparing interphase ratios of the chromosomal region that contains Six1 (BAC clone RP11-1042B17, or B17) to a region representing the near centromere of chromosome 14 as a copy number control (BAC clone RP11-324B11, or B11). Range denotes the range of signals per cell observed in all cells counted from the tumor sample; average denotes the average number of signals per cell. Gene amplification was noted if the ratio of Six1/control was >1.5; overrepresentation was noted if the Six1/control ratio was >1.3 but <1.5. Abbreviations: ND, not determined; SD, standard deviation. *Approximate fold increase in Six1 mRNA expression detected in the tumor samples as compared with the average mRNA expression of Six1 in normal breast samples. cSamples in which clusters of the Six1 gene were identified.

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Figure 4. 14q23 amplification correlates with Six1 overexpression in invasive ductal carcinomas. FISH (SIX1/Con), FISH analyses in one representative cell from several tumors in which Six1 gene amplification was detected [patient (Pt) 14, 31, 34, and 42] as well as in normal breast in which Six1 gene amplification was not observed (patient 3). The analyses were done as described in Fig. 1. Six1 in situ, Six1 expression as determined by in situ hybridization using an antisense Six1 digoxigenin-riboprobe for each corresponding complete core. Control in situ hybridizations using a sense Six1 digoxigenin-riboprobe did not yield any signal (not shown). The level of Six1 expression was scored by two individuals on a scale of 0 to 4 and used to quantitate Six1 overexpression as compared with control breast (see Table 2). H&E, H&E staining from each core represented.

state in many solid tumors (37). In addition, the amplified region of primarily expressed during embryonic mammary gland develop- 14q23 contains the PPKCH gene, which encodes PKC-D, a protein ment, is lost in the adult, differentiated mammary gland, and is kinase that has been implicated in various tumors, both positively reexpressed in breast cancer (4). Furthermore, the discovery that and negatively (56–59). Of additional interest in this locus is the RBP- additional Six family members may be amplified in breast cancers 1 gene. This gene encodes a retinoblastoma-binding protein that exhibiting Six1 gene amplification suggests that a coordinate up- promotes growth by inhibiting Rb (60). Interestingly, antibodies regulation of the genes may synergistically promote breast cancer, an against a heptameric peptide sequence within RBP-1 were found in issue that warrants further investigation. breast cancer patients, and work is under way to develop such peptide antigens as a potential vaccine against breast cancer (60). Acknowledgments Thus, it is possible that these genes, as well as others within the Received 12/1/2004; revised 1/10/2005; accepted 1/14/2005. 14q23 amplicon, cooperate with the Six family members to enhance Grant support: NIH grant 1R01CA095277-01, Susan G. Komen Breast Cancer the tumorigenic phenotype in cells in which they coamplify. Foundation (9862), American Cancer Society/University of Colorado Cancer Center, In closing, we have examined the prevalence of a genomic gain of and Avon Foundation (H.L. Ford). K.J. Reichenberger was supported by an institutional fellowship from the Department of Defense Breast Cancer Program, and R.D. Coletta the 14q23 region encompassing the Six1 gene in breast cancer. As by fellowships from the Coordenacao de Aperfeicoamento de Pessoal de Nivel Six1 is implicated in breast cancer progression (4, 5), its Superior (Brazil), the University of Colorado Cancer Center, the Cancer League of Colorado, and the W.M. Thorkildsen Foundation. amplification and subsequent overexpression may play an impor- The costs of publication of this article were defrayed in part by the payment of page tant role in the etiology of the disease. Determining both the charges. This article must therefore be hereby marked advertisement in accordance mechanisms and consequences of Six1 overexpression in breast with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Drs. Andrew Bradford and Paul Jedlicka for helpful comments and critical cancer remain an important issue to address, particularly as the gene reading of the manuscript, Dr. Jedlicka for help in analyzing tumor expression data, may serve as an excellent diagnostic and therapeutic target as it is and Dr. Peter Jones for contributing reagents needed to carry out the tissue array work.

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Kelly J. Reichenberger, Ricardo D. Coletta, Aline P. Schulte, et al.

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