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[Cancer Research 65, 2668-2675, April 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Gene Amplification Is a Mechanism of Six1 Overexpression in Breast Cancer

Kelly J. Reichenberger1, Ricardo D. Coletta2,4, Aline P. Schulte3, Marileila Varella-Garcia3 and Heide L. Ford1,2

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

Requests for reprints: Haide L. Ford, Obstetrics and Gynecology, Division of Basic Reproductive Sciences, University of Colorado Health Sciences Center, Fitzsimons Campus, Mail stop 8309, PO BOX 6511, Aurora, CO 80045. Phone: 303-724-3509; Fax: 303-724-3512; E-mail: heide.ford{at}uchsc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Six1 homeoprotein plays a critical role in expanding progenitor populations during normal development via its stimulation of proliferation and inhibition of apoptosis. Overexpression of Six1 is observed in several tumor types, suggesting that when expressed out of context, Six1 may contribute to tumorigenesis by reinstating properties normally conveyed on developing cells. Indeed, Six1 contributes to tumor cell proliferation both in breast cancer and in rhabdomyosarcomas, in which it is also implicated in metastasis. Whereas Six1 overexpression has been reported in several tumor types, the mechanism responsible for its overexpression has not previously been examined. Here we show that a change in gene dosage may contribute to Six1 mRNA overexpression. Significant Six1 gene amplification and overrepresentation occurs in numerous breast cancer cell lines as compared with normal mammary epithelial cells, and the changes in gene dosage correlate with increased Six1 mRNA levels. Of 214 human infiltrating ductal breast carcinomas examined for Six1 gene dosage, 4.7% show Six1 amplification/overrepresentation, and tumors that exhibit an increase in Six1 gene dosage overexpress Six1 mRNA. These data implicate Six1 gene amplification/overrepresentation as a mechanism of Six1 mRNA overexpression in human breast cancer.

Key Words: Six1 • homeobox genes • breast cancer • gene amplification • gene overrepresentation • overexpression


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Recent evidence suggests that many parallels exist between normal development and tumorigenesis. Both processes require changes in cellular proliferation, motility, invasion, apoptosis, and neovascularization, and thus it is not surprising that genes critical for normal development are often used by neoplastic lesions (1, 2). Indeed, many homeobox genes, which are known to be critical for specifying cell fate during development, display altered expression in cancer (2), and recent evidence suggests that their altered expression is not merely correlative but can play a causal role in tumor progression (2–4).

The Six family of homeobox genes is often misregulated in cancer (4–9). Members of the family encode proteins that are characterized by a divergent homeodomain, as well as a second conserved domain, the Six domain, which is amino-terminal to the homeodomain and is involved in interactions with cofactors as well as in DNA binding together with the homeodomain (10). Several of the Six family members, including Six1, Six3, and Six6, have been implicated in the process of proliferation that precedes differentiation (11–19), and it is this proliferative function that may in part contribute to their role in tumorigenesis (4, 5). Indeed, a number of the Six family members have been implicated in tumorigenesis. Six3 is expressed in human extraskeletal myxoid chondrosarcomas (9) and Six5 in borderline ovarian tumors (8). We previously showed that Six1 is overexpressed in breast cancer and that its overexpression leads to an increase in the proliferative capacity of breast cancer cells via an up-regulation of the tissue-restricted cyclin A1 (4, 5). We further showed that overexpression of Six1 results in an increase in tumor burden in a nude mouse model of breast cancer (4). Interestingly, overexpression of Six1 has since been observed in Wilms' tumors (6), and in alveolar rhabdomyosarcomas (7), in which it has been shown to be critical for the metastatic process (20). Together, this suggests that overexpression of Six1 may contribute to the establishment or progression of numerous tumor types.

Although the functional consequences of altered Six1 expression are being widely explored, the mechanism for Six1 overexpression in breast cancer has not previously been determined. Here we report that gene amplification and overrepresentation of Six1 occurs in breast cancer, and that overexpression of Six1 occurs in those tumors in which Six1 gene dosage is altered.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. The 21T series of cell lines (16N, 21PT, 21NT, 21MT1, and 21MT2) were cultured as previously described (21, 22) . MCF-10A cells were cultured in DMEM/F-12 medium supplemented with 5% fetal bovine serum (FBS), 0.1 µg/mL cholera toxin, 10 µg/mL insulin, 0.5 µg/mL hydrocortisone, 0.02 µg/mL epidermal growth factor, 2 mmol/L L-glutamine, and antibiotics. 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 mmol/L HEPES, 2 mmol/L L-glutamine, 0.1 mmol/L nonessential amino acids, 0.8 µg/mL insulin, and antibiotics. MCF7 cells were cultured as previously described (24). T47-D cells were cultured in DMEM supplemented with 15% FBS, 2 mmol/L L-glutamine, and antibiotics.

Quantitative real-time PCR and quantitative real-time RT-PCR. Genomic DNA was isolated using the DNeasy kit (Qiagen, Valencia, CA) and total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturers' protocols. Quantitative real-time PCR (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, 5'-CACCTCCCCAAAGTCCAGAC-3'; Six1 reverse primer, 5'-CCTGGCGTGGCCCATA-3'; Six1 TaqMan probe, 5'-CGGTCCTTCTGCTGCAGGGCATA-3'. Standard curves were used to determine relative levels of DNA content or target gene expression. DNA samples were normalized according to the levels of ß-actin and RNA samples were normalized according to the levels of 18S rRNA. DNA or RNA isolations were done independently for each experiment.

Northern and Southern blot analysis. RNA (15 µg) from each respective cell line was electrophoresed on a formaldehyde-agarose gel, transferred via capillary action, and probed with a radioactively labeled cDNA fragment of Six1, as previously described (5). Genomic DNA (10 µg) isolated from the cell lines was digested with EcoR1, electrophoresed on a 1% agarose gel, transferred, and probed with a radioactively labeled cDNA fragment of Six1 or ß-actin.

Fluorescence in situ hybridizations. DNA probes recognizing the Six1 amplicon were derived from BAC clones RP11-1042B17, RP11-307P22, and RP11-246E14 and were generated using Vysis SpectrumRed-conjugated dUTP and the Vysis Nick translation labeling kit following a modified version of the manufacturer's protocol (Downer's Grove, IL; ref. 26). A centromeric chromosome 14 probe is not available; thus, the control probe used was a near-centromeric chromosome 14 probe (14q11; RP11-324B11) labeled with Vysis SpectrumGreen-conjugated dUTP. Fluorescence in situ hybridization (FISH) was done on cell lines (26) and tissue 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, specifically, the T-BO-1, TARP breast and ovarian cancer array] and from Ambion (LandMark High Density Breast Specific Array Lots 013P09A and 072P08A, Austin, TX). Approximately 100 nuclei were analyzed per tissue core. Analysis was done by dividing 14q23 (Six1) by 14q11 (near-centromere 14 control) signals in interphase FISH experiments. Ratios that were >1.5 were considered amplified (28), and ratios between 1.3 and 1.5 were considered overrepresented (29). In the few cases in which two tissue cores from the same patient had different patterns, the more complex of the two patterns was used to define the patient.

RNA in situ hybridizations. Full-length Six1 was cloned into the pZERO2 plasmid (Invitrogen), which includes SP6 and T7 promoter sites to enable the generation of sense and antisense probes with the same template DNA. Human Six1 sense and antisense probes were generated using a DIG RNA labeling kit (Roche, Indianapolis, IN) following the manufacturer's protocol. RNA in situ hybridizations were done as previously described (30) on Ambion Landmark High Density Breast Specific Array (Lot 013P09A). In cases in which two cores from the same patient had different signal levels, the more complex of the two patterns was used to define the patient.

Statistical significance. One-way ANOVA was used to determine if cancer lines had differential Six1 expression as compared with normal lines. Post hoc comparisons were made using the Kruskal-Wallis multiple comparison z test. In all cases, P < 0.05 was considered statistically significant, and all cases of significance are indicated with an asterisk.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Six1 is amplified, overrepresented, and overexpressed in breast cancer cell lines. To identify a mechanism that may contribute to Six1 overexpression in breast cancer, we examined gene copy number in the 21T series of mammary carcinoma cell lines, a unique model for studying breast cancer progression. This series consists of normal and breast tumor cell lines derived from a single patient with breast cancer who had an infiltrating ductal carcinoma (21, 22, 31, 32). H16N2 (16N) was isolated and derived from the patient's normal adjacent breast tissue (32), 21NT and 21PT from her primary tumor, and 21MT1 and 21MT2 from her metastatic pleural effusions (21, 22, 31). Interestingly, the immortalized 16N mammary epithelial cell line expresses almost no Six1, whereas the primary and metastatic breast tumor lines express high levels of Six1 (Fig. 1A), thus making this series of cell lines ideal for examining the cause of Six1 overexpression in an isogenic background. Southern blot and qPCR analyses identified that the normal, immortalized 16N mammary epithelial cells have lower levels of Six1 DNA than the breast tumor lines (21NT, 21PT, 21MT1, and 21MT2), thus indicating that gene dosage is altered in the tumorigenic lines and that gene amplification or overrepresentation may play a causal role in Six1 mRNA overexpression in this patient (Fig. 1B and C). This was confirmed by FISH analysis on both metaphase spreads and interphase nuclei from each of the 21T series of cell lines using the control 14q11 probe (SpectrumGreen labeled) versus the Six1-specific probe (SpectrumRed labeled; Fig. 1D). FISH patterns observed in the metaphase spreads of the cancer lines 21NT, 21PT, 21MT1, and 21MT2 suggest that gene amplification is the mechanism by which Six1 gene dosage is altered, because clusters of Six1 signals are present (Fig. 1D). FISH analyses done on interphase nuclei was used to quantitate the number of Six1 gene copies per 14q11 copies per cell in the cell lines. An increased ratio of Six1 (red) to 14q11 (green) was observed in all of the tumorigenic lines as compared with the normal 16N cell line (Table 1). The Six1/control ratio in each of the tumorigenic cell lines was greater than the 1.5 defined to represent gene amplification (28), thus confirming that in contrast to the 16N normal mammary epithelial cell line, all four mammary tumor lines have amplified Six1.



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  		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

 
To determine if the increase in Six1 gene dosage in the 21T series is specific for this series of cell lines (all derived from the same patient) or whether it is a more general phenomenon in breast cancer, Southern blot, qPCR, and FISH analyses were done on additional normal, immortalized breast (MCF-10A) and cancer cell lines (MCF7, MDA-MB-231, MDA-MB-435, T47-D, and ZR-75-1). As shown by all three methods, MCF7, T47-D, and ZR-75-1 breast cancer cell lines exhibit an increase in Six1 DNA content as compared with the normal breast cell line MCF-10A via (data not shown; Fig. 2A and B). FISH analyses on both metaphase (Fig. 2B) and interphase cells (not shown) further show that the mode of increasing Six1 DNA in these cell lines is both gene amplification, which is due to tandem duplications, and overrepresentation, which is the result of imbalanced genomic gains due to chromosomal rearrangements. Overrepresentation, like gene amplification, leads to an increase in Six1 gene dosage, albeit at lower levels. Previous reports have suggested that interphase ratios (gene/control) between 1.3 and 1.5 represent overrepresentation (29), whereas those ≥1.5 generally represent amplification (28). By these criteria, Six1 is amplified in T47-D cells and overrepresented in MCF7 cells (Table 1), but is not amplified or overrepresented in ZR-75-1 cells, which fall just below the cutoff for overrepresentation (Six1/control = 1.2, Table 1). However, it should be noted that the low interphase ratio of Six1/control in ZR-75-1 cells occurs because of focal amplification, because only 43% of the scored cells had increased copies of Six1, and the remaining 57% of cells had no gain in Six1 DNA. Thus, the actual ratio of Six1/control per cell, when examining only the cells in which a gain in Six1 gene dosage occurs, is in fact considerably higher. This suggests that overrepresentation/amplification does occur in the ZR-75-1 mammary carcinoma cell line, although only focally.



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  		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 ß-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 (*).

 
To determine whether Six1 expression correlates with gene dosage in the above-mentioned normal and mammary carcinoma cell lines, qRT-PCR analyses were done (Fig. 2C). The breast cancer cell lines MCF7 and T47-D exhibit 8-fold and 3.7-fold increases in Six1 expression, respectively, as compared with the normal line MCF-10A (Fig. 2C). In contrast, the ZR-75-1 cell line, in which a low level of overrepresentation occurs in a percentage of cells, did not have a significant increase in Six1 mRNA levels. MDA-MB-231 and MDA-MB-435 cells, in which Six1 gene dosage was not altered, also do not exhibit statistically significant increases in Six1 expression as compared with MCF-10A cells (Fig. 2C). The correlation of Six1 overrepresentation with Six1 expression in numerous breast cancer cell lines suggests that gene dosage is an important mechanism for inappropriately up-regulating Six1 in breast cancer.

Six4 and Six6, in addition to Six1, are amplified in 21MT1 cells. The 14q23 chromosomal region contains a cluster of Six family members including Six1, Six4, and Six6 (ref. 33; Fig. 3A). Because numerous Six family members have been implicated in proliferation during normal development (11–19), it is possible that they may synergistically act to stimulate proliferation in a tumorigenic setting. To determine whether the amplicon contains Six4 and Six6 in addition to Six1, as well as to identify other genes that may be in the 14q23 amplicon and that may contribute to cancer, we walked down chromosome 14 in 21MT1 breast cancer cells (because Six1 is amplified in this cell line) via FISH analysis with numerous BAC clones (Fig. 3). Using SpectrumRed-labeled probes derived from various BAC clones, we mapped the 14q23 amplicon in the 21MT1 cell line and found that it extends ~5 Mb and contains ~35 genes, including Six1, Six4, and Six6 (Fig. 3).



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  		Figure 3. The Six1 amplicon in 21MT1 cells is ~5 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).

 
14q23 Status and Six1 expression in human breast tumors. To determine the prevalence of 14q23 amplification/overrepresentation in human breast cancer, we did FISH on multiple breast tumor tissue arrays using the control 14q11 (green) and the RP11-1042B17 probe (red), which extends across the region of 14q23 containing Six1 (Fig. 3). Of the 214 infiltrating ductal carcinomas examined, 4.7% (10/214) were positive for unbalanced 14q23 gains. Of the 10 cases where 14q23 gains were observed, 9 had Six1/control ratios ≥1.5, and 6 had obvious clusters of Six1, demonstrating that most of the gains were due to gene amplification (Table 2; Fig. 4A). Only 1 of 10 cases had a Six1/control ratio just under 1.5, falling in the category of overrepresentation as opposed to gene amplification (Table 2). However, this patient exhibited clusters of Six1 DNA signals in some cells, suggesting that focal gene amplification occurred in this patient's tumor (not shown). Similar to the situation described in the ZR-75-1 cell line above, the Six1/control ratio was thus reduced in this patient due to tumor heterogeneity.


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

 


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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.

 
To further determine whether 14q23 amplification/overrepresentation 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 analysis, Ambion 2) as this was the only array for which we had duplicate slides that were of high enough quality to perform both analyses. This array contained 90 high-quality, analyzable, primary infiltrating ductal carcinomas, 7 of which showed Six1 amplification/overrepresentation (8%). Six (86%) of seven overexpressed Six1 ~3-fold as compared with the average expression level in normal breast samples (n = 14), and the remaining 1 tumor expressed Six1 at levels 1.4-fold above the normal breast control (Table 2; Fig. 4). No staining was observed in duplicate samples hybridized to the Six1-specific sense probe (not shown). Together, this shows that the 14q23 genomic gain encompassing the Six1 gene correlates with Six1 expression, and suggests that Six1 gene amplification/overrepresentation leads to Six1 overexpression. It should be noted that tumors that did not have 14q23 genomic gains also showed Six1 overexpression, suggesting that other mechanisms are working to inappropriately increase expression of Six1. Altogether, Six1 overexpression (at a level of 3-fold or greater) was observed in 47 (52%) of 90 primary infiltrating ductal carcinomas, and in 8 (89%) of 9 metastases resulting from infiltrating ductal carcinomas. This correlates well with our previous study in which Six1 overexpression was first described in breast cancer (5).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have implicated Six1 in the etiology of several types of cancer, including breast cancer, Wilms' tumor, and rhabdomyosarcoma (4–7, 20), and it contributes to at least some of these cancers by controlling the cell cycle (4, 5) as well as by controlling properties important for metastasis (20). Our data suggest that gene amplification/overrepresentation is one mechanism responsible for Six1 overexpression in breast cancer. Amplification/overrepresentation of the region on 14q23 in which Six1 resides was found to correlate with Six1 expression in numerous breast cancer cell lines examined as well as in human breast infiltrating ductal carcinomas. Corroborating our finding, comparative genomic hybridization studies have shown that breast cancers (34, 35), chondrosarcomas (36), prostate cancer (37), and fibrosarcomas (38) show increased 14q23 DNA content. In contrast, other studies have observed a loss of 14q23 DNA in breast cancer (39, 40), gastrointestinal stromal tumors (41), and neuroblastomas (42) and when taken together with the formerly mentioned comparative genomic hybridization studies, suggest that both oncogenes and tumor suppressor genes may be found at 14q23. As 14q23 gains have not frequently and consistently been observed in human cancers, it may be necessary to use more precise analysis looking at specific genes to determine the role of this locus in breast and other cancers. As such, this report shows that when examined at a more refined location (encompassing the region on 14q23 in which Six1 resides), 14q23 gains do indeed occur in a small, albeit significant, percentage of breast cancer cases.

Because ~5% of infiltrating ductal carcinomas exhibit Six1 gene amplification/overrepresentation, whereas about 50% of primary infiltrating ductal carcinomas overexpress Six1, additional mechanisms must exist that contribute to its overexpression in breast tumors. Such a phenomenon has been reported for other genes that are critical in breast tumorigenesis. For example, the chromosomal locus 11q13, in which cyclin D1 resides, is amplified in up to 15% of breast cancers (43, 44), yet cyclin D1 is overexpressed in 35% to 45% of these cancers (45, 46). Additional mechanisms that may contribute to the differential expression of a gene in cancer include changes in histone acetylation or in DNA methylation (47–49), in mRNA stability (50), or in promoter activity (51), among others. Six1 was recently identified as a target of E2F1 (52), a transcription factor critical for the G1-S transition whose activity is inhibited by Rb (53, 54). In many human cancers, some aspect of the E2F/Rb pathway is altered (54). Thus, Six1 overexpression in breast cancer may occur as a result of a combination of gene amplification/overrepresentation and increased transcription due to an altered E2F/Rb pathway.

Although multiple mechanisms may be involved in Six1 overexpression, the importance of gene amplification as a mechanism of overexpression is clear. Recent studies show that gene amplification is highly correlated with mRNA overexpression (34, 55) and that, on the whole, as little as a 2-fold change in DNA copy number is associated with a corresponding 1.5-fold change in mRNA levels (34). In this study, 52% of all primary infiltrating ductal carcinomas overexpress Six1 (3-fold) as compared with the average expression in normal mammary gland, whereas 86% of primary infiltrating ductal carcinomas in which Six1 gene amplification is detected display a 3-fold overexpression of Six1. This suggests that gene amplification is indeed an important mechanism for Six1 overexpression.

Interestingly, both Six4 and Six6 are amplified along with Six1 in 21MT1 cells (Fig. 3) and therefore may be co-overexpressed with Six1 in a subset of breast cancers. Presently, it is known that overexpression of Six1 in breast cancer cell lines can increase proliferation via the reactivation of the tissue-restricted cyclin A1 (4); however, the role of Six4 and Six6 has not yet been examined. As numerous members of the Six family, including Six6, have been implicated in proliferative processes (4, 5, 10–19) , the coordinate overexpression of three Six family members in breast cancer may result in, among other things, a hyperproliferative phenotype contributing to tumorigenesis. It will therefore be important to determine whether Six4 and Six6 are co-overexpressed with Six1 in breast cancers that display 14q23 amplification/overrepresentation and, furthermore, to determine whether these genes, along with Six1, contribute to the tumorigenic process.

The amplicon on 14q23 in 21MT1 cells is ~5 Mb long (see Fig. 3) and contains at least 35 genes in addition to Six family members, including several that are implicated in cancer. Genes present in the amplicon that are associated with cancer include the hypoxia-inducible factor 1 gene (HIF-1A), which encodes a transcription factor that is overexpressed in many types of cancer and whose transcriptional targets include several genes responsible for adaptation to reduced oxygen or hypoxic conditions, a common state in many solid tumors (37). In addition, the amplified region of 14q23 contains the PPKCH gene, which encodes PKC-{eta}, a protein kinase that has been implicated in various tumors, both positively and negatively (56–59). Of additional interest in this locus is the RBP-1 gene. This gene encodes a retinoblastoma-binding protein that promotes growth by inhibiting Rb (60). Interestingly, antibodies against a heptameric peptide sequence within RBP-1 were found in breast cancer patients, and work is under way to develop such peptide antigens as a potential vaccine against breast cancer (60). Thus, it is possible that these genes, as well as others within the 14q23 amplicon, cooperate with the Six family members to enhance the tumorigenic phenotype in cells in which they coamplify.

In closing, we have examined the prevalence of a genomic gain of the 14q23 region encompassing the Six1 gene in breast cancer. As Six1 is implicated in breast cancer progression (4, 5), its amplification and subsequent overexpression may play an important role in the etiology of the disease. Determining both the mechanisms and consequences of Six1 overexpression in breast cancer remain an important issue to address, particularly as the gene may serve as an excellent diagnostic and therapeutic target as it is primarily expressed during embryonic mammary gland development, is lost in the adult, differentiated mammary gland, and is reexpressed in breast cancer (4). Furthermore, the discovery that additional Six family members may be amplified in breast cancers exhibiting Six1 gene amplification suggests that a coordinate up-regulation of the genes may synergistically promote breast cancer, an issue that warrants further investigation.


   Acknowledgments
 
Grant support: NIH grant 1R01CA095277-01, Susan G. Komen Breast Cancer Foundation (9862), American Cancer Society/University of Colorado Cancer Center, 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 by fellowships from the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (Brazil), the University of Colorado Cancer Center, the Cancer League of Colorado, and the W.M. Thorkildsen Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance 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 reading of the manuscript, Dr. Jedlicka for help in analyzing tumor expression data, and Dr. Peter Jones for contributing reagents needed to carry out the tissue array work.

Received 12/ 1/04. Revised 1/10/05. Accepted 1/14/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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