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[Cancer Research 63, 2179-2187, May 1, 2003]
© 2003 American Association for Cancer Research

Molecular Biology and Genetics

The Septin 9 (MSF) Gene Is Amplified and Overexpressed in Mouse Mammary Gland Adenocarcinomas and Human Breast Cancer Cell Lines1

Cristina Montagna, Myung-Soo Lyu, Kent Hunter, Luanne Lukes, William Lowther, Tricia Reppert, Bruce Hissong, Zoë Weaver2 and Thomas Ried3

Genetics Branch [C. M., T. Re., Z. W., T. Ri.], Laboratory of Population Genetics [M-S. L., K. H., L. L.], Laboratory of Tumor Immunology and Biology [W. L.], Center for Cancer Research, National Cancer Institute, and Molecular Immunology and Inflammation Branch, National Institute of Arthritis, Musculoskeletal, and Skin Diseases [B. H.], NIH, Bethesda, Maryland 20892


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The expression of polyomavirus middle T antigen under the control of the mouse mammary tumor virus promoter in transgenic mice results in the induction of aggressive mammary gland adenocarcinomas at an early age. We screened 26 tumors for chromosomal aneuploidies using SKY and CGH. In 70% of the tumor samples we could detect high-level copy number gains, which mapped to chromosome band 11E2, a region orthologous to human 17q25.3. We then identified a bacterial artificial chromosome clone that labeled double-minute chromosomes found in the tumor metaphases. This bacterial artificial chromosome clone showed sequence homology to a member of the septin gene family. Real-time PCR analysis revealed a consistently increased expression of septin 9 (Sept9), not only in polyomavirus middle T antigen-induced, but in a wide variety of mouse models of breast cancer. Six of 9 human tumor cell lines also revealed elevated expression levels of Sept9. The family of septin genes is involved in a plethora of cellular processes, including cytokinesis in yeast and vesicle transport, and possesses GTPase activity. We identified down-regulation of Thsp1- and Bax-regulated apoptotic response in those tumors with Sept9 overexpression, an effect that could be reversed by inhibiting Sept9 expression using transfection with small interference RNA. Our results now suggest that signaling via members of the septin family plays a novel and common role in breast tumorigenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mouse models of human breast cancer are used widely to study molecular pathways of tumorigenesis (1) . The overexpression of oncogenes known to be involved in human breast cancer, such as c-Myc, HER2/Neu, and Ccnd1, with tissue-specific promoters induces tumors in the mammary gland of transgenic mice (2 , 3) . Likewise, the inactivation of tumor suppressor genes via introduction of viral oncogenes or by conditional knockout using CreLox technology can promote tumorigenesis (4 , 5) . Human breast cancers, like other epithelial cancers, are characterized by a recurrent and conserved pattern of genomic imbalances (6) . These imbalances result in up- or down-regulation of oncogenes and tumor suppressor genes, respectively, located on these chromosomes or chromosomal regions. The strong conservation of specific chromosomal imbalances in human solid tumors suggests that these aneuploidies are crucial events in tumorigenesis. Therefore, it is reasonable to assume that the identification of such aneuploidies in mouse models of breast cancer and the comparison to their distribution in human tumors can contribute to the validation of respective tumor models. The detection of recurring regions of genomic imbalances also can provide entry points for the molecular cloning of cancer-associated genes. The advent of molecular cytogenetic tools has allowed the screening of the entire murine genome for such genetic alterations. In particular, CGH and SKY have facilitated the analysis of such changes in mouse tumors (7 , 8) . We used these techniques previously to characterize genomic imbalances in several models of human breast cancer. For example, the overexpression of c-Myc or HER2/Neu oncogenes under MMTV4 or endogenous promoters results in tumorigenesis; however, cytogenetic analysis revealed that secondary genomic alterations are required, most notably deletions that map to mouse chromosome 4 (band C-E), a region that is orthologous to human 1p32–36 (9 , 10) . However, conditional inactivation of the breast cancer susceptibility gene Brca1 requires amplification of terminal mouse chromosome 11, which in some cases includes the HER2/Neu oncogene in addition to losses on mouse chromosome 4 (4 , 9) .

Here we have analyzed the consequences of mammary gland-specific expression of the PyV-mT antigen on genomic stability with a particular emphasis on the development of secondary chromosomal and genetic aberrations. The expression of PyV-mT results in the formation of mammary tumors (11) . Middle T antigen interacts with growth factor signaling pathways via activation of the src-tyrosine kinases, phosphatidylinositol 3'-kinase, and phospholipase C. On ShcA it creates binding sites for Grb2, of which the function is enhanced by overexpression (12) . These pathways stimulate the mitogen-activated protein kinase cascade and promote cell division (13) . Consequently, the mice develop multifocal mammary gland adenocarcinomas by 5 weeks of age (11) . However, as in other murine models of breast cancer, it is not clear whether additional genetic aberrations are required for tumorigenesis and which molecular pathways are involved. The molecular cytogenetic and genetic characterization of 26 PyV-mT-induced mammary gland adenocarcinomas revealed a strictly conserved pattern of genomic imbalances, including high-level copy number increases that map to distal chromosome 11. The consequences of this amplification on gene expression levels were assessed using quantitative real-time PCR in this and other mouse models, and in cell lines derived from human breast carcinomas. The results suggest a novel signaling pathway involved in breast tumorigenesis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tumors and Cell Lines.
Mammary epithelial cell lines were derived from 15 different MMTV-PyV-mT primary tumors and 11 cell lines derived from their lung metastases. C-myc 83, brt-1, brt-5, brt-10, and pbrt-1 were derived from the MMTV-c-Myc transgenic model, and the Brca1 conditional knockout as reported previously (7 , 9) . FF2, 4, 5, 7, 8, 9, 10, 12, and 21 cell lines were derived from the Notch4 transgenic mice described earlier (14) . Metaphase chromosomes were prepared from the cell lines at passages 15–20 after Colcemid arrest (1 h; final concentration 0.1 µg/ml), and standard hypotonic treatment and fixation in methanol/acetic acid. The human breast cancer cell lines BT 549, UACC 812, Zr 75-30, UACC 893, T47D, Pc3, and MDA 157 (American Type Culture Collection, Manassas, VA), SUM159 (University of Michigan, Ann Arbor, MI), and MPE 600 (Vysis, Downers Grove, IL) were cultured as described.5

Molecular Cytogenetic Analysis.
SKY and CGH were performed essentially as described before. Details of the protocols can be found elsewhere.6 The following mouse BAC clones for FISH analysis were selected using information provided elsewhere:7 ,8 RP23-147O23, 284L12, 101J23, 341C5, 202G21, 369E6, 382B19, 99M13, 84C12, 28I4, 354K2, 333B13, 48A17, and 480B11 (Invitrogen, Carlsbad, CA). The clones were labeled by nick-translation and hybridized to tumor metaphase chromosomes according to standard procedures. Images were acquired using a DMRXA microscope (Leica, Wetzlar, Germany), connected to a Sensys CCD camera (Roper Scientific, Tucson, AZ) using Q-FISH software (Leica Microsystems, Cambridge, United Kingdom). Complete results of the SKY and CGH analysis and the derived tumor karyotypes can be retrieved from the National Cancer Institute/National Center for Biotechnology Information SKY and CGH website.9

Total mRNA from the human breast cancer cell lines SUM159, BT 549, UACC 812, MPE 600, Zr 75-30, UACC 893, T47D, Pc3, and MDA 157 was extracted using Qiagen mini kit (Qiagen, Valencia, CA).

Quantitative Analysis of Gene Expression.
For real-time PCR experiments, were 2 µg of total RNA reverse-transcribed from by using a first-strand cDNA synthesis kit (Invitrogen, Grand Island, NY) and analyzed with the ABI PRISM7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Primers and carboxyfluorescein/6-carboxytetramethylrhodamine real-time PCR probes (Synthegen, Houston, TX) for the cDNA-specific real-time quantitative PCR assay were designed using Primer express 1.5 software for human GRB2, MSF, and RAC3 genes, and for the mouse Bax, Grb2, HER2/Neu, Rac3, Sept9, and Thbs1 genes (see Supplemental Table 1). The custom designed primers were used along with commercially available primers for mouse glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA and 18S rRNA housekeeping controls (Applied Biosystems) to determine relative mRNA expression levels. cDNA derived from normal mouse brain and normal mammary glands were used for generating a standard curve and normalizing data. The normal mammary gland standard was arbitrarily designated as 1.0. cDNA from samples 143mt, 404mt, 2571mt, and pbrt-5 were hybridized to 96 gene cDNA arrays filters from the mouse cancer pathway finder and cell cycle series (SuperArray, Bethesda, MD) following the suppliers protocol.

siRNA Transfection.
siRNAs corresponding to pEGFP reporter gene and to Sept9 mRNAs were designed as recommended (15) , with two base overhangs (Xeragon, Germantown, MD). The following gene-specific sequences were used successfully: si-GFP antisense 5'-GAACUUCAGGGUCAGCUUGCCG-dTT-3'; Sept9 siRNA sense 5'-GUCCACUUUAAUCAAUACC dTT-3' and antisense 5'-GGUAUUGAUUAAAGUGGAC dTT-3'. Annealing for duplex siRNA formation was performed in annealing buffer [100 mM potassium acetate, 30 mM HEPES-KOH at (pH 7.4), and 2 mM magnesium acetate] for 1 min at 90°C, followed by 1 h at 37°C. Transfections of reporter plasmid and siRNA were performed with Oligofectamine (Invitrogen) in six-well plates. Briefly, 3 µl of oligofectamine diluted in Opti-MEM was applied to the siRNA duplex mixture (60 pmol in 3 µl annealing buffer) and incubated 25 min at room temperature. The siRNA duplex oligofectamine mixture was added to cultured cells (40–50% confluent). The cells were seeded the previous day in tissue culture medium with 10% fetal bovine serum but without antibiotics. Transfection was carried out for 48 h. mRNA was extracted with Qiagen mini kit (Qiagen), and expression level for Bax, Sept9, and Thsp1 was determined by real-time PCR.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To identify secondary genetic events required for PyV-mT-associated carcinogenesis in the mammary gland, we analyzed 26 cell lines established from primary tumors or tumor metastases by CGH and SKY. These screening tests for genomic imbalances and chromosomal translocations revealed three hotspots of genetic instability in this tumor model. We detected frequent losses of the distal part of chromosome 4 (10 of 26 cases), and copy number increases on chromosomes 11 and 15 (17 and 10 cases, respectively). The chromosome 11 gain was particularly striking, because high-level copy number increases (amplifications) were focused to just chromosome band 11E2. A summary of the genomic imbalances is presented in Fig. 1Citation and the results can also be viewed at the SKY/CGH website.9 SKY analysis revealed that different chromosomal events led to the genomic amplification of chromosome 11 band E2. Nine tumors showed jumping translocations (recipient chromosomes were 1, 4, 5, 6, 7, and X), and 4 tumors showed duplications of distal chromosome 11 (Fig. 2, A and B)Citation . Two tumors (158mt and 2571mt) revealed numerous dmins, a cytogenetic feature reflecting the amplification of oncogenes in solid tumors (8–20 dmin/cell in 50% of the cells). Mouse chromosome 11 contains a plethora of breast cancer-associated genes including the tumor suppressor genes Trp53 and Brca1, and the HER2/Neu oncogene, a member of the Egf receptor family. To determine whether these genes were deleted or amplified, we performed FISH analysis with gene-specific BAC clones. In each case the copy numbers were equal to the ploidy of the cell. c-Myc, another oncogene commonly up-regulated in breast carcinomas, was also present in the normal copy number on mouse chromosome 15 (Fig. 2, C and D)Citation . CGH analyses of human carcinomas suggested a second region of frequent amplification that maps to chromosome band 17q23 (16 , 17) . Candidate genes in this amplicon include Rad51c and Tbx2. Neither of these genes was involved in the copy number increases on the orthologous region of mouse chromosome 11 (Fig. 2, E and F)Citation . To create additional probes for band 11E2 we established a physical map of BAC clones specific for the cytogenetically identified region of copy number gain on chromosome 11E2. Eleven BAC clones spanning a region of 17 Mbp were selected and hybridized to tumor metaphase chromosomes derived from five different tumors representing the diversity of cytogenetic abnormalities observed. In some tumors (157mt and 404mt), the BAC clones (RP23-284L12 and 84C12) containing sequence homology for Sept9 and Rac3 were present in additional copy numbers. In other tumors (158mt and 2571mt), only the Sept9-specific clone showed copy number increases. Of note, only this clone specifically labeled the dmins identified in a subset of tumors with gain on chromosome 11E2 (Fig. 2, G and H)Citation . Therefore, we conclude that the minimally amplified region comprises exclusively BAC clone RP23-284L12, the clone that contains the Sept9 gene. Sequence analysis revealed that no other known genes are present in this genomic clone. It was important to determine whether the Sept9 expression level was increased as a result of its genomic amplification. Therefore, we designed primers for quantitative real-time PCR. Because of the genomic coamplification of the Rac3 gene in two tumors, primers specific for this gene were included in the analysis. Primers for the Grb2 gene were included as a positive control, as this gene is specifically up-regulated by PyV-mT. The results showed that Sept9, but not Rac3, is consistently present at substantially increased expression levels in all of the cases with a genomic amplification of 11E2, when compared with the expression level in the normal mammary gland (Fig. 3)Citation . To assess whether Sept9 gene amplification and overexpression is restricted to the PyV-mT model, we extended the transcription profiling to four additional models of human breast cancer where gain of chromosome 11 or amplification of the distal region of it are frequently observed. Myc 83, a tumor from a mouse model that overexpresses c-Myc under the control of the MMTV promoter, also revealed increased expression levels. Sept9 levels were increased in two of four mammary gland adenocarcinomas from Brca1 conditional knockouts as well. Additionally, we analyzed nine tumors from mice transgenic for Notch4. Increased transcript levels of Sept9 defined the majority of the tumors, sometimes also in the absence of genomic amplification. Our results indicate that Sept9 but not Grb2 or Rac 3 is the common target for mRNA overexpression in the region of genomic amplification. This suggests that cellular pathways that promote tumorigenesis independent of the oncogenic stimulus are dependent on, or at least benefit from, increased Sept9 levels. Human chromosome 17q, which is orthologous to mouse chromosome 11, is gained frequently in human breast carcinomas. In fact, this chromosome arm contains several amplicons, with either known (HER2/Neu on chromosome band 17q13) or suspected oncogenes (18) . However, in cytogenetic studies of human breast cancer we have also frequently observed increased copy numbers distal to chromosome band 17q23, as have other groups (19) . The MSF (Sept9) gene maps to band 17q25. Therefore, we extracted RNA from a series of breast cancer cell lines, some of which showed gain or amplification on band 17q25 (20) . The quantitative PCR clearly established that MSF (Sept9) is highly expressed in six of nine cell lines, and that the overexpression of this gene can occur in the absence of genomic copy number increase (Fig. 4)Citation . In two cell lines (MDA157, MPE600), MSF (Sept9) overexpression was accompanied by an increase in RAC3 expression. GRB2 mRNA levels were not increased. This finding establishes that abnormal expression of the MSF (Sept9) gene is not only involved in murine tumorigenesis but in human carcinomas as well.



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  		Fig. 1. Summary of genomic imbalances detected by CGH and SKY of 26 tumors induced by overexpression of PyV-mT under the control of the MMTV promoter. Genomic imbalances that result in gains are plotted on the right side of the ideogram, whereas losses are displayed on the left. Note the predilection of only few chromosomes or chromosomal bands to recurrent copy number losses (chromosome 4), or gains (chromosomes 11 and 15). A complete view of the aberrations in individual cases can be viewed elsewhere.9

 


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  		Fig. 2. Molecular cytogenetic characterization of PyV-mT induced tumors and high-resolution amplicon mapping. A, SKY of a representative metaphase from tumor 355mt. Note extra chromosome 11 material involved in an insertion. B, summary of chromosome rearrangements observed in several tumors revealed multiple possible mechanisms for the acquisition of copy number increases on chromosomes 11, including dmin chromosomes (top panel), duplications (middle panel), and different types of translocations (bottom panel). In all instances, chromosome translocations resulted in copy number increases of chromosome 11 sequences. Full karyotypes of all tumors can be viewed elsewhere.9 C–F, high-resolution mapping of candidate genes involved in mammary gland tumorigenesis. Different from other murine models for breast cancer, deletions of neither Trp53 nor Brca1 are required for tumorigenesis in PyV-mT induced tumors, as both copies are present (C). The gain of chromosomes 11 and 15 suggested the amplification of the HER2/Neu (chromosome 11) and Myc-oncogenes (chromosome 15). However, FISH analysis revealed normal copy numbers for these genes (D). Likewise, two additional candidate genes, Tbx2 and Rad51c, on the amplicon on chromosome 11 were not subject to copy number increases and did not label the dmin in the tumors (E and F). G and H, to determine more precisely the specific sequences amplified in the dmins, a BAC contig was assembled for chromosome band 11E2. Note that clone 284L12 is present in eight copies in tumor 404mt (G) and consistently labeled the dmins in tumor 158mt (H). The adjacent clone 382B19 (shown in green) was not involved. Clone 284L12 contains the Sept9 gene. The insert shows the 4',6-diamidino-2-phenylindole DNA counterstain of dmin chromosomes.

 


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  		Fig. 3. Real-time PCR-based quantification of gene expression in the PyV-mT-induced tumors and in tumors arising by overexpression of c-Myc, HER2/Neu, or Notch4, and conditional knockout of Brca1. * indicate the presence of ME2 genomic amplification. RNA extracted from normal mammary gland tissue and the cell line 143mt (which did not show genomic amplification of Sept9) were used as controls, and the standard expression level was assigned as 1. Note the significantly increased expression levels for Sept9 in 16 tumors from the four mouse models analyzed. In contrast, overexpression of Grb2 is only found in the PyV-mT tumors, whereas Rac3 overexpression is observed occasionally in the Notch4 tumors. With the exception of brt-10, where genomic amplification of 11E2 was not accompanied by increased expression of Sept9, copy number increases of chromosome band 11E2 were correlated to Sept9 overexpression. In the Notch4 transgenic mice we could detect increased transcription levels also in the absence of genomic amplification.

 


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  		Fig. 4. Evaluation of MSF (sept9) expression levels in human breast carcinomas. The results of quantitative real-time PCR are plotted. Six of eight cell lines showed expression levels that ranged from 2.2-fold (UACC 893 and Pc3) to >20.8-fold (MPE 600). MSF (sept9) amplification also occurred independently of genomic copy number levels on human chromosome 17q25 (in tumor UACC-812), the chromosomal mapping position of the MSF (sept9) gene. RAC3, a gene that maps close to MSF (sept9) was not amplified consistently.

 
Septins play a role in multiple cellular functions ranging from vesicle transport to cytokinesis. To identify in which cancer-related pathway the overexpression observed here exerts its function, we have used pathway finder array filters that allow one to query the expression levels of 96 genes simultaneously. One array contained genes involved in cell cycle regulation, and the second array contained genes that affect different cancer pathways. Four cell lines were tested, three of which were derived from the group of tumors induced by PyV-mT overexpression. One tumor, pbrt-5, was derived from conditional Brca1 knockouts. The latter and two of the PyV-mT models showed 2.8–12.3-fold increased expression levels of Sept9 (see Fig. 3Citation ). The two groups of tumors could be distinguished by the expression levels of Thsp1 and Bax, which increased only in the absence of Sept9 overexpression, i.e., the one PyV-mT cell line that did not overexpress Sept9 showed increased levels of Thsp1 and Bax (Fig. 5)Citation . The down-regulation of Thsp1 and Bax was confirmed in a larger series of tumors using quantitative reverse transcription-PCR (Fig. 6)Citation . We conclude that the down-regulation of Thsp1 and Bax is linked to the overexpression of Sept9. Up-regulation of both Thsp1 and Bax induces apoptosis (21 , 22) .



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  		Fig. 5. Down-regulation of the apoptosis genes Thsp1 and Bax. RNA from three PyV-mT tumors (143mt, 404mt, and 2571mt) and one Brca1 conditional knockout (pbrt-5) was hybridized to a cDNA filter containing 96 genes involved in apoptosis. Tumors 404mt, 2571mt, and pbrt-5 have increased expression levels of Sept9; tumor 143mt has not. Note the down-regulation of Bax and Thsp1 in the Sept9-positive tumors.

 


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  		Fig. 6. Quantitative real-time PCR analysis of Bax (A) and Thsp1 (B) levels as compared with Sept9 expression levels. The tumor number and induction mode is provided below the graphs. The * denote those tumors in which genomic amplification of Sept9 was observed. Note that increased expression levels of Sept9 correlate with reduced expression levels of Bax and Tshp1 in the majority of the tumors, independent of mode of tumor induction.

 
In addition to pathway dissection, another way of performing functional analysis of a gene is to inhibit its expression using interference RNA. To determine the consequences of Sept9 gene inactivation we designed siRNAs and transiently transfected cells with and without Sept9 overexpression. Successful transfection was determined by real-time PCR as the levels of detectable RNA decreased. mRNA expression decrease (28.8–58%) was observed in the three cell lines with Sept9 overexpression. Independent of that, we determined transfection efficiency using GFP siRNA on the same cells in combination with a GFP vector. Whereas GFP transfection alone revealed 40% positive cells, none of the cells showed green fluorescence when the GFP vector was combined with siGFP, indicating the successful transfection. Under the conditions used here, the inactivation of Sept9 expression induced no visible morphological changes, or changes on the growth kinetics of the cell populations, nor was DNA synthesis impaired as measured by bromodeoxyuridine incorporation. However, reduction of Sept9 expression by siRNA resulted in the up-regulation of Bax message in cell lines 404mt, 2571mt, and brt-5, whereas the expression levels in the cell line that did not overexpress Sept9 were reduced slightly (Fig. 7A)Citation . Treatment of the cells with Sept9 siRNA also caused the up-regulation of Thsp1 in cell lines 404mt, 2571mt, and brt-5, all three belonging to the Sept9-overexpressing group (see Fig. 7BCitation ). Thsp1 levels were down-regulated in cell line 143mt, which showed no a priori increase of Sept9 levels (Fig. 7B)Citation .



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  		Fig. 7. Consequences of Sept9 inhibition by siRNA on the expression levels of the apoptosis genes Bax and Thsp1. The tumor numbers are provided below the graphs. The * denote those tumors in which genomic amplification of Sept9 was observed. {blacksquare} indicate the expression levels of Sept9 before siRNA was added to the medium. In all tumors that showed increased Sept9 expression levels, siRNA resulted in a pronounced up-regulation of both Bax and Thsp1.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here we report the molecular cytogenetic characterization of 26 tumor samples derived by overexpression of the PyV-mT under the control of the MMTV promoter. As in other mouse models of human breast cancer, tumor formation is accompanied by, or requires, the acquisition of specific chromosomal aberrations. The vast majority of these aberrations result in genomic gains or losses. It has been firmly established that chromosomal regions of recurrent genomic imbalance point to the location of tumor suppressor genes in case of chromosomal loss or to oncogenes when genomic amplification is observed. Therefore, we were intrigued by the consistent copy number increase on mouse chromosome 11. Mouse chromosome 11 contains sequences orthologous to human chromosomes 22, 16, 5, and 17 (23) .10 In fact, most of human chromosome 17 (HSA17) resides on mouse chromosome 11 (MMU11). Many genes known to be involved in breast cancer map to this chromosome, including Trp53, HER2/Neu, and Brca1. In addition, CGH analyses of human breast cancers identified another region of frequent amplification that maps to chromosome band 17q23 (16 , 17) . Candidate genes in this region include Rad51c and Tbx2. Therefore, the significance of this chromosome prompted us to identify the critical region with higher resolution. On the basis of available sequence maps, we selected a set of BAC clones for high-resolution FISH mapping. This effort was greatly aided by the fact that in two of the tumors with amplification on chromosome 11, dmins were present in metaphase spreads. One of the BAC clones, RP23-284L12, labeled the dmin in two tumors (158mt and 2571mt) and translocated fragments of chromosome 11 in two additional ones (404mt and 157mt). Adjacent BAC clones, both distal and proximal of 284L12, labeled only the normal copies of chromosome 11 but not the dmin copies. In this regard, we were particularly interested in whether the amplicon on chromosome 11 contained genomic sequences for Grb2. Overexpression of Grb2 expedites tumor development in PyV-mT mice. A high level of GRB2 mRNA is also observed in human breast cancer cell lines, accompanied in some instances by genomic amplification (24 , 25) . The real-time PCR results showed clearly that Grb2 expression is elevated in most of the PyV-mT-derived tumor cell lines. However, the BAC clone containing the murine Grb2 gene was not amplified and did not label the dmins nor did it label the extra fragments of chromosome 11 involved in copy number increases. Therefore, we conclude that the genomic amplification on chromosome band 11E2 and the presence of dmins is not driven by the requirement for increased message of Grb2, but that Grb2 expression is up-regulated by mechanisms other than genomic amplification. Therefore, Sept9 is the only known gene in the minimal overlapping region of genomic amplification on mouse chromosome 11. One tumor, brt-10, which was derived from Brca1conditional knockout mutants, revealed genomic amplification of MMU11 band, which was not accompanied by increased expression levels of Sept9. This could be because the genomic amplicon actually does not contain Sept9, a possibility that is likely because the CGH ratio profile suggests that Sept9 is not included in the amplicon in this particular case (data not shown). Sept9 overexpression is not restricted to the PyV-mT model. In fact, even higher levels of Sept9 were found in MMTV-myc and MMTV-Notch 4 tumors, and in tumors arising in conditional mutants for Brca1. This and the observation that increased Sept9 levels were also present in the majority of human breast cancer cell lines included in this study suggest that this gene is involved rather ubiquitously in tumorigenesis in the mammary gland. Although tumorigenesis arises through different initial pathways in these models, the resulting overexpression of the same gene argues in favor of its importance in a downstream pathway.

In yeast, members of the septin family are required for mitosis-specific activation of the Gin4 kinase, and the loss of septin causes a mitotic delay (26) . In Drosophila, mutations in the septin genes, such as sep1 and pnut, cause the formation of multinucleated cells (27) . The septin family is conserved in humans. One human septin, NEDD5, localizes to the cleavage furrow, and functional inactivation of this septin by antibody injection results in the disruption of cytokinesis (28) . Therefore, it appears that one role of the septins relates to the successful completion of mitosis, a function that is conserved from yeast to human. In addition to this function, septins contain a conserved domain with GTPase activity (29) .

Several papers have reported that MSF (Sept9) maps to a chromosomal region that is consistently lost in ovarian carcinomas (30 , 31) . This led to the hypothesis that septin may function as a tumor suppressor gene in breast and ovarian carcinomas. This interpretation is not supported by our findings, because genomic gain and amplification of the MSF (Sept9) are observed consistently in quite different mammary gland adenocarcinomas and in cell lines established from human breast tumors. Most importantly, the genomic amplification is invariably accompanied by the overexpression of this gene in the tumors analyzed here. In some tumors, overexpression is observed even in the absence of genomic amplification. Also, the presence of this gene in the dmins strongly argues for its role as an oncogene rather than in tumor suppression. This notion is additionally corroborated by the results published by Risk et al. (32) , who actually excluded MSF (Sept9) from a commonly deleted region on human chromosome 17q25.

Translocation of two members of the septin genes to the MLL gene on chromosome 11q23 in acute myeloid leukemia has been reported from two groups (33 , 34) . Whereas evidence has not yet been established unambiguously, it seems that MLL alone is not sufficient for leukemogenesis. This is supported by the fact that these chromosomal translocations occur in frame, hence producing a fusion protein. This is entirely consistent with the paradigm of chromosomal translocations in leukemia, including the BCR-ABL fusion as a consequence of the Philadelphia chromosome (35) . Therefore, it seems reasonable to assume that septins, when activated or overexpressed, provide a growth advantage to cells, in particular to rapidly growing cancer cells.

The pathway blots and quantitative reverse transcription-PCR analyses that we have used to query how increased Sept9 levels interact in the signaling cascades in mammary tumor formation suggest that two proteins that mediate apoptotic response were selectively down-regulated as a consequence of Sept9 overexpression. In an attempt to understand the consequences of increased cellular septin levels in breast cancer cells, we used interference RNA to experimentally reduce septin levels in three cell lines where we observed overexpression as well as in a control one that revealed normal expression. Transfection with siRNA resulted in increased expression levels of Thsp1 and Bax, and in all of the tumors with elevated Sept9 levels, but not in the tumors without genomic amplification and overexpression of Sept9, additionally corroborating the functional significance of the genetic alteration. However, none of the investigated parameters that we assessed, such as morphology, DNA synthesis, or population doubling times were altered significantly. Whereas at the moment we are lacking a clear explanation for this phenomenon, we hypothesize that either the transfection efficiency of ~40% was too low to affect the cancer cell population as a whole or that redundant pathways that rescue septin deficiency compensate for Sept9 down-regulation.

In summary, we provide evidence for a generalized Sept9 overexpression in multiple and diverse mouse models of human breast cancer and in human breast cancer cell lines. On the basis of their known roles in cytokinesis and control of mitotic cell division, we suggest that increased septin levels might assist in the rapid mitotic turnover that is characteristic for malignant cells. This could be facilitated by the observed inhibition of Thsp1- and Bax-triggered apoptotic response.


   ACKNOWLEDGMENTS
 
We thank Buddy Chen for manuscript editing, Dr. Vittorio Sartorelli for advice on the design of the siRNA experiments, and Dr. Stan Lipkowitz for valuable discussion and comments.


   FOOTNOTES
 
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.

1 Supplemental data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org). Back

2 Present address: Avalon Pharmaceuticals, 19 Firstfield Road, Gaithersburg, MD 20878. Back

3 To whom requests for reprints should be addressed, at Genetics Branch, Center for Cancer Research/National Cancer Institute/NIH, 50 South Drive, Building 50, Room 1306, Bethesda, MD 20892-8010. Phone: (301) 594-3118; Fax: (301) 435-4428; E-mail: riedt{at}mail.nih.gov Back

4 The abbreviations used are: MMTV, mouse mammary tumor virus; PyV-mT, polyomavirus middle T antigen; BAC, bacterial artificial chromosome; FISH, fluorescence in situ hybridization; dmin, double min chromosome; siRNA, small interference RNA primer; GFP, green fluorescent protein; CGH, comparative genomic hybridization; sky, spectral karyotyping; Mbp, mega base pair. Back

5 Internet address: http://www.atcc.org/. Back

6 Internet address: www.riedlab.nci.nih.gov. Back

7 Internet address: http://www.ncbi.nlm.nih.gov/genome/guide/mouse/index.html. Back

8 Internet address: http://www.ensembl.org/Mus_musculus. Back

9 Internet address: http://www.ncbi.nlm.nih.gov/sky/skyweb.cgi. Back

10 Internet address: http://www.ncbi.nlm.nih.gov/Homology/index.html. Back

Received 11/ 5/02. Accepted 3/ 4/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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