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[Cancer Research 65, 10363-10370, November 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Transforming Acidic Coiled Coil 1 Promotes Transformation and Mammary Tumorigenesis

Megan Cully1,2, Jessica Shiu1,2, Roland P. Piekorz4, William J. Muller5, Susan J. Done2,3 and Tak W. Mak1,2

1 Campbell Family Institute for Breast Cancer Research; Departments of 2 Medical Biophysics and 3 Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; 4 Institute for Biochemistry and Molecular Biology II, Heinrich Heine University, Duesseldorf, Germany; and 5 Molecular Oncology Group, McGill University, Montreal, Quebec, Canada

Requests for reprints: Tak W. Mak, Campbell Family Institute for Breast Cancer Research, 620 University Ave, Suite 706, Toronto, Ontario, Canada M5G 2C1. Phone: 416-946-2234; Fax: 416-204-5300; E-mail: tmak{at}uhnres.utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transforming acidic coiled coil 1 (TACC1) is a putative oncogene located within a breast cancer amplicon found on human chromosome 8p11. Although TACC1 has been reported to transform fibroblasts, it is also down-regulated in a subset of mammary tumors treated with anthracyclin. Here, we show that ectopic TACC1 overexpression can cooperate with Ras to induce focus formation in murine fibroblast cultures and prevent death caused by overexpression of Pten or a dominant-negative form of protein kinase B (PKB)/Akt. In transgenic mice carrying TACC1 under the control of the mouse mammary tumor virus promoter, TACC1 expression reduced apoptosis during mammary gland involution, increased the penetrance of mammary tumors in a pten+/– background, and decreased the average age of mammary tumor onset in a mouse model based on a phosphatidylinositol 3'-kinase (PI3K)–decoupled mutant of polyoma middle T. Elevated levels of both phospho-PKB and phospho-extracellular signal-regulated kinase were found in mammary tissue containing the TACC1 transgene. Thus, TACC1 positively regulates the Ras and PI3K pathways, promotes Ras-mediated transformation, and prevents apoptosis induced by PI3K pathway inhibition. TACC1 also cooperates with tumorigenic mutations in the PI3K pathway and thereby plays an oncogenic role in tumor formation in the murine mammary gland.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Transforming acidic coiled coil 1 (TACC1) was originally identified as the sole coding sequence consistently found within the 8p11 human breast cancer amplicon (1). The TACC family of proteins is defined by a conserved COOH-terminal coiled coil domain, but little homology exists among TACC family members outside this domain. TACC family homologues in lower organisms, such as Caenorhabditis elegans (TAC-1) and Drosophila melanogaster (D-TACC), stabilize microtubules within the growing spindle during mitosis (2–5). Mutations in D-TACC result in either aneuploidy and polyploidy in early embryogenesis or a failure to initiate the first mitotic division (3). Similarly, mutations in TAC-1 cause microtubule-associated defects in meiosis and pronuclear migration that are associated with improper spindle function (2, 4, 5). TAC-1 and D-TACC interact with the microtubule-associated proteins of the XMAP215/Msps family at the centrosome, and this interaction is thought to stabilize the growing microtubules (2, 4–6). Localization of D-TACC to the centrosome is controlled by the mitotic kinase Aurora A (7).

Both the human and mouse TACC families each consist of three members. It is unclear which of these molecules is the functional homologue of D-TACC and TAC-1. The three mammalian TACCs display distinct localization patterns, although all three partially colocalize with microtubules (8). TACC2 and TACC3 colocalize strongly with the centrosomes during mitosis, and TACC3 overexpression seems to increase the stability of microtubules (8). In contrast, TACC1 is only very weakly associated with microtubules and has not been shown promote microtubule stability. However, the mammalian member of the XMAP215/Msps family, ch-TOG, has been shown to interact with TACC1 in a yeast two-hybrid screen, suggesting a potential role for TACC1 at the centrosome (9).

Alternative splicing gives rise to multiple isoforms of TACC1 and TACC2. The shorter splice variant of TACC2 (AZU-1) attenuates malignant conversion of mammary epithelial cells (10), whereas the function of the longer TACC2 isoform is undetermined. Mice in which both forms of TACC2 have been disrupted by gene targeting do not develop tumors, suggesting that AZU-1 may not be a critical tumor suppressor (11). The short isoform of TACC1 (TACC1s), which lacks exon 2 and exon 3, has yet to be explored, although expression of this variant is found in numerous transformed cell lines (12). The full-length isoform of TACC1, which retains exon 2 and exon 3, can transform fibroblasts (1) but was down-regulated in a cohort of mammary tumors treated with anthracyclins (12). Full-length TACC1 has also been shown to interact in vitro with a myriad of proteins, including transcription factors, microtubule-associated proteins, and components of the nonsense-mediated decay machinery (9, 12–14). The physiologic significance of these interactions remains unclear.

Mammary tumorigenesis can be induced in mice by a number of genetic mechanisms, including the overexpression of oncogenes, such as ErbB2 or the polyoma virus middle T antigen (PyMT; refs. 15, 16). PyMT expression driven by the mammary-specific murine mammary tumor virus (MMTV) promoter results in metastatic, multifocal mammary adenocarcinomas (15). At the molecular level, PyMT expression activates the intracellular signaling molecules Src homology and collagen (Shc), Src, and phosphatidylinositol 3'-kinase (PI3K), thus triggering both the extracellular signal-regulated kinase (ERK) and protein kinase B (PKB) signaling pathways that promote cell proliferation and survival (17). Mutations within the PyMT coding sequence ablating the interaction between PyMT and either Shc or PI3K result in hyperplasias that progress to tumors with a much longer latency than wild-type (WT) PyMT. These data highlight the critical roles played by Shc and PI3K in PyMT-mediated tumorigenesis (18).

Murine mammary tumorigenesis can also be induced by the loss of Pten. Mice heterozygous for pten mutations develop tumors in multiple organ systems, including the mammary glands (19). Tissue-specific homozygous deletion of pten in the mammary gland results in neoplasia as well as delayed involution (20). Conversely, overexpression of Pten in the mammary gland causes defects in lobuloalveolar development (21). These observations reinforce the importance of the PI3K pathway in maintaining homeostasis in the murine mammary gland. PI3K signaling is also crucial for human mammary tumorigenesis, as breast cancers often contain mutations in molecules acting within the PI3K pathway. Amplification of the receptor tyrosine kinase ErbB2 and loss of PTEN are commonly found in human breast tumors (22).

To test the role of TACC1 in PI3K signaling, cell lines and mice transgenic for TACC1 were generated. Strikingly, in mice, TACC1 expression enhances tumor formation induced by PyMT315/322 expression or pten heterozygosity. Our physiologic confirmation of TACC1 as an oncogene may lead to new strategies for cancer therapeutics designed to combat human breast cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
ß-Galactosidase cell survival assay. NIH-3T3 fibroblasts were infected with pBabe, pBabe-TACC1, or pBabe-TACC1s, and stable cell lines were generated by exposure of these cells to puromycin (Sigma, St. Louis, MO; 2 µg/mL) for 3 days. These stable cells were then transfected with pcDNA3.1 expressing Pten or PKB-AAA along with 1:10 (w/w) pcDNA3.1-ß-galactosidase vector using LipofectAMINE 2000 (Invitrogen, San Diego, CA). Cells were incubated for 48 hours, and viable, transfected cells were identified by staining for ß-galactosidase expression. Cells were first fixed with 2% paraformaldehyde in PBS for 5 minutes and washed twice with PBS. Cells were then incubated overnight with staining solution (1 mg/mL X-gal, 5 mmol/L potassium ferricyanide, 5 mmol/L potassium ferrocyanide, 2 mmol/L MgCl2 in PBS). Cells were counted per x5 visual field.

Focus formation assay. NIH-3T3 fibroblasts stably infected with pBabe, pBabe-TACC1, or pBabe-TACC1s were infected with virus containing pBabe expressing oncogenic Ras (Ras-V12). Ras-V12-infected cells (1 x 104) were plated into six-well plates and incubated for 10 days. Focus formation was assayed by crystal violet staining. Cells were washed once in PBS and then stained for 1 minute in crystal violet staining solution (25% ethanol, 1% formaldehyde, 0.125% NaCl, 0.25% crystal violet). Finally, cells were washed with warm tap water and air-dried.

Western blotting. Tissues were isolated from mice and flash-frozen in liquid nitrogen. Frozen tissues were lysed in CHAPS buffer [10 mmol/L Tris (pH 7.5), 1 mmol/L MgCl2, 1 mmol/L EGTA, 0.5% CHAPS powder (Sigma), 10% glycerol, 100 µmol/L NaVO3, 50 mmol/L NaF, protease inhibitor cocktail tablets (Boehringer Mannheim, Indianapolis, IN)], and total protein concentrations were normalized using the Bradford assay (Bio-Rad, Richmond, CA) before fractionation by SDS-PAGE and blotting. Antibodies recognizing the following proteins were used at 1:1,000 TACC1 (Upstate, Lake Placid, NY), tubulin (Upstate), PKB (Santa Cruz Biotechnology, Santa Cruz, CA), actin (Santa Cruz Biotechnology), FLAG (Sigma), Myc (Invitrogen), phospho-Foxo3a, phospho-PKB, phospho-ERK, phospho-mitogen-activated protein/ERK kinase 1 (MEK1), ERK, and cleaved caspase-3 (all from Cell Signaling Technologies, Beverly, MA). The TACC1 antibody does not recognize the short isoform of TACC1.

Generation and care of MMTV-TACC1 mice. Mice transgenic for human TACC1 under the control of the MMTV promoter (MMTV-TACC1 mice) were generated as follows. The human full-length TACC1 gene was cloned into a vector based on HE8 (23). The HCR promoter was replaced with the MMTV promoter using XhoI and SpeI, whereas the interleukin 8 cDNA was replaced with TACC1 cDNA (SpeI-NotI). This construct was linearized and injected into murine oocytes of the FvB genetic background according to standard protocols. Two independent lines of MMTV-TACC1 transgenic mice were generated and characterized; the phenotypes of both lines were identical. Mice were kept at the Animal Resources Centre at Princess Margaret Hospital in accordance with ethical guidelines.

Whole mount and involution analysis. Female MMTV-TACC1 mice were bred to FvB mice to generate litters. Following delivery, litters were normalized to eight pups each, and mothers were allowed to nurse for 10 days. On day 10, the pups were removed. This serves as day 0 of involution. Mammary tissue from the fourth inguinal gland was fixed onto slides in Carnoy's solution (75% ethanol, 25% glacial acetic acid) overnight. The slides were dehydrated in 70% ethanol and stained overnight with 0.2% carmine alum and 0.5% aluminum potassium sulfate. Slides were destained in 70% ethanol with 2% HCl for 3 to 4 hours and then dehydrated through increasing concentrations of ethanol before being immersed in toluene.

Terminal deoxynucleotidyl transferase–mediated nick-end labeling staining for apoptosis. Mammary glands were fixed in neutral buffered formalin and embedded in wax, and sections of 4 µm were prepared according to standard protocols. Terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) staining was done using the In Situ Cell Death Detection kit according to the manufacturer's instructions (Roche, Indianapolis, IN).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TACC1 positively regulates cell survival and transformation. Activation of the PI3K signaling pathway promotes cell survival, and inhibition of this pathway through forced expression of Pten induces cell death (24). Many of the prosurvival effects of PI3K are mediated through PKB activation (25). Mutation to alanine of PKB's two serine/threonine phosphorylation sites as well as the lysine residue within the ATP-binding pocket results in the generation of the dominant-negative mutant PKB-AAA. PKB-AAA expression causes cell death due to interference with the normal PKB signaling pathway (24). To test the effect of TACC1 on PKB-mediated survival signaling, NIH-3T3 cells stably overexpressing full-length TACC1 or TACC1s (and empty vector controls) were generated by retroviral infection (Fig. 1A). These cells were then transiently transfected with additional vectors expressing either Pten or PKB-AAA. A vector encoding ß-galactosidase was also transfected and used as a marker for transiently transfected cells. Transfection with Pten or PKB-AAA caused the death of NIH-3T3 cells transfected with empty pBabe vector. However, ectopic expression of TACC1 protected these cells from death induced by transient Pten or PKB-AAA expression (Fig. 1B; data are normalized to transfection with pcDNA3.1). TACC1s had no effect on cell death induced by either Pten or PKB-AAA. These data suggest that TACC1 can mediate cell survival in situations where normal PI3K signaling is blocked.



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  		Figure 1. TACC1 attenuates apoptosis mediated by Pten or PKB-AAA overexpression and promotes Ras-mediated transformation. A, generation of stable pBabe-TACC1 or pBabe-TACC1s cell lines. Lysates of NIH-3T3 fibroblasts infected with retrovirus carrying the indicated constructs were selected with puromycin (2 µg/mL) for 3 days and subjected to Western blotting and probed with anti-TACC1 or anti-FLAG antibodies. Expression from the pBabe vector is driven by the retroviral 5' long terminal repeat. Tubulin, loading control. B, increased cell survival. NIH-3T3 cells stably infected with retrovirus carrying empty pBabe vector, pBabe-TACC1, or pBabe-TACC1s were transiently transfected with pcDNA3.1 expressing either Pten or PKB-AAA along with 1:10 (w/w) pcDNA3.1-ß-galactosidase. Cells were stained with X-gal, and 10 visual fields (magnification, x5) were counted under the microscope. All counts were normalized to cells transfected with empty pcDNA3.1 vector plus 1:10 pcDNA3.1-ß-galactosidase (defined as 100% survival). Columns, mean from three independent experiments each done in triplicate; bars, SE. *, P = 0.03, statistically significant difference between Ras and Ras+TACC1 (Student's t test). C, reduced PKB and Foxo3a dephosphorylation. NIH-3T3 cells were infected with retrovirus carrying either pBabe or pBabe-TACC1. These cells were either left untreated (lanes 1-3) or starved in 0.5% serum for 18 hours (lanes 4-6). Duplicate results (lanes 3 and 6). D, increased focus formation. NIH-3T3 cells stably infected with pBabe, pBabe-TACC1, or pBabe-TACC1s were transiently transfected with either empty pBabe or pBabe-RasV12. Infected cells (1 x 104) were plated onto a six-well plate, incubated for 10 days, and stained with crystal violet. The number of foci per well of a six-well plate was counted. Columns, mean from three experiments each done in triplicate; bars, SE.

 
PKB dephosphorylation triggered by growth factor withdrawal or serum starvation can be prevented by activating the PI3K pathway. Interestingly, we found that TACC1 overexpression in NIH-3T3 cells could prevent dephosphorylation of PKB induced by serum starvation (Fig. 1C). Consistent with this observation, the phosphorylation of Foxo3a, a direct target of PKB, was up-regulated in serum-starved TACC1-overexpressing cells. Thus, overexpression of TACC1 in 3T3 fibroblasts is able to attenuate cell death in response to PKB down-regulation and is also able to prevent PKB dephosphorylation in response to serum withdrawal.

TACC1 has previously been reported to promote transformation (1). Whereas TACC1 alone is mildly transforming, TACC1 enhances Ras-induced focus formation (Fig. 1D). TACC1s was unable to promote focus formation either alone or in combination with RasV12. These data highlight the oncogenic potential unique to full-length TACC1 and suggest that regions encoded by exon 2 and exon 3 are critical to the role of TACC1 in transformation and cell survival.

Generation of MMTV-TACC1 mice. To investigate the role of TACC1 in mammary gland development and tumorigenesis in vivo, transgenic mice expressing full-length TACC1 under the control of the MMTV promoter were generated (Fig. 2A). Two transgenic lines expressing TACC1 were identified by Western blotting (Tg1 and Tg2). Both MMTV-TACC1 Tg1 and Tg2 mice expressed the human TACC1 gene at significant levels in mammary tissue (Fig. 2B), as well as in spleen, lymph nodes, and thymus (Fig. 2C, left). This previously described antibody was raised against the NH2-terminal region of TACC1 and cannot detect the short isoform of TACC1 (12, 13). The TACC1 antibody recognizes both endogenous and overexpressed TACC1; the bottom band corresponds to endogenous TACC1, whereas the top band corresponds to exogenous TACC1. No exogenous TACC1 expression was observed in purified populations of B or T cells (Fig. 2C, right), suggesting that the TACC1 protein seen in spleen, thymus, and lymph nodes was contributed by tissues of nonlymphoid origin. The TACC1 expression seen in these organs may be contributed by epithelial cells in these organs and seems specific to mice carrying the TACC1 transgene. TACC1 expression was not observed in liver, uterus, or salivary gland (data not shown). All subsequent experiments were done in both transgenic strains. Similar results were obtained from both strains.



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  		Figure 2. Generation of MMTV-TACC1 transgenic mice. A, schematic illustration of the MMTV-TACC1 construct showing the position of the ApoE intronic sequence, the human full-length TACC1 cDNA, and the SV40 polyadenylation signal. B, expression of TACC1 in the mammary glands of representative mice of two transgenic lines (Tg1 and Tg2). Western blot analysis shows both endogenous (bottom band) and transgenic (top band) expression of TACC1. Actin, loading control. C, expression of TACC1 in lymphoid organs. Western blot analysis shows expression of both endogenous and transgenic TACC1 in the indicated lymphoid organs and cells of the representative MMTV-TACC1 mice. Representative blots of three independent experiments.

 
Extracellular signal-regulated kinase and protein kinase B signaling pathways are hyperactive in MMTV-TACC1 mice. To investigate the role of TACC1 in mammary gland development, whole mount analysis was done on 10- to 12-week-old MMTV-TACC1 mice. Mammary glands from virgin MMTV-TACC1 mice were slightly larger than mammary glands from WT littermates and showed a slight increase in side branching (Fig. 3A-B). Impregnated MMTV-TACC1 mice showed no mammary gland abnormalities during gestation, were able to nurse their pups, and could give birth to multiple litters. No mammary gland hyperplasias or tumors had developed by 1 year of age in either virgin or multiparous MMTV-TACC1 mice despite multiple rounds of exposure to high levels of TACC1 in the latter.



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  		Figure 3. Effect of TACC1 overexpression on virgin murine mammary glands. A, whole mount analyses of the fourth inguinal mammary glands of 10-week-old virgin WT (left) and MMTV-TACC1 (right) female littermates. The mutants show a slight increase in side branching. Representative of glands examined from 12 mice (3 Tg1, 3 Tg2, and 6 WT littermate controls). B, higher magnification of side branching of the WT (left) and MMTV-TACC1 (right) mammary glands in (A). C, increased PKB and ERK phosphorylation. Western blot analysis of phospho-PKB and phospho-ERK levels in two lines of MMTV-TACC1 mice and an age-matched WT control (lane 1). D, increased Foxo3a and MEK1 phosphorylation. Western blot analysis of phospho-Foxo3a and phospho-MEK1 levels in Tg2 MMTV-TACC1 transgenic mice. Lanes 1 and 3, WT littermate controls for lanes 2 and 4, respectively. C and D, representative blots of three independent experiments.

 
Mammary glands are often able to compensate for hyperactive cell survival signaling and maintain normal development despite increased levels of active signaling molecules. For example, mammary glands engineered to overexpress a constitutively active form of PKB (PKB-DD) still develop normally (26). To investigate signaling in MMTV-TACC1 mammary glands, the phosphorylation status of various components of the PKB and ERK pathways was examined. Lysates of MMTV-TACC1 mammary glands contained elevated levels of both phosphorylated PKB (Fig. 3C) and its target Foxo3a (Fig. 3D), suggesting hyperactivation of the PKB signaling pathway. Similarly, phosphorylation levels of both ERK and its upstream kinase MEK1 were increased in the mutant glands (Fig. 3C-D). Thus, the mammary glands of mice overexpressing TACC1 contain hyperphosphorylated components of both the ERK and PKB signaling pathways while maintaining essentially normal development.

Decreased apoptosis in involuting mammary glands of MMTV-TACC1 mice. During pregnancy, the murine mammary gland undergoes significant morphologic changes. The mammary epithelium grows dramatically, expanding the ductal network and forming lobules. Each lobule consists of multiple alveoli, the milk-secreting compartments of the lactating mammary gland (27). Mammary glands from lactating MMTV-TACC1 mice are normal and contain lobuloalveolar structures (data not shown).

Upon parturition in WT mice, the mammary gland reverts to a virgin-like state through the process of involution characterized by widespread apoptosis and the sloughing off of dead cells into the luminal space. Involution is controlled by a number of cell signaling pathways, including the PKB pathway. MMTV-PKB-DD and mammary-specific pten–/– mice show delayed involution associated with decreased apoptosis and slower kinetics of tissue remodeling (20, 26, 28). As MMTV-TACC1 mice displayed elevated levels of phospho-PKB, the process of involution was investigated in these mice. Compared with WT littermate controls, MMTV-TACC1 mice showed a decrease in the number of cells in the luminal space on the first day of involution or i1 (Fig. 4A-C) and a corresponding decrease in TUNEL-positive nuclei in i1 MMTV-TACC1 mammary glands (Fig. 4B). Caspase-3 activation was also reduced in MMTV-TACC1 i1 mammary glands (Fig. 4D). Overexpression of TACC1 in the mammary gland can attenuate involution-mediated apoptosis.



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  		Figure 4. Effect of TACC1 overexpression on murine mammary gland involution. A, altered involution. H&E-stained sections on day 1 of involution of mammary glands of WT (left) and MMTV-TACC1 (Tg2; right) mice. The mutants show delayed involution. Arrows point to epithelial cells sloughed off into the luminal space. Similar results were obtained from Tg1. B, decreased apoptosis. TUNEL staining of mammary glands from WT (left) and MMTV-TACC1 (Tg1; right) mice on day 1 of involution. Similar results were obtained from Tg2. C, decreased sloughing off of epithelial cells. Quantification of epithelial cells in the luminal space (arrows in A and B). The total numbers of apoptotic epithelial cells (as determined by H&E staining) in 10 visual fields are shown for mammary glands from WT and MMTV-TACC1 mice at days 1 (i1), 2 (i2), and 3 (i3) of involution. Three mice from each group were used for analysis. *, P < 0.05 (Student's t test). D, decreased caspase-3 activation. Cleaved caspase-3 levels on day i1 in mammary glands from WT mice (lane 1) and both of MMTV-TACC1 mice (lanes 2 and 3).

 
In the later stages of involution, apoptosis is decreased and the gland is repopulated by adipocytes (27). During these stages, mammary glands from MMTV-TACC1 mice involute normally. Tissue mass during involution is unchanged in the MMTV-TACC1 mice. Although TACC1 overexpression in the mammary gland reduces apoptosis during the first day following parturition, this alteration is not sufficient to induce gross changes in mammary gland morphology.

TACC1 increases tumor formation in pten+/– mice. TACC1 has been implicated in cellular transformation (1), but a direct role for TACC1 in tumorigenesis in vivo has yet to be shown. At 1 year of age, MMTV-TACC1 mice do not develop tumors and mammary tissue does not seem hyperplastic, suggesting that TACC1 overexpression alone is not sufficient for tumorigenesis. Because data from both cell culture systems and involution studies suggests that TACC1 promotes PI3K-dependent signaling and cell survival (Figs. 1, 3, and 4), MMTV-TACC1 mice were backcrossed thrice to the C57Bl6 strain and then bred to the pten+/– strain. The genetic background of the resulting MMTV-TACC1;pten+/– mice was >90% C57Bl6. Background-matched control pten+/– mice started to develop mammary tumors by ~6 months of age, with a penetrance of 27% in mice 6 to 12 months old. In contrast, MMTV-TACC1;pten+/– mice developed mammary tumors at a 3-fold greater frequency than pten+/– mice (P = 0.005; Fig. 5A). Histologically, these tumors were adenocarcinomas indistinguishable from those arising in pten+/– mice (Fig. 5B-C). No differences in lymphoma or endometrial tumor formation were observed between pten+/– and MMTV-TACC1;pten+/– mice (Fig. 5A). TACC1 expression increases mammary tumor formation in the genetically susceptible pten+/– mice.



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  		Figure 5. TACC1 increases the incidence of mammary tumor formation in pten+/– mice. A, percentage of pten+/– (n = 22) or pten+/–;MMTV-TACC1 (n = 15) mice between the ages of 6 and 12 months showing the indicated tumor types at time of sacrifice. The mutants showed a significant increase in mammary tumor formation (P = 0.005, Student's t test). The genetic background of all mice used was >90% C57Bl6. Both Tg1 and Tg2 were used for these tumor studies. There was no significant difference between these two lines. Data cumulative from both Tg1 and Tg2. B, H&E stained section from a pten+/– mammary tumor. C, H&E-stained section from a pten+/–;MMTV-TACC1 mammary tumor.

 
TACC1 accelerates tumorigenesis induced by PyMT315/322 overexpression. PyMT is a potent oncogene, and MMTV-PyMT mice develop multifocal mammary adenocarcinomas with an average latency of 4 to 7 weeks (15, 18). Transgenic mice carrying the PI3K-decoupled PyMT315/322 gene under control of the MMTV promoter (MMTV-PyMT315/322 mice) develop focal carcinomas with an ~2.5-fold longer latency (18). When MMTV-TACC1 mice were crossed to MMTV-PyMT315/322 animals, the resulting MMTV-TACC1;MMTV-PyMT315/322 mice developed tumors with a shorter latency than their MMTV-PyMT315/322 littermates (Fig. 6A). Although MMTV-PyMT315/322 mice developed observable focal carcinomas in situ with a mean latency of 20.6 weeks, the mean latency period was only 18.0 weeks (P = 0.003, Student's t test) in MMTV-TACC1;MMTV-PyMT315/322 mice. No differences were observed in tumor grade or stage, and no metastases were observed in either group (data not shown). Tumors from both groups were also similar in size, although tumors in the doubly transgenic cohort had less necrosis (Fig. 6B). H&E staining of tissue sections from MMTV-PyMT315/322 and MMTV-TACC1;MMTV-PyMT315/322 mice showed that both the singly and doubly transgenic mice developed carcinomas, although the tumors arising in the doubly transgenic mice were more papillary in nature (P = 0.002; Fig. 6C-D). Thus, TACC1 overexpression increases the penetrance of tumor formation in pten+/– mice and accelerates the appearance of tumors in MMTV-PyMT315/322 mice.



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  		Figure 6. TACC1 accelerates tumor progression in MMTV-PyMT315/322 mice. A, Kaplan-Meier survival curves of WT (n = 8), MMTV-TACC1 (n = 7), MMTV-TACC1;PyMT315/322 (n = 16), and PyMT315/322 (n = 24) mice. Expression of TACC1 shortens the latency of tumors induced by PyMT315/322 expression. There was no significant difference between these two lines. Data cumulative from both Tg1 and Tg2. B, differing characteristics of mammary tumors in of PyMT315/322 and MMTV-TACC1;PyMT315/322 mice. Mitotic index refers to the number of mitotic cells in five visual fields at x40 magnification. % Necrosis and %tubule formation are estimated for the largest tumor in each mouse. C-D, H&E-stained sections from tumors of (C) PyMT315/322 and (D) MMTV-TACC1;PyMT315/322 mice. Tumors in MMTV-TACC1;PyMT315/322 are more papillary than tumors in PyMT315/322 mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, the role of TACC1 in tumor formation was examined using cell culture systems and MMTV-TACC1 transgenic mice. TACC1 enhances transformation induced by RasV12, increases tumor formation in pten+/– mice, and accelerates tumorigenesis mediated by PyMT315/322. Overexpression of TACC1 in mammary tissue activates both the ERK and PKB signaling pathways. Mild morphologic defects are present in virgin MMTV-TACC1 mammary tissue, and apoptosis is decreased during involution of transgenic glands. In cell culture systems, TACC1 is able to attenuate apoptosis induced by overexpression of either Pten or PKB-AAA. Consistent with these observations, TACC1 promotes malignancies in both the pten+/– and PyMT315/322 mammary tumor models.

Human TACC1 has also been shown to interact with Aurora A (13). Most recently, Aurora A has been found to promote Ras-mediated transformation in fibroblasts (29). Because TACC1 promotes Ras-mediated focus formation (Fig. 1), the cooperation between Ras and Aurora A that promotes focus formation may be due to the ability of Aurora A to phosphorylate and stabilize TACC1. This may represent a novel, spindle-independent role for TACC1 and Aurora A. Interestingly, conditional overexpression of Aurora A in the murine mammary gland also induces hyperplasias (30).

TACC1 has been described as transforming (1) and yet is down-regulated in anthracyclin-treated mammary tumors (12). It has been proposed that these two observations are not necessarily contradictory; if TACC1 is able to control microtubule stability during mitosis, either an increase or a decrease in TACC1 levels might contribute to chromosomal instability (31). Levels of TACC1 in newly diagnosed tumors have not been yet reported. As an alternative explanation to these apparently contradictory results, it is possible that TACC1 may promote both tumorigenesis and chemosensitivity. If true, overexpression of TACC1 might serve as a useful marker for chemosensitive tumors. Consistent with this hypothesis, we have found that TACC1 overexpression seems to promote paclitaxel-induced cell death.6 The tumorigenic properties of TACC1 may result from the ability of TACC1 to activate Ras and PKB, whereas the chemosensitivity of TACC1-overexpressing cells may be due to the interaction of TACC1 with microtubules and the mitotic apparatus.

One of the major roles of the PI3K pathway is to promote cell survival. Growth factor signaling through PI3K inhibits the activity of a number of molecules known to cause cell cycle arrest and apoptosis, including the Foxo family of transcription factors (32). Ectopic expression of TACC1 results in the phosphorylation and activation of PKB and Foxo3a in mammary tissue (Fig. 2C). In NIH-3T3 cells, TACC1 expression prevents dephosphorylation of PKB and Foxo3a in response to serum starvation (Fig. 1C). In addition, TACC1 protects NIH-3T3 cells from death induced by Pten or PKB-AAA overexpression (Fig. 1B). Similarly, MMTV-TACC1 mice show decreased apoptosis during involution (Fig. 4). Our results are consistent with reports by others that overexpression of activated PKB decreases apoptosis during involution (28), and that involution is delayed in MMTV/PKB-DD mice (26). Taken together, these data suggest a role for TACC1 in preventing cell death induced by PI3K pathway inhibition.

TACC1 expression results in increases in both ERK and PKB phosphorylation in mammary tissue. Both of these pathways can be activated by Ras, suggesting that TACC1 might activate Ras either directly or indirectly. The coiled coil domain of TACC1 may interact with other molecules containing coiled coil domains, such as Ras-GRF2, to promote Ras activation (33). TACC1 may promote transformation and mammary tumorigenesis through the activation of Ras.

We have shown using two independent tumor models that TACC1 synergistically enhances tumor formation induced by other genes. Tumor formation is accelerated in mice doubly transgenic for TACC1 and PyMT315/322. Moreover, these tumors are more papillary in nature and therefore histologically distinguishable from tumors arising in PyMT315/322 mice. The significance of this latter observation is currently unknown, as no underlying biochemical changes have been identified in either the Ras or PI3K pathways in MMTV-TACC1;PyMT315/322 mice.

In TACC1-expressing pten+/– mice, the penetrance of tumor formation was decreased, whereas the latency remained unchanged. PTEN mutations occur in multiple organ systems, including the breast (34), and TACC1 was originally identified within a breast cancer amplicon. Based on evidence presented here, it seems plausible that amplification of TACC1 can promote tumorigenesis in human mammary epithelial cells already containing a PTEN mutation.

Interestingly, unlike full-length TACC1, the short isoform of TACC1 (TACC1s) does not contribute to Ras-mediated transformation or PI3K-dependent cell survival. This finding highlights the importance of distinguishing between these two splice variants when reporting levels of TACC1 expression in tumors. There are at least six isoforms of TACC1 expressed in gastric tumors (35). Two isoforms, designated TACC1-D and TACC1-F, are specifically up-regulated in gastric tumors relative to adjacent normal tissue (35). TACC1-D and TACC1-F both contain an alternative exon 4a, which is absent from all other splicing isoforms of TACC1. TACC-D and TACC-F have not been found in normal tissues. The association of aberrant alternative splicing with human disease is significant; it has been estimated that >15% of disease-causing mutations alter normal splicing (36). The spectrum of diseases associated with abnormal splicing includes atypical cystic fibrosis, retinitis pigmentosa, and myotonic dystrophy (36). Mutations affecting the alternative splicing of numerous genes, including CD44 and FGFR1, have been associated with tumorigenesis (37). Abnormal alternative splicing may play a role akin to genomic instability in the progression of cancer. Dysregulated splicing may result in the production of oncogenic splice isoforms, which could in turn lead to malignant progression. The existence of alternatively spliced TACC1 isoforms found exclusively in tumors supports this hypothesis. In normal mammary epithelial cells, bands corresponding to full-length TACC as well as TACC1s are found, suggesting that both of these molecules are important to normal mammary gland function (12). The physiologic roles of the splice variants of TACC1 warrant further investigation.

In conclusion, the data presented here suggest that TACC1 is able to cooperate with Ras and PI3K signaling in focus formation, cell survival, and tumorigenesis. TACC1 is thus an important potential collaborator in neoplasia, most likely due to its ability to promote signaling through the ERK and PKB pathways. In addition, the divergent roles of TACC1 and TACC1s suggest that alternative splice variants of TACC1 and the mechanisms underlying the regulation of alternative splicing may play a crucial role in mammary carcinogenesis.


   Acknowledgments
 
Grant support: Canadian Institutes for Health Research (M. Cully).

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 James Ihle, Vuk Stambolic, and Ying-Ju Jang for helpful discussions and Mary Saunders for scientific editing.


   Footnotes
 
Note: M. Cully is currently at the Cancer Research UK London Research Institute, London, United Kingdom.

6 M. Cully, J. Shiu, and T.W. Mak, unpublished observations. Back

Received 5/19/05. Revised 9/ 2/05. Accepted 9/13/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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