The MMTV-c-ErbB2 (line 202) transgenic mice 6 were obtained from The Jackson Laboratory, Bar Harbor, ME. Details of the MMTV-myr-Akt1 mice have been previously described 11. These two FVB-derived transgenic lines were crossed and progeny genotypes were determined by PCR analysis. Virgin MMTV-c-ErbB2 and bitransgenic MMTV-c-ErbB2, MMTV-myr-Akt1 female mice were palpated weekly to detect the presence of mammary tumours starting at 60 days of age. Tumours were excised when they reached 1 cm in diameter and 1 cm deep, and normal, non-tumourigenic mammary tissue was harvested from the same animal at the time of tumour harvest. All mice were maintained in the Center for Comparative Medicine at the University of Colorado Denver – Anshutz Medical Campus, an Association for Assessment and Accrediation of Laboratory Animal Care-approved facility, and used in accordance with Institutional Animal Care and Use Committee-approved protocols.

DNA was extracted from 1.5 cm sections of tail and genotyping was performed using previously described protocols 11. Detection of the Myr-Akt1 transgene utilised a forward primer which anneals to the Akt1 sequence: (5'-GCCGCTACTATGCCATGAAGA-3') and a reverse primer which anneals to the HA (haemagglutin) epitope: (5'-GTAATCTGGAACATCGTATGGGTA-3'). Detection of the ErbB2 transgene utilised the forward primer (Neu-3) 5'-CGGAACCCACATCAGGCC-3' and the reverse primer (Neu-4) 5'-TTTCCTGCAGCAGCCTACGC-3' 17.

Total RNA isolated from tumour samples was subjected to single-strand cDNA synthesis using 2.5 μM random hexamers and 1 μg RNA. Amplification of the rat ERBB2 gene, nucleotides 1492 to 2117, was performed using forward primer AB2913, 5'-CGGAACCCACATCAGGCC-3', and reverse primer AB1310, 5'-TTTCCTGCAGCAGCCTACGC-3', as previously described 18. The PCR products were separated on a 2% agarose gel, the bands of interest (representing truncated ERBB2) were purified and then re-amplified using the same primers. Sequence analysis was conducted by the University of Colorado Cancer Center Sequencing Core using forward primer 1882, 5'-CACTACAAGGACTCGTCCT-3', and reverse primer 2133, 5'-CCAACGACCACCACTAAG-3'.

Dissected tumours and normal mammary tissue were fixed in 4% neutral buffer formalin, embedded in paraffin, sectioned (4 μm) and stained with haematoxylin and eosin. Histological sectioning and staining were performed by the Histology Service, Department of Pathology, University of Colorado School of Medicine. The mitotic index of tumours of each genotype was determined by counting the number of mitotic figures in 10 fields of view with a magnification of 500 and the data were presented as the mean of three tumours +/- standard deviation (SD).

Detection of apoptotic cells was performed by immunohistochemical staining with anti-active caspase-3 antibody (Cell Signaling Technologies, Beverly, MA, USA). For antigen retrieval, slides in citrate buffer were heated in a microwave for 20 minutes and allowed to cool before blocking with 10% normal goat serum. Slides were incubated overnight at 4°C with anti-activated caspase-3 antibody at a dilution of 1:100. Non-specific peroxidase activity was quenched with 1% hydrogen peroxide followed by secondary antibody (goat anti-rabbit) then tertiary Vector ABC (Vector Laboratories, Burlingame, CA, USA). Colour development was achieved by incubation with DAB followed by counterstaining with Gill's Haematoxylin. Cell counts were performed on a minimum of five fields of view per slide from three mice (total cells counted ranged from 1800 to 2500 per mouse).

Protein was extracted from frozen tumour tissue and normal tissue by grinding them to a powder under liquid nitrogen, resuspended in Mammary Gland Lysis Buffer (50 mM Tris (2-Amino-2-(hydroxymethyl)-1,3-propanediol) pH 7.4, 150 mM sodium chloride, 2 mM EDTA, 50 mM sodium fluoride, 1% Triton X-100, 1% deoxycholic acid, 0.1% sodium dodecyl sulfate [SDS], 1 mM dithiothreitol, 5 mM sodium orthovanadate, 100 μg/ml phenylmethanesulphonylfluoride and a complete protease inhibitor cocktail (Roche Applied Sciences, Indianapolis, IN, USA)), followed by Dounce homogenisation. Lysates were clarified by centrifugation and protein concentrations determined using the Bradford assay (BioRad, Hercules, CA, USA). Equal amounts of total protein per lane (2 to 50 μg) were resolved on SDS-polyacrylamide gels, transferred to polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore, Bedford, MA, USA) and immunoblotted with the desired antibody.

Anti-HA antibody was obtained from Roche Applied Sciences (Indianapolis, IN, USA). Anti-phospho-ErbB2 (Tyr877), anti-phospho-ErbB3 (Tyr1289), anti-phospho-retinoblastoma (Rb) (Ser780), anti-phospho-Akt (Ser473), anti-phospho-Src (Tyr416), anti-phospho-Gab2 (Tyr452), anti-phospho-Shc (Tyr313), anti-phospho-Shc (Tyr239/240), anti-Akt, anti-p15, anti-p27 and anti-cyclin D1 antibodies were obtained from Cell Signaling Technologies (Beverly, MA, USA). The anti-ErbB2, anti-ErbB3, anti-ErbB4, anti-EGFR, anti-Src, anti-Shc, anti-β-actin and anti-ERK1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-phospho-ErbB2 (Tyr1248) was obtained from Abcam (Cambridge, MA, USA). The anti-Rb antibody was obtained from BD Pharmingen (San Jose, CA, USA). The anti-phospho-ERK antibody was obtained from Promega (Madison, WI, USA). The anti-Gab2 polyclonal antibody was generously supplied by Dr. Haihua Gu (University of Colorado, Denver, USA). The glucose transporter 1 (GLUT1) polyclonal antibody was generated by Global Peptide (Fort Collins, CO, USA) by immunising rabbits with a peptide corresponding to the C-terminus of the human/mouse GLUT1 sequence (KTPEELFHPLGADSQV) and affinity purifying the resulting IgG.

Snap-frozen tissues were ground into a powder under liquid nitrogen then homogenised by sonication in chloroform-methanol to precipitate proteins and separate aqueous and lipid-soluble metabolites as described 19. The lyophilised aqueous and lipid extracts were dissolved in deuterated solvents and analysed using high-resolution ¹H-magnetic resonance spectroscopy (MRS) with the Bruker narrow-bore 500 MHz DRX system and an inverse TXI-5-mm probe (Bruker Biospin Inc., Fremont, CA, USA). The following ¹H-nuclear magnetic resonance (NMR) parameters with water suppression ('zgpr') were used: 500.12 MHz operating ¹H frequency; 40 accumulations; 0 dB power level; 55 dB irradiation power level; 7.5 μs pulse width; 12 ppm spectral width; and 12.8 second repetition time (fully relaxed). An external standard substance, trimethylsilyl propionic-2,2,3,3,-d₄ acid ([TMSP] 20 and 50 mM in heavy water) in a thin sealed glass capillary was placed into the NMR tubes during all experiments for metabolite quantification and as a ¹H chemical shift reference (at 0 ppm). After performing Fourier transformation, phase and base line corrections, each ¹H peak of corresponding metabolites was integrated using 1D WINNMR program (Bruker Biospin Inc., Fremont, CA, USA). The NMR peak assignment was confirmed by two-dimensional NMR spectra 19. All quantitative data sets from ¹H-MRS are reported as mean ± SD (n = 5 for each group). The p values (from analysis of variance [ANOVA]) below 0.05 were considered as statistically significant.

We have previously described the transgenic mice that express the activated myr-Akt1 transgene in the mammary gland which rarely develop tumours 11. MMTV-c-ErbB2 (line 202) mice express the wild type rat ERBB2 gene in the mammary gland and have been an extensively used model of mammary carcinomas 202122. To determine whether myr-Akt1 could accelerate mammary tumourigenesis, we crossed MMTV-c-ErbB2 (line 202) mice 6 with MMTV-myr-Akt1 mice 11. The MMTV-c-ErbB2 female mice developed single focal mammary tumours with a mean latency of 231 days (Figure 1a).

The bitransgenic MMTV-c-ErbB2, MMTV-myr-Akt1 mice developed single focal mammary tumours with a mean latency of 114 days, meaning the bitransgenic animals develop mammary tumours twice as fast as the MMTV-c-ErbB2 mice (Figure 1a). A total of 30 MMTV-c-ErbB2 mice and 22 bitransgenic mice were used to calculate mean tumour latency.

Tumours arising in the MMTV-c-ErbB2 mice usually display mutations in the wild type rat c-ERBB2 transgene resulting in a constitutively activated form of ErbB2 18. One possible means by which expression of myr-Akt1 could accelerate tumourigenesis in the MMTV-c-ErbB2 mice would be to bypass the apparent requirement for mutation of rat c-ERBB2. However, analysis of the tumours that appeared in the bitransgenic mice indicated that these tumours also contain the activating mutations in the rat c-ERBB2 allele (data not shown).

Tumour histology was evaluated using haematoxylin and eosin stained sections. The histology of the c-ErbB2 tumours was consistent with previous descriptions: they are solid tumours composed of uniformly sized and shaped cells with small stroma and no evidence of myoepithelial cells 623 (Figure 1b). Staining of tumours from the bitransgenic animals revealed two different types of tumours: those similar to c-ErbB2 tumours with a solid, uniform architecture (Figure 1c); and the second type of tumour demonstrated necrosis in areas of the tumour 10 to 20 cells away from vasculature, consistent with a tumour that outgrows its blood supply (Figure 1d, e). Very little necrosis is ever observed in tumours derived from MMTV-c-ErbB2 mice.

After initial detection, tumour volumes were determined by measuring the tumour dimensions with calipers to estimate tumour volume (volume = (length × width × width)/2). A comparison of tumour growth in mice of both genotypes revealed that tumour volume increased two to three times faster in the bitransgenic mice compared with tumours in the MMTV-c-ErbB2 transgenic mice (data not shown). The increased growth rate of tumours in the bitransgenic mice could result from either increased proliferation, decreased apoptosis or both. Measurement of the apoptotic rates in both tumour types by activated caspase-3 immunohistochemistry demonstrates that the tumours from the bitransgenic animals have an apoptotic rate half that of the tumours derived from the MMTV-c-ErbB2 animals (Figure 1f). The rate of proliferation, determined by quantitating the number of mitotic figures in tumour sections, demonstrated that tumours from bitransgenic animals had a higher proliferation rate than tumours from MMTV-c-ErbB2 animals (Figure 1g). Thus, the bitransgenic animals rapidly develop mammary tumours with a low rate of apoptosis and high rate of proliferation compared with the tumours from MMTV-c-ErbB2 animals, and at least half of these bitransgenic tumours exhibit extensive necrosis.

The expression of the transgenes was examined at the protein level in tumours (T) and normal mammary gland control (N) taken from tumour-bearing mice of both the MMTV-c-ErbB2 and MMTV-c-ErbB2, MMTV-myr-Akt1 genotypes. Mammary glands from FVB mice and MMTV-myr-Akt1 mice were used as controls. Immunoblotting with an anti-ErbB2 antibody demonstrated that the level of ErbB2 protein was dramatically increased in tumours of both origins compared with normal mammary tissue from the same mouse, from FVB mice or from myr-Akt1 transgenic mice (Figure 2a).

Expression of the HA-tagged myr-Akt1 transgene was only detected in tumour tissue from the bitransgenic animals (Figure 2b). The myr-Akt1 transgene in the bitransgenic tumours was phosphorylated at Ser473, indicating enzymatic activity, and can be distinguished from endogenous Akt because the myr-Akt1 transgene has a higher molecular weight (Figure 2c). Akt was also phosphorylated in c-ErbB2 tumours, consistent with previously published data 8 (Figure 2c). Immunoblot using anti-pan-Akt antibody demonstrates expression of endogenous Akt in both tumour types with the c-ErbB2 tumours expressing more Akt than the bitransgenic tumours (Figure 2d). An immunoblot with anti-β-actin antibody demonstrates equal sample loading (Figure 2e). All immunoblot data presented in the present study is representative of all tumour and normal gland pairs examined (n = 5 for c-ErbB2 and n = 7 for bitransgenic). The different levels of proteins expressed in tumour tissue versus normal tissue is probably due to both a difference in selective pressures brought on by tumourigenesis and a dramatic increase in epithelial cell content in tumour tissue: the virgin mammary gland is predominantly adipocytes and tumour tissue is predominantly epithelial in nature.

Mammary tumours from transgenic mice expressing activated mutants of ErbB2 also express elevated levels of total and tyrosine-phosphorylated ErbB2 and ErbB3 7. As previously shown in Figure 2, ErbB2 was increased in tumour lysates from both MMTV-c-ErbB2 and bitransgenic mice compared with normal tissue (Figure 3c). The extent of activating phosphorylation of ErbB2 was lower in the bitransgenic tumours than in the c-ErbB2 tumours, demonstrated by using phosphospecific antibodies to two different phosphorylation sites in ErbB2, Tyr877 and Tyr1248 (Figure 3a, b). Phosphorylation of Tyr877 is mediated by Src and contributes to the activation of the ErbB2 tyrosine kinase, and Tyr1248 is an autophosphorylation site 2425. Increased levels of ErbB3 (Figure 3e), Tyr1289-phosphorylated ErbB3 (Figure 3d), epidermal growth factor receptor (EGFR) (Figure 3f) and ErbB4 (Figure 3g) were readily detected in the c-ErbB2 tumours compared with normal tissue from the same mice.

While increased levels of EGFR and ErbB3 protein could be detected in the bitransgenic tumours when compared with normal tissue, the extent of the increase was dramatically reduced compared with that observed in the c-ErbB2 tumours. Most importantly, the phosphorylation of ErbB3 was very low at Tyr1289 (Figure 3d), which is an important phosphorylation site for PI3K recruitment and activation 26. ErbB4, the sole member of the EGFR family with an expression that bears positive prognostic value to breast cancer patients and suppresses proliferation and promotes apoptosis 2728, was not detected in the bitransgenic tumours (Figure 3g). These data suggest that expression of activated Akt1 in the MMTV-c-ErbB2 transgenic mice alters the requirement for overexpression and activation of ErbB3 and other EGFR family members in mammary tumours induced by ErbB2.

Mammary tumours expressing c-ErbB2 have been found to possess elevated levels of Src tyrosine protein kinase activity 2930. The Src protein binds to phosphotyrosine residues in the cytoplasmic tail of ErbB2, resulting in Src phosphorylation at Tyr416 and catalytic activation 3132. The amount of Tyr416-phosphorylated Src present in the c-ErbB2 tumours was dramatically increased in comparison to the bitransgenic tumours and normal tissue (Figure 4a), even though the bitransgenic tumours express more total Src than the c-ErbB2 tumours (Figure 4b). The reduced Src activation indicated by low Tyr416 phosphorylation levels in the bitransgenic tumours is corroborated by the reduced phosphorylation of ErbB2 at Tyr877 (Figure 3a), which is mediated by Src 2425. The reciprocal positive regulation of Src by ErbB2 and ErbB2 by Src is strongly diminished in the bitransgenic tumours (in addition to the reduced activation of ErbB3) indicating a general decline in plasma membrane tyrosine kinase signalling in the bitransgenic tumours.

The lack of activation/overexpression of EGFR family members and the lack of activation of Src in the bitransgenic tumours suggested that signalling mediated by secondary signalling molecules might also be attenuated in the bitransgenic tumours when compared with c-ErbB2 tumours. Gab2 is a scaffolding protein that can recruit the p85 subunit of PI3K when it is phosphorylated at Tyr452 33 which in turn activates PH-domain containing proteins such as Akt 34. The levels of Gab2 protein are elevated in both c-ErbB2 tumours and bitransgenic tumours compared with normal gland controls (Figure 4d), although the level is slightly higher in c-ErbB2 tumours than bitransgenic tumours. The amount of Tyr452-phosphorylated Gab2 is dramatically higher in the c-ErbB2 tumours compared with the bitransgenic tumours (Figure 4c) suggesting that expression of activated Akt1 in the bitransgenic animals attenuates phosphorylation of Gab2 and subsequent docking of PI3K, which occurs in c-ErbB2 animals.

It has recently been demonstrated that another secondary signalling molecule, ShcA, is required for ErbB2-mediated tumourigenesis and phosphorylation of Tyr313 in ShcA may be important for tumour cell survival, while phosphorylation of Tyr239/240 may be important for tumour vascularisation 35. c-ErbB2 tumours and bitransgenic tumours both demonstrate expression of ShcA (Figure 4g), particularly the p52 and the p46 isoforms while the p66 isoform was barely detectable. Both tumour types also appear to have similar levels of Tyr239/240-phosphorylated ShcA (with the bitransgenic tumours having slightly less phosphorylation than the c-ErbB2 tumours), indicating that activities regulated by phosphorylation of this site (perhaps in angiogenesis) are important in both tumour types (Figure 4f). However, the difference in phosphorylation of ShcA at Tyr313 is more dramatic between the two tumour types (Figure 4e), with the c-ErbB2 tumours having more ShcA phosphorylated at Tyr313 than the bitransgenic tumours suggesting that activities regulated by phosphorylation of this site (cell survival) may be less important in the tumours that express activated Akt1.

Oncogenic signalling often activates the Erk pathway, which is a known mediator of cell proliferation, cell survival, angiogenesis and cell migration (reviewed in 36). Dual phosphorylation of Erk1 and Erk2 (corresponding to Thr183 and Tyr185 of human Erk2) in the activation loop results in catalytic activation of kinase activity 37. Despite the attenuation of several signalling events in the bitransgenic tumours when compared with the c-ErbB2 tumours as discussed above, both tumour types maintain similar levels of phosphorylated/activated Erk (Figure 4h) indicating that transgenic activation of Akt1 does not bypass Erk signalling. Normal gland controls demonstrate various levels of Erk activation.

The decrease in the tumour latency observed in the bitransgenic tumours would predict that there would be a change in the expression/modification of cell cycle control proteins in the bitransgenic tumours compared with the c-ErbB2 tumours. Phosphorylation of Rb by active cyclin-dependent kinases inactivates Rb activity and is an important regulatory step in cell cycle entry 3839. Immunoblot analysis with a phospho-specific antibody against Rb (Ser780) demonstrated Rb phosphorylation in tumour lysates from both genotypes of mice and not in the normal tissue from the same mice (Figure 5a). The extent of Rb phosphorylation was greater in the c-ErbB2 tumours than in the bitransgenic tumours. This is surprising because the bitransgenic tumours have a shorter tumour latency and greater mitotic index which we predicted would correlate with more Rb phosphorylation and a more active cell cycle. However, Hutchinson et al. observed a similar phenomenon in their study: the NDL2 tumours (activated ErbB2) demonstrated a higher level of Ser780-phosphorylated Rb than the bitransgenic NDL2/Akt1DD tumours even though the bitransgenic animals had a shorter tumour latency 15. The total amount of Rb is almost the same in all tumours examined indicating differences observed in Rb phosphorylation are not in fact due to differences in total Rb levels (Figure 5b).

We also examined a second cell cycle control protein, cyclin D1. The D-type cyclins are synthesised during the G1 phase of the cell cycle after stimulation of cells with growth factors that stimulate cell cycle entry and are required for cell cycle progression 40. Immunoblot analysis revealed cyclin D1 in all samples with the bitransgenic tumours having a higher level of cyclin D1 than normal tissue, but the c-ErbB2 tumours containing more cyclin D1 than the bitransgenic tumours (Figure 5c). This data correlates with the Rb phosphorylation data which is consistent with the role of cyclin D1 in positively regulating cyclin dependent kinase activity and phosphorylation of Rb 40.

The bitransgenic tumours have a shorter latency than the c-ErbB2 tumours, but contain less phosphorylated Rb and less cyclin D1, which led us to examine some cell cycle inhibitors. p15 (INK4b), p27 (Kip1) and p21 (Cip1) are all inhibitors of cell cycle progression 41. Immunoblot analysis for p15 demonstrated high expression in the c-ErbB2 tumours, a lack of expression in all normal glands and very faint expression in the bitransgenic tumours (Figure 5d). The expression profile of p27 was similar with a lack of expression in all normal tissue, high expression in c-ErbB2 tumours and slightly lower level of expression in the bitransgenic tumours (Figure 5e). We were unable to detect p53 and p21 in any of the tumour and normal mammary tissue samples, though both were present in an irradiated control gland (data not shown). While c-ErbB2 tumours contain more positive markers of the cell cycle than the bitransgenic tumours (phosphorylated Rb and cyclin D1), they also express more negative regulators of the cell cycle (p15 and p27), which likely serves to balance cell cycle progression.

One of the hallmarks of tumours and tumour cell lines is the elevated level of glucose transport to support the high glycolytic rate of tumour cells 4243. Myr-Akt1 can stimulate the translocation of the GLUT1 glucose transporter to the cell surface of lymphoid cells cultured in the absence of growth factors as well as increase hexokinase activity, glucose consumption and lactate production, which suggests that Akt stimulates aerobic glycolysis and produces the so-called 'Warburg effect' 4445. Immunoblot analysis demonstrates elevated levels of GLUT1 (the major glucose transporter in cancer cells) in lysates from mammary tumours from both the MMTV-c-ErbB2 and the bitransgenic mice compared with levels in normal glands. However, GLUT1 protein expression was greater in the bitransgenic tumours (Figure 6a). Cell-line transfection using GLUT1 cDNA validates the elongated band identified by GLUT1 immunoblot and the reason for this elongated band is probably due to glycosylation of GLUT1 (data not shown).

To determine whether the increased level of GLUT1 correlated with an increase in glycolysis (increased glucose consumption and increased lactate production), we used MRS to quantitate lactate concentrations in extracts prepared from tumours because lactate is the end product of glycolysis. The mammary tumours examined in this study are of epithelial cell origin, so the use of mammary tissue from day two of lactation (L2) as a control tissue allows for the comparison of normal epithelium with tumour epithelium, whereas the normal virgin mammary gland is composed mostly of adipocytes 46. Compared with normal L2 tissue, there was a four-fold increase in the amount of lactate in the MMTV-c-ErbB2 tumours and a six-fold increase in the bitransgenic tumours (Figure 6c) (p < 0.00001 between L2 and both tumours and p < 0.02 between ErbB2 and bitransgenic tumours, all n = 5). This increase in tumour glycolytic activity was accompanied by significantly decreased intratumuor concentrations of glucose: 1.10 nmol/mg in the L2 mammary gland, 0.46 nmol/mg in ErbB2 tumours (p < 0.01) and 0.14 nmol/mg in bitransgenic tumours (p < 0.0004) (Figure 6d). This indicates high glucose utilisation due to the increased activity of glycolytic enzymes. Thus, the bitransgenic tumours appear to consume more glucose and secrete more lactate than the c-ErbB2 tumours or the normal epithelial cell-enriched lactating mammary gland.