Activated Akt1 accelerates MMTV-c-ErbB2 mammary tumourigenesis in mice without activation of ErbB3

Abstract

Introduction: ErbB2, a member of the epidermal growth factor receptor (EGFR) family, is overexpressed in 20% to 30% of human breast cancer cases and forms oncogenic signalling complexes when dimerised to ErbB3 or other EGFR family members.

Methods: We crossed mouse mammary tumour virus (MMTV)-myr-Akt1 transgenic mice (which express constitutively active Akt1 in the mammary gland) with MMTV-c-ErbB2 transgenic mice to evaluate the role of Akt1 activation in ErbB2-induced mammary carcinoma using immunoblot analysis, magnetic resonance spectroscopy and histological analyses.

Results: Bitransgenic MMTV-c-ErbB2, MMTV-myr-Akt1 mice develop mammary tumours twice as fast as MMTV-c-ErbB2 mice. The bitransgenic tumours were less organised, had more mitotic figures and fewer apoptotic cells. However, many bitransgenic tumours displayed areas of extensive necrosis compared with tumours from MMTV-c-ErbB2 mice. The two tumour types demonstrate dramatically different expression and activation of EGFR family members, as well as different metabolic profiles. c-ErbB2 tumours demonstrate overexpression of EGFR, ErbB2, ErbB3 and ErbB4, and activation/phosphorylation of both ErbB2 and ErbB3, underscoring the importance of the entire EGFR family in ErbB2-induced tumourigenesis. Tumours from bitransgenic mice overexpress the myr-Akt1 and ErbB2 transgenes, but there was dramatically less overexpression and phosphorylation of ErbB3, diminished phosphorylation of ErbB2, decreased level of EGFR protein and undetectable ErbB4 protein. There was also an observable attenuation in a subset of tyrosine-phosphorylated secondary signalling molecules in the bitransgenic tumours compared with c-ErbB2 tumours, but Erk was activated/phosphorylated in both tumour types. Finally, the bitransgenic tumours were metabolically more active as indicated by increased glucose transporter 1 (GLUT1) expression, elevated lactate production and decreased intracellular glucose (suggesting increased glycolysis).

Conclusion: Expression of activated Akt1 in MMTV-c-ErbB2 mice accelerates tumourigenesis with a reduced requirement for signalling through the EGFR family, as well as a reduced requirement for a subset of downstream signaling molecules with a metabolic shift in the tumours from bitransgenic mice. The reduction in signalling downstream of ErbB2 when Akt is activated suggest a possible mechanism by which tumour cells can become resistant to ErbB2-targeted therapies, necessitating therapies that target oncogenic signalling events downstream of ErbB2.

Figure 1

Bitransgenic MMTV-myr-Akt1, MMTV-c-ErbB2 mice have decreased mammary tumour latency, more aggressive tumour histology and decreased apoptosis compared with MMTV-c-ErbB2 mice. (a) Mammary tumour latency in bitransgenic MMTV-myr-Akt1, MMTV-c-ErbB2 mice and MMTV-c-ErbB2 mice. Sixty days after birth, bitransgenic MMTV-myr-Akt1, MMTV-c-ErbB2 and MMTV-c-ErbB2 mice were palpated weekly to monitor for the presence of mammary tumours. The graph shows the rate at which tumours were first detected for both genotypes. A total of 22 bitransgenic mice and 30 MMTV-c-ErbB2 mice were monitored and the graph shows the number of days to tumour detection versus the percentage of tumour-free mice. (b-e) Haematoxylin and eosin stained tumour sections. (b) Tumour derived from a MMTV-c-ErbB2 mouse. (c-e) Tumors derived from bitransgenic MMTV-myr-Akt1, MMTV-c-ErbB2 mice. (c) A bitransgenic tumour with histology similar to that of c-ErbB2 tumours. (d-e) Two different bitransgenic tumours demonstrating necrotic tumour tissue distal to a blood vessel (blood vessel marked with * and necrosis marker with **). ×200 original magnification. (f) Tumours from bitransgenic MMTV-myr-Akt1, MMTV-c-ErbB2 mice have less apoptosis than tumours from MMTV-c-ErbB2 mice. Apoptosis was quantitated by activated caspase-3 immunohistochemistry. The number of cells staining positively for activated caspase-3 was divided by the total number of cells counted to generate the apoptotic rate. (g) Tumours from bitransgenic mice have a higher proliferation rate than tumours from MMTV-c-ErbB2 mice. Proliferation rate was determined by counting the number of mitotic figures in 10 500× magnification fields of view and the data is presented as the mean +/- standard deviation for three tumours of each genotype.

Figure 2

Tumours from bitransgenic MMTV-myr-Akt1, MMTV-c-ErbB2 mice and MMTV-c-ErbB2 mice overexpress ErbB2 and phosphorylated Akt. Tumour (T) and normal mammary tissue (N) were harvested from tumour-bearing mice. Tumours from three different bitransgenic MMTV-myr-Akt1, MMTV-c-ErbB2 mice and three different MMTV-c-ErbB2 mice are represented with normal mammary tissue from the same animal. Additionally, normal mammary tissue was collected from control FVB and MMTV-myr-Akt1 mice and analysed as a control. Equal amounts of total protein was loaded per lane to a 10% sodium dodecyl sulfate polyacrylamide gel, transferred to polyvinylidene difluoride and probed with the following antibodies: (a) anti-ErbB2; (b) anti-HA to detect the HA epitope-tagged myr-Akt1 transgene; (c) anti-phospho-Akt (Ser473); (d) anti-pan-Akt; and (e) anti-β-actin to demonstrate equal loading of the gel.

Figure 3

Expression and activation of EGF receptor tyrosine kinase family members is decreased in tumours from bitransgenic MMTV-myr-Akt1, MMTV-c-ErbB2 mice compared with tumours from MMTV-c-ErbB2 mice. Lysates from tumour (T) and normal mammary tissue (N) were used for immunoblot analysis using the following antibodies: (a) anti-phospho-ErbB2 (Tyr877); (b) anti-phospho-ErbB2 (Tyr1248); (c) anti-ErbB2; (d) anti-phospho-ErbB3 (Tyr1289); (e) anti-ErbB3; (f) anti-EGFR; (g) anti-ErbB4; and (h) anti-β-actin to demonstrate equal loading of the gel.

Figure 4

Tumours from MMTV-myr-Akt1, MMTV-c-ErbB2 mice have decrease tyrosine kinase signalling compared with tumours from MMTV-c-ErbB2 mice. Lysates from tumour (T) and normal mammary tissue (N) were used for immunoblot analysis using the following antibodies: (a) anti-phospho-Src (Tyr416); (b) anti-Src; (c) anti-phospho-Gab2 (Tyr452); (d) anti-Gab2; (e) anti-phospho-Shc (Tyr313); (f) anti-phospho-Shc (Tyr239/240): (g) anti-Shc; (h) anti-phospho-Erk (corresponding to Erk2 phosphorylation at Thr183 and Tyr185); and (i) anti-Erk.

Figure 5

MMTV-c-ErbB2 tumours demonstrate different cell cycle proteins than tumours from bitransgenic MMTV-myr-Akt1, MMTV-c-ErbB2 mice. Lysates from tumour (T) and normal mammary tissue (N) were used for immunoblot analysis using the following antibodies: (a) anti-phospho-Rb (Ser780); (b) anti-Rb; (c) anti-cyclin D1; (d) anti-p15; and (e) anti-p27.

Figure 6

Metabolic activity is elevated in tumours from bitransgenic MMTV-myr-Akt1, MMTV-c-ErbB2 mice compared with tumours from MMTV-c-ErbB2 mice. Increased expression of the GLUT1 glucose transporter in bitransgenic MMTV-myr-Akt1, MMTV-c-ErbB2 tumours compared with the MMTV-c-ErbB2 tumours. Lysates from tumour (T) and normal mammary tissue (N) were used for immunoblot analysis using a polyclonal antibody against the c-terminus of GLUT1 (a) and actin-loading control (b). Intratumour concentration (nmol/mg tissue) of lactate (c) and glucose (d) in mammary glands at day 2 of lactation (L2), tumours from MMTV-c-ErbB2 and bitransgenic MMTV-myr-Akt1, MMTV-c-ErbB2 mice (all n = 5) calculated from ¹H-magnetic resonance spectroscopy.