Cdk2 is required for breast cancer mediated by the low-molecular-weight isoform of cyclin E

Abstract

Cyclin E activates Cdk2, controls centrosome duplication, and regulates histone gene transcription. Cyclin E is deregulated in cancer and appears as low-molecular-weight (LMW) isoforms that correlate strongly with decreased survival in breast cancer patients. Transgenic mice overexpressing LMW-cyclin E have increased incidence of mammary tumors and distant metastasis when compared with mice that had full-length cyclin E. To specifically test the requirement for Cdk2 in LMW-cyclin E-mediated mammary tumorigenesis, we generated transgenic mice, which expressed LMW-cyclin E in a Cdk2-deficient background. We found that mammary gland development proceeds relatively normally in these animals, indicating that Cdk2 kinase activity is largely dispensable for this process. However, Cdk2-deficient mice were completely resistant to LMW-cyclin E-mediated mammary tumors. Cdk2 wild-type or heterozygous mice succumbed to mammary tumors with mean latencies of 16 and 19.5 months, respectively, but Cdk2 nullizygous littermates did not display tumors through 24 months. Similarly, continuous administration of two different Cdk inhibitors significantly delayed LMW-cyclin E-induced mammary tumor progression. Triple transgenic mice generated in a p53 heterozygous background also displayed no tumors. Finally, we found that Cdk2 silencing induced cell death in LMW-overexpressing breast cancer cell lines, but not in cell lines lacking LMW expression. Our findings establish a requirement for Cdk2 in LMW-cyclin E-mediated mammary tumorigenesis, arguing that human breast tumors overexpressing LMW-cyclin E are prime candidates for anti-Cdk2 therapy.

Figure 1. Hormone induced proliferation in Cdk2-/- mammary glands

A. A cohort of mice were left untreated (No hormones) or treated acutely (48–50 h) with estradiol benzoate (E, 1 μg, Sigma, St. Louis, MO) and P (1 mg, Sigma) in 100 μl sesame oil via a single interscapular subcutaneous injection behind the neck once (E+P, 48 hrs) or 3 times, 48 hours apart (E+P, 3× 48 hrs). Two hours before sacrifice, the mice were injected with BrdUrd (0.03 mg/g of body weight) intraperitoneally. Following treatment, the contralateral inguinal gland was removed (Cdk2+/+ mice, n = 3; Cdk2+/- mice, n = 3; Cdk2-/- mice, n = 3, 11–32 weeks of age at day 0 of experiment) and whole-mount were prepared. B. and C. Proliferation is quantified by BrdU staining.

Figure 2. Resistance of Cdk2-/- mice to LMW cyclin E-induced breast cancer

Percentage of mammary tumor free mice among MMTV-LMW cyclin E-T1; Cdk2+/+; MMTV-LMW cyclin E-T1; Cdk2+/- ; MMTV-LMW cyclin E-T1; Cdk2-/- females in A. and MMTV-LMW cyclin E-T1; p53+/-; Cdk2+/+; MMTVLMW cyclin E-T1; p53+/-; Cdk2+/-; MMTV-LMW cyclin E-T1; p53+/-; Cdk2-/- females in B. Whereas LMW-cyclin E-T1 mice in Cdk2+/- or Cdk2+/+ background succomb to mammary tumors with mean latencies of 16 and 19.5 months respectively, their LMW cyclin E-T1; Cdk2-/- littermates do not form mammary tumors (P=0.05 Cdk2+/+ versus Cdk2-/-; P=0.0252 Cdk2+/- versus Cdk2-/-; P=0.1457 Cdk2+/+ versus Cdk2+/-, Logrank test). Similarly, MMTV-LMW cyclin E T1/p53+/- transgenic mice in Cdk2+/- or Cdk2+/+ background succomb to mammary gland tumors between 7 and 13 months with 100% penetrance, their MMTV-LMW cyclin E-T1;p53+/-; Cdk2-/- littermates are resistant to tumor formation (P<0.01 Cdk2+/+ versus CDk2-/-; P=0.011 Cdk2+/- versus Cdk2-/-, P=0.5072 Cdk2+/+ versus Cdk2-/-, Logrank Test).

Figure 3. Cdk2 absence does not prevent LMW cyclin E expression in the mammary gland

(A) Immunohistochemistry for human cyclin E protein in mammary glands of MMTV-LMW cyclin E-T1; p53+/-; Cdk2+/+ and MMTV-LMW cyclin E-T1; p53+/-; Cdk2-/-. (B) Cyclin E-associated kinase assays of mammary glands protein lysates from age-matched wild-type (Non-Tg), MMTV-LMW cyclin E; p53+/- mice in Cdk2+/+, Cdk2+/- or Cdk2-/- background was carried out with a cyclin E antibody (HE12) using histone H1 as substrate. The histogram show the relative activities after densitometric scanning (arbitrary units).

Figure 4. Roscovitine and meriolin treatment delay LMW cyclin E-induced breast cancer

A: Tumor-free survival after roscovitine treatment. Randomly selected MMTV-LMW cyclin E-T1; p53+/- mice were treated either with DMSO (n=7) or with roscovitine (75mg/kg, twice daily, n=7) for 1 week. B. Randomly selected MMTV-LMW cyclin E-T1; p53+/- mice were treated either with DMSO (n=7) or with meriolin 5 (1mg/kg, for two series of 5 days with a 2-day break in between, n=7).

Figure 5. Cdk2 inhibition causes apoptosis and inhibition of cell growth in LMW cyclin E overexpressing breast cancer cell lines

A. Two breast cancer cell lines expressing LMW cyclin E, HCC1806 and HCC1569, and 2 breast cancer cell lines with no LMW cyclin E expression, ZR75-1 and UACC812 were harvested at 48 hours and immunoblotted for cyclin E. B. 100,000 cells for each cell lines were plated at day 0, transfected with scrambled siRNA (Neg) or siRNA against Cdk2 (#1 and #2). Samples were harvested at 48 hrs and counted using a coulter counter. C. FACS analysis of the same cells as in B. (left panel) and western blot analysis for Cdk2 and actin (rigth panel). The levels of proteins were measured by densitometric scanning of the corresponding bands and normalized using actin values. The values indicated at the bottom (in %) were compared with the values obtained with scrambled siRNA transfected cells set at 100%. D. pTRE-LMW Cyclin E (T2) stably transfected MCF-7 cells were incubated with or without doxycycline (Dox, 1 ⌠g/mL) for 24 h then transfected with 21-bp double strand siRNA against Cdk2 or with a negative control siRNA. Cells were collected 48 hours later and used for counting or western blot analysis with cyclin E (T2), Cdk2 and actin antibodies. E. Effect of the expression of LMW Cyclin E (T2) induced by doxycycline in MCF-7 cells on cyclin-dependent kinase inhibitors response. pTRE-LMW Cyclin E–transfected MCF-7 cells (2,500 cells per well) were incubated in 96-well plates for 24 hrs, incubated with or without doxycycline (Dox, 1 ⌠g/mL) for 24 h, and cells were then treated with either diluent (DMSO) or CVT-313, or meriolin 5, or roscovitine at 8 different concentrations for 48 hrs and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetraolium bromide (MTT) metabolization was measured. Absorbance was measured at 590 nm. The mean absorbance values of diluent-treated samples were taken as 100%. Absorbance values of the cells treated with different drugs are plotted as percentages with respect to the mean value of the diluent-treated samples. A least-squares fit was obtained to estimate the IC50. Means and 95% confidence intervals of an experiment representative of three independent experiments performed in quadruplicate are shown.