Cyclin E amplification/overexpression is a mechanism of trastuzumab resistance in HER2+ breast cancer patients

Abstract

Clinical benefits from trastuzumab and other anti-HER2 therapies in patients with HER2 amplified breast cancer remain limited by primary or acquired resistance. To identify potential mechanisms of resistance, we established trastuzumab-resistant HER2 amplified breast cancer cells by chronic exposure to trastuzumab treatment. Genomewide copy-number variation analyses of the resistant cells compared with parental cells revealed a focal amplification of genomic DNA containing the cyclin E gene. In a cohort of 34 HER2(+) patients treated with trastuzumab-based therapy, we found that cyclin E amplification/overexpression was associated with a worse clinical benefit (33.3% compared with 87.5%, P < 0.02) and a lower progression-free survival (6 mo vs. 14 mo, P < 0.002) compared with nonoverexpressing cyclin E tumors. To dissect the potential role of cyclin E in trastuzumab resistance, we studied the effects of cyclin E overexpression and cyclin E suppression. Cyclin E overexpression resulted in resistance to trastuzumab both in vitro and in vivo. Inhibition of cyclin E activity in cyclin E-amplified trastuzumab resistant clones, either by knockdown of cyclin E expression or treatment with cyclin-dependent kinase 2 (CDK2) inhibitors, led to a dramatic decrease in proliferation and enhanced apoptosis. In vivo, CDK2 inhibition significantly reduced tumor growth of trastuzumab-resistant xenografts. Our findings point to a causative role for cyclin E overexpression and the consequent increase in CDK2 activity in trastuzumab resistance and suggest that treatment with CDK2 inhibitors may be a valid strategy in patients with breast tumors with HER2 and cyclin E coamplification/overexpression.

Fig. 1.

Generation and characterization of trastuzumab-resistant cell lines. (A) Trastuzumab-sensitive breast cancer cell line BT474 was made resistant by persistent exposure to increasing concentrations of trastuzumab (Results). BT474 and BT474R cells were treated with trastuzumab at the indicated concentrations for 6 d and proliferation was measured. BT474R cells were subsequently injected s.c. in nude mice and animals were treated twice weekly with 10 mg/kg trastuzumab. Resistant tumors were excised and new trastuzumab-resistant cell lines (BT474R2) established. (B) Genomewide copy-number analyses of BT474R cells compared with parental controls. Gain of region 19q12 (Left) and loss of region 14q31 (Right). The amplification on 19q12 locus encodes the gene for cyclin E1 (CCNE1). Genetic analysis of BT474R2 cells gave identical results. (C) Western blot analyses of BT474 and BT474R cells. Cells were exposed to 100 nM trastuzumab for 48 h. Whole-cell extracts were analyzed with the indicated antibodies.

Fig. 2.

Cyclin E amplification/overexpression in HER2⁺ breast tumors and clinical trastuzumab resistance. (A) FISH analyses showing a representative case of a patient with no amplification of CCNE1 (patient 1) and with amplification of CCNE1 (patient 2). (B) Representative immunohistochemistry showing a tumor with negative nuclear staining for cyclin E (patient 1) and a tumor with positive nuclear staining for cyclin E (H score > 30, patient 2). (C) Clinical benefit rate of cyclin E positive (green, n = 18) vs. cyclin E negative (blue, n = 16) patients treated with trastuzumab-based therapy. Breslow test: P = 0.02. (D) PFS of cyclin E positive (green, n = 18) vs. cyclin E negative (blue, n = 16) patients. Median PFS was 4 mo for cyclin E positive patients and 14 mo for cyclin E negative patients. Breslow test: P = 0.002.

Fig. 3.

BT474 and BT474R sensitivity to in vitro cyclin E manipulation. (A) Quantification of crystal violet staining of parental BT474 cells infected with empty vector control (BT474) compared with two independent pools stably expressing full-length cyclin E (BT474-A and BT474-B). Cells were treated for 8 d with 100 nM trastuzumab. Experiments were performed in triplicate. (Student's t test; *P = 0.001, **P = 0.001). (B) Western blot analyses showing cyclin E overexpression in BT474-A and BT474B. (C) Quantification of crystal violet staining of BT474 or BT474R cells upon siRNA-mediated knockdown of cyclin E (siCCNE1) or scrambled siRNA control (SCB). Cells were treated with 10 nM trastuzumab for 8 d. Experiments were performed in triplicate. (Student's t test; *P = 0.013, **P = 0.0003, ***P = 0.15) (D) Western blot analyses of BT474 and BT474R cells transfected with siRNA-targeting cyclin E or control. Whole-cell extracts were probed with indicated antibodies.

Fig. 4.

BT474 and BT474R sensitivity to CDK2 inhibition. (A) Quantification of crystal violet staining of BT474 or BT474R cells treated for 8 d with trastuzumab, CYC065, or the combination (T+C) at the indicated concentrations. Proliferation assays were performed in triplicate. (B) Western blot analysis of BT474 parental and resistant cell lines exposed to trastuzumab, CYC065, or the combination for 48 h at the indicated concentrations. Whole-cell extracts were analyzed with the indicated antibodies. Similar results were obtained comparing BT474 with BT474R2. (C) Cell death of BT474 or BT474R cells as delineated by sub-G1 population following treatment of trastuzumab, CYC065, or the combination for 48 h at the indicated concentrations. Similar results were obtained comparing BT474 with BT474R2. (D) Cell death of BT474 or BT474R cells as delineated by annexin V staining following treatment of trastuzumab, CYC065, or the combination for 48 h at the indicated concentrations. Similar results were obtained comparing BT474 with BT474R2. (E) Tumor growth inhibition in response to trastuzumab, CYC065, and the combination of the two agents. Student's t test was used to compare tumor sizes between the groups and data are expressed as mean ± SE *P = 0.0019 vs. trastuzumab; **P = 0.00085 vs. trastuzumab on day 12. The experiment was repeated two times with similar results.

Fig. 5.

(A) In unperturbed conditions HER2-dependent activation of AKT inhibits the cyclin E/CDK2 inhibitor p27. (B) Inhibition of HER2 suppresses AKT activity leading to increased levels of p27 and inhibition of the cyclin E/Cdk2 activity. (C) Trastuzumab-resistant cells harboring an amplification of cyclin E renders these cells insensitive to the negative regulation by p27.