Statin-induced anti-proliferative effects via cyclin D1 and p27 in a window-of-opportunity breast cancer trial

Abstract

Purpose: Cholesterol lowering statins have been demonstrated to exert anti-tumoral effects on breast cancer by decreasing proliferation as measured by Ki67. The biological mechanisms behind the anti-proliferative effects remain elusive. The aim of this study was to investigate potential statin-induced effects on the central cell cycle regulators cyclin D1 and p27.

Experimental design: This phase II window-of-opportunity trial (Trial registration: ClinicalTrials.gov NCT00816244 , NIH) included 50 patients with primary invasive breast cancer. High-dose atorvastatin (80 mg/day) was prescribed to patients for two weeks prior to surgery. Paired paraffin embedded pre- and post-statin treatment tumor samples were analyzed using immunohistochemistry for the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and the cell cycle regulators cyclin D1 and p27. Corresponding frozen tumor sample pairs were analyzed for expression of the genes coding for cyclin D1 and p27, CCND1 and CDKN1B, respectively.

Results: Forty-two patients completed all study parts, and immunohistochemical evaluation of ER and PR was achievable in 30 tumor pairs, HER2 in 29 tumor pairs, cyclin D1 in 30 tumor pairs and p27 in 33 tumor pairs. The expression of ER, PR and HER2 did not change significantly following atorvastatin treatment. Cyclin D1 expression in terms of nuclear intensity was significantly decreased (P = 0.008) after statin treatment in paired tumor samples. The protein expression of the tumor suppressor p27, evaluated either as the fraction of stained tumor cells or as cytoplasmic intensity, increased significantly (P = 0.03 and P = 0.02, respectively). At the transcriptional level, no significant differences in mRNA expression were detected for cyclin D1 (CCND1) and p27 (CDKN1B). However, CCND1 expression was lower in tumors responding to atorvastatin treatment with a decrease in proliferation although not significantly (P = 0.08).

Conclusions: We have previously reported statin-induced anti-proliferative effects in breast cancer. This study suggests that cell cycle regulatory effects may contribute to these anti-proliferative effects via cyclin D1 and p27.

Figures

Figure 1

The cell cycle, and the main actions of cyclin D1 and p27. Cyclin D1 regulates the G1/S-phase transition, binds and activates Cdk4/Cdk6 to phosphorylate the retinoblastoma (pRb) protein. Phosphorylation of Rb leads to separation from E2F, and allows the transcription of proliferation genes [21]. In G0 and early G1, p27 inhibits CDK2-cyclin E, and in S-phase CDK2-cyclin A. In G1 there is a decrease in p27, allowing CDK2-cyclin E and CDK2-cyclin A to activate the transcription of genes acquired for the G1-S-transition [25]. P27 also interacts with CDK4/6-cyclin D comprehensively, p27 acting as both an inhibitor and as a required assembly factor for the complex, depending on the growth state of the cell [24].

Figure 2

Change in tumor expression of cyclin D1 from baseline (i.e., before atorvastatin treatment) to time of surgery (i.e., after atorvastatin treatment). A) Fraction of stained nuclei; B) Nuclear intensity; C) Cytoplasmic intensity. To reduce the problem of completely overlapping lines in the spaghetti plot, for each pair of pre/post-treatment samples, a random number from a uniform distribution over the interval [−0.15, 0.15] was added, shifting the corresponding line at most 15%, upwards or downwards, of a step on the integer-valued score scale.

Figure 3

Change in tumor expression of p27 from baseline (i.e., before atorvastatin treatment) to time of surgery (i.e., after atorvastatin treatment). A) Fraction of stained nuclei; B) Nuclear intensity; C) Cytoplasmic intensity. To reduce the problem of completely overlapping lines in the spaghetti plot, for each pair of pre/post-treatment samples, a random number from a uniform distribution over the interval [−0.15, 0.15] was added, shifting the corresponding line at most 15%, upwards or downwards, of a step on the integer-valued score scale.

Figure 4

Correlation between change in Ki67 and change in cyclin D1 (cytoplasmic intensity) after treatment with atorvastatin. Marker color and filling represents immunohistochemical scoring of cyclin D1 cytoplasmic intensity in the pre-treatment samples; filled red circles (strong cyclin D1 intensity), red empty circles (moderate cyclin D1 internsity), green empty circles (week cyclin D1 intensity), green filled circles (no cyclin D1 expression).

Figure 5

Expression of CCND1 and CDKN1B pre- and post-atorvastatin treatment, divided into tumors responding with a decrease or increase in proliferation (Ki67) following statin treatment. A) Pre-treatment CCND1 expression, B) Post-treatment CCND1 expression, C) Pre-treatment CDKN1B expression, D) Post-treatment CDKN1B expression.