Inhibition of the transition of ductal carcinoma in situ to invasive ductal carcinoma by a Gemini vitamin D analog

Abstract

Ductal carcinoma in situ (DCIS) is a nonmalignant lesion of the breast with the potential to progress to invasive ductal carcinoma (IDC). The disappearance and breakdown of the myoepithelial cell layer and basement membrane in DCIS have been identified as major events in the development of breast cancer. The MCF10DCIS.com cell line is a well-established model, which recapitulates the progression of breast cancer from DCIS to IDC. We have previously reported that a novel Gemini vitamin D analog, 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124) is a potent inhibitor of the growth of MCF10DCIS.com xenografted tumors without hypercalcemic toxicity. In this study, we utilized the MCF10DCIS.com in vivo model to assess the effects of BXL0124 on breast cancer progression from weeks 1 to 4. Upon DCIS progression to IDC from weeks 3 to 4, tumors lost the myoepithelial cell layer and basement membrane as shown by immunofluorescence staining with smooth muscle actin and laminin 5, respectively. Administration of BXL0124 maintained the critical myoepithelial cell layer as well as basement membrane, and animals treated with BXL0124 showed a 43% reduction in tumor volume by week 4. BXL0124 treatment decreased cell proliferation and maintained vitamin D receptor levels in tumors. In addition, the BXL0124 treatment reduced the mRNA levels of matrix metalloproteinases starting at week 3, contributing to the inhibition of invasive transition. Our results suggest that the maintenance of DCIS plays a significant role in the cancer preventive action of the Gemini vitamin D BXL0124 during the progression of breast lesions.

Conflict of interest statement

Conflicts of Interest: Authors have no potential conflicts of interest to disclose.

©2014 American Association for Cancer Research.

Figures

Figure 1

BXL0124 inhibits tumor growth and inhibits progression to IDC in MCF10DCIS.com subcutaneous xenografts in nu/nu mice. A. Average tumor volume at weekly time points is shown, * p < 0.05 (n=5 per group). Tumor volume (V; cubed centimeters) was calculated using the equation V = D*d2/2 where D (centimeters) and d (centimeters) are the largest and smallest perpendicular diameters. B. Average final bodyweight at autopsy is shown (n=5 per group) C. Serum calcium determination to assess hypercalcemic toxicity is shown (n=5 per group) D. A representative hematoxylin and eosin (H&E) staining showing the progression of MCF10DCIS.com subcutaneous xenografts in nu/nu mice from weeks 1, 2, 3, and 4 (40x). DCIS quantification, * p < 0.05 (n=4 per group).

Figure 2

BXL0124 treatment decreases the proliferation of MCF10DCIS.com tumors at week 4. A. A representative immunohistochemical analysis of PCNA in tumor samples from weeks 1, 2, 3, and 4 is shown (100x). PCNA-positive staining is found in the nucleus of the cells. B. Four tumors from each group were blinded and quantified, three representative areas from each tumor were quantified for the intensity of PCNA staining, mean +/- S.E.M. The staining intensities were scored from 0+ (negative staining) to 3+ (the strongest staining), * p < 0.05.

Figure 3

BXL0124 treatment maintains vitamin D receptor levels in MCF10DCIS.com tumors. A. A representative immunohistochemical analysis for VDR from weeks 1, 2, 3, and 4 is shown (40x). VDR-positive staining is found in the cytoplasm and nucleus of the cells. Whole tumor mounts of VDR expression from week 4 is shown as a contracted view. B. Four tumors from each group were blinded and quantified and three representative areas from each tumor were quantified for the intensity of VDR staining, mean +/- S.E.M. The staining intensities were scored from 0+ (negative staining) to 3+ (the strongest staining), * p < 0.05. C. The protein level of VDR was increased in the tumors of week 4 BXL0124 treated mice as shown by western blot analysis. Five xenograft tumors from each group were combined for pooled samples. β-actin was used as a loading control.

Figure 4

Treatment with BXL0124 inhibits progression to IDC by maintaining the myoepithelial cell layer. A representative immunofluorescence staining for tumor samples from weeks 1, 2, 3, and 4 with the myoepithelial cell marker smooth muscle actin (SMA, shown in red) and the epithelial cell marker pancytokeratin (panCK, shown in green) is shown (200x). Nuclei were stained with TO-PRO-3 (blue). Expanded magnification is shown for specific areas from week 4 tumors. Scale bars represent 100 μm.

Figure 5

Treatment with BXL0124 maintains the basement membrane. A representative immunofluorescence staining with the myoepithelial cell marker SMA (shown in red) and the basal membrane marker laminin 5 (shown in green) on tumors from weeks 1, 2, 3, and 4 is shown (200x). Nuclei were stained with TO-PRO-3 (blue). Expanded magnification is shown for specific areas from week 4 tumors. Scale bars represent 100 μm.

Figure 6

BXL0124 inhibits the mRNA expression levels of the matrix metalloproteinases during DCIS to IDC progression. A. qPCR analysis of MMPs and VDR mRNA levels in MCF10DCIS.com tumor samples from weeks 3 and 4 is shown, mean +/- S.E.M., Cycle numbers are shown in parenthesis: MMP2 (25), MMP9 (28), MMP14 (23), MMP15 (28), MMP16 (25), and VDR (26). Statistical significance refers to the respective week control, * p < 0.05, ** p < 0.01, *** p < 0.001 (n=3-5 per group). B. A representative immunofluorescence staining with the myoepithelial cell marker, SMA (shown in red), and VDR (shown in green) on tumors from weeks 3 and 4 are shown (200x). Nuclei were stained with TO-PRO-3 (blue). Scale bars represent 100 μm.