Enpp1: a potential facilitator of breast cancer bone metastasis

Abstract

Bone is the most common site of breast cancer metastasis and once established, it is frequently incurable. Critical to our ability to prevent and treat bone metastasis is the identification of the key factors mediating its establishment and understanding their biological function. To address this issue we previously carried out an in vivo selection process to isolate murine mammary tumor sublines possessing an enhanced ability to colonize the bone. A comparison of gene expression between parental cells and sublines by genome-wide cDNA microarray analysis revealed several potential mediators of bone metastasis, including the pyrophosphate-generating ectoenzyme Enpp1. By qRT-PCR and Western analysis we found that expression of Enpp1 was elevated in human breast cancer cell lines known to produce bone metastasis in animal models compared to non-metastatic and normal mammary epithelial cell lines. Further, in clinical specimens, levels of Enpp1 were significantly elevated in human primary breast tumors relative to normal mammary epithelium, with highest levels observed in breast-bone metastasis as determined by qRT-PCR and immunohistochemical analysis. To examine the potential role of Enpp1 in the development of bone metastasis, Enpp1 expression was stably increased in the breast cancer cell line MDA-MB-231 and the ability to colonize the bone following intracardiac and direct intratibial injection of athymic nude mice was determined. By both routes of administration, increased expression of Enpp1 enhanced the ability of MDA-MB-231 cells to form tumors in the bone relative to cells expressing vector alone, as determined by digital radiography and histological analysis. Taken together, these data suggest a potential role for Enpp1 in the development of breast cancer bone metastasis.

Figure 1. Expression of Enpp1 in breast cancer cell lines.

Enpp1 (A, B) mRNA and (C) protein expression was determined in (A) NT2.5 murine breast cancer cells and bone sublines, and (B, C) immortalized normal human mammary epithelial cell lines and human breast cancer cell lines by (A, B) qRT-PCR and (C) Western analysis. (A, B) Data are representative of three independent experiments performed in triplicate and expressed as the mean ± s.e.m (**p<0.01, ***p<0.001). (C) Protein expression was determined by Western analysis performed on equal amounts of protein from total cell lysates.

Figure 2. Expression of Enpp1 in human primary breast cancer and bone metastasis tissues.

Enpp1 (A) mRNA and (B–D) protein expression in human normal mammary epithelium (N), primary breast tumor (T), and breast cancer bone metastasis (BBM) was determined by (A) qRT-PCR and (B–D) IHC analysis. (A) Data are representative of three independent experiments performed in triplicate (*p<0.05 for T versus N). (B–D) Proteins were identified using DAB (brown). Sections were counterstained with hematoxylin and visualized by light microscopy (200X). N – Normal, T – Tumor.

Figure 3. Generation of breast cancer cells with increased Enpp1 expression and enzymatic activity.

MDA-MB-231 cells were stably transduced with Enpp1 or empty vector (EV). Enpp1 (A) protein expression was determined by Western analysis performed on equal amounts of protein from total cell lysates. (B) Nucleotide phosphodiesterase activity was determined using the nucleotide derivative p-nitrophenyl thymidine 5′-monophosphate (pNP-TMP) as a substrate. Data are representative of three independent experiments performed in triplicate and expressed as the mean ± s.e.m (**p<0.01).

Figure 4. Effect of Enpp1 on the development of bone metastasis.

MDA-MB-231 cells stably expressing Enpp1 or empty vector (EV) were injected into the (A, B) tibia (n = 9/group) and (C, D) left cardiac ventricle (n = 5/group) of athymic nude mice and digital radiographic imaging was performed at weekly intervals. (A, C) Osteolytic area and (B, D) tumor area were measured on radiographic images and histological sections, respectively (*p<0.05, **p<0.01). Representative images are shown at 4 weeks following tumor cell administration. Black outlined areas on histological images indicate areas of tumor.