Increased expression of preprotachykinin-I and neurokinin receptors in human breast cancer cells: implications for bone marrow metastasis

Abstract

Neuropeptides are implicated in many tumors, breast cancer (BC) included. Preprotachykinin-I (PPT-I) encodes multiple neuropeptides with pleiotropic functions such as neurotransmission, immune/hematopoietic modulation, angiogenesis, and mitogenesis. PPT-I is constitutively expressed in some tumors. In this study, we investigated a role for PPT-I and its receptors, neurokinin-1 (NK-1) and NK-2, in BC by using quantitative reverse transcription-PCR, ELISA, and in situ hybridization. Compared with normal mammary epithelial cells (n = 2) and benign breast biopsies (n = 21), BC cell lines (n = 7) and malignant breast biopsies (n = 25) showed increased expression of PPT-I and NK-1. NK-2 levels were high in normal and malignant cells. Specific NK-1 and NK-2 antagonists inhibited BC cell proliferation, suggesting autocrine and/or intercrine stimulation of BC cells by PPT-I peptides. NK-2 showed no effect on the proliferation of normal cells but mediated the proliferation of BC cells. Cytosolic extracts from malignant BC cells enhanced PPT-I translation whereas extracts from normal mammary epithelial cells caused no change. These enhancing effects may be protein-specific because a similar increase was observed for IL-6 translation and no effect was observed for IL-1alpha and stem cell factor. The data suggest that PPT-I peptides and their receptors may be important in BC development. Considering that PPT-I peptides are hematopoietic modulators, these results could be extended to understand early integration of BC cells in the bone marrow, a preferred site of metastasis. Molecular signaling transduced by PPT-I peptides and the mechanism that enhances translation of PPT-I mRNA could lead to innovative strategies for BC treatments and metastasis.

Figure 1

In situ hybridization for PPT-I, NK-1, and NK-2 mRNA in breast biopsies. PPT-I, NK-1, and NK-2 mRNA was studied by in situ hybridization with tissues from paraffin-embedded breast biopsies. A representative stain from different benign (n = 21) and malignant (n = 25) tissues is shown at ×1,000 magnification. Tissues were hybridized with oligonucleotides specific for NK-1 (A), NK-2 (B and D), or β-PPT-I (C). E represents benign sections hybridized for NK-1 or β-PPT-I, and F shows the staining pattern with sense oligonucleotides from benign or malignant tissues.

Figure 2

SP-IR in in vitro translation reactions. Poly(A) RNA from IL-1α-stimulated BM stroma was used as substrate in in vitro translation. Reactions contained cytosolic extract from malignant or normal mammary epithelial cells. Lanes: 1, no RNA; 2, Luciferase RNA; 3, poly(A) RNA with no extract; 4, T-47D extract; 5, BT-474 extract; 6, ZR-75–30 extract; 7, DU4475 extract; 8, MCF-12A extract. SP was immunoprecipitated and then characterized in Western blot.