Conditional knockdown of integrin beta-3 reveals its involvement in osteolytic and soft tissue lesions of breast cancer skeletal metastasis

Abstract

Integrin β3 (ITGB3) is probably related to skeletal metastasis, which is the most frequent complication in breast cancer progression. We aimed to define its role and suitability as target for anti-metastatic therapy. We generated two MDA-MB-231 cell clones with conditional miRNA-mediated ITGB3 knockdown for analyzing the resulting effects in vitro regarding mRNA expression, proliferation and migration, as well the impact on skeletal metastasis in a nude rat model. Furthermore, ITGB3 levels were analyzed in exosomes from plasma of rats with skeletal metastases, and from MDA-MB-231 cells incubated with these vesicles, as well as from exosomes secreted by cells with conditional ITGB3 knockdown. This inhibition of ITGB3 expression decreased cellular proliferation and more distinctly inhibited cellular migration. Reduction and even complete remissions of respective soft tissue and osteolytic lesions were detected after ITGB3 knockdown in vivo. Furthermore, ITGB3 levels were increased in exosomes isolated from plasma of rats harboring MDA-MB-231 lesions as well as in respective cells incubated with these vesicles in vitro. ITGB3 was distinctly decreased in exosomes from cells with ITGB3 knockdown. The observed in vitro and in vivo anti-ITGB3 effects can be explained by downregulation of specific genes, which have roles in angiogenesis (NPTN, RRM2), tumor growth (NPTN), energy metabolism (ISCA1), cytokinesis (SEPT11), migration (RRM2, STX6), cell proliferation, invasiveness, senescence, tumorigenesis (RRM2) and vesicle trafficking (SEPT11, STX6). ITGB3 has a role in breast cancer skeletal metastasis via gene expression modulation, as mirrored for ITGB3 in exosomes, thus it could serve as target for anti-metastatic therapy.

Keywords: Breast cancer; Conditional ITGB3 knockdown; Doxycycline; Integrins; Skeletal metastasis.

Fig. 1

Gene cassettes integrated into the genome of MDA-MB-231 breast cancer cells. Scheme of two cassettes, which were transduced into MDA-MB-231 breast cancer cells. The upper cassette (transduced initially) contains: hPGK (human phosphoglycerate kinase promoter); tTA (tetracycline-controlled transactivator); IRES (internal ribosome entry site); eGFP (enhanced green fluorescent protein). The lower cassettes contains luciferase (firefly luciferase); P_tet-bi (bidirectional tet-regulated promoter); Cherry (red fluorescent protein mCherry); FRT and F3 (wild-type and mutant Flp—recombinase target sites); miRNA—artificial microRNA targeting specifically ITGB3 mRNA. The left bottom part illustrates the conditions in absence of doxycycline: tTA binds and activates the bidirectional promoter P_tet-bi, which leads to expression of firefly luciferase, red fluorescent protein mCherry and of the miRNA targeting ITGB3 mRNA. The right bottom part shows the consequences of doxycycline presence: tTA cannot bind to its respective promoter, it remains inactive and the expression of the subsequent genes is switched off

Fig. 2

Regulation of the red fluorescent protein mCherry by doxycycline and ITGB3 mRNA and protein levels following conditional knockdown. a Flow cytometry analysis of I3 and I5 cell clones after cultivating them for 6 days in media with or without doxycycline. mCherry expression was stimulated after removal of the doxycycline from the media of the cells. b qPCR analysis of the mRNA levels of ITGB3 in cell clones I3 and I5, cultivated for 6 days in media with or without doxycycline. Integrin β3 mRNA was significantly inhibited upon stimulated expression of specific miRNA. c Western blot analysis of ITGB3 in I3 and I5 cells after cultivating them in media with or without doxycycline for 6 days. d Quantitation of the western blot analysis

Fig. 3

Cellular functions of breast cancer cell clones upon 6 days of conditional miRNA mediated ITGB3 knockdown a Cellular proliferation determined by MTT assay; b cellular migration; these assays were run with two controls: the parent cell clone and an additional control clone (Ic), which contains the gene cassette without miRNA sequence. The conditional ITGB3 knockdown provoked slight, but significant inhibition of proliferation in I5 cells only, whereas migration was decreased in both cell clones I3 and I5, yet more pronounced in I5 cells. ***p < 0.001 versus controls

Fig. 4

Breast cancer skeletal metastasis inhibition after ITGB3 knockdown a The line graph (x-axis: time (in days) after tumor cell injection (tumor cell inj.); y-axis: bioluminescence in photons/second/cm²/steradian) shows quantitation of bioluminescence imaging (BLI) signals from control rats (red color) and rats treated with a miRNA against integrin β3 (green color). Below the graph, a time line is given, indicating the (relative) time of tumor cell injection, and of magnetic resonance imaging (MRI; indicating the volume of soft tissue lesions) and volume computed tomography (VCT; indicating the volume of osteolytic lesions) measurements, as indicated by arrows. Below this time line, the mean values and corresponding ranges are given for the treated rats, only. The breast cancer skeletal metastases of experimental rats were monitored initially after discontinuation of the doxycycline intake (the time point is indicated with a dashed line). The cells of the specific cell clone I5 were injected into the saphenous artery of the first four rats (rat 1 to rat 4). The other three rats (rat 5 to rat 7) received cells from the parent cell clone and these rats were used as controls. The difference in light emission between miRNA treated and control rats was highly significant (p < 0.001) as indicated by red asterisks. b BLI images (left panel) and VCT images (middle and right panels) show the status of rat 1 at 20 and 28 days (upper panel) and at 30 and 49 days after tumor cell inoculation (lower panel), respectively. Clearly, the luminescence signal is no longer visible at the later time point, and the bones show re-calcification

Fig. 5

Protein levels of ITGB3 in exosomes. a Levels of ITGB3 in exosomes isolated from plasma of healthy rats (exo 1) and from plasma of rats with breast cancer skeletal metastasis (exo 2 and exo 3). The latter exosomes show higher levels of this protein. b ITGB3 in MDA-MB-231 breast cancer cells incubated with exo 1 or exo 2 for 72 h. The levels of this protein were increased after incubating the cells with exosomes isolated from plasma of rats with breast cancer lesions (exo 2). c ITGB3 levels in exosomes isolated in vitro from: (1) parent cell clone, cultivated in medium with doxycycline (+ dox control), (2) I5 cells cultivated in medium with dox (+ dox, specific control), (3) parent cell clone, cultivated in medium without dox (-dox control), (4) I5 cells cultivated in medium without dox (-dox, anti-ITGB3 effects). ITGB3 decreased twofold in exosomes derived from cells with conditional integrin β3 knockdown

Fig. 6

Gene modulation upon integrin beta-3 knockdown (ITGB3 KDN) Downregulation of selected genes after 3 and 6 days of ITGB3 miRNA-mediated inhibition. The gene expression of the cell clone with conditional ITGB3 knockdown was compared to that of the same cell clone cultivated in media with doxycycline (the corresponding control). The dashed line indicates a fold change without gene expression modulation, the asterisks indicate a significantly altered fold change (p < 0.001). The genes include NPTN (neuroplastin), ISCA1 (iron-sulfur cluster assembly 1), SEPT11 (septin 11), STX6 (Syntaxin 6), and RRM2 (ribonucleotide reductase M2 polypeptide). The right side of the figure shows the respective gene functions