Bone sialoprotein-αvβ3 integrin axis promotes breast cancer metastasis to the bone

Abstract

The underlying mechanisms of breast cancer cells metastasizing to distant sites are complex and multifactorial. Bone sialoprotein (BSP) and αvβ3 integrin were reported to promote the metastatic progress of breast cancer cells, particularly metastasis to bone. Most theories presume that BSP promotes breast cancer metastasis by binding to αvβ3 integrin. Interestingly, we found the αvβ3 integrin decreased in BSP silenced cells (BSPi), which have weak ability to form bone metastases. However, the relevance of their expression in primary tumor and the way they participate in metastasis are not clear. In this study, we evaluated the relationship between BSP, αvβ3 integrin levels, and the bone metastatic ability of breast cancer cells in patient tissues, and the data indicated that the αvβ3 integrin level is closely correlated to BSP level and metastatic potential. Overexpression of αvβ3 integrin in cancer cells could reverse the effect of BSPi in vitro and promote bone metastasis in a mouse model, whereas knockdown of αvβ3 integrin have effects just like BSPi. Moreover, The Cancer Genome Atlas data and RT-PCR analysis have also shown that SPP1, KCNK2, and PTK2B might be involved in this process. Thus, we propose that αvβ3 integrin is one of the downstream factors regulated by BSP in the breast cancer-bone metastatic cascade.

Keywords: bone sialoprotein; breast cancer; gene expression; metastasis; αvβ3 integrin.

Figure 1

Expression of bone sialoprotein (BSP) and αvβ₃ integrin in primary tumors of 3 breast cancer patients. A, H&E and immunohistochemical staining of serial sections of formalin‐fixed paraffin‐embedded in situ breast cancer tissue. Patient 3 (P3), without breast cancer metastasis, showed both low expression of BSP and αvβ₃ integrin, whereas P1 and P2 with breast cancer metastasis highly expressed both BSP and αvβ₃ integrin. Arrows indicate in situ breast cancer B, Correlation analysis of metastatic sites and BSP scores, αv integrin scores, and β3 integrin scores

Figure 2

Establishment of αv and β3 integrin‐overexpressing cells. A, Schematic of ITGAV and ITGB3 overexpression vectors. B, Micrograph of cells after transfection. C, RT‐PCR assessment of ITGAV and ITGB3 gene overexpression after transfection. D,E, Western blot assessment of ITGAV and ITGB3 gene overexpression after transfection. GAPDH is the negative control. Quantification was carried out using ImageJ software. Results are expressed as mean ± SD, n = 3. * P < .05, ** P < .01. αv, 231BO‐BSPi‐αv; αvβ3, 231BO‐BSPi‐αvβ3; αvE, 231BO‐BSPi‐αv‐EGFP; β3, 231BO‐BSPi‐β3; β3M, 231BO‐BSPi‐β3‐mCHER; BSPi, 231BO cells with IBSP gene silenced; E, 231BO‐BSPi‐EGFP; EM, 231BO‐BSPi‐EGFP‐mCHER; M, 231BO‐BSPi‐mCHER

Figure 3

Effect of αv and β3 integrin overexpression on proliferation, migration, and invasion of human breast cancer cells in vitro. A, Cell growth curves detected by CCK‐8 assay. B,C, Wound healing assay to detect migration ability. D, Cell invasion ability determined by Transwell assay. E,F, Cell apoptosis detected by flow cytometry. Results are expressed as mean ± SD, n = 3. * P < .05, ** P < .01. αv, 231BO‐BSPi‐αv; αvβ3, 231BO‐BSPi‐αvβ3; β3, 231BO‐BSPi‐β3; BSPI, 231BO cells with IBSP gene silenced; E, 231BO‐BSPi‐EGFP; EM, 231BO‐BSPi‐EGFP‐mCHER; M, 231BO‐BSPi‐mCHER

Figure 4

Left panels, micro‐computed tomography photographs of legs of mice inoculated with different breast cancer cells. Right panels, H&E staining of tissue slices of brain, lung, and legs. Arrows indicate metastases. αv, 231BO‐BSPi‐αv; αvβ3, 231BO‐BSPi‐αvβ3; β3, 231BO‐BSPi‐β3; BSPI, 231BO cells with IBSP gene silenced; E, 231BO‐BSPi‐EGFP; EM, 231BO‐BSPi‐EGFP‐mCHER; M, 231BO‐BSPi‐mCHER

Figure 5

αv‐siRNA and β3‐siRNA could decrease the migration and invasion ability, and promote apoptosis, of 231BO breast cancer cells. A, RT‐PCR assessment of ITGAV and ITGB3 gene knockdown by siRNA in 231BO cells. B,D, Western blot assessment of ITGAV and ITGB3 gene knockdown by siRNA in 231BO cells. C,E, Wound healing assay to detect migration ability. F, Cell invasion ability determined by Transwell assay. G,H, Cell apoptosis detected by flow cytometry. Results are expressed as mean ± SD, n = 3. * P < .05, ** P < .01

Figure 6

Results of RNA sequencing. A, Expression of ITGB3,PTK2B,SPP1, and KCNK2 mRNA in breast cancer cells by RNA sequencing. B, Expression of PTK2B,SPP1, and KCNK2 mRNA in breast cancer cells by RT‐PCR. C,D, Protein levels of KCNK2, PTK2B, and osteopontin (OPN; encoded by SPP1 gene) in breast cancer cells by western blot analysis. E, Cluster analysis chart of ITGB3, PTK2B, SPP1, KCNK2 and their relative genes. 231, breast cancer cells MDA‐MB‐231; 231BO, bone‐seeking breast cancer cells MDA‐MB‐231BO; β3, 231BO cells transfected by IBSP‐siRNA and β3 overexpressing vector; E, Control cells of β3 cells, established from 231BO cells after IBSP‐siRNA and EX‐EGFP‐Lv151 vector transfection. Results are expressed as mean ± SD, n = 3. * P < .05, ** P < .01

Figure 7

Correlation analysis of gene expression in The Cancer Genome Atlas dataset. A, Correlation analysis of IBSP,ITGAV,ITGB3,SPP1,KCNK2, and PTK2B gene expression. *P < .05, **P < .01, ***P < .001. B, Relationship between IBSP,ITGAV, and ITGB3 expression and bone metastasis

Figure 8

Schematic illustration how bone sialoprotein (BSP) and αvβ3 integrin might promote breast cancer bone metastasis. In breast cancer cells, BSP expression could influence the level of αv and β3 integrin, which in turn promote the expression of each other. Then BSP is released into the extracellular environment, and the αv and β3 integrin are transferred to the membrane, where BSP binds to αvβ3 through its RGD motif. Simultaneously, they could regulate the expression of osteopontin (OPN), KCNK2 and PTK2B. OPN can also binds to αvβ3. All these factors together regulate breast cancer bone metastasis