Tumor- and Osteoblast-Derived Periostin in Prostate Cancer bone Metastases

Abstract

Exploring the biological function of periostin (POSTN) in prostate cancer (PCa) bone metastasis is of importance. It was observed that the expression of POSTN was high in PCa, especially highest in PCa metastasized to bone. In this study, we found that inhibiting POSTN in PCa cells could significantly alleviate PCa bone metastasis in vivo, suggesting POSTN is a promising therapeutic target. Since, due to the secreted expression of POSTN in osteoblasts and PCa, we hypothesized the positive feedback loop between osteoblasts and PCa mediated by POSTN in PCa bone metastasis. The in vitro experiments demonstrated that osteoblast-derived POSTN promoted PCa cell proliferation and invasion and PCa cell-derived POSTN promotes proliferation of osteoblasts. Furthermore, we found that POSTN regulated PCa and osteoblast function through integrin receptors. Finally, ¹⁸F-Alfatide II was used as the molecule probe of integrin αvβ3 in PET-CT, revealing high intake in metastatic lesions. Our findings together indicate that targeting POSTN in PCa cells as well as in the osteoblastic may be an effective treatment for PCa bone metastasis.

Keywords: bone metastasis; integrin receptor; osteoblast; periostin; prostate cancer.

Figure 1

The association between POSTN and metastasis in PCa was verified by the TGCA database. (A) The outcome of OPLS-DA. C, control; LR, local recurrence; BR, biochemical recurrence; DM, distant metastases. In this figure, the points inside the red line spheroid means a least significant difference test at a 95% confidence level. (B) Volcano plot showing differentially expressed proteins. Red color represents a high relative expression level; blue color represents a low relative expression level. (C) Relative mRNA expression of POSTN in the DM group vs. control group. ^** p < 0.01. (D) POSTN expression between normal and patients verified by the Oncomine database. (E) Survival curve analysis of disease-free survival (DFS). DFS time between higher-expression level and lower-expression level of POSTN in PCa. Red line displays the cases with highly expressed POSTN, and blue line reveals the cases with lowly expressed POSTN. HR, hazard ratio. p(HR) = 0.041.

Figure 2

POSTN knockdown suppresses the bone metastasis capability of PCa cells. (A) MDA PCa 2b cells or POSTN-knockdown MDA PCa 2b cells were injected in the intramedullary cavity of the tibia. Shown are representative whole-animal luciferase imaging of mice 5 weeks postsurgery (n = 8/group). (B) Bioluminescent curve of tumor development [related to (A)]. (C) MDA PCa 2b cells or POSTN-knockdown MDA PCa 2b cells were injected into the left ventricle in nude mice. Representative whole-animal luciferase imaging of mice 60 days postsurgery (n = 8/group). (D) Metastasis-free survival [related to (C)]. ^* p < 0.05.

Figure 3

The high-confidence networks of POSTN were constructed by multiomics. (A) The top 1 enrichment network of Metacore™. The ITGAV-B3 heterodimer presented integrin αVβ3. (B) The interaction network of POSTN by IPA. Dumbbell shape: transcription regulator; boomerang shape: growth factor; the arrow direction: activating.

Figure 4

Osteoblast-derived POSTN promoted proliferation of PCa cells. (A) PC-3 and MDA PCa 2b cells were incubated with various concentrations of osteoblast (hFOB)-conditioned medium (OBCM) for 48 h before proliferation was assessed. (B) PC-3 and MDA PCa 2b cells were incubated with 30% OBCM supplemented with various concentrations of POSTN monoclonal antibody for 48 h before proliferation was assessed. (C) hFOB cells were transfected with control shRNA lentivirus (Lv-NC) or POSTN shRNA lentivirus (POSTN shRNA). The expression of POSTN was detected by real-time PCR. (D) hFOB cells were transfected with control shRNA lentivirus (Lv-NC) or POSTN shRNA lentivirus (POSTN shRNA). The expression of POSTN was detected by Western blotting. (E) The levels of POSTN in hFOB cell medium were detected by ELISA. (F) PC-3 and MDA PCa 2b cells were incubated with 30% OBCM from control or POSTN-knockdown hFOB cells. Proliferation was assessed after 48 h. ^* p < 0.05 compared with control; ^# p < 0.05 compared with the OBCM-treated group.

Figure 5

Osteoblast-derived POSTN promotes invasion of PCa cells. (A) PC-3 and MDA PCa 2b cells were incubated with various concentrations of osteoblast (hFOB)-conditioned medium (OBCM), and cell invasion was measured by Transwell^® assays. (B) PC-3 and MDA PCa 2b cells were incubated with 30% OBCM supplemented with various concentrations of POSTN monoclonal antibody, and cell invasion was measured by Transwell^® assays. (C) PC-3 and MDA PCa 2b cells were incubated with 30% OBCM from control or POSTN-knockdown hFOB cells. Cell invasion was measured by Transwell^® assays. ^* p < 0.05 compared with the control; ^# p < 0.05 compared with the OBCM-treated group.

Figure 6

PCa cell-derived POSTN promotes proliferation of osteoblasts. (A) hFOB cells were incubated with various concentrations of PC cell (MDA PCa 2b)-conditioned medium (PCCM) for 48 h and proliferation was assessed. (B) hFOB cells were incubated with 30% PCCM supplemented with various concentrations of POSTN monoclonal antibody for 48 h, and proliferation was assessed. (C) MDA PCa 2b cells were transfected with control shRNA lentivirus (Lv-NC) or POSTN shRNA lentivirus (POSTN shRNA). The expression of POSTN was detected by real-time PCR. (D) MDA PCa 2b cells were transfected with control shRNA lentivirus (Lv-NC) or POSTN shRNA lentivirus (POSTN shRNA). The expression of POSTN was detected by Western blotting. (E) The levels of POSTN in MDA PCa 2b cell medium were detected by ELISA. (F) hFOB cells were incubated with 30% PCCM from control or POSTN-knockdown MDA PCa 2b cells. Proliferation was assessed after 48 h. ^* p < 0.05 compared with the control; ^# p < 0.05 compared with the OBCM-treated group.

Figure 7

POSTN functions in PCa cells and osteoblasts via integrin receptors. (A) Immunofluorescence staining of αVβ3 and αVβ5 in MDA PCa 2b and hFOB cells. (B) MDA PCa 2b cells were incubated with 30% OBCM and αVβ3 or αVβ5 antibody. Cell proliferation was assessed. (C) MDA PCa 2b cells were incubated with 30% OBCM and αVβ3 or αVβ5 antibody. Invasions were assessed. (D) hFOB cells were incubated with 30% PCCM and αVβ3 or αVβ5 antibody. Cell proliferation was assessed. ^# p < 0.05 compared with the OBCM- or PCCM-treated group.

Figure 8

¹⁸F-Alfatide II as the molecule probe of integrin αvβ₃ revealed high intake in primary and metastatic lesions through the PET-CT detection in a case of 82-year-old man. (A) Gross appearance of coronary position detected by PET-CT. The darker the color was, the higher the intake of tracer in the corresponding body part. (B) The direction cross focused on the primary lesion in prostate. The first row were CT views centered on high intake lesion of PCa from three different directions in the pelvis, which revealed the adjacent organs and structure. The second row were the PET-CT views detected by ¹⁸F-Alfatide II as the molecule probe of integrin αvβ₃ centered on high intake lesion of PCa from three different directions. As the main function of bladder should be storing urine and the tracer was discharged by urine, it was rational that there was darker high-intake region in the bladder. However, the abnormal area of high-intake tracer appearing in the prostate suggested high-level affinity of probe which might be closely correlated to malignant cancer. The third row was combined with the figures of the first and second rows. The lighter the color shows the higher the intake of tracer in prostate. (C) The direction cross focused on the metastatic lesion in the middle part of the pubis from three differential directions. The first row was the view of regular CT, which revealed the bone structure and surrounding tissues. There was an unusual small spot of osteoproliferation in the direction cross. The second row demonstrated the abnormal high-intake lesion of ¹⁸F-Alfatide II in the left side of the middle part of the pubis, respectively, which was highly coincident with the “unusual small spot”, The third row was the combination of the first and second row, which systematically reported the accurate location of metastatic lesion in pubis and relative extent of tracer intaking. (D) The direction cross focused on the metastatic lesion on the right side of the lumbar 1 vertebra. The first row was regular CT view in the slice of abdomen. Compared with the adjacent vertebrae, the direction cross revealed high density in bone cortex, which was consistent with the characteristics of osteoblastic metastasis in PCa. In the second row, from the sight of ¹⁸F-Alfatide II, except for the rich blood supply organs like kidneys, spleen, and liver, the strange high intake of probe in the vertebra presented metastatic lesion. In the third row, the emerge of both first and second rows in the same slice then provided the reliable evidence of metastatic lesion form the aspects of position and signal strength.

Figure 9

Another clinical replicate case from PET-CT in the case of 76-year-old man. (A) Gross view of coronary position detected by PET-CT. Similarly, the darker the color was, the higher the intake of tracer in the corresponding body part. Since the tracer was excreted through urine, the organs of urinary system were perceptibly high take of tracer. However, the prostate indicated abnormal high intake with dark tracer accumulation, consistent with local lesion of PCa. At the lower right corner of the picture, the spinning black “wire” should be the indwelling urine catheter and the urine with tracer inside. The bladder of the patient was not full, for the existence of urine catheter led the urine into the urine collection bag instantly. (B) The direction cross focused on the enlarged lymph node on the right side of the external iliac vessel. From the view of regular CT, the first row exhibited enlarged lymph node in right side of the external iliac vessel with the same density of soft tissue, and it displayed its neighboring tissues and bone structure. The specific nature of enlarged lymph node was unclear. The high-intake lesion on the right side of the external iliac vessel was also detected by the probe of ¹⁸F-Alfatide II in the second row from three directions, which suggested convincing evidence of lymphatic metastasis of PCa. Meanwhile, there were other two high intake points in the same slice of axial direction. As the association of the first and second rows, the third row demonstrated the highlighting point on the right side of the external iliac vessel was enlarged lymph node of metastasis while the other two points were a primary tumor lesion in the prostate and a section of urine catheter in vitro according to their position. Therefore, except for the tracing of bone metastatic lesion, the utilization of detecting lymph node of metastasis by the probe of ¹⁸F-Alfatide II should also be available. (C) The direction cross focused on the metastatic lesion in the lumbar 1 vertebra. The first, second, and third rows corresponded to the view of regular CT, PET-CT (¹⁸F-Alfatide II), and mergence in three directions in the lesion of the lumbar 1 vertebra. The first row presented the high density of bone cortex in the lumbar 1 vertebra, which also tallied with the feature of bone metastasis in PCa. The second row showed the abnormal high intake lesion of ¹⁸F-Alfatide II in the lumbar 1 vertebra, hinting the malignant tendency of this lesion. As the mergence of the first and second rows, the third row displayed the abnormal lesion in the lumbar 1 vertebra through high brightness signal. (D) The direction cross focused on the metastatic lesion behind the left ilium. The first, second, and third rows separately revealed the observed lesion behind the left ilium in the view of regular CT, PET-CT (¹⁸F-Alfatide II), and combination of regular CT and PET-CT in three directions. In the first row, we could find the unusual bone hyperplasia behind the left ilium. Then, the high intake of tracer in the views of PET-CT (¹⁸F-Alfatide II) revealed malignant metastatic lesion. The third row provided the combination of the first and second rows that we could discover corresponding region of another bone metastatic lesion.