HMGA1B/2 transcriptionally activated-POU1F1 facilitates gastric carcinoma metastasis via CXCL12/CXCR4 axis-mediated macrophage polarization

Abstract

Tumor-associated macrophages (TAMs) in the tumor microenvironment contribute to poor prognosis in gastric cancer (GC). However, the underlying mechanism by which TAMs promote GC progression and metastasis remains elusive. Expression of POU1F1 was detected in 60 matched GC-normal tissue pairs using qRT-PCR and immunohistochemistry (IHC) analysis. The correlation between POU1F1 and the clinical-pathological factors of GC patients were further assessed. Cell proliferation was monitored by CCK-8, colony formation, and 5-Ethynyl-2'-deoxyuridine (EdU) incorporation assays. Cell migration and invasion were assessed by transwell assays. The impact on angiogenesis was evaluated by tube formation assay. Xenograft model was generated to investigate the role of POU1F1 on tumor growth and lung metastasis in vivo. GST pull-down and Co-immunoprecipitation (Co-IP) were used to study the interaction between HMGA1B/2 and POU1F1. Chromatin immunoprecipitation (ChIP) and dual luciferase reporter assays were performed to investigate the transcriptional regulation of POU1F1. Flow cytometry was performed to detect the surface expression of macrophage markers. Upregulated POU1F1 observed both in GC tissues and cell lines was positively correlated with poor prognosis. Knockdown of POU1F1 inhibited cell proliferation, migration, invasion, and angiogenesis in vitro, and suppressed tumor growth in vivo. HMGA1B/2 transcriptionally activated-POU1F1. POU1F1 promoted GC progression via regulating macrophage proliferation, migration, polarization, and angiogenesis in a CXCL12/CXCR4-dependent manner. POU1F1 also promoted GC metastasis in lung by modulating macrophage polarization through CXCL12/CXCR4 axis in vivo. HMGA1B/2-upregulated POU1F1 promoted GC metastasis via regulating macrophage polarization in a CXCL12/CXCR4-dependent manner.

Fig. 1. Upregulated POU1F1 is associated with poor prognosis in GC.

A Expression of POU1F1 in GC and paired adjacent normal tissues were determined by IHC. B The mRNA level of POU1F1 in GC and paired adjacent normal tissues were determined by qRT-PCR assay. C Expression patterns of POU1F1 in T1 or T2/above and different TNM stages were determined by qRT-PCR analysis. D Kaplan–Meier curves for overall survival of GC patients. E, F The mRNA and protein levels of POU1F1 in GC cell lines and GES-1 cells were examined using qRT-PCR and western blot analysis, respectively. GAPDH served as an internal control. Data were representative images or were expressed as the mean ± SD of n = 3 experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2. Knockdown of POU1F1 inhibits cell proliferation, migration, invasion, and angiogenesis in GC cells.

A The mRNA level of POU1F1 was determined by qRT-PCR analysis. B The protein level of POU1F1 was determined by western blot. GAPDH served as a loading control. C Cell proliferation was monitored by CCK-8 assay. D Colony forming ability was assessed by colony formation assay. E DNA synthesis and cell proliferation was monitored by EdU incorporation assay. Red, EdU; Blue, DAPI. F The capacity of cell migration and invasion were assessed by transwell system. G In vitro angiogenesis was monitored by tube formation assay. Data were representative images or were expressed as the mean ± SD of n = 3 experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. Knockdown of POU1F1 inhibits tumor growth and metastasis in vivo.

A The photos of xenograft tumors. B Quantitative analysis of tumor size. C Quantitative analysis of tumor weight. D Histopathological analysis of xenograft tumors. The histopathological changes were determined by H&E staining. The immunoreactivities of POU1F1 and Ki67 were assessed by IHC analysis. E The mRNA level of POU1F1 in xenograft tumors were determined by qRT-PCR. GAPDH served as an internal control. F The photos of lung tissues. G Histopathological analysis of metastatic nodules in lung. The histopathological changes were determined by H&E staining. H Quantitative analysis of metastatic nodule numbers. Data were representative images or were expressed as the mean ± SD of n = 3 experiments. *P < 0.05, **P < 0.01.

Fig. 4. HMGA1B/2 upregulates POU1F1 transcriptionally.

A In vitro interactions between POU1F1 and HMGA1B or HMGA2 were determined by GST pull-down assay. B In vivo interactions between POU1F1 and HMGA1B or HMGA2 were determined by co-IP. Whole-cell lysates served as an input control. C The enrichment of HMGA1 or HMGA2 at POU1F1 promoter was assessed by ChIP assay. Normal IgG served as a negative control. The non-immunoprecipitated chromatin served as an input control. D POU1F1 promoter region containing sequence between nt −1321 and +15 was cloned into pGL-3 Basic vector. Luciferase activity was determined by dual luciferase reporter assay. Renilla luciferase activity served as an internal control. E The protein level of POU1F1 was determined by western blot. GAPDH served as a loading control. F The correlations between POU1F1 and HMGA1B or HMGA2 in GC were determined by Pearson correlation analysis. Data were expressed as the mean ± SD of n = 3 experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5. POU1F1 promotes GC progression via regulating macrophage proliferation, migration, polarization, and angiogenesis.

A The immunoreactivity of CD163 was assessed by IHC analysis. B Kaplan–Meier curves for overall survival of GC patients. C The correlation between CD163 and POU1F1 in GC was determined by Pearson correlation analysis. D Cell surface CD14, CD11b, F4/80, and CD11c expression were analyzed by flow cytometry. E Cell viability of macrophages was monitored by CCK-8 assay. F Cell migration of macrophages was determined by transwell migration assay. The mRNA (G) and protein (H) levels of CD163 and CD206 were detected by qRT-PCR and western blot, respectively. CD11b served as an internal control. (I) The immunoreactivity of CD31 was assessed by IHC analysis. J The correlations between CD31 and CD163 in GC were determined by Pearson correlation analysis. K Cell viability of HUVECs was monitored by CCK-8 assay. L In vitro angiogenesis was monitored by tube formation assay. M The protein level of VEGF was determined by western blot. GAPDH served as a loading control. Data were representative images or were expressed as the mean ± SD of n = 3 experiments. *P < 0.05, **P < 0.01.

Fig. 6. CXCL12/CXCR4 axis is involved in POU1F1-induced macrophage polarization.

A The mRNA level of CXCL12 was determined by qRT-PCR. GAPDH served as an internal control. B The correlation between CXCL12 and POU1F1 in GC was determined by Pearson correlation analysis. C The correlations between CXCL12 and CD206, CD163, CCR2, or CD204 in GC were determined by Pearson correlation analysis. D Cell migration of macrophages was determined by transwell migration assay. E In vitro angiogenesis was monitored by tube formation assay. F The mRNA levels of CD163 and VEGFA were determined by qRT-PCR. GAPDH served as an internal control. G The protein levels of p-CXCR4, CXCR4, p-Akt, Akt, p-VEGFR2, and VEGFR2 were determined by western blot. GAPDH served as a loading control. Data were representative images or were expressed as the mean ± SD of n = 3 experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7. POU1F1 promotes GC metastasis in lung by modulating macrophage polarization through CXCL12/CXCR4 axis.

A The photos of xenograft tumors. B Quantitative analysis of tumor size. C Quantitative analysis of tumor weight. D The photos of lung tissues. Quantitative analysis of metastatic nodule numbers. E Histopathological analysis of metastatic nodules in lung. The histopathological changes were determined by H&E staining. The immunoreactivities of CD31, CD163, and POU1F1 were assessed by IHC analysis.