Calmodulin 2 Facilitates Angiogenesis and Metastasis of Gastric Cancer via STAT3/HIF-1A/VEGF-A Mediated Macrophage Polarization

Abstract

Background: Tumor-associated macrophages (TAMs) are indispensable to mediating the connections between cells in the tumor microenvironment. In this study, we intended to research the function and mechanism of Calmodulin2 (CALM2) in gastric cancer (GC)-TAM microenvironment.

Materials and methods: CALM2 expression in GC tissues and GC cells was determined through quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). The correlation between CALM2 level and the survival rate of GC patients was assessed. The CALM2 overexpression or knockdown model was constructed to evaluate its role in GC cell proliferation, migration, and invasion. THP1 cells or HUVECs were co-cultured with the conditioned medium of GC cells. Tubule formation experiment was done to examine the angiogenesis of endothelial cells. The proliferation, migration, and polarization of THP1 cells were measured. A xenograft model was set up in BALB/c male nude mice to study CALM2x's effects on tumor growth and lung metastasis in vivo. Western Blot (WB) checked the profile of JAK2/STAT3/HIF-1/VEGFA in GC tissues and cells.

Results: In GC tissues and cell lines, CALM2 expression was elevated and positively relevant to the poor prognosis of GC patients. In in-vitro experiments, CALM2 overexpression or knockdown could facilitate or curb the proliferation, migration, invasion, and angiogenesis of HUVECs and M2 polarization of THP1 cells. In in-vivo experiments, CALM2 boosted tumor growth and lung metastasis. Mechanically, CALM2 could arouse the JAK2/STAT3/HIF-1/VEGFA signaling. It was also discovered that JAK2 and HIF-1A inhibition could attenuate the promoting effects of CALM2 on GC, HUVECs cells, and macrophages.

Conclusion: CALM2 modulates the JAK2/STAT3/HIF-1/VEGFA axis and bolsters macrophage polarization, thus facilitating GC metastasis and angiogenesis.

Keywords: angiogenesis; calmodulin 2; cancer; gastric cancer; tumor-associated macrophages.

Figure 1

CALM2 up-regulation was correlated with poorer GC prognosis. (A) CALM2 expression in GC and paired para-carcinoma tissues was figured out through IHC staining. (B) qRT-PCR determined the mRNA level of CALM2 in GC tissues of patients in different clinical phases. (C) The protein level of CALM2 in GC tissues of 12 different GC tissues was evaluated through Western Blot. (D) Kaplan-Meier curve analyzed the correlation between the high or low profile of CALM2 and the survival rate of 31 patients with GC. p = 0.0315. (E) CALM2 mRNA expression in the human gastric epithelial cell line GES-1 and gastric cancer cell lines SGC7901, BGC823, MKN45, HGC27, as well as AGS was confirmed via qRT-PCR. (F) Western Blot measured the protein profile of CALM2 in different GC cell lines. Statistics were displayed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

CALM2 overexpression strengthened GC cell proliferation, migration, and invasion. CALM2 overexpression plasmids or negative vectors were transfected into AGS and MKN45 cells. (A) qRT-PCR ascertained the mRNA expression of CALM2 in AGS and MKN45 cells. (B) Western Blot determined the protein level of CALM2 in AGS and MKN45 cells. (C) The proliferation of AGS and MKN45 cells was monitored by CCK8. (D) The colony formation capability of AGS and MKN45 cells was verified via colony formation experiment. (E, F) Transwell evaluated the migration and invasion of AGS and MKN45 cells. (G) Western Blot was adopted to examine the EMT-concerned proteins of the cells. Data were presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 (vs. the Vector group). NSP > 0.05 (vs. Blank group).

Figure 3

CALM2 inhibition hampered GC cell proliferation, migration, and invasion. Si-NC, si-CALM2#1, si-CALM2#2, or si-CALM2#3 was transfected into AGS and MKN45 cells. (A) qRT-PCR checked the mRNA expression of CALM2 in AGS and MKN45 cells. (B) The protein level of CALM2 in AGS and MKN45 cells was examined through Western Blot. (C) The proliferation of AGS and MKN45 cells was monitored through CCK8. (D) Colony formation assay determined the colony formation ability of AGS and MKN45 cells. (E, F) Transwell measured these cells’ migration and invasion. (G) EMT-associated proteins in AGS and MKN45 cells were examined via Western Blot. Statistics were displayed as mean ± SD (n=3). *P < 0.05, **P < 0.01, ***P < 0.001 (vs. the Si-NC group). NS P > 0.05 (vs. Blank group).

Figure 4

CALM2 overexpression aggravated GC cell-mediated angiogenesis and macrophage polarization. (A) IHC was operated to assess the profiles of CD163, CD206, and CD11b in GC tissues (data from the Human Protein Atlas). (B) CALM2’s affinities with CD163, CD206, and CD11b in GC tissues were analyzed through Person regression analysis. THP1 cells stimulated by PMA were co-cultured with the conditioned medium of GC cells. (C) CCK8 monitored macrophage proliferation. (D) Transwell investigated macrophage migration. (E, F) The profiles of M2-type macrophage surface receptors CD206, CD163, and CD11b and M1-type macrophage markers CD80, CD86, and iNOS were figured out via Western Blot. (G) The mRNA expressions of M2-type macrophage-concerned factors CXCL12, IL-4, IL-13, IL-10, and VEGFA were confirmed through qRT-PCR. HUVECs were co-cultured with the conditioned medium of GC cells. (H) The viability of HUVECs was examined by CCK8. (I) HUVEC angiogenesis was checked by tubule formation experiment. (J) HUVECs’ migration was assessed through the wound scratch test. Statistics were presented as mean ± SD (n=3). *P < 0.05, **P < 0.01, ***P < 0.001 (vs. the Blank group). &P < 0.05, &&P < 0.01 (vs. the CM+Vector group).

Figure 5

CALM2 overexpression or knockdown could facilitate or hinder tumor growth and metastasis in vivo. The nude mice were transplanted with AGS cells transfected with CALM2 overexpression plasmid or si-CALM2. After 35 days of neoplasm transplantation, the animals were sacrificed, with their tumors isolated. (A) The picture of the xenograft tumor. (B) Quantitative analysis of tumor size. (C) Quantitative analysis of tumor weight. (D) Quantitative analysis of the amount of lung metastatic nodules. (E–H) IHC analyzed and evaluated the profiles of Ki67, CD31, VEGFA, CD163 in the tumors. Statistics were displayed as mean ± SD (n = 5). ***P < 0.001 (vs. the Vector group or the Si-NC group).

Figure 6

CALM2 overexpression activated JAK2/STAT3/HIF-1/VEGFA profile in GC tissues and cells. (A) String (https://string-db.org/) was taken for analyzing the potential pathway regulated by CALM2. (B). JAK2/STAT3/HIF-1/VEGFA was discovered to be an underlying downstream pathway of CALM2. (C) IHC ascertained the protein profiles of p-JAK2, p-STAT3, and HIF-1A in the mice’s tumor tissues. (C) Western Blot confirmed the protein profile of the JAK2/STAT3/HIF-1/VEGFA signaling axis in AGS and MKN45 cells with CALM2 overexpression or down-regulation. Data were exhibited as mean ± SD (n=3). **P < 0.01, ***P < 0.001 (vs. the Vector or Si-NC group).

Figure 7

JAK2 or HIF-1 inhibition reduced CALM2’s impact on GC cell proliferation, migration, and invasion. The JAK2 inhibitor LY2784544 (1 μM) and HIF-1 inhibitor SYP-5 (10 μM) were applied to AGS and MKN45 cells with CALM2 overexpression for treatment. (A) Western Blot determined the profile of the JAK2/STAT3/HIF-1/VEGFA signaling axis in AGS and MKN45 cells. (B) The proliferation of those cells was monitored by CCK8. (C) Colony formation assay examined the colony formation of AGS and MKN45 cells. (D, E) Transwell measured the cells’ migration and invasion. (F) EMT-correlated proteins in AGS and MKN45 cells were examined through Western Blot. Statistics were displayed as mean ± SD (n=3). *P < 0.05, **P < 0.01, ***P < 0.001 (vs. the Vector group). NSP > 0.05, &P < 0.05, &&P < 0.01, &&&P < 0.001 (vs. the CALM2 group).

Figure 8

JAK2 or HIF-1 inhibition mitigated CALM2-mediated macrophage polarization and angiogenesis. The JAK2 inhibitor LY2784544 (1 μM) and HIF-1 inhibitor SYP-5 (10 μM) were deployed to treat AGS and MKN45 cells with CALM2 overexpression. The conditioned medium was collected and cultivated along with macrophages and HUVECs for 24 hours. (A) Western Blot was done to figure out the profile of the JAK2/STAT3/HIF-1/VEGFA axis in macrophages. (B, C) Western Blot also ascertained the protein profiles of CD206, CD163, CD11b, CD80, CD86, and iNOS in macrophages. (D) qRT-PCR disclosed the mRNA expressions of M2-type macrophage-concerned factors CXCL12, IL-4, IL-13, IL-10, and VEGFA. (E) Western Blot verified the profile of the JAK2/STAT3/HIF-1/VEGFA pathway in HUVECs. Data were presented as mean ± SD (n=3). NSP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 (vs. the CM+CALM2 group).