New insights into the role of the CHI3L2 protein in invasive ductal breast carcinoma

Abstract

Chitinase-like proteins have multiple biological functions that promote tumor growth, angiogenesis and metastasis. Expression of CHI3L2, which is similar in structure to CHI3L1, is detected in glioma cells and tumor-associated macrophages (TAMs) in glioma and breast cancer. However, its exact role remains unclear. We analyzed the expression of CHI3L2 in 74 invasive ductal breast carcinoma (IDC) tumors, breast cancer and macrophages cell cultures using immunohistochemistry, immunofluorescence, Western blot and PCR methods. Clinicopathologic data were included in the analysis. The results obtained show that CHI3L2 expression decreases with increasing degree of tumor grade and negative status of estrogen (ER) and progesterone receptors (PR). Furthermore, CHI3L2 is significantly and positively correlated with phosphorylation of STAT-3 and ERK1/2 signaling pathways, but negatively correlated with macrophage infiltration. CHI3L2 is expressed both in the cytoplasm of cancer cells and in macrophages and may regulate STAT-3 and ERK1/2 phosphorylation in breast cancer cell lines. Analysis of the clinicopathologic data revealed that CHI3L2 levels had no effect on patient survival. CHI3L2 expression may be specific for cancer cells in IDC and involved in cross-talk with the tumor microenvironment. Our study has shown that IDC cancer cells express the CHI3L2 protein, possibly indicating a novel function of this protein.

Keywords: CHI3L1; CHI3L2; Chitinase-like proteins; ERK1/2; Invasive ductal breast carcinoma; STAT-3.

Fig. 1

Immunohistochemical reactions show differential expression of CHI3L2 in the cancer cells, CD68 pan-macrophage markers and CD206-positive TAMs, magnification 100x.

Fig. 2

Analysis of immunohistochemical expression of CHI3L2, CD68 and CD206 in tumors of different malignancy grade (G1, G2, G3; G0: non-malignant breast tissue lesion (NBTLs); Kruskal–Wallis test followed by Dunn test; statistically significant p-values were < 0.05.

Fig. 3

Analysis of immunohistochemical expression and correlations of CHI3L2 and A) angiogenic markers: CHI3L1 (Spearman correlation), CD34 and VEGFA (Kendall’s tau-b correlation); B) analysis of expression of CHI3L2, CD68 and CD206 (Spearman correlation); statistically significant p-values were < 0.05.

Fig. 4

Analysis of immunohistochemical expression and its correlation with estrogen receptor (ER; ER⁺ N = 45, ER^- N = 21), progesterone receptor (PR, PR⁺ N = 40, ER^- N = 26) and human epidermal growth factor receptor 2 (HER2, HER2⁺ N = 40, HER2^- N = 26) status was performed using the Mann–Whitney U test. Expression was expressed as 0 for negative and 1 for positive receptors. Statistically significant p-values were < 0.05.

Fig. 5

Expression of CHI3L2 in IDC tumors. Immunofluorescence reactions visualized by confocal microscopy showed the expression of CHI3L2 (green; yellow arows) in cancer cells and CD206 (red; white arroheads) as a characteristic feature of TAMs. Co-expression of CHI3L2 and CD206 was also observed (magenta arrows). The nucleus was stained with DAPI (blue). Images from single fluorescent channels is showed in Fig. 1S supplemental material.

Fig. 6

Western blot analysis of CHI3L2 expression in IDC in A tumors of different malignancy grade: G1 (n = 3), G2 (n = 9) and G3 (n = 10), with expressions of pSTAT-3, STAT-3, pERK1/2 and ERK1/2; B statistical analysis (data from densitometric analysis of Western blots experiments provided in triplicates) on CHI3L2, pSTAT-3 and pERK1/2 expressions in G1-G3 grade tumors (Kruskal–Wallis test, post-hoc Dunn’s test). Raw data from densitrometric analysis is showed in Fig. 2S supplemental material.

Fig. 7

Spearman’s correlations of CHI3L2 and pSTAT-3 and pERK1/2 expressions from Western blot analysis; statistically significant p-values were < 0.05. Western blots were performed in triplicate.

Fig. 8

Western blot analysis of CHI3L2, pSTAT-3, STAT-3, pERK1/2 and ERK1/2 in breast cancer cell lines, macrophages (M) and macrophages in co-culture with MCF-7 (M/MCF7), MDA-MB-468 (M/M468), Me16C cells (M/Me16C) and MDA-MB-231 (M/M231) cells (the characteristics of breast cancer cell lines are shown in Table 1).

Fig. 9

Breast cancer cells models with different status of CHI3L2 after transfection with CHI3L2 siRNA: (A) changing in STAT-3 and ERK1/2 phosphorylation; Western blot analysis; (B) analysis of CHI3L2 mRNA in MDA-MB-321 and (C) BT-549 cells during trnasfection experiments provided in triplicates in ddPCR analysis; and (D) with presence of recombinant CHI3L2 protein in MDA-MB-468 and BT474 cells ; Western blot analysis; Kruskal–Wallis test followed by Dunn test

Fig. 10

PCR analysis of CHI3L2 mRNA expression level in breast tumors. Different tumor grades: G1 (n = 67), G2 (n = 168), G3 (n = 54); G1 vs G2 p = 0.083; G2vsG3 p = 0.12, G1vs G3 p = 0.9 (Kruskal–Wallis test, post-hoc Dunn’s test); status of estrogen (ER), progesterone (PR) and HER2 receptor; 0: negative expression, 1: positive expression of the receptors; CHI3L2 vs PR p = 0.44, CHI3L2 vs ER p = 0.46 (Mann–Whitney U-test); statistically significant p-values were < 0.05.