Targeting Tn Antigen Suppresses Aberrant O-Glycosylation-Elicited Metastasis in Breast Cancer

Abstract

The Tn antigen, a truncated O-glycan representing aberrant mucin-type O-glycosylation, is frequently observed in human breast cancer. However, the functional role of Tn antigen in breast cancer metastasis remains insufficiently investigated. This study aimed to elucidate the expression profile of Tn antigen in breast cancer and its potential as a therapeutic target for inhibiting metastasis. Immunohistochemical staining was performed to determine the levels of Tn antigen expression in breast cancer tissues and its clinical relevance was analyzed accordingly. Tn-positive breast cancer cell lines were generated through disruption of the Cosmc gene. The functional roles of Tn antigen in breast cancer metastasis were studied in both in vitro and in vivo models. Western blotting and immunofluorescence staining were employed to investigate the molecular mechanisms by which Tn antigen promotes breast cancer metastasis. Our findings revealed that Tn antigen was prevalent in breast carcinomas, particularly within metastatic lesions. Tn antigen expression was positively correlated with lymph node metastasis and poorer patient survival. Tn antigen-expressing breast cancer cells exhibited enhanced invasiveness and metastasis, along with significant activation of EMT and FAK signaling pathways. Targeting Tn-positive cells with HPA (Helix pomatia agglutinin) demonstrated the suppression of invasive and metastatic capabilities, EMT program, and FAK signaling in vitro, as well as reduced pulmonary metastasis in a xenotransplant mouse model. This study reveals that Tn antigen-mediated aberrant O-glycosylation plays a contributing role in breast cancer metastasis, which may serve as a potential therapeutic target in clinical practice.

Keywords: HPA; O‐glycosylation; Tn antigen; breast cancer; metastasis.

FIGURE 1

Representative immunohistochemistry staining of Tn antigen in primary breast cancer tissues and distant metastasis tumor tissues. (A) Tn antigen was absent in normal breast tissues, whereas it showed positive expression in primary breast cancer tissues, lung metastasis, and spinal metastasis. All scale bars are 100 μm. (B) Positive staining of Tn antigen was quantified. Error bars indicate the mean ± SD, with significant differences indicated (**p < 0.01).

FIGURE 2

Tn antigen promotes migration and invasion properties. (A, B) The significant increased migration and invasion in Tn‐positive breast cancer cells. The histograms illustrate the quantification of migrating and invading cells, indicating a statistically significant difference (***p < 0.001) (C). The wound healing assays demonstrated a significantly higher motility in Tn‐positive cells compared to Tn‐negative cells.

FIGURE 3

Breast tumors expressing Tn antigen exhibit increased metastatic capacity. (A) Photographs of lung metastases formation in tail vein injection mouse models. (B) Representative HE staining of mouse lung tissue sections (original magnification ×40 and ×200) were shown. (C) Quantities of lung metastases in breast tumor. (D) The analysis of survival rates in mice models with metastatic tumors indicated a notable decrease in survival among those injected with Tn‐positive cells.

FIGURE 4

Tn antigen promotes epithelial‐mesenchymal transition in breast cancer cells. Western blot assay revealed a decrease in the expression of epithelial markers, namely E‐cadherin and ZO‐1, along with an increase in the expression of mesenchymal markers, including ZEB‐1, Vimentin, and the transcription factors Snail and Slug. The clear background is caused by the Bio‐Rad imaging system, which helps highlight the detected bands.

FIGURE 5

Tn antigen facilitates FAK phosphorylation and the formation of cellular protrusion. (A) The immunofluorescence images of F‐actin staining indicated a significant increase in protrusions formation (white arrow) in Tn‐positive cells. (B) Western blot analysis of FAK, phosphorylated FAK (p‐FAK), phosphorylated paxillin (p‐paxillin), and phosphorylated p130cas (p‐p130cas) were determined. The clear background, is caused by the Bio‐Rad imaging system, which helps highlight the detected bands.

FIGURE 6

The metastatic capacity of Tn‐positive cells is significantly reduced through the administration of HPA treatment. (A, B) The properties of cell migration and invasion in Tn‐positive cells with HPA and the control lectin PNA were analyzed by Transwell assays. The histograms illustrate the quantification of migrating and invading cells, indicating a statistically significant difference (***p < 0.001). (C) In the NOD/SCID mice lung metastasis model, the combination of tail vein injection of Tn‐positive MDA‐MB‐231 cells with HPA treatment effectively inhibited the in vivo metastatic process, in comparison to the control group treated with PNA. (D) Quantification of lung metastatic nodules in the NOD/SCID mouse model injected with Tn‐positive MDA‐MB‐231 cells, comparing HPA‐treated mice to the PNA‐treated control group (*p < 0.05).

FIGURE 7

HPA treatment inhibits protrusion formation and EMT activation. (A) The F‐actin staining images revealed the application of HPA treatment resulted in the inhibition of protrusions on the surface of breast cancer cells when compared with the control PNA treatment. (B) Western blot analysis showed the inhibitory effects of HPA on the FAK signaling pathway and the EMT program in contrast to PNA. The clear background, is caused by the Bio‐Rad imaging system, which helps highlight the detected bands.