This study suggests that PANX1 is highly expressed in TNBC and is a prognostic factor associated with poor prognosis in TNBC. PANX1 promotes TAN infiltration by increasing exATP levels in TNBC, and TANs highly express ENTPD1/NT5E, which synergistically contributes to an immunosuppressive environment with high exADO levels and promotes TNBC progression.
Malignant tumors are known as "never healing wounds", and chronic inflammation is one of the key features of malignancy. Chronic inflammatory processes are involved in tumorigenesis and tumor progression. Purine nucleosides (ATP and ADO) exert a strong immunomodulatory ability in the TME, and exATP/exADO can regulate local immune responses by activating immune cell purinergic P2 receptors. Previous studies have focused on the role of the nucleotidases ENTPD1 and NT5E in exADO production and the establishment of an immunosuppressive microenvironment (25, 26). However, the source and regulatory mechanism of exATP in the TME, which is the substrate for exADO, has not been as well studied.
Stewart et al. found that PANX1 expression was required for breast development during lactation and that high PANX1 expression was associated with worse clinical outcomes in breast cancer (27). In other malignant tumors, PANX1 exhibited procarcinogenic effects in hepatocellular carcinoma (28), testicular carcinoma (29) and melanoma (30) and anticarcinogenic effects in glioma (31) and rhabdomyosarcoma (32). The above results suggest that the role of PANX1 in tumorigenesis might be complex and might depend on the tumor type and stage. In this study, we investigated the effect of PANX1 in TNBC primary lesions. We found that PANX1 was highly expressed in TNBC and could increase the exATP level in the TME and that high PANX1 expression was associated with poor prognosis in TNBC. These prognosis results are consistent with those in previous breast cancer studies (29, 33). However, previous studies have primarily focused on the effects of PANX1 on breast cancer cells (33), and the role of PANX1 in the formation of the immunosuppressive microenvironment has not been fully explored. In vitro studies had shown that PANX1 promoted the activation of NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome and increased the level of interleukin 1β (IL-1β) in the local microenvironment (34).
In this study, we found that the abundance of infiltrating neutrophils was significantly higher in the PANX1 high expression TNBC and that the genes coexpressed with PANX1 were related to granulocyte chemotaxis. In the early stage of tumor development, as the first immune cells to enter the tumor microenvironment, neutrophils mediate subsequent immune responses and regulatory processes (35, 36). The exATP secreted by PANX1 is considered to be an important damage-associated molecular pattern (DAMP) signal (37) that could lead to neutrophil recruitment (8). exATP could exacerbate the local immune response by mediating NLRP3 inflammasome activation and IL-1β secretion via the P2Y7 receptor (P2X7R) on neutrophils (38). exATP could also delay neutrophil apoptosis via the P2Y11 receptor (P2Y11R) (39). This study also demonstrated that TANs expressing high levels of ENTPD1/NT5E could promote the hydrolysis of exATP to exADO to aggravate the immunosuppressive TME. This is consistent with the results of studies of inflammatory states (40, 41). Chen et al. demonstrated that neutrophils were chemotactic to exATP and hydrolyzed exATP to exADO by NT5E to promote cell migration (41, 42). In addition, exADO inhibits neutrophil adhesion and the release of TNF-α and chemokines from LPS-stimulated neutrophils (43). Neutrophil-expressed adenosine A2A receptor (A2AR) could inhibit the neutrophil recruitment cascade (44). Previous studies also revealed that the purinergic receptor P2Y6 receptor (P2Y6R) (45), adenosine A2B receptor (A2BR) (46), and adenosine A3 receptor (A3R) (47) on neutrophils are involved in the regulation of neutrophil extracellular traps (NETs) in inflammatory states. Whether exATP/exADO can regulate NETs or the N1/N2-like subtype transition of TANs through purinergic receptors and thus affect the development and metastasis of breast cancer requires further investigation.
In this study, we demonstrated that ENTPD1 and NT5E expression was higher in the PANX1 high expression TNBC and PANX1 upregulated exADO levels in the TME. PANX1 is an important immunomodulator in the TME. When the results of single-cell transcriptome data analysis were combined with TCGA-BRCA data analysis in TNBC, we found that PANX1 expression was negatively correlated with the infiltration levels of CD8+ T cells and NK cells and was positively correlated with the infiltration levels of Tregs. The levels of CAFs were also higher in PANX1 high expression TNBC. A similar phenomenon has been observed during renal injury. Higher PANX1 expression caused an increase in exATP, which was further catabolized by ENTPD1 and then converted to adenosine by NT5E (48). PANX1 plays an important role in the maintenance of chronic inflammation. Targeting PANX1 could inhibit tumor-associated inflammatory responses and reverse the local immunosuppressive microenvironment. Therefore, in-depth investigations of PANX1 may help elucidate tumor-inflammation interactions.
In summary, the high expression of PANX1 in TNBC was a poor prognostic factor and positively correlated with the exADO level. PANX1 could promote TAN infiltration through exATP secretion, and the high expression of ENTPD1/NT5E in TANs could synergistically establish an immunosuppressive TME with high exADO levels. In this study, the relationship between high exATP/exADO levels and TANs was investigated to elucidate the properties of PANX1 and its ability to reshape the metabolic-immunosuppressive TME and provide new targets and strategies for TNBC treatment.