PAFAH1B3, as one of the top 50 overexpressed metabolic enzymes in human cancers,[18] is involved in multiple types of tumors, but the roles of PAFAH1B3 in LUAD have not been clarified. In this research, we identified PAFAH1B3 as a promising oncogene in LUAD by analyzing public datasets and clinical samples, as well as by performing functional assays in vitro. We also found that PAFAH1B3 was correlated with immune infiltrates in LUAD.
First, we determined that PAFAH1B3 was significantly overexpressed in both LUAD tissues and LUAD cells. Then, we revealed that high PAFAH1B3 expression was significantly correlated with distant metastasis, advanced TNM stage and poor prognosis, indicating that PAFAH1B3 upregulation is positively related to LUAD progression. Furthermore, we confirmed PAFAH1B3 as an independent prognostic risk factor for LUAD through multivariate Cox regression analysis. Similarly, previous studies have confirmed that there is high expression of PAFAH1B3 in HSCC tissues by IHC analyses, and high expression of PAFAH1B3 is positively correlated with poor prognosis of HSCC patients.[13] Thus, we believe that PAFAH1B3 has potential clinical value in LUAD.
Next, we conducted in vitro experiments. We found that the suppression of PAFAH1B3 expression in LUAD cells inhibited clone formation and proliferation and caused G1 phase arrest in LUAD cells. These results indicate that PAFAH1B3 is a novel potential oncogene in LUAD. A previous study demonstrated that knockdown of PAFAH1B3 impaired the proliferation, migration, and invasiveness of breast carcinoma cells by elevating tumor-suppressing signaling lipids.[11] Moreover, PAFAH1B3 loss in vivo sensitized leukemia cells to tyrosine kinase inhibitor (TKI) treatment via the platelet activating factor (PAF)/platelet activating factor receptor (PAFR) signaling pathway, and PAF/PAFR has been found to exhibit beneficial effects in some cancers.[7, 12] PAFR suppresses the inflammation and neoplastic transformation induced by chemical carcinogens.[19] PAFAH1B3 might promote malignant progression of LUAD through the PAF/PAFR signaling pathway. More studies are needed to further study the changes in downstream signaling pathways and tumor signaling lipids that occur after silencing PAFAH1B3.
Invasion is one step of metastasis. Our results demonstrated that downregulation of PAFAH1B3 significantly inhibited the invasive ability of A549 and H1299 cells. As EMT is considered the first step of metastasis,[20] we further studied the mechanism through which PAFAH1B3 facilitates LUAD invasion. Through loss-of-function analysis in LUAD cells, we clearly demonstrated that PAFAH1B3 knockdown upregulated the protein expression of the epithelial marker E-cadherin but downregulated the expression of the mesenchymal marker N-cadherin and EMT-inducing transcription factors (Snail/Slug).
EMT is a process by which differentiated epithelial cells transition into motile mesenchymal cells, and it involves some key events, including loss of intercellular junctions, loss of cell polarity and acquisition of enhanced cell invasion and migration capacities.[21] Tumor cells that have undergone EMT may be resistant to chemotherapy and immunotherapy, acquire stem cell properties and escape immune surveillance.[22] EMT plays a central role in the development and progression of lung cancer, and targeting EMT signaling may be a new therapeutic strategy.[23]
EMT is characterized by loss of the epithelial marker E cadherin and increased expression of the mesenchymal marker N-cadherin.[22] E-cadherin expression can be repressed by EMT-related transcription factors, including SNAIL, SLUG and TWIST. snail1 and slug, belonging to the snail family, are encoded by snail1 and snail2, respectively, and they can bind to the promoter region of E-cadherin and repress its transcription.[24, 25] Here, for the first time, we demonstrated that PAFAH1B3 might promote EMT by activating Snail/slug, which might lead to the downregulation of E-cadherin, resulting in enhanced invasion and mobility of LUAD cells. However, further studies of the pathways related to activation of Slug/snail by PAFAH1B3 and studies determining whether there are other transcription factors involved in the PAFAH1B3-mediated reduction in E-cadherin are needed.
Immune cells have been increasingly recognized to be involved in carcinogenesis and cancer progression in recent years; for example, tumor-infiltrating T cells in particular have been found to significantly influence prognosis and therapeutic outcomes.[26] We demonstrated that the infiltration score was notably lower in the PAFAH1B3-high group than in the PAFAH1B3-low group, indicating that increased expression of PAFAH1B3 was correlated with a lower immune infiltration level. Furthermore, our results showed that the PAFAH1B3-high expression group had significantly enriched tumor-promoting cells, such as nTregs, exhausted T cells and neutrophils. Neutrophils are considered the most significant predictor of poor survival in solid tumors.[27] In a mouse model of LUAD, neutrophil infiltration promoted tumor growth, increased the ability of cancer cells to metastasize and elevated the expression of snail, which in turn accelerated neutrophil accumulation.[28] These results are consistent with our results showing that neutrophils were upregulated in the PAFAH1B3 high-expression group and that snail was reduced when PAFAH1B3 was knocked down. Exhausted T cells can be observed in chronic infections and cancer, and elevated expression of inhibitory receptors on exhausted T cells facilitates CD8 + T cell failure and immune evasion.[29] nTregs, a subpopulation of T cells, are increased in the tumor microenvironment and are associated with poor survival in lung cancer.[30] In addition, we also found that CD4 + T cells, Tc cells, DCs, Tfh cells and MAIT cells were notably enriched in the PAFAH1B3-low group, which is consistent with their positive role in antitumor processes.[31–34] Tc cells are the main cells that participate in cancer cell killing and elimination of tumors.[31] With the help of special DCs, CD4 + T cells can relay helpful signals to enhance the antitumor properties of cytotoxic T lymphocytes.[32] These results may partly account for the poor clinical outcomes of LUAD patients with PAFAH1B3 overexpression, and more experiments are needed to confirm the association between PAFAH1B3 expression and tumor-infiltrating immune cells.
According to the above experiments and public dataset analysis, we found that PAFAH1B3 plays an oncogenic role in the progression of LUAD and might be a reasonable target for LUAD therapy. Further validation of the tumorigenic effect of PAFAH1B3 in mice is needed. In the future, specific biological agents targeting PAFAH1B3 could be developed to benefit LUAD patients with high PAFAH1B3 expression.