Panoply™ Human CHRM4 Knockdown Stable Cell Line

Nerve growth factor (NGF) promotes malignant tumor progression. However, the functional role and regulatory mechanisms of NGF in the development of neuroendocrine prostate cancer (NEPC) remain unclear. Here, researchers show that androgen deprivation therapy (ADT)-stimulated transcription factor ZBTB46 upregulates NGF levels through its mediated NGF transcriptional activation. Following ADT treatment, NGF regulates NEPC differentiation through physical interaction with the G-protein-coupled receptor cholinergic receptor muscarinic 4 (CHRM4). Pharmacological NGF blockade and NGF knockdown significantly inhibited CHRM4-mediated NEPC differentiation and AKT-MYCN signaling activation. CHRM4 stimulation correlated with ADT resistance and was significantly associated with elevated NGF in samples from patients with high-grade and small-cell neuroendocrine prostate cancer (SCNC). These findings reveal a role for NGF in NEPC development, linked to ZBTB46 upregulation and CHRM4 accumulation. The NGF-CHRM4 axis may become a therapeutic target to inhibit the progression of NEPC.

To investigate the downstream signaling of CHRM4 after ADT, experiments were performed in LNCaP and C4-2 cells transfected with CHRM4 shRNA and further treated with CSS-containing medium. Western blot analysis showed that after ADT, CHRM4 accumulation was associated with elevated p-AKT and MYCN levels. This effect was abolished in CHRM4 knockdown cells, regardless of ADT treatment (Figure 1i). Furthermore, GSEA confirmed that tissues with high NGF and CHRM4 expression were positively correlated with gene signatures responsive to activated AKT and MYCN-targeted signaling pathways. These results are consistent with the notion that NGF upregulates CHRM4 and, through CHRM4, activates AKT-MYCN signaling. Furthermore, ectopic CHRM4 cDNA expression upregulated neuroendocrine markers in LNCaP and C4-2 cells, whereas CHRM4 knockdown reduced neuroendocrine marker expression in PC3 cells. Notably, in PC3 cells, CHRM4 and neuroendocrine markers accumulated after NGF protein treatment. However, even in cells treated with NGF protein, CHRM4 knockdown cells showed decreased expression of neuroendocrine markers (Figure 1j). The researchers also found that CHRM4 and neuroendocrine marker levels were upregulated in MDV3100-resistant C4-2 cells. However, decreased levels of neuroendocrine markers were observed in CHRM4 knockdown cells. These results suggest that CHRM4 upregulation is associated with NEPC differentiation in an NGF-dependent manner.

Figure 1. i Protein levels of CHRM4, p-AKT, AKT, and MYCN in LNCaP and C4-2 cells after stable knockdown of CHRM4 and culture in medium containing CSS for 1 week. j Relative mRNA levels of CHRM4, CHGA, CHGB, SYP, and ENO2 in PC3 cells after stably expressing NC or CHRM4 shRNA vectors and treating the cells with 100 ng/ml NGF protein in medium containing CSS for 1 week. (Chen W Y, et al., 2021)