Panoply™ Human CHRM4 Over-expressing Stable Cell Line

Current treatment options for prostate cancer primarily focus on targeting androgen receptor (AR) signaling. Inhibiting AR may activate neuroendocrine differentiation and lineage plasticity pathways, thereby promoting the development of neuroendocrine prostate cancer (NEPC). Understanding the regulatory mechanisms of AR has important clinical implications for this most aggressive form of prostate cancer. Here, researchers elucidate the tumor suppressor role of AR and discover that activated AR directly binds to regulatory sequences of the muscarinic acetylcholine receptor 4 (CHRM4) and downregulates its expression. CHRM4 is highly expressed in prostate cancer cells after androgen deprivation therapy (ADT). CHRM4 overexpression may drive neuroendocrine differentiation in prostate cancer cells and is associated with immunosuppressive cytokine responses in the prostate cancer tumor microenvironment (TME). Mechanistically, CHRM4-driven AKT/MYCN signaling upregulates interferon α17 (IFNA17) expression in the prostate cancer TME following ADT. IFNA17 mediates feedback mechanisms in the tumor microenvironment by activating the CHRM4/AKT/MYCN signaling-driven immune checkpoint pathway and neuroendocrine differentiation of prostate cancer cells.

To investigate the role of CHRM4 in the progression of NED in prostate cancer, the researchers overexpressed or knocked down CHRM4 in AR-positive C4-2 cells and AR-negative PC3 cells, respectively. CHRM4 overexpression increased the mRNA and protein levels of NE markers in C4-2 cells compared to cells transfected with EVs (Figure 1A, C). Conversely, mRNA and protein levels of NE markers were significantly reduced in CHRM4-kD PC3 cells compared to control cells (Figure 1B, C). Furthermore, in analysis of a prostate cancer dataset, CHRM4 overexpression was positively correlated with a NEPC-responsive gene signature (Figure 1D). Next, the researchers assessed the role of CHRM4 in prostate cancer cells and found that CHRM4-overexpressing C4-2 cells exhibited increased cell migration and invasion through Matrigel (Figure 1E). In contrast, these effects were attenuated in CHRM4-kD PC3 cells (Figure 1F). The relevance of CHRM4-mediated proliferation was further assessed in C4-2 and PC3 cells. CHRM4-overexpressing cells showed upregulated proliferation compared to EV-expressing cells (Figure 1G), while cells with CHRM4-KD showed downregulated proliferation compared to cells expressing NC (Figure 1H). When mice were subcutaneously injected with PC3 cells containing CHRM4-KD, the researchers observed a significant reduction in tumor size and weight in mice bearing CHRM4-KD cells compared to mice bearing control cells (Figures 1I-K). These findings suggest that inhibition of CHRM4 may reduce the expression of NE markers, tumor growth, and functional features of malignant progression, and may contribute to the development of NED in prostate cancer.

Figure 1. Increased CHRM4 is associated with oncogenic features and neuroendocrine differentiation in prostate cancer. (Wen Y C, et al., 2023)