Characterization of the role of cholinergic receptor muscarinic type 3 (CHRM3) in driving cancer cell plasticity in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) represents a substantial global health burden. Apart from the limited choice of systemic treatments in advanced disease, key contributors to its dire prognosis include therapeutic resistance and recurrence, which are linked to the presence of a stem cell-like state in cancer cells, i.e., cancer stemness. In HCC, PROM1/CD133 marks a specific subset of cancer cells (cancer stem cells, CSCs) that are less differentiated and possess enhanced stem-like features, including self-renewal and multi-lineage differentiation to fuel tumor progression and heterogeneity. Understanding cancer stemness, particularly in the identification of actionable molecular targets, is essential for translating this knowledge into clinical benefits. The cholinergic receptor muscarinic type 3 (CHRM3) was identified as a potentially actionable regulator of cancer stemness in HCC based on its preferential expression in dissociated Prom1/CD133+ single cells but suppressed expression in Prom1/CD133-lineage-depleted tumors, performed on an NRAS/AKT hydrodynamic tail vein injection mice HCC model. In mice, CHRM3 expression is associated with tumor progression on both the transcriptomic and proteomic levels. Furthermore, the overexpression of CHRM3 in human HCC was then validated by immunohistochemistry on clinical HCC samples as well as the TCGA-LIHC and GSE94660 (HBV-associated human HCC) datasets. Subsequent functional experiments revealed that CHRM3-knockout in the SNU423 cell line abolished its spheroid-forming capacity and tumor-initiating potential with minimal impact on proliferative rate. Likewise, the administration of darifenacin, a small molecule inhibitor of CHRM3, restricted the spheroid-forming capacity in the SNU423, Huh7 and MHCC97L cell lines. Given that chemoresistance represents a closely associated feature of CSCs, cells were then subjected to darifenacin and sorafenib co-treatment in order to examine the sensitizing effect of CHRM3 inhibition. Indeed, from percent inhibition determined by the XTT assay and the Annexin V-apoptosis assay, a significant synergism was observed by calculation of the excess over Bliss and excess over HAS scores. In summary, this study presents in vitro evidence suggesting the possible role of CHRM3 in the regulation of CD133 CSCs through a series of functional experiments. Nonetheless, further in vivo assays and the elucidation of underlying molecular mechanisms are necessary to establish CHRM3 as a definite player in maintenance of a more cancer stemness state in HCC.