Cholinergic signaling influences the expression of immune checkpoint inhibitors, PD-L1 and PD-L2, and tumor hallmarks in human colorectal cancer tissues and cell lines

Background

Cancer cells express immunosuppressive molecules, such as programmed death ligands (PD-L)1 and PD-L2, enabling evasion from the host’s immune system. Cancer cells synthesize and secrete acetylcholine (ACh), acting as an autocrine or paracrine hormone to promote their proliferation, differentiation, and migration.

Methods

We correlated the expression of PD-L1, PD-L2, cholinergic muscarinic receptor 3 (M3R), alpha 7 nicotinic receptor (α7nAChR), and choline acetyltransferase (ChAT) in colorectal cancer (CRC) tissues with the stage of disease, gender, age, risk, and patient survival. The effects of a muscarinic receptor blocker, atropine, and a selective M3R blocker, 4-DAMP, on the expression of immunosuppressive and cholinergic markers were evaluated in human CRC (LIM-2405 and HT-29) cells.

Results

The increased expression of PD-L1, M3R, and ChAT at stages III-IV was associated with a high risk of CRC and poor survival outcomes independent of patients’ gender and age. α7nAChR and PD-L2 were not changed at any CRC stages. Atropine and 4-DAMP suppressed the proliferation and migration of human CRC cells; induced apoptosis; and decreased PD-L1, PD-L2, and M3R expression in CRC cells via the inhibition of EGFR and the phosphorylation of ERK.

Conclusions

The expression of immunosuppressive and cholinergic markers may increase the risk of recurrence of CRC. These markers might be used in determining prognosis and treatment regimens for CRC patients. Blocking cholinergic signaling may be a potential therapeutic strategy for CRC through anti-proliferation and anti-migration via the inhibition of EGFR and the phosphorylation of ERK. These effects allow the immune system to recognize and eliminate cancer cells.