Bedside to bench: role of muscarinic receptor activation in ultrarapid growth of colorectal cancer in a patient with pheochromocytoma

Abstract

An elderly man with long-standing, nonresectable pheochromocytoma had rapid development of rectal adenocarcinoma despite close endoscopic surveillance. We determined that the patient's colorectal cancer overexpressed muscarinic receptor subtype 3, whereas his pheochromocytoma expressed choline acetyltransferase, an enzyme required to produce acetylcholine, which is a muscarinic receptor agonist. These findings suggested that acetylcholine release from the pheochromocytoma stimulated rapid growth of the rectal neoplasm. As proof of principle, we found that culture media conditioned by pheochromocytoma cells stimulates proliferation of a human colon cancer cell line, an effect attenuated by atropine, a muscarinic receptor inhibitor. Our observations provide both clinical and laboratory evidence that muscarinic receptor agonists promote the growth of colorectal neoplasia.

Keywords: ChAT; M3R; PCR; choline acetyltransferase; muscarinic receptor subtype 3; polymerase chain reaction.

Figure 1. Endoscopic Findings

Panel A shows our patient’s rectal polyp prior to initial polypectomy. Panel B displays the site six-months later when additional polypectomy revealed residual adenoma. Panel C shows a polypectomy scar after an additional three months. Multiple biopsies obtained at this time showed no adenomatous tissue. Panel D demonstrates the rectal adenocarcinoma found 28 months after the unremarkable sigmoidoscopy with biopsy (white arrows outline the proximal tumor margin).

Figure 2. Histopathological and Immunohistochemical Evaluation of Pheochromocytoma

Histopathological evaluation of the pheochromocytoma showed connective tissue with clusters and isolated pleomorphic tumor cells with abundant and granular cytoplasm (Panel A, H&E stain). Immunohistochemical staining for choline acetyltransferase (ChAT) shows strong cytoplasmic staining of most tumor cells in this field (Panel B, anti-ChAT stain). Two of five additional archival specimens stained positively for ChAT (Panels C & D, anti-ChAT stain). ChAT = choline acetyltransferase.

Figure 3. M3 muscarinic receptor (M3R) gene and protein expression

Panel A shows muscarinic receptor subtype gene expression in our patient’s colorectal cancer compared to adjacent normal colon tissue. The dashed line represents equivalence. Results were obtained by real-time PCR using the 2^−ΔΔCt comparative method using GAPDH as the relative control. Panel B shows immunohistochemical staining for M3R (see Methods) and reveals weak staining in normal colon mucosa (Panel B1) whereas robust staining is observed in a fragment of the tubular adenoma removed by endoscopic biopsy prior to progression to cancer (Panel B2). Staining for M3R in an endoscopic biopsy of the moderately well-differentiated colorectal cancer (Panel B3) was less intense than that observed in the polyp (Panel B2) but substantially greater than that observed in normal colon (Panel B1). M3R = muscarinic receptor subtype 3.

Figure 4. Results of Conditioned Media Experiments

Media exposed to PC-12 pheochromocytoma cells (‘conditioned media’) stimulates proliferation of H508 human colon cancer cells. H508 cell proliferation was determined by absorbance at 490 nm as described in Methods. The proliferative effects of ‘conditioned media’ were significantly attenuated in the presence of 5 μM atropine. Data represent means and standard errors from three independent experiments. At = atropine.