PURPOSE: In patients with peritoneal metastasis (PM) from gastric cancer (GC), chemotherapy is the treatment of choice. Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) are still being debated. This randomized, controlled, open-label, multicenter phase III trial (EudraCT 2006-006088-22; ClinicalTrials.gov identifier: NCT02158988 ) explored the impact on overall survival (OS) of HIPEC after CRS. PATIENTS AND METHODS: Adult patients with GC and histologically proven PM were randomly assigned (1:1) to perioperative chemotherapy and CRS alone (CRS-A) or CRS plus HIPEC (CRS + H). HIPEC comprised mitomycin C 15 mg/m² and cisplatin 75 mg/m² in 5 L of saline perfused for 60 minutes at 42°C. The primary end point was OS; secondary endpoints included progression-free survival (PFS), other distant metastasis-free survival (MFS), and safety. Analyses followed the intention-to-treat principle. RESULTS: Between March 2014 and June 2018, 105 patients were randomly assigned (53 patients to CRS-A and 52 patients to CRS + H). The trial stopped prematurely because of slow recruitment. In 55 patients, treatment stopped before CRS mainly due to disease progression/death. Median OS was the same for both groups (CRS + H, 14.9 [97.2% CI, 8.7 to 17.7] months v CRS-A, 14.9 [97.2% CI, 7.0 to 19.4] months; P = .1647). The PFS was 3.5 months (95% CI, 3.0 to 7.0) in the CRS-A group and 7.1 months (95% CI, 3.7 to 10.5; P = .047) in the CRS + H group. The CRS + H group showed better MFS (10.2 months [95% CI, 7.7 to 14.7] v CRS-A, 9.2 months [95% CI, 6.8 to 11.5]; P = .0286). The incidence of grade ≥3 adverse events (AEs) was similar between groups (CRS-A, 38.1% v CRS + H, 43.6%; P = .79). CONCLUSION: This study showed no OS difference between CRS + H and CRS-A. PFS and MFS were significantly better in the CRS + H group, which needs further exploration. HIPEC did not increase AEs.

Cholinergic signaling via the muscarinic M3 acetylcholine receptor (M3R) is involved in the development and progression of colorectal cancer (CRC). The present study aimed to analyze the blocking of M3R signaling in CRC using darifenacin, a selective M3R antagonist. Darifenacin effects were studied on HT-29 and SW480 CRC cells using MTT and BrdU assays, Western blotting and real time RT-PCR. In vivo, blocking of M3R was assessed in an orthotopic CRC xenograft BALB/c^nu/nu mouse model. M3R expression in clinical tumor specimens was studied by immunohistochemistry on a tissue microarray of 585 CRC patients. In vitro, darifenacin decreased tumor cell survival and proliferation in a dose-dependent manner. Acetylcholine-induced p38, ERK1/2 and Akt signaling, and MMP-1 mRNA expression were decreased by darifenacin, as well as matrigel invasion of tumor cells. In mice, darifenacin reduced primary tumor volume and weight (p < 0.05), as well as liver metastases, compared to controls. High expression scores of M3R were found on 89.2% of clinical CRC samples and correlated with infiltrative tumor border and non-mucinous histology (p < 0.05). In conclusion, darifenacin inhibited components of tumor growth and progression in vitro and reduced tumor growth in vivo. Its target, M3R, was expressed on the majority of CRC. Thus, repurposing darifenacin may be an attractive addition to systemic tumor therapy in CRC patients expressing M3R.

Innervation interacts with enteric immune responses. Chronic intestinal inflammation is associated with increased risk of colorectal cancer. We aimed to study potential extrinsic neuronal modulation of intestinal tumor development in a mouse model. Experiments were performed with male ApcMin/+ or wild type mice (4 weeks old, body weight approximately 20 g). Subgroups with subdiaphragmatic vagotomy (apcV/wtV), sympathetic denervation of the small intestine (apcS/wtS) or sham operated controls (apcC/wtC) were investigated (n = 6-14 per group). Three months after surgical manipulation, 10 cm of terminal ileum were excised, fixed for 48 h in 4% paraformaldehyde and all tumors were counted and their area determined in mm2 (mean ± standard error of the mean (SEM)). Whole mounts of the muscularis of terminal ileum and duodenum (internal positive control) were also stained for tyrosine hydroxylase to confirm successful sympathetic denervation. Tumor count in ApcMin/+ mice was 62 ± 8 (apcC), 46 ± 11 (apcV) and 54 ± 8 (apcS) which was increased compared to wildtype controls with 4 ± 0.5 (wtC), 5 ± 0.5 (wtV) and 5 ± 0.6 (wtS; all p < 0.05). For ApcMin/+ groups, vagotomized animals showed a trend towards decreased tumor counts compared to sham operated ApcMin/+ controls while sympathetic denervation was similar to sham ApcMin/+. Area covered by tumors in ApcMin/+ mice was 55 ± 10 (apcC), 31 ± 8 (apcV) and 42 ± 8 (apcS) mm2, which was generally increased compared to wildtype controls with 7 ± 0.6 (wtC), 7 ± 0.4 (wtV) and 7 ± 0.6 (wtS) mm2 (all p < 0.05). In ApcMin/+ groups, tumor area was decreased in vagotomized animals compared to sham operated controls (p < 0.05) while sympathetically denervated mice showed a minor trend to decreased tumor area compared to controls. Extrinsic innervation of the small bowel is likely to modulate tumor development in ApcMin/+ mice. Interrupted vagal innervation, but not sympathetic denervation, seems to inhibit tumor growth.

The relevance of estrogen receptor (ER) expression in pancreatic ductal adenocarcinoma (PDAC) is largely unknown. Clinical trials targeting ER with selective estrogen receptor modulators in pancreatic cancer did not show any benefit. Here, we analyze the impact of recently characterized ER isoform beta on survival in a cohort of patients with resected PDAC. Eighty-four patients having undergone pancreatic resection for PDAC at a single institution were identified. Tissue microarrays were constructed of archival tumor specimens. The expression of ER beta was determined by immunohistochemistry and quantified by a system established for estrogen receptor expression in breast cancer. ER beta expression was then correlated with clinicopathological parameters, and univariate and multivariate survival analyses were performed. Nuclear expression of ER beta was found in 31% of tumors. No significant correlation was found between ER beta expression and TNM status, tumor grade, age or sex. Univariate analysis revealed nodal metastasis and the expression of ER beta as factors correlating with a shorter overall survival and disease free survival. When comparing ER beta expression in patients surviving more than 24 months with those who died from the tumor within 12 or 24 months, respectively, a significantly lower ER beta expression was found in the long term survivors. In multivariate analysis, ER beta expression was demonstrated to be an independent predictor of shorter overall survival. In resected PDAC, expression of ER beta seems to correlate with poor prognosis. These data may help to identify patients who may benefit from additional systemic therapy including selective estrogen receptor modulators.

Purpose: Currently, the exact role of estrogen receptor (ER) signaling in pancreatic cancer is unknown. Recently, we showed that expression of phosphorylated ERβ correlates with a poor prognosis in patients with pancreatic ductal adenocarcinoma (PDAC). Here, we hypothesized that raloxifene, a FDA-approved selective ER modulator (SERM), may suppress PDAC tumor growth by interfering with ERβ signaling. To test this hypothesis, we studied the impact of raloxifene on interleukin-6/glycoprotein-130/signal transducer and activator of transcription-3 (IL-6/gp130/STAT3) signaling. Methods: Human PDAC cell lines were exposed to raloxifene after which growth inhibition was assessed using a BrdU assay. ER knockdown was performed using siRNAs specific for ERα and ERβ. The effects of raloxifene on IL-6 expression and STAT3 phosphorylation in PDAC cells were assessed by ELISA and Western blotting, respectively. In addition, raloxifene was administered to an orthotopic PDAC tumor xenograft mouse model, after which tumor growth was monitored and immunohistochemistry was performed. Results: Raloxifene inhibited the in vitro growth of PDAC cells, and this effect was reversed by siRNA-mediated knockdown of ERβ, but not of ERα, indicating ER isotype-specific signaling. We also found that treatment with raloxifene inhibited the release of IL-6 and suppressed the phosphorylation of STAT3 ^Y705 in PDAC cells. In vivo, we found that orthotopic PDAC tumor growth, lymph node and liver metastases as well as Ki-67 expression were reduced in mice treated with raloxifene. Conclusions: Inhibition of ERβ and the IL-6/gp130/STAT3 signaling pathway by raloxifene leads to potent reduction of PDAC growth in vitro and in vivo. Our results suggest that ERβ signaling and IL-6/gp130 interaction may serve as promising drug targets for pancreatic cancer and that raloxifene may serve as an attractive therapeutic option for PDAC patients expressing the ERβ isotype.

Interleukine-6 plays a key role in the progression and poor survival in pancreatic ductal adenocarcinoma (PDAC). The present study aimed to clarify if targeting the interleukin-6/glycoprotein-130 signaling cascade using the small-molecule gp130 inhibitor SC144 or raloxifene, a non-steroidal selective estrogen receptor modulator, enhances paclitaxel efficacy. MTT/BrdU assays or TUNEL staining were performed to investigate cell viability, proliferation and apoptosis induction in L3.6pl and AsPC-1 human pancreatic cell lines. In vivo, effects were studied in an orthotopic PDAC mouse model. Tumor specimens were analyzed by qPCR, immunohistochemistry and ELISA. Combination of paclitaxel/raloxifene, but not paclitaxel/SC144, enhanced proliferation and viability inhibition and increased apoptosis compared to single treatment in vitro. Synergy score calculations confirmed an additive influence of raloxifene on paclitaxel. In the PDAC mouse model, both combinations of raloxifene/paclitaxel and SC144/paclitaxel reduced tumor weight and volume compared to single-agent therapy or control. Raloxifene/paclitaxel treatment decreased survivin mRNA expression and showed tendencies of increased caspase-3 staining in primary tumors. SC144/paclitaxel reduced interleukin-6 levels in mice’s tumors and plasma. In conclusion, raloxifene or SC144 can enhance the anti-tumorigenic effects of paclitaxel, suggesting that paclitaxel doses might also be reduced in combined chemotherapy to lessen paclitaxel side effects.